Immobilized Enzymes and

their application in Medicine.

Dr. Gul Shahnaz
Immobilization of enzymes
Immobilization of enzymes (or cells) refers to the technique of confining/anchoring the enzymes (or cells) in or
on an inert support for their stability and functional reuse.
Immobilized enzymes possess higher resistance to environmental changes and can be recovered/recycled easily when compared to the
free forms.
Problems and Limitations Associated with Industrial Enzymes
Enzyme immobilization methods
 Immobilization strategies entail fixing or entrapping enzymes within solid support materials.
Researchers have suggested several support materials, besides many beneficial approaches for the
immobilization of enzymes.

 The main function of the support is to stabilize the structures of enzymes and accordingly preserve their
efficacy to a great extent by rendering them more resistant to the surrounding environments.

 Enzyme immobilization permits an easy recovery of both the used enzymes and their support materials,
and this is particularly beneficial in the food, medical, and pharmaceutical applications.

 Enzymes in their immobilized forms possess much higher stability and are also easier to handle when
compared to their free forms.

 Additionally, the enzymatic reaction can occur in a nonaqueous medium where the solid supports
preserve the enzyme’s constituents and make them stronger, which enhances their catalytic activity and
renders them reusable for several times.

 Another benefit of the immobilization process is that the catalysts can alter from homogeneous to
heterogeneous forms after the enzymatic binding, which assists in separating the enzymes, producing
products with high purity
 Adsorption
• The enzyme here are adhere to carrier matrix due to hydrophobic
effects or by forming salt links.
• Binding is strong but may be weakened by substrate, pH etc.

• Commonly used matrices :-

ion exchange matrices, porous carbon,clays, hydrous metal oxides, polymeric
aromatic resins etc.
 Covalent binding
• The enzyme here are attached to certain matrix by forming covalent
bonds
• Binding is very strong. It generally occurs with side chain of the
enzyme. Lysine is very useful, as they are very reactive, usually
exposed to surface and very rarely occur at active site of enzyme.
• Commonly used matrices :-
agarose, cellulose, poly acrylamides etc.
• Activation by CNBr
 Cont….
• Activation by Ethyl chloroformate
 Cont….
• Immobilization of enzyme using Glutaraldehyde
Methods for immobilization

c) Entrapment d) Membrane confinement

 Entrapment

• The enzyme here are entrapped with in suitable gels or fibers,

with or with out covalent bonds.

• Commonly used matrices :-

Cellulose acetate fibers, calcium alginate etc.
Membrane confinement

• The enzyme molecule here are confined to semipermiable

membrane. Thus in a aqueous phase beg of membrane is free to
move with out allowing enzyme to come out.
 Advantages
• Enzymes are costly, Immobilization permits their repeated use.
• The product can be easily separated without any additional cost.
• Immobilized enzymes can be used in nonaqueous system.
• Continuous production system can be used.
• Thermo stability of some enzyme can be increased by immobilization.
• Enzyme can be used at much higher concentration then free enzyme.
Disadvantages

• Additional cost.
• It sometime affects stability and activity of enzyme.
• This approach can not be used when one of the substrates is
insoluble.
• Some immobilization methods restricts the diffusion of substrate.
Applications of immobilized enzymes

Enzyme Substrate Product

Glucose isomerase D-glucose HFCS
Invertase Sucrose Invert sugar
Lipase Vegetable oil Cocoa butter substitute
Lactase Milk and whey Lactose free milk &whey
Nitrile hydratase Acrylonitrile Acrylamide
Raffinase Raffinose Raffinose free solution
Glucoamylase Dextrins D-glucose
Genetic Engineering and its
Application in Medicine

Dr. Gul Shahnaz

 What is genetic engineering?

Genetic engineering is the direct modification of an

organism’s genome, which is the list of specific traits
(genes) stored in the DNA.
Changing the genome
enables engineers to give
desirable properties to
different organisms.
Organisms created by
genetic engineering
are called genetically modified organisms (GMOs).
 History of GMO Development
1973: created first genetically
modified bacteria
1974: created GM mice
1982: first commercial
development of GMOs
(insulin-producing bacteria)
1994: began to sell
genetically modified food
2003: began to sell GMOs as
pets (Glofish)
 What is the GMO process?
• All genetic changes affect the protein synthesis of the
organism.
• By changing which proteins are produced, genetic
engineers can affect the overall traits of the organism.
• Genetic modification can be completed by a number of
different methods:
• Inserting new genetic
material randomly or in
targeted locations
• Direct replacement of
genes (recombination)
• Removal of genes
• Mutation of existing genes
 Gene Therapy
How is it done?
Growth hormone and therapy
• Growth hormone, also known as
somatotropin, is a protein hormone of about
190 amino acids that is synthesized and
secreted by cells called somatotrophs in the
anterior pituitary. It is a major participant in
control of several complex physiologic
processes, including growth and metabolism.
Growth hormone is also of considerable interest
as a drug used in both humans and animals.
Recombinant Protein
(Insulin)
 Production of recombinant insulin

Attemps to produce insulin by recombinant DNA technology started in late 1970s.

The basic technique consisted of inserting human insulin gene and the promoter gene of
lac operon on to the plasmids of E. coli.

By this method human insulin was produced.

It was in July 1980, seventeen human volunteers were, for the first time, administered recombinant insulin for
treatment of diabetes at Guy's Hospital, London. And in fact, insulin was the first ever pharmaceutical product
of recombinant DNA technology administered to humans.

Recombinant insulin worked well, and this gave hope to scientists that DNA technology could be successfully
employed to produce substances of medical and commercial importance. An approval, by the concerned
authorities, for using recombinant insulin for the treatment of diabetes mellitus was given in 1982.

And in 1986, Eli Lilly company received approval to market human insulin under the trade name Humulin.
 Production of recombinant insulin

Isolation of plasmid and Human insulin insert the human insulin gene Formation of r-DNA
gene into the plasmid.

Put the “recombinant” bacteria Return the plasmid to the bacteria

Harvest and purify the Recombinant bacteria use in large fermentation tanks (Transformation)
substance for use as a the gene to begin
medicine. producing human insulin.
 Production of recombinant insulin

The orginal technique of insulin synthesis in E. coli has undergone

several changes, for improving the yield. e.g. addition of signal
peptide, synthesis of A and B chains separately etc.

The procedure employed for the synthesis of two insulin chains A

and B is illustrated in Fig.

The genes for insulin A chain and B chain are separately inserted to
the plasmids of two different E. coli cultures. The lac operon system
(consisting of inducer gene, promoter gene, operator gene and
structural gene Z for β-galactosidase) is used to express both the
genes.

The presence of lactose in the culture medium induces the synthesis

of insulin A and B chains in separate cultures.

The so formed insulin chains can be isolated, purified and joined

together to give a full-fledged human insulin.
Source: Adopted from Biochemistry (U. Satyanarayna)
What is Protein therapeutics?

It is currently estimated that there are 25,000–40,000 different

genes in the human genome, viewed from the perspective of
disease mechanisms, as disease may result
when any one of these proteins contains
mutations or other abnormalities, so it
gives a tremendous opportunity for
Protein therapeutics to alleviate
these disease.
Why protein therapeutics?

Proteins cannot be mimicked by simple chemical compounds.

There is often less potential for protein therapeutics to interfere

with normal biological processes and cause adverse effects.

It is often well tolerated and are less likely to elicit immune responses.

Provide effective replacement treatment without the need for gene therapy

Time of protein therapeutics may be faster

History and Development

2002 and beyond BIOTECHNOLOGY IMPROVEMENT

1992–1999 BIOTECHNOLOGY INDUSTRY

1986–1991 MORE BIOTECHNOLOGY SUCCESSES

Pre-1986 A STAR IS BORN

The Evolution of Protein Therapeutics : A Timeline

1953 First accurate model of DNA suggested

1982 Human insulin, created using recombinant DNA technology

1986 Interferon alfa and muromonab-CD3 approved

1993 CBER's Office of Therapeutics Research and Review (OTRR) formed

1997 First whole chimeric antibody, rituximab, and first humanized
antibody, daclizumab, approved

2002 Market for biotechnology products represents approximately $30

billion of $400 billion in yearly worldwide pharmaceutical sales

2006 An inhaled form of insulin (Exubera) approved, expanding protein

products into a new dosage form.
 THE IMMUNE SYSTEM

The Latin term “IMMUNIS” means EXEMPT, referring to

protection against foreign agents.

DEFINITION: - The integrated body system of organs, tissues,

cells & cell products that differentiates self from non – self &
neutralizes potentially pathogenic organisms.
(The American Heritage Stedman's Medical Dictionary)

The Immune System consists of

1. Innate Immunity Primary Response
2. Acquired Immunity Secondary Response
CELLS OF THE IMMUNE SYSTEM
FUNCTIONING OF THE IMMUNE SYSTEM
HUMORAL (ANTIBODY MEDIATED) IMMUNE RESPONSE CELL MEDIATED IMMUNE RESPONSE
ANTIGEN (1ST EXPOSURE)
ENGULFED BY

MACROPHAGE ANTIGENS
FREE
ANTIGENS DISPLAYED
BECOMES BY
DIRECTLY
INFECTED
ACTIVATE APC CELLS
ACTIVATE
STIMULATES

HELPER CYTOTOXIC
B CELLS T CELLS
STIMULATES STIMULATES T CELL

MEMORY
GIVES RISE TO HELPER T GIVES RISE TO
CELLS
STIMULATES STIMULATES
STIMULATES

ANTIGEN (2nd EXPOSURE)

PLASMA MEMORY STIMULATES ACTIVE
MEMORY
CELLS B CELLS T CELLS CYTOTOXIC T
CELL

SECRETE ANTIBODIES
 IMMUNOTHERAPY

Treatment of the disease by Inducing, Enhancing or

Suppressing the Immune System.

Active Immunotherapy: - Passive Immunotherapy: -

It stimulates the body’s own
immune system to fight the
disease.
It does not rely on the body to
attack the disease, instead they
use the immune system
components ( such as
antibodies) created outside the
body.
 What are antibodies
An antibody is a protein used by the immune system to identify and neutralize foreign objects
like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target.

Monoclonal antibodies (mAb) are antibodies that are identical because they were produced
by one type of immune cell, all clones of a single parent cell.

Polyclonal antibodies are antibodies that are derived from different cell lines.

Isotypes
According to differences in their heavy chain constant domains, immunoglobulins are
grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE.

IgG: IgG1 (66%), IgG2 (23%), IgG3 (7%) and IgG4 (4%) , blood and tissue liquid.
IgA:IgA1 (90%) and IgA2 (10%), stomach and intestines
IgM: normally pentamer, ocassionally hexamer, multiple immunoglobins linked with disulfide
bonds
IgD:1% of proteins in the plasma membranes of B-lymphocytes, function unknown
IgE: on the surface of plasma membrane of mast cells, play a role in immediate hypersensitive
and denfensive for parasite
 How do monoclonal antibody drugs work

1- Make the cancer cell more visible to the immune

system.
The immune system attacks foreign invaders in your body, but it doesn't
always recognize cancer cells as enemies. A monoclonal antibody can be
directed to attach to certain parts of a cancer cell. In this way, the antibody
marks the cancer cell and makes it easier for the immune system to find.

The monoclonal antibody drug Rituximab attaches to a specific protein (CD20)

found only on B cells, one type of white blood cell. Certain types of lymphomas
arise from these same B cells. When Rituximab attaches to this protein on the
B cells, it makes the cells more visible to the immune system, which can then
attack. Rituximab lowers the number of B cells, including your healthy B cells,
but your body produces new healthy B cells to replace these. The cancerous B
cells are less likely to recur.
RITUXIMAB (Rituxan)

• A recombinant chimeric murine/human antibody directed against the

CD20 antigen, a hydrophobic transmembrane protein located on normal
pre-B and mature B lymphocytes.

• Following binding, rituximab triggers a host cytotoxic immune response

against CD20-positive cells.

• Rituximab is used in the treatment of B-cell Non-

Hodgkin's lymphomas.
• MECHANISM OF ACTION:

• Complement fixation
• ADCC
• Direct cytotoxicity via apoptotic pathway activated by binding to CD20.
 FDA-APPROVED MONOCLONAL ANTIBODIES TARGETS and INDICATIONS
Antibody Trade Name Target Antigen FDA Indication

Rituximab Rituxan CD20 B-NHL

90Y-Ibritumomab Zevalin CD20 B-NHL

131I-tositumomab Bexxar CD20 B-NHL

Alemtuzumab Campath CD52 CLL

Gemtuzumab Mylotarg CD33 AML

ozogamicin
Cetuximab Erbitux EGFR metastatic
CRC
Panitumumab ABX-EGF EGFR metastatic
CRC
Trastuzumab Herceptin HER2/neu Metastatic
Br CA
Production of Recombinant Vaccine
 Production of recombinant Vaccine

The recombinant vaccines are an important group of therapeutic products.

A number of vaccines are now available for animals, and human which is going to have a major impact in the
healthcare industry.

One of the initial vaccines produced by rDNA method involves the cloning of the surface antigen of the
hepatitis B virus (HBsAg) in the yeast S. cerevisiae under the control of the alcohol dehydrogenase promoter.

A number of recombinant vaccines are now commercially prepared by the recombinant DNA technology,
where only the outside coat protein of the microorganism is expressed in the host to create the vaccine.

The expressed protein can then be purified from the recombinant host and used for inoculation.

This method has the advantage of safe delivery of antigen without transferring the actual disease-causing
microbe to the host. Currently recombinant vaccines for the hepatitis B virus, herpes type 2 viruses, and
malaria is under trial for use in future.
Table: list of diseases along with
the pathogenic organisms for
which recombinant vaccines are
developed

Source: Adopted from Biochemistry (U. Satyanarayna)

Pharmaceutical
Biotechnology
Dr. Gul Shahnaz
What Is Biotechnology and What Does It Mean to You?

• Biotechnology – using living organisms, or the products

of living organisms, for human benefit to make a product
or solve a problem

• Historical Examples
– Fermentation
– Selective breeding
– Use of antibiotics
 Why are new drugs needed?

 unmet medical need; new diseases (Corona Virus, Swine flu; AIDS,
Alzheimer’s; obesity); low efficacy (dementia, cancer); side effects
(antidepressants, antipsychotics)
 downstream health costs; (Alzheimer’s; spinal injury)
 cost of therapy; (Interleukins)
 sustain industrial activity; pharmaceutical industry employs
thousands and makes a massive contribution to overseas earnings);
patent expiry
 Drug resistance:
 The Long Road to a New Medicine

* 10-15 Year Process

Registration
New
Full Clinical
Data Medicine
Development Analysis

Candidate Medicine Tested in

Studies in 100-300 3-10,000 Patients (Phase III)
Patients – POC
(Phase II) Large Amounts of
Candidate Medicine
Synthesized Extensive
Safety
Studies
Studies in Healthy
Volunteers – safety Candidate
and POM (Phase I) Formulations
Exploratory Developed

Development

Early
Safety
Project Team Synthesis of Studies
and Plans Screening
Compounds

Discovery
Idea
 Involves high cost and time

~100 Discovery Approaches

7,000,000
Compounds Screened

Preclinical
Pharmacology

Preclinical Safety
1-2
Clinical Pharmacology
& Safety
Products

Discovery Exploratory Development Full Development

Phase I Phase II Phase III

0 5 10 15

Idea 10 - 15 Years Drug

Structure Based Drug
Design

Determine Protein Structure

Identify Interaction Sites

Discovery or design of De Novo Design 3D Database
molecules that interact
with biochemical targets Evaluate Structure
of known 3D structure
Synthesize Candidate
Test Candidate

Lead Compound
Drug Targets
Molecule or structure within the organism linked to a particular disease, whose activity can be
modified by a drug.
Currently used drug targets

J. Drews Science 287, 1960 -1964

(2000)
 What Is Biotechnology and What Does It
Mean to You?
• Example of Biotechnology – Selective Breeding
(a) (b)

Normal zebrafish "Casper" zebrafish – made

by selective breeding
• What feature of Casper makes it a "model organism" to study migration
of cancer cells compared to wildtype fish?
 What Is Biotechnology and What Does It
Mean to You?
• Based on this tree,
can you become
successful in the
biotech industry
only studying
biology?
What Is Biotechnology and What Does It Mean to You?

• Modern Examples
– Gene cloning
– Genetic engineering
– Recombinant DNA technology
– Human Genome Project
 1.1 What Is Biotechnology and What Does It
Mean to You?
• Example of "modern" biotechnology:
– recombinant DNA technology started modern biotech as an industry
• Examples of applications
– development of disease-resistant plants
– food crops that produce greater yields
– "golden rice" engineered to be more nutritious
– genetically engineered bacteria that can degrade environmental pollutants
• Work in groups to come up with more examples of
applications
 1.1 What Is Biotechnology and What Does It
Mean to You?
• Use genetically modified cultured cells to make protein of
interest
• Products of Modern Biotechnology
– Example of proteins created by gene cloning called
recombinant proteins
Functional Genomics and Proteomics
Proteomics studies biological systems based
on global knowledge of protein sets
(proteomes).
Functional genomics studies biological
functions of proteins, complexes, pathways
based on the analysis of genome sequences.
Includes functional assignments for protein
sequences.
Genome Transcriptome Proteome Metabolome
“-ome”
CGTCCAA
CTGACGT
DNA Genome “Genomics”
CTACAAG
TTCCTAA DNA sequencing
GCT

Transcriptome
RNA
cDNA arrays
Cell
functions

Proteins Proteome “Proteomics”

2D PAGE, HPLC

Reactome, the chemical reactions involving a nucleotide

 Protein Chemistry/Proteomics
Protein Chemistry
• Individual proteins
• Complete sequence analysis
• Emphasis on structure and
function
• Structural biology
Proteomics
• Complex mixtures
• Partial sequence analysis
• Emphasis in identification by
database matching
• System biology
Why are we studying proteins?

Proteins are the mediators of functions in the cell

Deviations from normal status denotes disease

Proteins are drug/therapeutic targets

 Proteomics and biology
/Applications
Protein Expression Profiling
Identification of proteins in a particular
Proteome Mining sample as a function of a particular
Identifying as many as state of the organism or cell
possible of the proteins in Post-translational
your sample modifications
Identifying how and
where the proteins are
modified
Functional
proteomics

Protein-protein
interactions Protein-
Protein quantitation network mapping
Determining how the
or differential proteins interact with
Structural
analysis each other in living
Proteomics systems
Tools of Proteomics

Protein separation technology

Simplify complex protein mixtures
Target specific proteins for analysis

Mass spectrometry (MS)

Provide accurate molecular mass measurements
of intact proteins and peptides

Database
Protein, EST, and complete genome sequence
databases

Software collection
Match the MS data with specific protein
sequences in databases
The Proteome

The proteome in any cell represents a subset of all possible gene

products
Not all the genes are expressed in all the cells.
It will vary in different cells and tissue types in the same organism and
between different growth and developmental stages
The proteome is dependent on environmental factors, disease, drugs,
stress, growth conditions.

• Cycle of Proteins
• Proteins as Modular Structures – motifs, domains
• Functional Families
• Genomic Sequences
• Protein Expression /Protein level
 Life cycle of a protein
Information found in DNA is used for
synthesis of the proteins

mRNA Protein Folding Translocation

to specific subcellular or
extracellular compartments

Posttranslational Processing
Proteolytic Cleaveage
Degradation Acylation
Methylation
Damage
-free radicals Phosphorylation
Sulfation
Selenoproteins
Environmental Ubiquination
-chemicals Glycolisation
radioactiivty
 Molecular Structures
Primary structure a chain of amino acids Amino acids vary in their ability
to form the various secondary
Secondary structure three dimensional form, formally structure elements.
defined by the hydrogen bonds of the polymer

Amino acids that prefer to adopt helical conformations in

-helices proteins include methionine, alanine, leucine, glutamate
and lysine ("MALEK" in amino acid 1-letter codes)

The large aromatic residues (tryptophan, tyrosine and

-sheets phenylalanine) and Cβ-branched amino acids (isoleucine,
valine and threonine) prefer to adopt -strand
conformations.

Confer similar properties or functions when

they occur in a variety of proteins
 Sequence alignment

Sequence alignment is a way of arranging primary sequences (of

DNA, RNA, or proteins) in such a way as to align areas sharing
common properties.

The degree of relatedness, similarity between the A software tool used for general
sequences is predicted computationally or sequences alignment tasks is
statistically ClustalW
 General workflow of proteomics
analysis
separation
proteins digestion
MALDI, MS/MS

digestion

Identification
peptides
(LC)-MS/MS

ESI-MS
Electrospray Ionization tandem MS

MALDI-TOF
Matrix Assisted Laser Desorption
Ionization –Time of Flight
 Transcriptomes
• Genome: all of hereditary information encoded in
the DNA (or RNA)
• Transcriptome: set of all mRNAs ("transcripts”)
produced from a genome
• Term can be applied to:
complete set of transcripts for a given organism
specific subset of transcripts present in a
particular cell type or under specific growth
conditions
• Transcriptome varies because it reflects genes that
are actively expressed at any given time
 Genomics, ‘Omics & Technology
• Molecular biology: major scientific discipline for
past ~50 years
• Genomics = “analysis of genomes”: became
important science during 1990’s
• Analyses of various other biological molecules have
developed into their own scientific disciplines; e.g.
Metabolomics = “analysis of metabolites”, etc.
• Transcriptomics/Proteomics: developed during past
10-15 years
• Bioinformatics: has developed as major branch of
science - enables efficient analysis of data from
“omics” experiments
 Genomics & Technology
• Significance of “omics” coincides with dramatic
improvements in different technologies:
molecular biology: increased range of
approaches for purification and manipulation of
proteins and nucleic acids
computers: required for gathering and analysis
of data
internet: allows data to be shared, quickly and
easily
• All developments have increased speed and cost-
effectiveness - available to much wider audience
 Transcriptomics

• Transcriptomics uses high-

throughput techniques
based on DNA microarrays
• For further details about
microarrays see Lucchini et
al., Microbiology, 147, 1403-
1414 (2001)

Nelson & Cox, “Lehninger, Principles of

Biochemistry”, 4th edn, 2004, p. 328
 Transcriptomics

Nelson & Cox, “Lehninger, Principles of

Biochemistry”, 4th edn, 2004, p. 328

SYLICA 'Omic Technologies – Bowater Feb 2013

 Transcriptomics

Nelson & Cox, “Lehninger, Principles of

Biochemistry”, 4th edn, 2004, p. 328

SYLICA 'Omic Technologies – Bowater Feb 2013

 Transcriptomics

Nelson & Cox, “Lehninger, Principles of

Biochemistry”, 4th edn, 2004, p. 328

SYLICA 'Omic Technologies – Bowater Feb 2013

Transcriptomics

Nelson & Cox, “Lehninger, Principles of

Biochemistry”, 4th edn, 2004, p. 328
Transcriptomics

Nelson & Cox, “Lehninger, Principles of

Biochemistry”, 4th edn, 2004, p. 328
 Transcriptomics
•Experiments performed
under different
conditions
•Determines effect of
conditions on
expression

•Produces huge amount

of data
•Lots of repeats required
- expensive
Nelson & Cox, “Lehninger, Principles of
Biochemistry”, 4th edn, 2004, p. 328
 Polymerase Chain Reaction (PCR)
• Used to amplify DNA in the test tube
– Can amplify regions of interest (genes) within DNA
– Can amplify complete circular plasmids
• Mix together
– Target DNA
– Primers (oligonucleotides complementary to target)
– Nucleotides: dATP, dCTP, dGTP, dTTP
– Thermostable DNA polymerase
• Place the mixture into thermocycler
– Melt DNA at ~95°C
– Cool to ~ 50–60°C, primers anneal to target
– Polymerase extends primers in 5’3’ direction
– After a round of elongation is done, repeat steps
 General Steps of PCR

SYLICA 'Omic Technologies – Bowater Feb 2013

 General Steps of PCR
•Repeat steps 1–3 many times:
Photolitographic Synthesis of DNA
 DNA Microarrays: Applications

DNA microarrays allow simultaneous screening of

many thousands of genes: high-throughput screening
• Genome-wide genotyping
– Which genes are present in this individual?
• Tissue-specific gene expression
– Which genes are used to make proteins?
• Mutational analysis
– Which genes have been mutated?

SYLICA 'Omic Technologies – Bowater Feb 2013

 Adaptations to PCR
• Reverse Transcriptase PCR (RT-PCR)
– Used to amplify RNA sequences
– First step uses reverse transcriptase to convert
RNA to DNA
• Quantitative PCR (Q-PCR)
– Used to show quantitative differences in gene
levels
qPCR
 Proteomes
• Proteome: set of all proteins produced under a given
set of conditions
• Term can be applied to:
complete set of proteins for a given organism
specific subset of proteins present in a particular
cell type or under specific growth conditions
• Proteome varies because it reflects genes that are
actively expressed at any given time
• Proteomics analyses many samples using 2D-
electrophoresis and mass spectrometry
• High-throughput, but less than transcriptomics
 Separation techniques
Separation techniques used with intact proteins

1D- and 2D-SDS PAGE

Separating intact proteins to take
Preparative IEF isoelectric advantage of their diversity in physical
focusing properties

HPLC

Separation techniques for peptides

MS-MS
HPLC (MudPIT)
SELDI

Differential display proteomics

Difference gel electrophoresis (DIGE)
Isotope-coded affinity tagging (ICAT)
 Enrichment /Fractionation

For the detection of low-abundance proteins, a separation of

complex mixtures into fractions with fewer components is
necessary

•Enrichment from larger volumes Selective precipitation

Selective centrifugation
Preparative approaches

•Combination of 2DE with LC

•Multi-dimensional LC
 Protein extraction
Detergents: solubilize membrane proteins-
separation from lipids

Reductants: Reduce S-S bonds

Denaturing agents: Disrupt protein-protein

interactions-unfold proteins

Enzymes: Digest contaminating molecules (nucleic

acids etc)

Protease inhibitors

Aim: High recovery-low contamination-compatibility with separation method

 Protein digestion
Trypsin
Why digest the protein?
Cleaves at lysine and
Accuracy of mass measurements
arginine, unless either is
Suitability followed by proline in C-
terminal direction
Sensitivity

The ideal protein digestion approach

would cleave proteins at certain
specific amino acid residues to yield
fragments that are most compatible Good activity both in gel digestion and
with MS analysis. in solution

Peptide fragments of between Other enzymes with more or

6 – 20 amino acids are ideal less specific cleavage:
for MS analysis and database Chymotrypsin
comparisons. Glu C (V8 protease)
Lys C
Asp N
 Separation by LC
Salt
gradient UV detector

column

EC detector
waste

Number of peaks indicates the complexity of starting material

Peak position (i.e. elution time) may provide qualitative information about the sample
(comparison with standards)

Peak area may provide information on relative concentration of components.

If coupled to MS protein identification (MW) can be provided

modified:www.dcu.ie/chemistry/ssg/images/Te
chni7.gif
 Multidimensional HPLC

Mud PIT
Multidimensional Protein Identification Techniques or Tandem HPLC
the combination of dissimilar separation modes will allow a greater resolution
of peptides in mixture.

Ion-exchange Reversed phase

•Reversed phase, hydrophobicity

•Ion exchange, net positive/negative charge
•Size exclusion, peptide size, molecular weight
•Affinity chromatography, interaction with specific
functional groups
 Gel Electrophoresis
• Electrophoresis separates molecules by size
• Resolution is limited

Berg, Tymoczko & Stryer, “Biochemistry”,

6th edn, 2006, p. 71
 Gel electrophoresis
Classical process
High resolving power: visualization of thousands of protein forms
Quantative
Identifying proteins within proteome
Up/ down regulation of proteins
Detection of post-translational modifications
Coomassie blue stained gels

Protein fixing and staining or blotting

General detection methods (staining)
Organic dye – and silver based methods Coomassie blue, Silver
Radioactive labeling methods
Reverse stain methods
Fluorescence methods (Supro Ruby)
Ruby red
Gel scanning
(storage of image in a database)
Silver stained
 Isoelectric point

•Proteins are amphoteric molecules

i.e. they have both acidic and basic functional groups

•pI= isoelectric point, is where the protein does not have any net
charge

•The protein charge depends on the pH of the solution.

 Isoelectric Focusing
• Electrophoresis across a pH gradient
• Proteins migrate to their isoelectric pH

Berg, Tymoczko & Stryer, “Biochemistry”,

6th edn, 2006, p. 73
 1st dimension
IsoElectric Focusing, IEF
Immobilized pH gradients (IPGs)
A pH gradient is generated by a limited
number of well defined chemicals
(immobilines) which are co-
polymerized with the acrylamide
matrix.

Migration of proteins in a pH
gradient: protein stop at pH=pI

Loading quantities (18 cm strip)

Analytical run: 50-100 μg
Individual strips: Use narrow range IPG strips to
Micropreparative runs: 0,5 – 10 mg
focus on particular pI range
24,18,11,7 cm long
3 mm wide
0,5 mm thickness
 2nd dimension
pI
The strip is
loaded onto a
SDS gel
pH 10 Mw
pH 3

Staining !

Proteins that were

separated on IEF gel
are next separated in
the second dimension
based on their
molecular weights.
 Two-dimensional Gel
Electrophoresis
• Protein sample
initially fractionated
in one dimension by
isoelectric focusing
• SDS-PAGE
performed
perpendicular to
original direction
• Separates proteins
according to pI and
mass Berg, Tymoczko & Stryer, “Biochemistry”,
6th edn, 2006, p. 74
SYLICA 'Omic Technologies – Bowater Feb 2013
Two-dimensional Gel
Electrophoresis

• Proteins from E.
coli separated by
2D-electrophoresis
• >1,000 proteins
can be resolved

Berg, Tymoczko & Stryer, “Biochemistry”,

6th edn, 2006, p. 74
 Limitations/difficulties with the 2D gel
Reproducibility
Samples must be run at least in triplicate to rule out effects from
gel-to-gel variation (statistics)

Small dynamic range of protein staining as a detection

technique- visualization of abundant proteins while less abundant
might be missed.

Posttranscriptional control
Co-migrating spots forming a complex mechanisms
region
Incompatibility of some proteins with the
first dimension IEF step (hydrophobic
proteins)
Streaking and smearing
Marginal solubility leads to protein
precipitation and degradation- smearing
(Glycolysation, oxidation)
Weak spots and background
 A Mass Spectrometer
source analyzer detector

The sample has to be introduced into the ionization source of the instrument. Once inside the
ionization source the sample molecules are ionized, because ions are easier to manipulate than
neutral molecules.

These ions are extracted into the analyzer region of the mass spectrometer where they are separated
according to their mass (m)-to-charge (z) ratios (m/z).

The separated ions are detected and this signal sent to a data system where the m/z ratios are stored
together with their relative abundance for presentation in the format of a m/z spectrum.

The analyzer and detector of the mass spectrometer, and often the ionization source too, are
maintained under high vacuum to give the ions a reasonable chance of traveling from one end of the
instrument to the other without any hindrance from air molecules.
 ..consists of..

source analyzer detector

Source -produces the ions from the sample (vaporizationMALDI, Matrix-Assisted Laser Desorption and
/ionization) Ionisation

ESI, ElectroSpray Ionisation

Mass Anlyzer - resolves ions based on their
mass/charge (m/z) ratio
Generate different, but
Detector –detection of mass separated ions complementary information
 Mass Spectrometry
• MALDI-TOF mass spectrometry
• Protein sample is ionized and exposed to electrical field
• Ions travel according to size

Berg, Tymoczko & Stryer, “Biochemistry”,

6th edn, 2006, p. 94

SYLICA 'Omic Technologies – Bowater Feb 2013

 MALDI
Matrix Assisted Laser Desorption and Ionisation
laser
ions
Peptides co-crystallised with matrix
++
+
+
- +
+ Produces singly charged protonated molecular
-
-
ions
High throughput
Single proteins

Rapid procedure, high rate of sample

throughput
large scale identification (“first look at a
sample”)
 TOF
Time of flight

Measures the time it takes for the ions to

fly form one end to other and strike the
detector.
The speed with which the ions fly down
the analyzer tube is proportional to their
m/z values.
The greater the m/z the faster they fly
Separate ions o f different m/z based on
flight time
Fast
Requires pulsed ionization
 MALDI-TOF
Matrix-assisted laser desorption ionization-time of flight

++
+ +
+ - +
-
-
TOF analyzer

Quick, easy, inexpensive Low reproducibility and repeatability of

single shot spectra (Averaging)
Highly tolerant to contaminents
Low resolution
High sensitivity
Matrix ions interfere in the low max range
Good accuracy in mass determination
Compatible with robotic devices for
high-throughput proteomics work
Best suited to measuring peptide masses
 MALDI-TOF Mass Spectrum
• MALDI-TOF gives good estimates of molecular weights
• Can be used to identify a few proteins within a mixture

Berg, Tymoczko & Stryer, “Biochemistry”,

6th edn, 2006, p. 94
 MALDI-TOF data
Every peak corresponds to the exact mass (m/z) of a peptide ion

112.1
234.4

Peak List = List of masses

890.5
1296.9
1876.4
= fingerprint

1987.5
…….

Modified from http://plantsci.arabidopsis.info/pg/day3practical1.ppt

 Proteomic Analysis by Mass
Spectrometry
• Proteins separated by 2D
electrophoresis
• Single proteins eluted
• Digestion with trypsin
will give fragments with
unique set of sizes
• Sizes identified by mass
spectrometry and
matched to database
• Allows identification of
unknown proteins Berg, Tymoczko & Stryer, “Biochemistry”,
6th edn, 2006, p. 95
 mAbs

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/kmab20

High concentration formulation developability

approaches and considerations

Jonathan Zarzar, Tarik Khan, Maniraj Bhagawati, Benjamin Weiche, Jasmin

Sydow-Andersen & Alavattam Sreedhara

To cite this article: Jonathan Zarzar, Tarik Khan, Maniraj Bhagawati, Benjamin Weiche, Jasmin
Sydow-Andersen & Alavattam Sreedhara (2023) High concentration formulation developability
approaches and considerations, mAbs, 15:1, 2211185, DOI: 10.1080/19420862.2023.2211185

To link to this article: https://doi.org/10.1080/19420862.2023.2211185

© 2023 Genentech, Inc. Published with

license by Taylor & Francis Group, LLC.

Published online: 16 May 2023.

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MABS
2023, VOL. 15, NO. 1, 2211185
https://doi.org/10.1080/19420862.2023.2211185

REVIEW

High concentration formulation developability approaches and considerations

Jonathan Zarzara, Tarik Khanb, Maniraj Bhagawatic, Benjamin Weichec, Jasmin Sydow-Andersenc,
and Alavattam Sreedharaa
a
Pharmaceutical Development, Genentech Inc, South San Francisco, CA, USA; bPharma Technical Development Europe, F. Hoffmann-La Roche Ltd,
Basel, Switzerland; cLarge Molecule Research, Pharma Research and Early Development (pRED), Roche Diagnostics GmbH, Penzberg, Germany

ABSTRACT ARTICLE HISTORY

The growing need for biologics to be administered subcutaneously and ocularly, coupled with certain Received 27 January 2023
indications requiring high doses, has resulted in an increase in drug substance (DS) and drug product (DP) Revised 21 April 2023
protein concentrations. With this increase, more emphasis must be placed on identifying critical physico- Accepted 2 May 2023
chemical liabilities during drug development, including protein aggregation, precipitation, opalescence, KEYWORDS
particle formation, and high viscosity. Depending on the molecule, liabilities, and administration route, Aggregation; developability;
different formulation strategies can be used to overcome these challenges. However, due to the high high concentration
material requirements, identifying optimal conditions can be slow, costly, and often prevent therapeutics formulations; in-silico;
from moving rapidly into the clinic/market. In order to accelerate and derisk development, new experi­ physical instability;
mental and in-silico methods have emerged that can predict high concentration liabilities. Here, we subcutaneous
review the challenges in developing high concentration formulations, the advances that have been made administration; viscosity
in establishing low mass and high-throughput predictive analytics, and advances in in-silico tools and
algorithms aimed at identifying risks and understanding high concentration protein behavior.

Introduction
Monoclonal antibodies (mAb), and other protein-based thera­
peutics, have become a huge success, as exemplified by the more
than 100 different antibodies approved in the United States.1,2
The growing need for biologics to be administered subcuta­
neously and ocularly, which limits the total volume of drug that
can be delivered, coupled with certain indications requiring high
doses, has resulted in a growing need for high concentration (>50
mg/mL) formulations. While chemical liabilities such as deami­
dation and isomerization are independent of the protein concen­
tration, physical instabilities such as aggregation are protein
concentration dependent. Due to this, high concentration mAb
formulations can pose substantial manufacturing, stability, and
delivery challenges.3 As protein concentrations have increased, so
has the occurrence of physical instability (e.g., opalescence, aggre­
gation, particles) and viscosity challenges.4 In addition, as mole­
cule formats have become more complex, high concentration
instabilities have proven to be more challenging.
Two general approaches are used to mitigate physical instabil­
ities and viscosity issues. The first is to optimize the protein
formulation during development by finding conditions that mini­
mize potential liabilities (for example by changing the formulation
pH and/or testing various excipients), thereby ensuring acceptable
drug product manufacturing, administration conditions, and shelf
life. However, this approach does not allow the use of platform
formulations, as each molecule would require its own unique
formulation, nor does it help with challenges during manufactur­
ing where the protein may not be in its final formulation. The
process to optimize the formulation is often slow and may result in
a formulation that does not meet the intended shelf life, especially
in the case where there is a need to balance multiple competing
degradation pathways. Furthermore, formulation development
can only improve high concentration behavior of biomolecules
to a certain extent. While some liabilities can be addressed effi­
ciently (e.g., agitation-induced aggregation, particle formation),
others such as aggregation due to thermal unfolding and viscosity
require the selection of suitable molecules for the intended pur­
pose before technical development is started. As such, the second
approach is to use a developability assessment program during the
candidate selection phase to identify potential liabilities.4–6 Early
identification of such liabilities provides the opportunity to select
a different variant or to attempt to fix the liability using protein
engineering approaches. However, screening high concentration
liabilities requires large amounts of protein and therefore limits
the number of candidates that can be screened within a reasonable
time period and with limited resources.7 In addition, while there
has been success at identifying and removing chemical liabilities
(such as deamidation and isomerization), mitigating physical
instabilities is more complex. In these cases, there is generally no
single amino acid that needs to be replaced, as physical instabilities
are the result of protein-protein surface interactions. As such, the
mechanisms behind aggregation and viscosity and approaches to
predict, and therefore mitigate, such behavior have been the sub­
ject of much interest. Here, we review some of the recent work
aimed at understanding, measuring, predicting, and mitigating
high concentration instabilities and viscosity challenges. For
a comprehensive summary of the other aspects of developability,
we refer you to a recent review.6

CONTACT Jonathan Zarzar zarzar.jonathan@gene.com Pharmaceutical Development, Genentech Inc, 1 DNA Way, South San Francisco, CA 94080, USA
This article has been corrected with minor changes. These changes do not impact the academic content of the article.
© 2023 Genentech, Inc. Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the
posting of the Accepted Manuscript in a repository by the author(s) or with their consent.
2 J. ZARZAR ET AL.

Aggregation reported increased aggregation of an IgG2-trehalose formula­

tion when stored at −20°C. The glass transition temperature
Antibody aggregation is a substantial concern, as aggregates
(Tg’) of the matrix was determined to be −29°C. Interestingly,
are generally viewed as immunogenic, may be hyper-potent,
aggregation in this formulation increased with time at −20°C
and conversely can reduce overall efficacy.8 Aggregation
but not at −40°C or −10°C. This is most likely because of the
observed on stability may limit product shelf-life, which com­
crystallization of freeze-concentrated trehalose from the frozen
plicates supply chains. Aggregation occurs when a protein
solute at temperatures higher than the Tg’ of the matrix. Lack
forms high molecular weight species either through weak
of aggregation at −10°C was attributed to lower cryoconcen­
nonspecific interactions (self-association) or via covalent
tration of the protein at these temperatures, as well as higher
bonds (covalent-aggregation).9,10 In either case, it can lead to
mobility of the formulation that allows for any unfolded pro­
the formation of soluble (e.g., dimers, trimers) or insoluble
teins to refold back into native state. Connolly and others16
aggregates that can manifest as particulates. Protein aggrega­
expanded on the frozen instability studies and found that faster
tion can be influenced by a number of factors, such as mole­
cooling rates (>100°C/min) result in trehalose crystallization
cular properties; formulation composition, including active
and protein aggregation. They also reported that at lower
pharmaceutical ingredient (API) concentration; agitation;
cooling rates of≤1°C/min, trehalose remains predominantly
temperature; light; and contact with certain materials.11
amorphous and there is no effect on protein stability.
While controlling processing conditions, storage, shipment,
Interestingly, the authors found that phase distribution of
and handling may help reduce aggregation, it may not fully
amorphous and crystalline trehalose dihydrate in frozen solu­
mitigate aggregation. In those cases, other factors such as
tions depends on the ratio of trehalose to mAb. Based on these
molecular properties and formulation composition are impor­
observations, the authors found that an optimum ratio of 0.2
tant tools in reducing stability risks.
to 2.4 trehalose-mAb (w/w) ratio is ideal for frozen storage.
Optimization of formulations to prevent protein aggrega­
Bluemel et al.17 report an interesting cryoconcentration
tion is typically performed by leveraging prior experience
effect on mAb instability. Samples with lower mAb concentra­
combined with trial and error. In a typical study, the protein
tion showed increased formation of high molecular weight
is formulated in a variety of buffers, surfactants, excipients,
species (i.e., aggregates). In contrast, higher concentrated sam­
and pH levels and stressed via elevated temperature, agitation,
ples led to more subvisible particles (SVPs) when the samples
and freeze/thaw conditions to identify the optimal formulation
were stored above Tg’, that is, at −20°C or at −10°C. The
with minimum aggregation propensity. However, the propen­
authors went on to show that the protein and small excipients
sity of a molecule to aggregate at elevated temperatures may
freeze-concentrate differently, resulting in different local pro­
not be predictive of the propensity to aggregate at the intended
tein to stabilizer ratios within a container, ultimately leading to
storage temperature due to differences in degradation path­
mAb instability under these storage conditions.18
ways. Recently, Bunc et al. were able to predict the aggregate
Interestingly, Bluemel et al.19 have recently reported on the
fractions after three years of storage at 5°C using accelerated
use of computational fluid dynamic (CFD) simulations of
temperature degradation.12 The authors used a branched
mAb solutions. They conclude that CFD simulations are
kinetic model that breaks the stability behavior into a low
a promising tool to describe large-scale freezing and thawing
temperature and a high temperature pathway. Using this
of mAb solutions and could potentially save time and
model, the authors were able to predict the aggregation of six
resources. In addition to these instability pathways,
antibodies and resolved the curvature that is typically observed
a relatively new hypothesis on the interactions of proteins
when applying an Arrhenius model. The data also suggested
with air bubbles leading to aggregation has been proposed by
that at low temperature, aggregation is triggered by chemical
Salnikova et al.20 Authelin et al. recently published a detailed
modifications, while unfolding is the main driver at high
perspective covering these topics.21 In summary, higher pro­
temperatures.
tein concentrations in the DS can result in frozen instabilities
and have to be carefully evaluated.
Aggregation during frozen storage
Viscosity
The challenges of protein aggregation are not limited to hand­
ling at drug product (DP) storage temperature of 2–8°C and/or Self-association of mAbs increases with concentration and can
handling during typical production (e.g., 18–25°C). Frozen result in high viscosity, which challenges the capability of the
storage (</= −20°C) is a standard practice for protein drug ultrafiltration diafiltration (UFDF) unit operation during
substance (DS), and the storage temperature and storage mate­ manufacturing, resulting in slow operation and/or an unac­
rial must be carefully selected. Various stress factors that con­ ceptable pressure in the system. If the viscosity is only slightly
tribute to protein instability during DS processes have recently elevated, it is possible to mitigate it using single pass tangential
been reviewed by Das et al.13 and will not be reviewed herein. flow filtration (TFF) or increasing the recirculation tempera­
However, while the general assumption is that frozen storage ture to decrease the viscosity. However, increasing the recircu­
increases the stability profile, high concentration mAb formu­ lation temperature can also result in an increase in aggregation
lations have been found to be susceptible to instabilities such as and chemical degradation. High viscosity can also affect the
increased aggregation during frozen storage. Lazar et al.14 have filling operation, including the accuracy and fill rate. If the fill
shown that mAbs have a potential to undergo cold denatura­ rate is slowed enough, or stopped due to an interruption,
tion at storage temperatures near −20°C at pH 6.3. Singh et al.15 drying at the filling needles could occur, which can lead to

aggregates/particulates in the final drug product or yield loss
due to the need to flush the filling lines. During administra­
tion, high viscosity can prevent the use of a syringe and needle,
in particular for subcutaneous administration with a pre-filled
syringe. Above a certain viscosity, the injection force required
to expel the solution might be too high for certain populations
(e.g., geriatric populations) and/or dose volumes. It is impor­
tant that instructions for use and other training material
clearly highlight any product needs for warming the DP to
room temperature prior to injection, if needed to reduce visc­
osity to a level that is convenient for administration. Although
the use of autoinjectors or other devices can overcome some
limitations imposed by high viscosity, they can add complex­
ity, lengthen development time if an autoinjector must be
available during pivotal clinical trials, and increase the cost of
goods. Therefore, substantial effort has gone into the develop­
ment of high concentration protein formulations with low
viscosity. One approach is to use excipients that can help
lower the viscosity. The other approach is to use experimental
and computational approaches to measure and predict viscos­
ity in order to help select molecules with low viscosity.
Viscosity reducing formulations
Viscosity is the result of noncovalent intermolecular interac­
tions that result in the formation of a molecule network. As
a result, any molecule or excipient that has the ability to
disrupt the network can decrease the viscosity of the sample.
Charge-based interactions tend to have the biggest impact, as
they affect molecules over longer ranges compared to hydro­
phobic interactions. Sodium chloride can lower the viscosity of
a formulation by shielding protein charge and therefore
decreasing electrostatic driven protein-protein interactions
(PPI). In contrast, it has been hypothesized that arginine
works by direct binding to the protein surface.22 This can
result in both charge shielding, as well as a reduction in
hydrophobic forces by interacting with aromatic residues.
While typically salts such as sodium chloride decrease the
viscosity, there have been reports of it increasing the
viscosity.23,24 As such, each salt has to be evaluated on a case-
by-case basis, making evaluation a very slow and tedious
process. Caffeine has also shown promise at reducing viscosity,
and in some cases performed better than both sodium chloride
and arginine.24 Results obtained by Zeng et al.24 suggested that
formulating with caffeine reduces the attractive PPIs, leading
to lower viscosity. Using rigid body docking simulations, the
authors found that caffeine may preferentially bind to posi­
tively charged groups and aromatic groups on the protein
surface.
Other excipients have also been evaluated alone, or in
combination, to address viscosity and stability issues.
Recently, Banik et al.25 reported that the use of excipients
such as carnitine HCl, (S)-(+)-camphorsulfonic acid, and
ornithine HCl resulted in varying effects to viscosity and self-
interaction parameters that were also pH dependent. These
findings again highlight how the charge distribution on mole­
cules can have a substantial impact on what excipients are
most effective in reducing viscosity. Proline is an example of
another excipient that has been shown to reduce viscosity and
MABS 3
is used in the 200 mg/mL polyclonal human IgG drug product
Hizentra.26 Other amino acids and excipients that have hydro­
phobic and charged properties have also been shown to lower
viscosity in certain cases, furthering the understanding that
disrupting API network formation is key to lowering
viscosity.27,28
Other viscosity-lowering approaches use the formation of
nanoclusters or microparticles that restrict the viscosity effects
to interparticle interactions, rather than intermolecular inter­
actions. This approach creates two separate dominant forms of
molecular interactions, those within the particles, which do
not have a major influence on viscosity, and between the
surfaces of the particles, which is less than that of soluble
monomers. In one example, nanoclusters were formed by
lyophilizing a mAb formulation containing trehalose as
a crowding agent and reconstituting it in a buffer close to the
mAb’s isoelectric point using a lower volume than the pre-
lyophilization volume. This induced macromolecular crowd­
ing and led to the formation of nanoclusters that revert back to
monomer upon injection.29,30 Other particle-based technolo­
gies, using techniques such as electrospraying and microglas­
sification offered by companies like Elektrofi and Lindy
Biosciences, claim concentrations >400 mg/mL may be
reached. However, these microparticle approaches may require
substantial process development and require the use of non-
aqueous solvents for the DP to be administered.
Experimental measurement and prediction of
viscosity, solubility, and aggregation
High concentration behavior is influenced by a complex
dynamic of different molecular interactions (prominently
hydrophobic and electrostatic). Proximal energy effects caused
by molecular crowding in high concentrated protein solutions
further complicate the prediction of thhe behavior based on
single parameters.31 One of the major liabilities for molecules
intended for high concentration formulation is molecular self-
interaction. Depending on the exact nature of those interac­
tions and the underlying cause, a strong tendency to self-
interact can lead to high viscosity, opalescence, aggregation,
and/or particle formation. Colloidal interactions in a native,
chemically modified, or (partially) unfolded state can have
distinct consequences for a protein in solution. Furthermore,
molecules are exposed to different unfavorable conditions
during manufacturing and administration that can trigger
degradation via distinct pathways. Given the complexity of
underlying molecular mechanisms of self-interaction and the
variety of external stressors, it is clear that there is no simple
one-size-fits-all solution for the prediction of high concentra­
tion behavior in biopharmaceutical development. Even small-
scale experimental evaluation of viscosity, solubility, and
aggregation under specific conditions requires large mass
amounts of protein due to the need to formulate at high
concentration and the volume required by the analytics. For
example, the accepted method for evaluating the viscosity
behavior of a liquid dosage form at high concentration
involves formulating the protein at multiple concentrations
and generating a viscosity curve using a cone and plate rhe­
ometer. Due to the high mass requirements (~25–75 mg),
4 J. ZARZAR ET AL.
screening multiple variants can be slow and/or cost
prohibitive.4 As such, there is much interest in methods that
can increase throughput with lower mass requirements
(Table 1).
Molecular self-interaction in the formulation is determined
by three major factors: 1) the inherent physicochemical char­
acteristics of the protein, 2) the concentration of the protein in
solution, and 3) the presence of other components in the
solution (e.g., excipients). While the desired final concentra­
tion is mostly determined by the required clinical dose and the
route of administration, both molecule selection and formula­
tion development (or early evaluation of formulation suitabil­
ity) can be guided using assays predicting high concentration
behavior. In the following section, we discuss small scale (low
mass) options of these assays and give an overview of available
tools for the characterization of observed instabilities promi­
nent in these solutions.
Viscosity and self-interaction
The first set of methods for assessing self-interaction propen­
sity of proteins at low concentrations is based on assessing
the second virial coefficient (termed A2 or B22), which
describes the weak pairwise interaction between two solute
molecules in a solvent. A small-scale study of three mAbs
demonstrated that A2 can be an effective low concentration
(<20 mg/ml) predictor of protein aggregation and viscosity at
high concentrations.32 A less tedious alternative to measuring
A2, depending on the available instruments, is to measure its
component, the diffusion interaction parameter, kD, which is
amenable to high-throughput assessment using dynamic light
scattering (DLS).33,34 Using a panel of 29 mAbs (28 IgG1 and 1
IgG4) Connolly et al.33 demonstrated that kD is a useful pre­
dictor of viscosity in buffer systems of both high and low ionic
strengths. Kingsbury et al.34 expanded on this study by mea­
suring kD for 59 mAbs (44 IgG1, 4 IgG2, and 11 IgG4) and
trying to correlate the outcome with their high concentration
opalescence and viscosity behavior. The authors found that kD
measured under dilute concentrations in a low ionic strength
His-HCl buffer could very well predict high concentration
opalescence and viscosity liabilities. They could also set a kD
cutoff of 20 mL/g as a threshold below which most of the
antibodies in their panel tended to show high concentration
opalescence or viscosity.
A second family of methods that have proven useful for
measuring the self-interaction between protein molecules
using extremely low amounts of protein fall under the
umbrella of self-interaction nanoparticle spectroscopy
(SINS)-based techniques. These approaches take advantage
of the extreme sensitivity of the localized surface plasmon
resonance spectrum of gold nanoparticles (AuNP) to the
inter-nanoparticle distance to report on the self-interaction
of test proteins that are immobilized on the AuNP-surface.35
The extremely low protein concentrations (1–100 μg/ml) and
mass (μg scale) needed, the applicability with unpurified
samples, and amenability to high-throughput analysis make
these techniques suitable for application at very early project
phases, including during binder screening. One of the first
reported variants of a SINS method used for the
characterization of antibodies was affinity-capture SINS
(AC-SINS).36,37 In a seminal work, Lie et al.37 optimized
the experimental steps of AC-SINS and used it to measure
the self-interaction propensity of more than 400 mAbs.
Using the known high concentration properties of a small
subset of these mAbs, the authors claimed to predict the
developability of the entire panel. However, no attempt was
made to correlate these predictions with actual analysis at
high protein concentration for the entire panel. The biggest
drawback of AC-SINS is that it is only applicable under
physiological buffer conditions (PBS, pH 7.4), which prohi­
bits testing of proteins in formulation buffers at pH 5–6.
Attempts to overcome this limitation have focused on engi­
neering the surface properties of the AuNPs by functionaliz­
ing them with polymers that sterically or electrostatically
stabilize the nanoparticles. A recently developed method in
this area, termed PEG-stabilized SINS (PS-SINS), uses poly­
ethylene glycol (PEG) functionalization to extend the applic­
ability of the method to a wider pH range.38
A complementary method, which uses poly-L-lysine (PLL)
functionalization (termed charge stabilized SINS, CS-SINS)
to achieve a similar extension in applicability, was used to
analyze the behavior of a panel of 56 antibodies composed of
molecules with IgG1, IgG2, and IgG4 frameworks.39 The
authors showed that the behavior of IgG1 and IgG2 mAbs
in the CS-SINS assay correlated very well with their high
concentration viscosity and opalescence properties.
However, this correlation was much weaker for IgG4
mAbs, which shows that different molecular pathways con­
tribute to high concentration liabilities even for closely
related molecule types.
An alternative method to study protein self-interaction
in high throughput is based on biolayer interferometry
(BLI). Initially described by Sun et al.,40 Domnowski et al.41
implemented the method on an Octet HTX system, thereby
substantially increasing the throughput. This allows the
ranking of molecules in a standard buffer system, as well
as the early evaluation of different formulations and their
impact on self-interaction. The broad buffer compatibility
of the self-interaction (SI-) BLI technology and the com­
mercial availability of sensor tips with different capture
functionalities are advantages of this approach. However,
only indirect correlations to high concentration properties
have been established.
One early approach for the determination of self-
interaction parameters, such as the second virial coefficients,
is self-interaction chromatography (SIC). Using
a chromatographic resin functionalized with a protein of inter­
est, the retention time of the protein can be correlated to its
self-interaction propensity.42,43 While both SINS and SI-BLI
are assays designed with high-throughput applications in
mind, SIC can be used to characterize the self-interaction of
a protein in more detail, detecting heterogeneous behavior and
even sampling fractions of broad peaks to better understand
the heterogeneity. Furthermore, an in-depth characterization
of the influence of solution components is possible to guide
formulation or process development for a low number of
molecules.44 However, the preparation of a specifically func­
tionalized column resin for SIC is both skill and labor intensive

and requires relatively large amounts of protein. Therefore,
SIC is less suitable for ranking many molecule candidates
during developability assessment.
In contrast, cross-interaction chromatography (CIC) cir­
cumvents the need for a specific column preparation for each
individual mAb.44,45 Attachment of a polyclonal IgG mixture
or unrelated mAbs to the resin allows a higher throughput of
mAb candidates to be evaluated. The relatively large amount of
sample needed to specifically functionalize a column resin for
SIC is also omitted, making CIC a more suitable option for
early molecule assessment. Nevertheless, a good inverse corre­
lation between retention time on the column and protein
solubility was shown for a series of mAbs evaluated by Jacobs
et al.45 Potential association of test molecules with the general
structural features of IgGs caused by Fab-Fc or Fc-Fc interac­
tions can explain these observations. However, it must be
considered that a specific case of self interaction, caused by
mutual association of features within the complementarity-
determining regions (CDRs), will be missed using CIC. This
disadvantage can become more relevant when evaluating new
formats, especially when the constant part of the molecules
compared to the variable part comprises a smaller percent of
the overall construct (e.g., Fab fragments). While SIC is
a powerful tool to characterize one or few final candidates
with regards to the exact influence of the solvent, CIC can be
used during early selection of candidates in molecule
discovery.
Solubility
Solubility, or propensity to precipitate, is an important factor
in the formulation of stability and API behavior upon admin­
istration to patients. Determining a molecule’s solubility in
a low volume assay can be a useful tool for increasing the
number of molecules that can be screened in early project
stages. This can be accomplished in a high-throughput manner
by inducing precipitation at low concentration by the addition
of ammonium sulfate (AS) or polyethylene glycol (PEG).
While PEG mostly acts via the excluded volume effect (“mole­
cular crowding”), AS potentially alters the interaction behavior
of molecules.46 Therefore, PEG is considered to be the pre­
ferred precipitant in such experiments. The general principle is
to add different concentrations of the precipitant and measure
the free soluble protein concentration after separation of solu­
ble and insoluble fractions.47 The resulting midpoint concen­
tration of PEG is a good indicator for protein solubility and
correlates well with other predictors such as kD.48 An alter­
native study describes using microscopy to qualitatively assess
precipitation and other effects such as phase separation or
using turbidimetry as a direct readout for precipitation with­
out the need for prior separation of insoluble fractions.49
Advantages of this method include the low protein concentra­
tion required (in the range of 1 mg/mL for IgGs) and the
potential for high-throughput application with an optimized
small-scale assay and a suitable readout.49 Scannell et al.48 also
stated that PEG precipitation might be superior to DLS-based
kD determination when working with ionic excipients.
Therefore, early formulation screening for challenging mole­
cules could benefit from this approach. In contrast to most of
MABS 5
the above mentioned self-interaction assays, and similar to
light scattering-based kD determination, PEG/AS precipita­
tion does not require the immobilization of the interacting
molecules and thus avoids potential artifacts arising from this.
Aggregation and characterization of observed species
Low volume and/or low concentration stability studies are
a standard part of the developability assessment for protein
therapeutics. Aggregation liabilities can be identified using
a combination of freeze-thaw stress, mechanical stress, and
high-temperature stress conditions using DP storage and phy­
siological conditions. Interestingly, in a study of 137 mAbs
from different stages of clinical development, aggregation was
observed at 40°C (stress stability) only weakly correlated to
other parameters such as self-interaction.50 Therefore, the
specific evaluation of stability in small-scale experiments is
still necessary to cover this important aspect of developability
assessment. The design of such studies requires careful con­
sideration since, at high concentration, molecular crowding
(or molecular proximity) effects lead to a shift in prominence
of different forces that influence molecular interactions. Short-
range forces such as van der Waals and hydrophobic interac­
tion become more important factors of protein-protein inter­
actions at high protein concentrations. In contrast, protein-
protein interactions at lower concentrations (and therefore
longer molecular distances) are dominated by longer range
charge or dipole effects.31 As such, choosing suitable condi­
tions and a relevant concentration for accelerated stability
assessment is crucial to avoid missing molecule liabilities that
only become apparent at higher concentrations (>100 mg/mL).
Furthermore, specific routes of administration can result in
a high local concentration under physiological conditions, and
efforts should be made to assess the potential aggregation risk
under physiological conditions. Given the complexity and
diversity of the physiological environment to which a protein
therapeutic can be exposed, small-scale stability conditions
tailored to reflect this could become a prerequisite for devel­
opability assessments in the future.51 The relevance of confor­
mational stability (routinely determined by thermal unfolding
and the calculation of the denaturation midpoint temperature,
Tm) as compared to colloidal stability for protein aggregation,
is controversial. Jain et al.50 did not report any correlation
between Tm and accelerated stability results, indicating that
under common small-scale stress conditions (and in the range
of Tm values represented in the mAb test-set), this parameter
has no significant impact on aggregation.
Regardless of the study design, detailed characterization of
the observed impurities/degradation products (prominently
high molecular weight species and/or particles) is required.
This endeavor can be challenging and requires the use of
a specialized toolkit of analytical methods. Apart from the low-
resolution biophysical methods used for investigation of pro­
tein oligomerization and complex formation, such as DLS or
more time-consuming experimental techniques like analytical
ultracentrifugation, the most established and representative
method for assessment of species of different molecular
weights in the biopharmaceutical industry is size exclusion
chromatography (SEC). While SEC alone does not provide
6 J. ZARZAR ET AL.
an accurate size of the observed species, coupling it to a multi-
angle light scattering (MALS) detector enables determination
of the molar mass and concentration, which are proportional
to the light scattered by the particles.52 Despite the wide and
validated applicability of the SEC method for release and
stability monitoring, further investigation of aggregation phe­
nomena may require orthogonal techniques to SEC to gain
additional or more precise data that is typically not necessary
for release and stability monitoring.
Such orthogonal techniques include native mass spectro­
metry (MS) coupled to liquid chromatography for separation.
Modern ultra-high-mass-range (UHMR) mass spectrometers
allow acquisition of high-quality mass spectra in order to
determine molar masses of up to several MDa with superior
mass resolution and accuracy.53 Nevertheless, native MS is
a gas-phase technique, which requires volatile buffers and has
a low tolerance for salts and detergents.52
Another orthogonal method, mass photometry (MP), can
be used for the analysis of proteins and protein complexes
under native buffer conditions using very low sample quanti­
ties. This is achieved by the use of interferometric scattering
microscopy, which enables detection and quantification of
light scattered by single particles.52 The free choice of buffers
for investigation of observed impurities, as well as its high
sensitivity are advantages of this technology. MP provides
quantitative, label-free detection and mass determination of
biomolecules. In addition, MP makes studying stoichiometry,
energetics and dynamics of protein complexes possible.54
While the low sample consumption of MP is an advantage,
MP is only applicable at nanomolar or lower concentrations
and is thus unable to detect unstable high molecular weight
species that dissociate at such low concentrations.
Furthermore, the resolution of MP is lower than that of MS
and, while sufficient to detect aggregation/multimerization,
can fail to resolve species originating from degradation of
the API.
To complement these methods and cover the full size range
of possible aggregates, techniques for investigation of visible
and subvisible particles such as optical microscopy, light
obscuration, membrane microscopy, flow imaging, conductiv­
ity-based particle counter, and fluorescence microscopy, are
available.55 Additionally, laser diffraction, as well as DLS,
nanoparticle tracking analysis, or turbidimetry/nephelometry
can be used to assess the size of particles. Den Engelsman
et al.55 have made a comprehensive comparison of these meth­
odologies regarding their power of observations, their advan­
tages, and disadvantages.
Current state of the experimental toolbox for high
concentration behavior prediction
While high-throughput predictive assays with impressive cor­
relations to unwanted high concentration solution behavior of
proteins have been developed, it is currently impossible for
a single assay to predict which liability will ultimately be
manifested.38,39,41,49 Furthermore, most assays have been
developed for standard IgG formats. Due to the complexity
of the underlying molecular mechanism of self-association, the
limitation of these assays becomes relevant for alternative
molecule formats (e.g., Fabs, DutaFabs, fusion proteins,
Contorsbodies).56,57 In fact, Starr et al.39 observed differences
in the correlation between CS-SINS score and diffusion inter­
action parameter [kD] when evaluating different IgG sub­
classes. It would not be surprising if similar differences are
observed for more diverse formats. Similarly, Domnowski et ­
al.,41 while showing a good correlation between response rate
in SI-BLI to kD by DLS, observed exceptions that indicate that
the exact method setup (e.g., molecules immobilized vs free in
solution) could bias the obtained results. While for early mole­
cule selection, these exceptions are acceptable, a late develop­
ability assessment of a limited number of candidates could
benefit from a confirmation using a second orthogonal
method. Additional studies are needed to determine which
assays have broader general applicability or can be quickly
adjusted to format needs and which approaches might be too
specific to keep pace with a rapidly evolving drug discovery
pipeline.
In-silico approaches for viscosity and aggregation
prediction
In addition to the development of high-throughput analytics to
measure and understand high concentration liabilities, there
have been many advances in the use of computational
approaches. Studies have suggested that electrostatic and
hydrophobic interactions both play a role in viscosity, solubi­
lity, and aggregation of proteins.58–61 In particular, it has been
shown that negative charge on the variable fragment (Fv) of
a IgG1 mAb correlates with high viscosity, presumably due to
intermolecular interactions with corresponding positive
charge patches on Fc regions.62 This has led to the develop­
ment of various methods that quantify the charge and hydro­
phobicity of proteins and then attempt to correlate those
results to viscosity and aggregation. The first generation of
methods relied on protein sequence to calculate properties of
interest, such as the isoelectric point or hydrophobic content,
and predict high concentration behavior. One such approach
correlated the viscosity of 14 antibodies with the net charge at
the formulation pH, charge symmetry, and the hydrophobicity
index.62 In that study the authors showed that hydrophobicity
and charge distribution affect the solution viscosity, while
increased charge decreases it. Similar sequence-based
approaches, including methods such as AGGRESCAN,
PASTA, TANGO, and Zyggregator, have been used to predict
protein aggregation.63–67 However, these methods are not anti­
body specific, tend to have high levels of false positives for
mAbs, and ignore the contribution of neighboring residues.
This led to the creation of structure-based prediction methods
that take into account the three-dimensional (3D) protein
structure to gain a better representation of surface patches
that could result in protein-protein interactions. One such
method is AGGRESCAN3D (A3D), which combines the
aggregation propensity of amino acids obtained from
AGGRESCAN with structural information from the 3D pro­
tein structure to identify aggregation prone regions.68,69 By
taking into account only residues that are surface exposed
and within a spherical region around the residue’s central
carbon, A3D is able to lower the false positive rate, as it ensures

only residues that are able to interact are taken into account.
Another method known as the spatial charge map (SCM) uses
homology modeling to build the 3D structure of the Fv region
and calculates the spatial summation of charge of the surface
exposed residues.70 While SCM has been shown to differenti­
ate between high and low viscosity molecules using three
different datasets, the cutoff between the two populations
varied each time. This could result from differences in experi­
mental conditions between datasets or the method picking up
differences in molecule frameworks. In addition, SCM does
not account for the effect of buffer on viscosity, and thus this
method cannot be used to determine if the high viscosity can
be fixed via formulation changes.
Similar approaches can be taken to calculate hydrophobi­
city of an antibody with one common method being the spatial
aggregation propensity (SAP). SAP measures the hydrophobi­
city of a region by taking into account the surface exposure and
hydrophobic nature of particular amino acids and those prox­
imal to that residue.7 Compared with purely sequence-based
approaches, SAP is able to determine hydrophobic patches
created by multiple hydrophobic residues that are near each
other in the folded state. To demonstrate the applicability of
SAP, Chennamsetty et al. used two antibodies with high
hydrophobic scores, identified the regions responsible for the
hydrophobicity, and introduced single or multiple point muta­
tions to lower the hydrophobic nature.7 After incubating the
mutants at elevated temperatures, they demonstrated that the
new variants had higher thermal stability as measured by
the percent monomer still present after thermal stress. In
addition, functional analysis demonstrated that they preserved
the antigen-binding activity when mutations were not in the
CDR. Such a technique can prove useful for selecting as well as
optimizing therapeutic candidates.71 Another method known
as Aggscore takes into account the distribution of hydrophobic
and electrostatic patches on the protein surface.72 As opposed
to SAP, Aggscore has a scoring function that takes into account
the intensity and relative orientation of the respective surface
patches. However, Aggscore was not trained on antibodies and
while the authors correlated Aggscore to hydrophobic interac­
tion chromatography (HIC), CIC, and standup mono-layer
adsorption chromatography (SMAC), the method was not
evaluated against high concentration properties such as visc­
osity, aggregation, or opalescence.
Building on SAP, Lauer et al. devised the Developability
Index (DI), which incorporates the competing effects of elec­
trostatic and hydrophobic interactions.73 Using a dataset of 12
antibodies of different subtypes, the authors were able to gen­
erally predict the aggregation at elevated temperatures and
across a range of pH. While the method provides a general
trend of aggregation propensity, the validation dataset is lim­
ited, and it does not give insight into other high concentration
liabilities such as viscosity or opalescence. Similarly, Raybould
et al. developed the Therapeutic Antibody Profiler (TAP) to
provide developability rules similar to the Lipinski rules for
small-molecule development.74 To develop TAP, Raybould
et al.74 modeled the variable domain of clinical stage
antibodies50 and used them to determine five metrics that
were thought to be important in developability. The metrics
include the length of the CDRs, the extent and magnitude of
MABS 7
surface hydrophobicity, positive and negative CDR charge and
charge asymmetry. The authors then demonstrated that TAP
was able to flag both an affinity-matured molecule that exhib­
ited high levels of aggregation due to a large surface hydro­
phobic patch, as well as another antibody that exhibited poor
expression. Using such tools, residues of interest could be
identified and used to introduce mutations that either add or
remove charge or change a hydrophobic patch and thereby
improve a molecule’s developability.
Beyond calculating the charge and hydrophobicity of
a molecule, it has become increasingly common to calculate
additional descriptors that can correlate with high concentra­
tion liabilities. For example, Li and coworkers modeled the Fv
regions and calculated molecular descriptors to understand
and predict the viscosity of antibodies.75 The authors used
a panel of 11 antibodies, all formulated under-standardized
conditions and found that the charge, zeta-potential, and pI of
the Fv region correlated with the viscosity of highly concen­
trated solutions. In addition, at 150 mg/mL, molecules with
high viscosity had negatively charged patches on the Fv region,
which could interact with the positively charged IgG1 Fc
regions. This was similar to Vikas et al.’s results showing Fv
charge distribution affected viscosity.62 However, using only
the Fv to understand high concentration liabilities disregards
the effect of the constant regions, which have been shown to
engage in transient intermolecular networks.76,77 It is also
possible that the introduction of Fc mutations (for example
for half-life extension) can destabilize the Fc and alter protein-
protein interactions.78 To account for this, there has been
a shift to modeling the whole antibody. In one example,
Tomar et al. predicted the concentration-dependent viscosity
curve from the combination of experimental data and homol­
ogy-based structural models.79 Using molecular descriptors
calculated on 16 mAbs, the authors found that the hydropho­
bic surface area of the full-length antibody, as well as the
charges on the VH, VL, and hinge region could be used to
predict the viscosity curves. Similar to other works, the robust­
ness of the method still needs to be determined since the
results are limited to a small dataset and a single formulation
condition.
While homology modeling the whole antibody further
improves model accuracy, molecules are inherently dynamic
and that motion can result in different descriptor values when
compared to those from a single static structure. To account
for this, it is becoming common to use molecular dynamics
(MD) to capture the side chain fluctuations and understand
the impact of conformational dynamics on antibody proper­
ties. In addition, MD can be used to identify key residues that
may be involved in protein-protein interactions and propose
mutations to disrupt such interactions.80 Furthermore, MD
can also identify additional types of interactions such as
cation-π and/or π-π, which may contribute to high concentra­
tion behavior.80 However, doing full atom molecular dynamics
on a complete antibody is computationally intensive and hence
limits the speed and throughput as a screening tool. With the
introduction of even larger complex structures, the limitation
becomes even more pronounced. One way to overcome this
limitation is to coarse grain (CG) model the molecule, which
allows the simplification of an antibody to a reduced system
8 J. ZARZAR ET AL.
composed of 8–16 beads, with each bead containing a single
charge representing the summation of all partial charges in
that domain.76,77,81–83 Not only does this allow the modeling of
the whole antibody, but also the study of interactions between
multiple copies of a protein or even multiple proteins. In one
study, a 12 and 26 bead model was constructed to model two
antibodies that had different viscosity behavior at high protein
concentrations.77 Using such an approach, the authors studied
the impact of domain-level charge-charge interactions on the
two antibodies and visualized the type of interaction (Fab-Fab
or Fab-Fc). Interestingly, the antibody that had high viscosity
at high concentration formed dense clusters, whereas the anti­
body with low viscosity at high concentration did not. To
further understand the mechanism behind network formation,
the authors investigated the impact of charge swap mutants on
the viscosity of the two antibodies and found differences in the
number of Fab-Fab interactions across the variants.82 Similar
results were found by Buck et al. when they studied four
different antibodies using CG models.76,77,81–83 In this case,
the authors used an asymmetrical model that is more repre­
sentative of the structure in solution and found that molecules
with higher viscosity also had a higher number of pairwise
interactions.76 While these studies highlight the importance of
electrostatic interactions in network formation, hydrophobic
interactions will likely play an important role when the inter­
molecular distance decreases. Izadi et al. expanded the pre­
vious work and developed a CG model that not only accounts
for higher-order electrostatic multipole moments, but also for
hydrophobicity.83 After tuning the model with data from three
antibodies, the authors predicted the viscosity behavior of 12
different antibodies and demonstrated that the model could be
useful in predicting the effect of ionic strength on the viscosity
of solutions. Such an approach could be useful to determine
the likelihood that a high concentration formulation could be
developed in the case where no low viscosity variants exist.
In addition to the use of MD and coarse grain modeling,
there has been an increased interest in using machine learning
(ML) to predict high concentration liabilities as well as for­
mulation fixes for those liabilities.84–91 After removing high
correlated descriptors, Lai et al. used a dataset of 21 approved
antibodies to develop an aggregation model.87 In that model,
the positive SCM values and the solvent-accessible surface area
Table 1. Overview of small-scale assays to prediction solution behavior of proteins in high concentration formulations.
Method Parameter
AUC ks
DLS/SLS Kd/A2 (self-interaction)
SINS Wavelength shift (self-interaction)
SIC/CIC Retention time (self-interaction)
SI-BLI Binding response rate (self-interaction)
Scissor turbidity
PEG/ammonium sulfate Equilibrium protein concentration as function
precipitation of precipitant concentration (solubility)
Small scale stability (F/T,
temperature, mechanical
stress)
(*) relevant concentration of proteins has to be used, long term high concentration exposure under physiological conditions becomes more relevant in e.g.,
Ophthalmology.
(SASA) of the CDR-H2/H3 region correlated with the aggre­
gation rate. The SAP value of the CDR-H3 was also identified
as being important, which agreed with previous studies that
charge and hydrophobicity are key factors. Comparison of the
two-factor linear model with the dynamic mode of A3D
showed that the new model performed much better for this
limited dataset (prediction coefficient of 0.71 vs 0.28). In
a subsequent study, Lai et al. measured the viscosity of 27
commercially approved mAbs and used the experimental
data, along with computationally derived descriptors, to
develop a decision tree to classify antibodies into high and
low viscosity.85 The authors found that antibody net charge
and Fv amino acid composition drive viscosity. In particular,
they found that the number of hydrophilic and hydrophobic
residues in the Fv region are important. Expanding the dataset
to predict the aggregation propensity of 20 pre-clinical and
clinical stage antibodies gave mixed results.88 In the case of
aggregation, the authors found that positive SCM values and
the solvent-accessible surface area of hydrophobic residues on
the variable fragment were the most important, which agreed
with their previous results. However, for viscosity, the authors
found that the model developed with commercial antibodies
did not adequately predict the viscosity of the clinical-stage
antibodies.88 A potential explanation is that the original model
was trained on too small of a dataset, resulting in feature
selection that is not representative of a large population of
antibodies. The smaller the dataset, the higher the risk of
overfitting ML models, resulting in models that are not pre­
dictive when applied to a new dataset. Since datasets of high
concentration molecules are limited, this becomes a recurring
problem in predicting high concentration liabilities. In addi­
tion, given the expense of producing such data, it is likely that
this will remain a bottleneck for some time. To try to overcome
this, the authors combined both commercial and clinical-stage
antibodies to produce a model trained on 47 molecules. In that
model, the two most important parameters are the number of
hydrophobic residues and net charges on the light chain vari­
able region. More data will be required to determine if the new
model accurately predicts high concentration behavior.
Another model developed by Cloutier et al. used an elastic
net (EN) and support vector machine (SVM) model to predict
antibody-excipient interactions for various excipients
Predictive for Material need Throughput References
33
viscosity high low
25,33,34,98
Viscosity, aggregation/ medium low/medium
particle formation
36,39
Viscosity, opalescence low high
42–45,99
medium/high low/medium
40,41
Viscosity, opalescence low high
100,101
Precipitation upon high low
injection
48,49
low/medium medium/high
Aggregation, particle medium/high low
formation (*)
 MABS 9

including sucrose, arginine hydrochloride, and sodium formulations. Not only is the number of potential format
chloride.91 In that study, the aggregation and viscosity beha­ combinations increasing,94 but in-silico modeling of such for­
vior of antibodies was correlated with the relative strength and mats still remains relatively unexplored.
weakness of the protein-excipient interactions. The authors Building on the theme of more data, it is becoming
proposed that such an approach can be used in combination increasingly common to use multi-attribute monitoring
with SAP and SCM to determine when to mutate the antibody (MAM) methods for the residue level quantification of
to correct such liabilities. antibodies.95–97 The use of such methods provides deeper
In summary, many advances in predicting molecule aggre­ characterization of product variants and could be useful in
gation and viscosity have been made. Although the approach furthering our understanding of protein aggregation, in
behind each method varies, charge and hydrophobicity are particular during long-term and accelerated storage.
typically found to play a role in these high concentration When coupled with automation and low volume analytics,
liabilities. In addition, the limited datasets in combination the resulting data could be used to build various models
with the small number of problematic molecules in those that filter molecules with unfavorable properties from
datasets, have resulted in models that fail to have wide applic­ entering development. Eventually, the use of computational
ability. However, computational models are expected to tools could increase the number of variants that are eval­
improve and eventually help identify high risk molecules uated, further increasing the probability of technical suc­
before they enter development. In the cases where no viable cess. Ultimately, a fundamental understanding of high
alternative exists, computational approaches can provide concentration liabilities will be required, especially as mole­
insight into possible engineering solutions. Two recent reviews cule formats become more complex.
provide an in-depth discussion of some of the recent advances
in computational assessments for developability.92,93
Acknowledgments
The authors would like to thank Jasper Lin and Trevor Swartz for their
Conclusion and future outlook careful review and comments.
With the focus on higher protein concentrations and the
introduction of more complex molecule formats, high concen­ Disclosure statement
tration instabilities have been observed more frequently. If
predictive assessments, whether experimentally or computa­ No potential conflict of interest was reported by the authors.
tionally, are to be applied, additional data to build and validate
models will be required. However, due to the high protein
masses and the labor-intensive process needed to generate Funding
high concentration data, most studies conducted to date have The author(s) reported that there is no funding associated with the work
been carried out at low concentration. For example, the exten­ featured in this article.
sive survey conducted by Jain et al.50 looked at biophysical
properties found in clinical stage and commercial antibodies
with the assumption that antibodies that reached that stage of Acronyms
clinical trials have characteristics amenable to therapeutic A3D AGGRESCAN3D
development. However, most of those molecules were not AC-SINS affinity-capture SINS
developed at high concentration and thus the high concentra­ API active pharmaceutical ingredient
tion data do not exist. It is possible that, as the protein con­ AS ammonium sulfate
AUC analytical ultracentrifugation
centration of those molecules increases, additional factors AuNP gold nanoparticles
relevant to developability will be uncovered while documented BLI biolayer interferometry
thresholds for each property will need recalibrating. CDR Complementarity-determining regions
Building a deeper understanding of high concentration CFD computational fluid dynamics
liabilities will require systematic data of molecules at high CG coarse grained
CIC cross-interaction chromatography
concentration. While automation can help decrease the experi­ CS-SINS charge stabilized SINS
mental burden, many limitations exist, including the high DI developability index
protein masses required by the standard analytics. DLS dynamic light scattering
Identification of low volume high-throughput analytics could DS drug substance
decrease this burden, but additional work is required to estab­ DP drug product
EN elastic network
lish correlations between low mass, high-throughput assay Fv Fragment variable
results, and high concentration aggregation. As such, the kD diffusion interaction parameter
need for large amounts of protein remains. In addition, auto­ MALS multi-angle light scattering
mation of the sample preparation becomes difficult due to the MAM multi-attribute monitoring
increase in viscosity that is often observed at higher protein MD molecular dynamics
ML machine learning
concentrations, and sample-to-sample variation in viscosity MP mass photometry
adds to the complexity. The move to novel formats also adds MS mass spectrometry
complexity to the process of developing high concentration PEG polyethylene glycol
10 J. ZARZAR ET AL.

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OPEN ACCESS
EDITED BY
Ayaz Shahid,
Western University of Health Sciences,
United States
REVIEWED BY
Jifa Zhang,
Sutro Biopharma, Inc., United States
Marco Cavaco,
Universidade de Lisboa, Portugal
*CORRESPONDENCE
Paolo Macor,
pmacor@units.it
RECEIVED 07 August 2023
ACCEPTED 07 September 2023
PUBLISHED 18 September 2023
CITATION
Riccardi F, Dal Bo M, Macor P and
Toffoli G (2023), A comprehensive
overview on antibody-drug conjugates:
from the conceptualization to
cancer therapy.
Front. Pharmacol. 14:1274088.
doi: 10.3389/fphar.2023.1274088
COPYRIGHT
© 2023 Riccardi, Dal Bo, Macor and
Toffoli. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original author(s)
and the copyright owner(s) are credited
and that the original publication in this
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distribution or reproduction is permitted
which does not comply with these terms.
Frontiers in Pharmacology
TYPE Review
PUBLISHED 18 September 2023
DOI 10.3389/fphar.2023.1274088
A comprehensive overview on
antibody-drug conjugates: from
the conceptualization to cancer
therapy
Federico Riccardi 1, Michele Dal Bo 1, Paolo Macor 2* and
Giuseppe Toffoli 1
1
Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, Aviano,
Italy, 2Department of Life Sciences, University of Trieste, Trieste, Italy
Antibody-Drug Conjugates (ADCs) represent an innovative class of potent anti-
cancer compounds that are widely used in the treatment of hematologic
malignancies and solid tumors. Unlike conventional chemotherapeutic drug-
based therapies, that are mainly associated with modest specificity and
therapeutic benefit, the three key components that form an ADC (a
monoclonal antibody bound to a cytotoxic drug via a chemical linker moiety)
achieve remarkable improvement in terms of targeted killing of cancer cells and,
while sparing healthy tissues, a reduction in systemic side effects caused by off-
tumor toxicity. Based on their beneficial mechanism of action, 15 ADCs have been
approved to date by the market approval by the Food and Drug Administration
(FDA), the European Medicines Agency (EMA) and/or other international
governmental agencies for use in clinical oncology, and hundreds are
undergoing evaluation in the preclinical and clinical phases. Here, our aim is to
provide a comprehensive overview of the key features revolving around ADC
therapeutic strategy including their structural and targeting properties,
mechanism of action, the role of the tumor microenvironment and review the
approved ADCs in clinical oncology, providing discussion regarding their toxicity
profile, clinical manifestations and use in novel combination therapies. Finally, we
briefly review ADCs in other pathological contexts and provide key information
regarding ADC manufacturing and analytical characterization.
KEYWORDS
antibody-drug conjugate, antibodies, linkers, payloads, target therapy, cancer treatment
1 Introduction
Cancer is a multi-stage progression that transforms normal healthy cells into
malignant lesions (Cooper, 2000). It is the leading cause of death worldwide,
accounting for nearly 10 million deaths in 2020, and one of the most challenging
diseases to treat (Sung et al., 2021). Conventional therapeutic strategies are grouped
into chemotherapy, radiotherapy, immunotherapy and surgery, with the former being the
main approach for treating various cancers (Dan et al., 2018). Although most remarkable
goals have been achieved with small cytotoxic drugs, they had several drawbacks that
limited their efficacy, including a low therapeutic index and a high off-tumor effect
(Senapati et al., 2018). Usually, the low effectiveness of chemotherapy provoked a high
incidence of severe side effects in patients, mainly caused by the non-specific action of
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chemotherapeutic drugs on rapidly dividing normal cells (Senapati
et al., 2018). Therefore, one of the hot topics in the field concerns
the discovery of new chemical agents with enhanced therapeutic
efficacy and that preferentially ablate tumor-derived cells, without
harming the body itself (Casi and Neri, 2012). In the field of
immunotherapy, several monoclonal antibodies (mAbs) have been
clinically approved as they showed therapeutic benefits in both
hematologic malignancies and solid tumors by selectively targeting
cancer cells and by activating direct and/or indirect killing
mechanisms (Weiner et al., 2009; Kimiz-Gebologlu et al., 2018;
Castelli et al., 2019). However, issues including tissue accessibility,
poor pharmacokinetics and lame interactions with the immune
system have led to the need to exploit newer, safer and more
effective targeted therapies (Chames et al., 2009; Talotta et al.,
2019). In 1907, German nobel laureate Paul Erlich postulated that
there could exist compounds that would selectively target
pathogenic microbes, such as bacteria, while sparing normal
cells, a concept gone down in history as “zauberkugel” (magic
bullet) (Schwartz, 2004). From his hypothesis, an innovative
therapeutic approach, known as Antibody-Drug Conjugate
(ADC), has arisen in the oncology field and was firstly 40 used
years ago to treat patients with advanced cancer (Ford et al., 1983).
ADCs are an emerging class of pharmacological compounds that
combine the potency of anti-cancer drugs (often called payloads)
with the specificity of mAbs to the tumor site, thus combining
chemotherapy and immunotherapy. They are composed of three
portions, a mAb, an organic spacer and a cytotoxic drug. Ideally,
they use the mAb targeting ability to take the cytotoxic agent,
which is bound to the mAb via a stable linker, to the cancerous cells
or to the cellular components of the tumor microenvironment
(TME) where it can exert its anti-tumor activity and lead to cell
death (Baah et al., 2021; Mckertish and Kayser, 2021; Fu et al.,
2022). Compared with standard chemotherapy, this strategy offers
several advantages including better drug tolerability, cytotoxicity
even at low concentrations, drug stability in the bloodstream and
in lysosomes, reduced off-target effects, and systemic toxicity, all
features that contribute to the expansion of its therapeutic
potential (Khongorzul et al., 2020; Drago et al., 2021; Mckertish
and Kayser, 2021; Fu et al., 2022). Beginning with the first ADC,
which was approved by the Food and Drug Administration (FDA)
in 2000 (Norsworthy et al., 2018), and given the ever-evolving
technology of mAbs, linkers, and payloads, by April
2023 13 different ADCs have been FDA-approved for clinical
use for both solid and hematologic malignancies, setting the
stage for a new era of targeted cancer therapy (Dumontet et al.,
2023). In addition, a few studies have already addressed the
potential use of ADCs in non-oncologic context, including
infections (O’Leary et al., 2023; Tvilum et al., 2023) and auto-
immune disorders (Lee et al., 2017; Yasunaga et al., 2017), with
promising results showing the possibility of expanding the use of
ADCs in various diseases. In this review, we aim to summarize the
current knowledge of ADCs and address some key points about
their molecular properties, their interaction with tumor mass and
TME, their clinical use, toxicities and combinate regimens in
cancer treatment. Finally, we will provide an overview on
current research on ADCs in other diseases and address the
main challenges and limitations in their production and
characterization.
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FIGURE 1
Schematic representation of the modular components of an
Antibody-Drug Conjugate (ADC). ADC consists of a monoclonal
antibody (blue), a linker (blue lines) and the cytotoxic drugs (gray/red).
In this picture, the representative ADC on the left has a Drug-to-
Antibody ratio equal to 4. A brief description of the basic function of
each module is indicated on the right.
2 ADCs consist of antibodies linked to
cytotoxic payloads via a linker
2.1 Antibodies form the scaffold that guides
ADCs to target cells
An ADC is a synthetic molecule with pharmacological
activity comprising three blocks: a selective mAb, a stable
linker, and a potent cytotoxic drug (Figure 1). Together, they
ensure tumor-specific targeting and efficient ablation of
malignant cells by creating a “new” compound with enhanced
therapeutic efficacy.
2.1.1 Full size antibodies are the most used as ADCs
scaffolds
An antibody (Ab) or immunoglobulin (Ig) is a Y-shaped
glycoprotein produced by plasma cells that presents an intrinsic
selective ability to bind to its target (Baah et al., 2021; Pettinato,
2021). Several Abs used in oncology, upon interaction with their
target antigens, possess the capacity to influence the biological
activity of the tumor mass by modulating survival-related
pathways and/or activating potent immune effector functions
through three main mechanisms: antibody-dependent cell-
mediated cytotoxicity (ADCC), in which the Fc portion of
bound Ab is recognized by Natural Killer (NK) cell Fc-
receptor and activates the release of lytic factors, complement-
dependent cytotoxicity (CDC), in which the interaction between
the Fc region and C1q triggers the classic pathway of the
complement system leading to cell lysis (Macor and Tedesco,
2007; Macor et al., 2018) and antibody-dependent cellular
phagocytosis (ADCP), a mechanism that relies on active
macrophages to engulf tumor cells (Peters and Brown, 2015;
Chen et al., 2020; Vozella et al., 2021). Similarly, tumor-targeting
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Abs in ADCs shall be monoclonal and ensure high target
specificity and binding affinity, long half-life in plasma,
minimal immunogenicity combined with low cross-reactivity,
and allow efficient internalization as well as induce direct/
indirect killing effects (Peters and Brown, 2015; Dean et al.,
2021; Liu and Chen, 2022). According to the amino acid
sequence of their heavy chain constant regions, human Igs are
classified into 5 isotypes or classes (IgM, IgG, IgA, IgE, and IgD),
with IgG being the most abundant in serum. Based on further
amino acid variations, this isotype can be subdivided into
4 subtypes (IgG1, IgG2, IgG3, and IgG4). IgG1 consists of the
variable heavy (VH) and light (VL) domains in the N-terminal
portion of the antibody, the C-domains of the light chains
constant region (CL) and the heavy chain constant regions
(CH1, CH2, CH3) and the hinge region between the CH1 and
CH2 domains (Chiu et al., 2019). To date, its backbone is the
most used in ADC preparations because of its serum half-life
(~21 days), high Fcγ receptors avidity, thereby having strong
immune activation of Fc-dependent pathways, and more potent
complement activation (Yu et al., 2020). Beside these indirect
cytotoxic mechanisms, upon the interaction with specific
antigens on malignant cells, IgG1-based mAb can also exert
direct killing effects by blocking pathways associated to cancer
cell proliferation, metastasis and invasiveness (Natsume et al.,
2009; Drago et al., 2021). As for the other subclasses, IgG2 plays a
role in the response against bacterial capsular polysaccharides
but exhibits low Fcγ receptor avidity, low plasma concentration
and tends to form covalent dimers that likely lead to aggregation
and ADC inefficacy (Zhang et al., 2018). IgG3 protects the body
from a range of intracellular bacteria, parasites, and viruses. They
are potent mediators of effector functions, including enhanced
ADCC and CDC responses, but they are also the subtype with the
shortest circulating half-life (Stapleton et al., 2011; Hoffmann
et al., 2018), limiting their suitability for ADCs. The IgG4 subtype
has a similar half-life to IgG1 and IgG2 but is less efficient at
triggering the C1q-related pathway because it has only
intermediate affinity for the Fc receptor on phagocytic cells
(Spiess et al., 2013; Fu et al., 2022). However, the ability to
induce a poor immune response results in a more favorable
safety profile compared to the IgG1 backbone, making
IgG4 ADCs suitable in cases where antibody-mediated
cytotoxicity is not desired (Herbener et al., 2018).
2.1.2 Antibody-fragments represent a new and
innovative approach in ADC strategy
A growing number of studies raises questions regarding the
usage of full-size IgG moiety in the treatment of solid cancers and
points to some critical limitations, particularly on its size
(Deonarain et al., 2018; Richards, 2018). As IgG1, which is the
most used in ADC synthesis, is quite large (150 kDa) it may hinder
the distribution of the drug in the tumor mass and consequently
affect in a negative manner ADC pharmacokinetics and the
therapeutic outcome. Although this issue may find a partial
solution in the tumor-associated leaky vasculature, which shall
still allow sufficient pharmacological benefit due to the retention
and permeability effect (EPR) (Ferl et al., 2006), its efficiency and
the microdistribution of the compound in the tumor depend on
many factors, such as ADC preparations and molecular and
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cellular signatures of the mass (Deonarain et al., 2018;
Richards, 2018). Other problems related to scaffold size
include systemic accumulation and slowed target-independent
clearance rate (Adams et al., 2001; Jain, 2001; Thurber and Dane
Wittrup, 2012). Because of these limitations, researchers are
seeking new “miniaturized” versions of natural antibodies
(also known as antibody-fragments) as a new and smaller
drug-conjugatable alternative to expand the ADC therapeutic
benefits. These fragments are produced either by proteolytic
cleavage of full-size antibodies or by recombinant protein
engineering and primarily retain the binding capacity of full-
size IgG through the VH and VL regions (Brinkmann and
Kontermann, 2017; Kholodenko et al., 2019). They present
engineered scaffolds that lack the CH2 domain and Fc region
and include three different formulations: the Fab format, a
~50 kDa structure in which VL and VH are bound to CL and
CH1, respectively, and linked by a disulfide bond between the
chains, the single-chain variable fragment (scFv), a ~27 kDa
structure in which VH is linked to VL by a short peptide
linker, and the diabody, a non-covalent ~55 kDa dimer scFv
consisting of the VH and VL regions linked by a small peptide
linker (Xenaki et al., 2017; Deonarain et al., 2018; Kholodenko
et al., 2019). In addition, small immunoproteins (SIPs) composed
of dimerized scFvs through a CHε4 domain are a fragmentated
format developed against fibronectin and other vascular antigens
(Perrino et al., 2014). By preserving the targeting capacity of the
full-size antibody and combining it with smaller and dynamic
formats, antibody-fragments have the potential to overcome
some of the major drawbacks of full-size Ig moieties and may
represent an innovation in the treatment of solid tumors. To date,
promising data showed a remarkable improvement in stability,
tumor targeting and penetration and epitope accessibility,
particularly in cancers that are still difficult to reach via
conventional IgG-based ADCs (Dennis et al., 2007; Thurber
et al., 2008). In addition, smaller formats should be better
tolerated by the body and produce fewer adverse effects
mainly because they do not show cross-reactivity caused by
the interaction with Fc receptor and targeted immune cells
(Cilliers et al., 2018; Gogesch et al., 2021). If these benefits
should be assessed to a greater extent, significant weaknesses
can be identified. For example, because of their smaller size and
the absence of the Fc domain, without which they cannot trigger
the neonatal Fc receptor rescue pathway, antibody-fragments are
degraded more rapidly and potentially do not remain in the body
long enough to exert sufficient anti-tumor activity. From this
perspective, one solution might be to control the dosing regimen
by administering higher and/or more frequent doses to achieve a
therapeutic effect, but too little is known about the behavior of
these formats in the clinic and extensive efforts are underway to
improve their feasibility (Deonarain et al., 2018; Deonarain and
Xue, 2020). Moreover, the absence of the Fc domain severely
limits the possibility of activating the immune system, thus losing
an important partner in the fight against the tumor progression.
Besides these formulations, scaffolds not based on antibodies are
currently being explored (Luo et al., 2022; Kaplon et al., 2023).
Nevertheless, though the Ab moiety properties must be carefully
considered, the choice of linker and payload are equally
important to create the most suitable therapeutic ADC.
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2.2 Linkers are sequences that connect
antibodies to payloads by a chemical bond
In the development of the ADC strategy, linkers represent the
technology that ensures a bridge between the mAb and the
cytotoxic payload. They are the most modifiable part of an
ADC and influence its biophysical and functional properties
such as stability, potency, efficacy, and toxicity. The two main
purposes of linkers are to prevent the premature release of the
cytotoxic drug in the blood circulation and to ensure its efficient
release at the target site (Lu et al., 2016; Bargh et al., 2019).
Depending on the release mechanisms of the payloads, linkers
can be broadly classified into two classes: cleavable and non-
cleavable. Recent advances in linker chemistry, including
formulations not currently used in clinical ADCs, such as
biorthogonal, photo-responsive and Fe(II)-cleavable linkers,
are discussed in Su et al., 2021.
2.2.1 Cleavable linkers are versatile and widely
employed in ADCs
Cleavable linkers are most commonly used in the synthesis of
ADCs and are designed to disengage the cytotoxic drug in response
to changes in environmental conditions (pH, redox potential, GSH
concentration, or enzymatic activity) that occur between the
bloodstream, the tumor cells and the TME niche. They are stable
under physiological conditions and, following the internalization of
the ADC into the tumor cell, they are rapidly cleaved to ensure
selective release of the cytotoxic preparation (Peters and Brown,
2015; Tsuchikama and An, 2018). In addition, these linkers are often
cleaved in the TME because of its higher acidity and oxidative stress,
making them the most used preparation to affect large solid masses
barely impenetrable to full-size antibodies (Ponziani et al., 2020).
Cleavable linkers are commonly divided into chemical (hydrazone
and disulfide bonds) and enzyme (peptide bonds and glucuronide)
cleavage linkers (Bargh et al., 2019). Hydrazone linkers are an
example of acid-labile linkers used mainly in hematologic
malignancies. They are usually stable in the physiological
pH range of the blood circulation and undergo hydrolysis within
the acidic microenvironment of the endosomes and lysosomes
(pH 4.8–6.2) of the tumor cell (Jain et al., 2015). Similarly,
linkers based on disulfide bonds are stable in the bloodstream
alkaline environment, but payload release is sensitive to
glutathione (GSH), a metabolite whose concentration is much
higher in the cytoplasm of cancer cells (Balendiran et al., 2004;
Estrela et al., 2006). However, both linkers raised concerns about
their non-targeted cytotoxicity (Baah et al., 2021; Fu et al., 2022).
Peptide bonds ensure that ADC remains integral in the circulation
and enable the release of the cytotoxic drug upon interaction with
lysosomal proteases (Tsuchikama and An, 2018), such as cathepsin
B, which is generally overexpressed in several tumor cell types
(Gondi and Rao, 2013). Peptide linkers are associated with
improved serum stability and anticancer activity compared
chemical linkers (Lu et al., 2016; Tang et al., 2019; Khongorzul
et al., 2020). In addition, linkers based on glucuronide bonds,
another type of enzyme-sensitive chemical bridge, are commonly
used in ADCs design and rely on cleavage by β-glucuronidase, the
level of which is often high in the tumor cellular microenvironment
(Kostova et al., 2021).
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2.2.2 Non-cleavable linkers avoid non-specific
payloads release
Non-cleavable linkers include maleimidocaproyl (MC) and 4-
maleimidomethyl cyclohexane-1-carboxylate (MCC) structures and
consist of chemical structures that are not fragmented by enzymatic
degradation. They are inert to conventional chemicals but allow the
release of the cytotoxic drug once the mAb has been completely
catabolized by the lysosome. In this way, they release their toxic
payload into the tumor target cells without harming normal healthy
cells. Due to their chemical synthesis, these linkers offer some
advantages over the cleavable alternative, including lower toxicity
and longer half-life in plasma (Baah et al., 2021; Fu et al., 2022). On
the other hand, their limitations are mainly related to their
mechanism of action as they are strongly dependent on an
efficient intracellular trafficking and on cellular components with
high-expression and internalizing antigens (Lu et al., 2016;
Tsuchikama and An, 2018).
2.3 Payloads consist of potent cytotoxic
agents against tumor cells
As described above, the linker serves as a spacer to connect the
mAb to the payload, a cytotoxic drug that must be released in the
tumor site to properly exert its pharmacological effects. To be
suitable as payloads in ADCs, chemicals shall ideally have low
molecular weight and immunogenicity, high stability in the blood
circulation and endosomal/lysosomal pathways, and high
cytotoxicity (Peters and Brown, 2015; Khongorzul et al., 2020;
Baah et al., 2021; Mckertish and Kayser, 2021). Because
intravenous administration has shown that only a very small
fraction of ADC reaches the tumor (0.1%–2%) (Chari, 2008;
Hughes, 2010; Beck et al., 2017), their payloads must be 100- to
1000-fold more effective than the drugs used in chemotherapeutics
as free small molecules (Baah et al., 2021). Given that the goal of the
ADC strategy is to achieve potent cytotoxic activity, an important
attribute to consider in the design process of these compounds is the
Drug-to-antibody ratio (DAR), a value that indicates the average
number of chemical molecules conjugated to the mAb. For current
conjugation methods based on lysine side-chain amidation or
mainly on the reduction of cysteine intermediate-chain disulfide
bonds, the common DAR values range from 0 (lowest value) to 8
(highest value) (Dan et al., 2018; Wagh et al., 2018; Sun Kang et al.,
2021). Nevertheless, in vivo experiments have demonstrated that a
high DAR value negatively correlates with Ab pharmacokinetics.
Although a low DAR implies the loading of poor number of drug
molecules and consequently lower therapeutic efficacy, it is worth
noting that an average DAR of 2 to 4 results in an ADC with greater
anti-cancer activity/efficacy compared to an ADC with higher DAR,
likely because the latter is more rapidly cleared from the body when
compared to the corresponding average-conjugated counterparts
(Hamblett et al., 2004; Strop et al., 2015). Nowadays, cytotoxic
payloads usually act as DNA-damaging agents or tubulin inhibitors,
but novel potential drugs under investigation include inhibitors of
B-cell lymphoma-extra large (Bcl-xL) anti-apoptotic protein, RNA
and Niacinamide phosphate ribose transferase (NAMPT) inhibitors,
and carmaphycins, inhibitors of proteasome activity (Wang et al.,
2023).
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2.3.1 DNA-damaging drugs act as crosslinkers,
alkylators and topoisomerase inhibitors
The available agents that induce cell death by damaging DNA
can act up to picomolar concentrations (Hartley, 2011) and affect
both proliferating and non-proliferating cells, so they can potentially
contribute to the ablation of the tumor mass by affecting tumor-
initiating cells (Cheung-Ong et al., 2013; Bornstein, 2015). From a
mechanistic point of view, these chemicals alter the double helix in
different ways, e.g., by inducing single/double strand breaks,
alkylation and cross-linking of the DNA minor groove, or by
inhibiting Topoisomerase I/II and thus replication. Some of them
include amanitins (naturally byciclic octapeptides that inhibit RNA
Polymerase II action and disrupt RNA and protein synthesis),
calicheamicins (DNA-interactive antitumor antibiotics, that cause
DNA double-strand breaks and inhibit replication), duocarmycins
(natural DNA minor groove alkylating molecules), and
pyrrolobenzodiazepines (highly potent DNA minor groove
crosslinking agents). Two other compounds that have been used
in first-generation ADCs that are worth mentioning are
camptothecin (DNA topoisomerase I inhibitor at the replication
bubble) and doxorubicin (antibiotic molecule that damages DNA by
intercalating into it and generating free radicals) (Sau et al., 2017; Fu
and Ho, 2018; Baah et al., 2021; Mckertish and Kayser, 2021).
2.3.2 Tubulin-targeting agents block the mitotic
fuse formation and the cell cycle
Tubulin inhibitors block the rapid proliferation of tumor cells at
the G2/M cell cycle stage by binding tubulin subunits, leading to cell
death by apoptosis. This class includes maytansinoids and
auristatins, a family of tubulin-inhibiting cytotoxins that arrest
cells in metaphase. The auristatin derivatives monomethyl
auristatin E (MMAE) and the less toxic F (MMAF) (Park et al.,
2019) are commonly used as payloads in ADC design and exert their
function by blocking tubulin polymerization, thereby perturbing
microtubule growth and causing cell cycle arrest (Sau et al., 2017;
Baah et al., 2021; Mckertish and Kayser, 2021). Looking at the
mechanism in detail, microtubule formation involves either
nucleation or assembly of the αβ-tubulin heterodimer into a
microtubule seed in the cytoplasm (Francisco et al., 2003;
Goodson and Jonasson, 2018). As auristatin, by interfering with
GTP hydrolysis on the β subunit, causes an excessive and sustained
growth of microtubules, they lose the ability to shorten and separate
sister chromatids in anaphase, causing cells to freeze in metaphase of
mitosis (Waight et al., 2016).
2.4 The development of ADC therapeutic
strategy goes through three generations of
compounds
In the last years the ADC development path brought into the
marketplace a dozen ADCs against various hematologic and solid
malignancies. Generally, these ADCs have been divided into three
generations (first, second, and third) according to the type of mAb,
the chemistry of the linker, the mechanism of action as well as the
relationship between DAR and the conjugation method (Sau et al.,
2017; Fu et al., 2022). Representing the first attempt at a novel
therapeutic strategy, the first-generation ADCs caused acute adverse
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effects, such as hematotoxicity, and morbidity in patients, mainly
due to the mAbs backbone, linker chemistry, and target non-
specificity rather than the drug itself (Sau et al., 2017). Originally,
these biologics utilized mouse-derived and then chimeric mAbs
conjugated via unstable linkers to the few weakly potent drugs
available in chemotherapy. Unfortunately, after administration,
these mAbs were recognized as non-self from the body and
inevitably triggered an immune response through the formation
of human anti-mouse antibodies (HAMA), often leading to serious
immunogenicity problems (Kim and Kim, 2015). In addition, the
chemistry of acid-labile linkers, which are quite unstable at
bloodstream pH, led to uncontrolled release of payloads (Beck
et al., 2017), such as calicheamicin, duocarmycin, and
doxorubicin, whose potency was in any case too low to cause
cancer cell death. In this context, stochastic conjugation to
random lysines did not allow to control DAR and resulted in
heterogeneous mAbs mixtures containing unconjugated, partially
conjugated, and overconjugated mAbs in unknown proportions,
which negatively affected ADC efficacy, limited tumor penetration,
and resulted in a narrow therapeutic window (Lucas et al., 2018).
Furthermore, antigens were selected even though they lacked tumor-
specific expression, resulting in severe systemic off-target effects
(Sau et al., 2017; Fu et al., 2022). As expected, based on the
limitations of the first-generation ADCs, the second-generation
ADCs offered some implementations aimed at improving
compound efficacy and largely reducing off-target toxicity. To
limit potential side effects associated with the mAb backbone as
much as possible, humanized mAbs were preferred over mouse-
derived or chimeric mAbs due to the lower immunological response
upon administration (Lambert and Chari, 2014). Cleavable linkers
have been replaced by the non-cleavable alternative to ensure the
stability of ADCs in the blood and to reduce the premature and
dangerous release of drugs (Sau et al., 2017; Tsuchikama and An,
2018). In addition, far more potent cytotoxic chemicals have been
discovered and selected to induce cell death by attacking DNA
structure (disrupting its double helix conformation) and tubulin
polymerization (disrupting mitotic fusion formation) (Carter and
Senter, 2008; Senter, 2009). Due to the low amount of ADC in situ,
the IgG1 subtype was preferred over IgG4 because it has better
targeting abilities and better conjugation capabilities (Lambert and
Chari, 2014). However, despite the introduction of improvements in
linker stability and higher drug cytotoxicity, the main weaknesses of
this generation of ADCs were still heterogeneous DAR (4–8), rapid
clearance for high DAR drugs, off-target toxicity, and drug
resistance effects (Sau et al., 2017). What makes the third
generation of ADCs the best (so far) is based on fully human
mAbs, avoiding the disadvantage of immunogenicity, and
optimization in terms of linker stability, payload cytotoxicity, and
site-specific conjugation. This new method consists in an evolution
of previous ones and was introduced to address heterogeneous
DARs and consequently improve ADC pharmacokinetics and
utility (Fu et al., 2022). To this end, the synthesis of recombinant
Abs bearing engineered cysteine residues enabled the precise
bioconjugation of various drugs, resulting in the so-called
THIOMAB drug conjugates (TDCs). The THIOMAB antibody
technology platform results in a highly stable and effective ADC,
whose DAR value is almost uniform (from 2 to 4) and is associated
with fewer systemic side effects, improved drug activity, toxicity, and
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FIGURE 2
Mechanism of action of ADC strategy. The major steps of the
process are indicated on the figure. Basically, following ADC-target
interaction on the surface of cancer cell (step 1), this complex
undergoes receptor-mediated endocytosis and enters the
endosomal/lysosomal pathway until the payload is released in the
cytoplasm (steps 2, 3a and 4). Then, the drug can exert its killing
activity either damaging DNA structure in the nucles or derange
mitotic fuse polymerization (step 5), leading to cell death by apoptosis.
A fraction of ADCs binds to FcRn receptors in the early step on
endosomal/lysosomal pathway and get transported out of the cell
(step 2a and 3b).
efficacy (Junutula et al., 2008). In addition, despite the potential
toxicity due to the high potency of the payloads, these ADCs have
lower immunogenicity and hydrophilic linkers, giving patients a
more chance to counteract cancer progression (Baah et al., 2021; Fu
et al., 2022).
2.5 Binding to specific tumor-related targets
triggers internalization and cytotoxicity of
ADCs
2.5.1 Choosing the right targeting antigen is critical
for ADCs killing
Considering that one of the advantages of ADC is that a potent
cytotoxic drug can be delivered specifically to cancer cells, the choice
of target antigen must be the first consideration in developing this
strategy. To take advantage of the maximal therapeutic index of
ADCs, the ideal antigen should be a cell surface structure (such as
proteins, glycoproteins, or aberrant gangliosides) that is highly or
predominantly expressed on tumor cells compared to healthy,
normal cells, or at least abundant on malignant, disease-
associated cells (Damelin et al., 2015; Peters and Brown, 2015).
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Ideally, the target proteins should be tumor-specific antigens
(TSAs), which are present only on cancer cell types, and/or
tumor-associated antigens (TAAs), which are proteins that are
highly overexpressed in tumors but rare or sparsely present in
normal tissue. In conjunction with this feature, the epitope of the
antigen should ideally face the extracellular matrix (rather than the
internal site) to ensure easier accessibility and interaction with ADC
after diffusing from blood vessels (Tipton et al., 2015; Zhao et al.,
2020). In addition, to avoid undesirable systemic side effects and
safety issues, the target antigen should be a protein that is anchored
to the membrane and not secreted into the blood circulation. If this
were the case, ADC would promote unwanted binding outside the
tumor and thus reduce the anticancer effect on malignant cells
(Ritchie et al., 2013; Zhao et al., 2020). Nevertheless, after the ADC
interaction, the optimal antigen should ensure proper
internalization of the antigen- ADC complex into the endosomal/
lysosomal pathway, leading to drug release and ultimately
cytotoxicity. It should be noted that the speed and efficiency of
the internalization process strictly depend on the nature of the
target, the type of the epitope, and the payload conjugated to ADC
(Carter and Senter, 2008; Donaghy, 2016; Fu et al., 2022). Finally,
since the tumor mass and its surrounding microenvironment (TME)
are tightly coupled and constantly communicate with each other,
TME components have been targeted as novel potential ADC targets
(see below in the text) (Andersen, 2023).
2.5.2 The mechanism of action of ADCs requires
internalization to exert antitumor cytotoxicity
The presence of a mAb targeting an antigen that is either
specifically present on cancer cell types or highly overexpressed
during carcinogenesis is a fundamental requirement for the ADC
strategy (Alley et al., 2010; Damelin et al., 2015). With this in mind,
the mechanism of action of ADCs is quite simple and allows for
therapeutic action against the disease as well as potent on-target
cytotoxicity (Figure 2) (Peters and Brown, 2015; Khongorzul et al.,
2020; Drago et al., 2021; Samantasinghar et al., 2023). After
administration, usually by intravenous injection to preserve drug
functionality (Nolting, 2013), the mAb portion of the ADC binds to
the target antigen on cancer cells, and after internalization of the
antigen- ADC complex by receptor-mediated endocytosis, the newly
formed early endosome matures into a late endosome that
ultimately fuses with the lysosome. In this cellular compartment,
acidic or redox conditions combined with the presence of proteases
(cathepsin B, plasmin, etc.) allow the detachment of the cytotoxic
payload from its mAb carrier, whereupon the drug diffuses into the
cell and leads to cell death by attacking DNA structure or
microtubule polymerization (Peters and Brown, 2015;
Khongorzul et al., 2020; Drago et al., 2021; Samantasinghar et al.,
2023). Of note, the IgG subtype that is widely employed in ADC
synthesis can be rescued from the endosomal/lysosomal degradation
pathway and recycled outside cells through interaction with the
neonatal Fc receptor (FcRn), an IgG receptor involved in the
regulation of IgG turnover. Therefore, this FcRn-mediated
transcytosis into the extracellular space, although involving only
a small percentage of the internalized ADC -complex, can
potentially enhance the clearance of ADC and thus reduce its
therapeutic index (Xu, 2015). On the other hand, if the small
molecule is permeable to the cell membrane, it can partially
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diffuse back into the extracellular matrix and enter neighboring cells
regardless of the expression level of the antigen, causing a “bystander
effect” (Staudacher and Brown, 2017). This phenomenon, by
altering components of the TME, such as neovascular endothelial
cells and/or cancer-associated fibroblasts (CAFs), could further
enhance the killing effect of ADC, especially in cancer lesions
with high heterogeneous target expression (Gerber et al., 2009;
Purcell et al., 2018; Szot et al., 2018). Moreover, the therapeutic
strategy of ADCs involves other killing mechanisms to ensure
efficacy against cancer. In several contexts, it has been shown
that the interaction of the mAb with its specific target can
directly cause potent inhibition of downstream signaling
pathways triggered by antigen receptor stimulation (Albanell
et al., 2003; Vu and Claret, 2012; Marei et al., 2022). While the
Fab fragment of the carrier is bound to the target epitope on the
malignant cell, the Fc portion of the same mAb can interact with the
FcR on NK cells and macrophages, triggering ADCC and ADCP,
respectively, as well as the C1q component of the complement
system, triggering CDC (Junttila et al., 2011; Tai et al., 2014; Redman
et al., 2015).
2.6 The TME offers new potential targets to
ADCs strategy
Most of the ADCs in preclinical and clinical development target
TAAs or TSAs localized mainly on the cancer mass (Sau et al., 2017;
Tong et al., 2021). Compared to hematologic malignancies, solid
tumors thrive in a complex and dynamic entity called the TME,
whose composition can vary widely depending on the tumor type.
Key features of the TME generally include abundant extracellular
matrix, stromal cells (e.g., cancer-associated fibroblasts, CAFs), new
and abnormal blood vessels, and immune cells, the latter capable of
infiltrating the tumor mass and exerting pro- and anti-tumor
functions (Anderson and Simon, 2020; Baghban et al., 2020).
Particularly in the early stages of tumor growth, a bidirectional
and complex interaction exists between cancer cells and the TME
components through the release of soluble factors that promote the
survival of the tumor mass, its local invasion and subsequent
metastatic spread (Visser and Joyce, 2023). In this sense, the
TME supports the basic needs of the tumor through a neo-
angiogenesis program that removes metabolic waste and, most
importantly, restores oxygen and nutrient supply to the mass
(Anderson and Simon, 2020; Visser and Joyce, 2023; Wang et al.,
2023). Given the close relationship between these two entities, TME-
associated antigens (TMAs), i.e., proteins that are dysregulated on
non-malignant cells within the TME, offer new potential targets for
the treatment of solid tumors as they differentiate from more
traditional tumor antigens (Andersen, 2023). Major TMAs
include chemokines and cytokines, transcription factors,
metabolic enzymes, and checkpoint molecules. Some of their
advantages lie in their overexpression on endothelial, stromal,
and immune cells, whereas they are rare or very low in healthy
tissues, and in their easier accessibility to ADCs when administered
into the bloodstream, especially to antigens present on neo-vessels
or stromal cells. Furthermore, because TME components are distinct
from cancer cells, they are less susceptible to resistance mechanisms
caused by inefficient DNA repair mechanisms (Agrafiotis et al.,
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10.3389/fphar.2023.1274088
2022; Ceci et al., 2022; Andersen, 2023). The development of agents
against these antigens not only weakens the tumor mass but also
provides the opportunity to modulate the TME itself, making it less
immunotolerant and more susceptible to tumor ablation (Andersen,
2023). Preclinical and clinical evidence suggest that cell types/factors
belonging to the tumor extracellular matrix and neo-blood vessels
may be valuable choices for new ADC target antigens (Peters and
Brown, 2015; Xiao and Yu, 2021). For instance, an ADC targeting
stromal cells may cause cell death by altering the concentration of
growth factors in the tumor niche or induce hypoxia and nutrient
deprivation by binding to an antigen on the neo-vasculature (Jain,
2005; Mahadevan and Von Hoff, 2007; Xiao and Yu, 2021). In this
regard, it is worth mentioning the effect of the “binding site barrier”
(BSB), a phenomenon that occurs between mAb and cell
populations near blood vessels and retains part of the ADC near
them, reducing the penetration of Ab into the tumor mass (van
Dongen, 2021). However, most TMAs have been identified on cells
of the immune system and targeting them offers an innovative anti-
cancer therapeutic approach achieved by promoting effector cell
proliferation, the anti-cancer cytokine/chemokine production, and
overall survival to create a new immune-hostile tumor niche with
reduced neo angiogenesis (Gajewski et al., 2006; Labani-Motlagh
et al., 2020; Andersen, 2023; Del Prete et al., 2023). To date, some
TMAs targeted by novel ADCs include CD74, an MHC class
chaperone II targeted by the ADC STRO-001, currently in phase
I in the treatment of B-cell malignancies (NCT03424603) (Le et al.,
2023) and CCR7, a chemokine receptor targeted by the novel ADC
JBH492 in non-Hodgkin lymhoma and chronic lymphocytic
leukemia patients (NCT04240704). In addition, Camidanlumab
tesirine, also known as ADCT-301, is an ADC in phase 1/2 for
the treatment of classical Hodgkin’s lymphoma (cHL) and non-HL
(NCT02432235) (Hamadani et al., 2021), and CD276, an immune
checkpoint overexpressed during pathologic angiogenesis and an
interesting candidate target of different ADCs in advanced solid
tumors (NCT04145622, NCT03729596 and NCT03595059) (Ceci
et al., 2022; Ziogas et al., 2023).
2.7 Cancer creates different ways to escape
the effectiveness of ADCs
A well-known feature of cancers is their ability to overcome the
efficacy of therapeutic approaches, making them susceptible to
various mechanisms of resistance. The evasion mechanisms
developed by malignant cells can be divided into down-/high-
regulation of antigen, the presence of drug efflux pumps, defects
in lysosomal functions, and deregulation of signaling pathways
involved in cell cycle progression and apoptotic dysregulation
(Shefet-Carasso and Benhar, 2015; Loganzo et al., 2016; García-
Alonso et al., 2018). In this context, an association between antigen
levels and the efficacy of ADC treatment with brentuximab vedotin
was observed, whose multiple treatment cycles correlated with
CD30 downregulation and consequently stronger tumor
resistance to MMAE (Chen et al., 2015). In another study, the
cancer cell line JIMT1-TM showed long-term resistance to the drug
after repeated administration of anti-human epidermal growth
factor receptor 2 (anti-HER2) transtuzumab maytansinoid, as the
level of HER2 protein decreased (Loganzo et al., 2015). Nevertheless,
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upregulation of CD33 antigen in the blood limited gemtuzumab
ozogamicin penetration into the bone marrow, suggesting that
elevated CD33 levels still negatively affect treatment and likely
reduce drug exposure (van der Velden et al., 2004). Another
non-negligible mechanism of resistance relies on a family of
transmembrane proteins called ABC transporters (Zheng et al.,
2021). These transmembrane proteins act as drug efflux pumps,
causing various chemicals, including those used as payloads, to be
excreted from the cancer cell, making it resistant or at least less
susceptible to treatment (Zheng et al., 2021). This mechanism has
been observed in AML cells, in which overexpression of the ABC-
family member MDR1 made them resistant to gemtuzumab
ozogamicin (Matsumoto et al., 2012; Senter and Sievers, 2012)
and in breast carcinoma cells, in which cyclic dosing of TDM-1
induced an increase in ABCC1 transporter levels (Loganzo et al.,
2015). Another escape mechanism involves lysosomal acidification.
After administration of ADC and internalization, linkers are cleaved
by lysosomal acid hydrolases to subsequently release the cytotoxic
agent into the cytoplasm of cancer cells. However, upon persistent
treatments malignant cells may acquire the ability to disrupt this
process by altering the pH of the lysosomal compartment to slow the
catabolic activity of their proteases, a process that has been
demonstrated in HER2-positive breast cancer clones resistant to
long-term T-DM1 (Ríos-Luci et al., 2017; García-Alonso et al.,
2018). Nevertheless, perturbations in signaling pathways involved
in cell cycle regulation and alterations in apoptotic regulation may
also modulate tumor cell sensitivity to ADC (Collins et al., 2019). In
T-DM1-resistant HER2-positive breast cancer cells, an increase in
cyclin B levels, a protein required for the G2/M cell cycle transition,
has been observed. This upregulation at the protein level could affect
cell cycle dynamics by altering sensitivity to treatment with ADC
(Sabbaghi et al., 2017). Moreover, in AML cells, activation of a
related pathway (PI3K/Akt) was associated with lower efficacy of
gemtuzumab ozogamicin and a deletion in the PTEN pathway was
associated with trastuzumab low effectiveness (García-Alonso et al.,
2018). Of note, in the same hematologic tumor, overexpression of
members of the anti-apoptotic BCL-2 and BCL-X families plays a
role in sensitivity to gemtuzumab ozogamicin (Godwin et al., 2020).
2.8 Clinically approved ADCs for the
treatment of hematologic and solid
malignancies
The ADC strategy represents a highly successful therapeutic
alternative in cancer treatment. The excellent ability to deliver a
pharmacological compound in situ by drawing the mAb of choice
along with various linkers and chemical alternatives represented an
innovative development compared to mAb-only based therapy and
traditional chemotherapy. As mentioned earlier, although the
development of ADC remains challenging in terms of drug
safety, efficacy, and targeting, the development of new and more
precise technologies, as well as the identification of new targets and
components, has led to an explosion in the use of ADC in clinical
oncology (Drago et al., 2021; Dumontet et al., 2023; Fuentes-Antrás
et al., 2023). To date, hundreds of ADCs are in clinical trials, and
15 of them have been approved by the FDA, the European Medicines
Agency (EMA), and/or other government agencies and launched
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into the marketplace for the treatment of hematologic malignancies
and solid tumors. Over the past 23 years, the following ADCs have
been developed for the treatment of hematologic tumors:
®
Gemtuzumab ozogamicin (Mylotarg ), Brentuximab vedotin
® ®
(Adcetris ), Inotuzumab ozogamicin (Besponsa ), Polatuzumab
vedotin ®
(Polivy ), Belantamab mafodotin (Blenrep ), ®
®
Loncastuximab tesirine (Zynlonta ) and Moxetumomab
®
pasudotox (Lumoxiti ), an ADC, which uses an immunotoxin
rather than a chemotherapeutic agent as a payload. The ADCs
currently approved for solid tumor therapy are Ado-trastuzumab
®
emtansine (Kadcyla ), Fam-trastuzumab deruxtecan (Enhertu ), ®
®
Enfortumab vedotin (Padcev ), Sacituzumab govitecan
® ®
(Trodelvy ), Tisotumab vedotin-tftv (Tivdak ), Mirvetuximab
®
soravtansine (ELAHERE ), Disitamab vedotin (Aidixi ), and ®
®
Cetuximab sarotalocan (Akalux ) (Fu et al., 2022; Kaplon et al.,
2023; Yao et al., 2023). A brief description of each agent is provided
below and key characteristics are listed in Table 1. In addition,
Supplementary Table 1 provides the recruiting clinical trials on the
use of novel ADCs being investigated for the treatment of cancer.
2.8.1 Hematologic malignancies
2.8.1.1 Gemtuzumab ozogamicin
Gemtuzumab ozogamicin (Mylotarg ; Pfizer) was the first ADC
®
ever developed and clinically approved by the FDA in 2000 and by
the EMA in 2018. As a first-generation ADC, it was based on a
humanized anti-CD33 IgG4 antibody linked to the DNA-interactive
agent calicheamicin (or ozogamicin) via a hydrazone-cleavable
linker bound to surface lysines (average DAR 2–3). It was
indicated for the treatment of relapsed/refractory acute myeloid
leukemia (AML) though it was withdrawn in 2010, but reapproved
at a lower dose in 2017, because patients suffered severe toxicity
problems likely due to the higher dose (Fu et al., 2022). Following
administration, Mylotarg binds to the CD33 transmembrane
glycoprotein on AML cells, and upon internalization, a precursor
of calicheamicin is released through hydrolysis of its linker. The
active form of the drug then exerts a cytotoxic activity by binding to
DNA and breaking its conformation to cause cell cycle arrest and cell
death. Of note, the hydrophobic nature of calicheamicin enables a
bystander killing of cells in the TME that are negative for the
CD33 target antigen (Coats et al., 2019; Kayser and Levis, 2022).
2.8.1.2 Brentuximab vedotin
®
Brentuximab vedotin (Adcetris ; Seagen, Takeda Pharma) was
approved by the FDA in 2011 and by the EMA in 2012 as
monotherapy for the treatment of systemic anaplastic large cell
lymphoma (sALCL) and in 2018 in combination with chemotherapy
for relapsed/refractory Hodgkin lymphoma (HL). It consists of a
chimeric IgG1 targeting CD30, a cell membrane protein of the
tumor necrosis factor receptor family, on cancer cells and is cysteine
conjugated to MMAE (DAR equals 4) through a protease-cleavable
linker (Mckertish and Kayser, 2021). After interacting with its target,
Adcetris enters the endosomal/lysosomal pathway via clathrin-
dependent endocytosis, where its linker is cleaved by acid
hydrolases to release MMAE in the cytoplasm. The drug
interferes with microtubule polymerization and induces apoptosis
and cell death. Like calicheamicin, MMAE exerts its cytotoxic effect
on neighboring CD30-negative cells using the bystander killing
effect, suggesting that the efficacy of Adcetris in heterogeneous
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TABLE 1 FDA/EMA approved ADCs in clinical oncology. e early; m metastatic; AML acute myeloid leukemia; HL Hodgkin lymphoma; sALCL systemic Anaplastic
Large Cell Lymphoma; B-cell prec. L B-cell precursor leukemia; DLBCL diffuse large B-cell lymphoma; MM multiple myeloma; HCL hairy cell leukemia; BC breast
cancer; UC urothelial cancer; NSCLC non-small-cell lung cancer; GC gastric cancer; GOJ gastro-oesophageal junction cancer; TNBC triple negative breast cancer; CC
cervical cancer; OC ovarian cancer; HNSCC Head and neck squamous cell carcinoma. The detailed description of the treatment for each disease is described in the
text. *: withdraw from market in 2022. **: Aidixi® and Akalux® have not received FDA/EMA approval yet but Akalux® is approved by PMDA (Pharmaceuticals and
Medical Devices Agency of Japan) and Aidixi® by NMPA (National Medical Products Administration of China).

ADC Manufacturer Trade name® Target FDA/EMA approval Cancer

Gemtuzumab ozogamicin Pfizer Mylotarg CD33 2000(2017)/2018 AML

Brentuximab vedotin Seagen, Takeda Pharma Adcetris CD30 2011/2012 HL; sALCL

Inotuzumab ozogamicin Pfizer Besponsa CD22 2017 B-cell prec. ALL

Polatuzumab vedotin Genentech Polivy CD79b 2019/2020 DLBCL

Belantamab mafodotin GlaxoSmithKline Blenrep BCMA 2020* MM

Loncastuximab tesirine ADC Therapeutics Zynlonta CD19 2021/2022 large B-cell prec. L

Moxetumomab pasudotox AstraZeneca Lumoxiti PE38 2018 HCL

Ado-Trastuzumab Emtansine Genentech Kadcyla HER2 2013 HER2+ e/m BC

Enfortumab vedotin Astellas Pharma US, Seagen Padcev NECTIN4 2019/2022 mUC

Fam-trastuzumab Deruxtecan Daichii Sankyo Enhertu HER2 2019/2021 HER2+ BC; NSCLC; GC/GOJ

Sacituzumab govitecan Gilead Sciences Trodelvy TROP2 2020/2021 TNBC; mUC

Tisotumab vedotin-tftv Seagen Tivdak TF 2021 (only FDA) CC

Mirvetuximab Soravtansine ImmunoGen ELAHERE FRα 2022 (only FDA) OC

Disitamab Vedotin Remegen Aidixi** HER2 — UC; GC

Cetuximab Sarotalocan Rakuten Medical Akalux** EGFR — HNSCC

lymphomas in vivo may be related to this effect (Katz et al., 2011;
Scott, 2017).
2.8.1.3 Inotuzumab ozogamicin
®
Inotuzumab ozogamicin (Besponsa ; Pfizer) was approved by
the FDA and EMA in 2017. It targets CD22, an antigen expressed on
relapsed/refractory B-cell precursors in acute lymphoblastic
leukemia (ALL). It consists of a humanized IgG4 mAb linked to
calicheamicin via an acid-cleavable linker attached to lysine residues.
It has a DAR, ranging from 5 to 7. From a mechanistic perspective, it
acts similarly to gemtuzumab ozogamicin in that it is based on the
same Ab backbone and loaded with the same drug (Dahl et al., 2016;
Garrett et al., 2019; Lanza et al., 2020).
2.8.1.4 Polatuzumab vedotin
®
Polatuzumab vedotin (Polivy ; Genentech) contains a
humanized IgG1 anti-CD79b, a component of the B-cell
receptor conjugated to MMAE via the same organic bridge
used in the synthesis of brentuximab vedotin. The conjugation
method was via engineered cysteines utilizing the THIOMAB
system and has a DAR of 3.5. The clinical use of Polivy has been
approved by the FDA in 2019 and by the EMA in 2020 for the
treatment of adult patients with relapsed/refractory diffuse large
B-cell lymphoma (DLBCL) in combination with bendamustine
and rituximab (anti-CD20 mAb) (Sehn et al., 2022; Varma et al.,
2022). Upon administration, this agent is internalized into cancer
cells and proteolytically cleaved to release MMAE, which causes
apoptotic cell death by inhibiting tubulin polymerization (Deeks,
2019; Kaplon et al., 2020).
2.8.1.5 Belantamab mafodotin
®
Belantamab mafodotin (Blenrep ; GlaxoSmithKline) was
approved by the FDA and EMA for the treatment of refractory/
relapsed multiple myeloma in 2020 (MM) but was withdrawn in
2022 because it did not meet FDA standards (https://www.myeloma.
org/news-events/withdrawal-blenrep-us-market). The mAb portion
of this ADC is unique in that it consists of a humanized Fc-
afucosylated IgG1, a modification that enhances binding and
cytotoxicity of the ADC. From the mAb backbone, a cysteine-
bound, non-cleavable linker bridges the mAb with the cytotoxic
payload MMAF. Its DAR is 4. The target of Blenrep is B cell
maturation antigen (BCMA), a member of the tumor necrosis
factor receptor family that is overexpressed in mature B
lymphocytes and plasma cells (Chen et al., 2020; Yu et al., 2020).
As with other ADCs, BCMA targeting internalizes ADC and degrades
mAb to release MMAF, a tubulin inhibitor, into the cytoplasm, where
it blocks cancer cell cycle progression and leads them to death
through apoptosis (Seckinger et al., 2017; Kaplon et al., 2020).
2.8.1.6 Loncastuximab tesirine
®
Loncastuximab tesirine (Zynlonta ; ADC Therapeutics) targets
CD19 and received accelerated approval from the FDA in 2021 and
from the EMA in 2022 for relapsed/refractory B-cell lymphoma after
two or more lines of systemic therapy, including DLBCL not
otherwise specified, DLBCL from low-grade lymphoma, and
high-grade B-cell lymphoma. Zynlonta targets CD19, a
transmembrane protein commonly expressed in all B cell
lineages, and consists of a humanized IgG1 mAb linked to
SG3199 (a dimeric PBD alkylating agent) via an enzymatically

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Riccardi et al.
cleavable linker (DAR of 2–3) (Lee, 2021). The payload exerts its
pharmacological benefit by irreversibly binding to DNA and
generating a strong adduct that inhibits DNA synthesis and
causes cell death. Because SG3199 exhibits cytotoxicity in the
picomolar range, it is the most toxic drug currently available on
the market ADC. Currently, Zynlonta is the only anti-CD19 drug
approved ADC for relapsed/refractory DLBCL as a single agent
(Zammarchi et al., 2018; Fu et al., 2022).
2.8.1.7 Moxetumomab pasudotox
®
Moxetumomab pasudotox (Lumoxiti ; AstraZeneca) is not
widely considered an ADC because its payload consists of a
fragment of Pseudomonas aeruginosa exotoxin A called PE38.
However, since it uses the same targeting mechanism based on
mAb, we would still like to consider it as part of the ADC
biocompound family. It was approved by the FDA in 2018 for
patients with refractory/relapsed hairy cell leukemia (HCL) who
have not received at least two systemic therapies. It was granted
marketing authorization in the EU in 2021. Lumoxiti is based on an
anti-CD22 mouse IgG1 mAb carrying a cleavable linker bound to
the immunotoxin PE38. After interaction, internalization, and
cleavage, PE38 is released into the cell cytoplasm and acts by
blocking translation and inducing cell apoptosis (Kreitman and
Pastan, 2011; Kreitman, 2019; Kang, 2021).
2.8.2 Solid tumors
2.8.2.1 Trastuzumab-based ADCs
Since FDA approval of rituximab for the treatment of non-HL in
1997 (Leget and Czuczman, 1998), several mAbs have been
investigated and achieved approval in clinical oncology.
Considering the mAbs that form the scaffold of approved ADCs,
trastuzumab is an example of an anti-cancer molecule used in
combination therapy or alone, and also represents the carrier
motif in two preparations ADC. Trastuzumab (Herceptin ) is a ® T-DM1 showed a significant 50% improvement in invasive disease-
humanized IgG1mAb that binds to the extracellular domain of
HER2, a tyrosine kinase receptor that is upregulated in 20% of
breast cancer (BC) patients, preventing its homodimerization and
thereby blocking its intracellular signaling (Greenblatt and
Khaddour, 2023). Because of its role in cell growth, survival, and
differentiation, HER2+ breast cancers tend to grow and spread more
aggressively than HER2 negative tumors (Iqbal and Iqbal, 2014).
Trastuzumab was approved by the FDA in 1998 for the treatment of
HER2+ BC. It improved overall survival (OS) and progression-free
survival (PFS) (Albanell and Baselga, 1999; Goldenberg, 1999), but
its administration was also associated with risk of cardiac toxicities,
such as left ventricular ejection fraction (LVEF) decline and
congestive heart failure (Bouwer et al., 2020). In the U.S. it is
approved for HER2+ BC in adjuvant therapy (with
anthracyclines and taxane) and for metastatic HER2+ BC in
monotherapy or in combination with chemotherapeutics, tyrosine
kinase inhibitors (TKIs), and immunotherapy. It is also used in a
combination regimen for HER2+ gastric cancer (Dumontet et al.,
2023). Trastuzumab is administered by intravenous infusion, and
the dosing regimen can be adjusted depending on the stage of tumor
growth (Greenblatt and Khaddour, 2023). Nowadays, the
trastuzumab backbone has been used for the synthesis of two
FDA- and EMA-approved ADCs, ado-trastuzumab emtansine
®
(T-DM1, Kadcyla ; Genentech) and fam-trastuzumab deruxtecan
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®
(T-Dxd, Enhertu , Daiichi Sankyo, Astrazeneca), which have
improved the OS in the second and third-line settings and are
currently used for the treatment of HER2+ early/metastatic and
HER2+ low BC, respectively (Ferraro et al., 2021; Rassy et al., 2022).
2.8.2.1.1 Ado-trastuzumab emtansine
®
Ado-trastuzumab emtansine (Kadcyla ; Genentech) is based on
a humanized IgG1 linked to emtansine via a non-cleavable linker
attached to the lysine residues. Its average DAR is 3.5 (Mckertish and
Kayser, 2021). After interaction with HER2 antigen, Kadcyla is
internalized by endocytosis and reaches the lysosome, where
IgG1 is completely proteolytically degraded. Subsequently, lysine-
MCC-DM1, a DM1-containing metabolite, is released into the
cytosol, where it disrupts the microtubule network and causes
cell death. Interestingly, lysine-MCC-DM1 has similar toxicity to
DM1 but cannot exert its pharmacological effect via the bystander
killing effect due to its charge at neutral pH (Barok et al., 2014;
Lambert and Chari, 2014). T-DM1 received FDA approval in
2013 for the treatment of advanced HER2+ BC based on data
from the EMILIA clinical trial (Phase III). This study evaluated
the efficacy of T-DM1 compared to capecitabine and lapatinib in
patients with HER2+ BC previously treated with transtuzumab and
taxane chemotherapy. Results based on 911 included patients were
favorable for T-DM1, whose administration resulted in an
improvement in objective response rate (ORR) (43.6% vs. 30.8%),
median PFS (9.6 months vs. 6.4 months; p < 0.001), and median OS
(29.9 vs. 25.9 months, p < 0.001) after a median follow-up of
47.8 months (Blackwell et al., 2012; Diéras et al., 2017). A few
years later, positive results from the KATHERINE clinical trial
(phase III) established the novel use of T-DM1 as adjuvant
therapy for patients with early-stage HER2+ BC with residual
disease after neoadjuvant treatment (taxane and trastuzumab).
Among the 1,486 patients who met criteria, those who received
free survival (IDFS) at 3 years compared with patients treated with
trastuzumab alone in the control arm (88.3% vs. 77%, p < 0.001)
(von Minckwitz et al., 2019; Wedam et al., 2020; Mamounas et al.,
2021). From the data collected in these two studies, T-DM1 exhibits
stronger therapeutic efficacy compared to chemotherapeutic agents
and trastuzumab alone in HER2+ early or metastatic BC, likely
because this ADC preserves the antineoplastic functions of
trastuzumab and adds a novel cytotoxic effect (Ferraro et al.,
2021). Remarkably, the superior benefit of T-DM1 was associated
with manageable side effects, mostly grade 1 or 2, as only a small
percentage of included patients reported elevations in liver enzymes
aspartate transaminase (AST) and alanine transaminase (ALT) and
thrombocytopenia (Diéras et al., 2017).
2.8.2.1.2 Fam-trastuzumab deruxtecan
®
Fam-trastuzumab Deruxtecan (Enhertu ; Daiichi Sankyo) is the
second ADC HER2-targeting drug approved by the FDA. T-Dxd was
approved by the FDA in 2019 and by the EMA in 2021 for the
treatment of unresectable or metastatic HER2+ breast cancer (after
patients have received two or more anti-HER2 therapies), non-small
cell lung cancer, and for locally advanced or metastatic HER2+ gastric
or gastroesophageal junction adenocarcinoma, after a trastuzumab-
based therapy. It consists of a humanized IgG1-mAb carrying Dxd, a
more potent DNA topoisomerase I inhibitor than SN-38, as a
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cytotoxic payload via an enzymatically cleavable linker. It has a DAR
of 7 or 8 (Mckertish and Kayser, 2021; Fu et al., 2022; Dumontet et al.,
2023). After internalization of the HER2-Enhertu complex and
cleavage, Dxd blocks DNA topoisomerase I, an enzyme that
controls and alters the topological state of DNA during
transcription, leading to cell death. Compared to Kadcyla, which
has the same target, Enhertu has several improvements related to the
novel cysteine-conjugated peptide linker, higher DAR and
cytotoxicity of the drug, which is more potent and hydrophobic to
increase the bystander killing effect on neighboring cells (Kaplon
et al., 2020; Shitara et al., 2021). This is essential for extending
cytotoxic activity to cells with low or heterogeneous HER2 levels.
In this sense, the bystander-killing effect achieved by T-Dxd allows for
a better therapeutic response and thus greater cytotoxicity in tumors
refractory to its T-DM1 counterpart (Ferraro et al., 2021). In 2019,
T-Dxd received accelerated FDA approval based on positive results
from the single-arm DESTINY -Breast01 trial (Phase II). In this study,
a cohort of 184 female patients with HER2+ metastatic BC who had
received two or more prior lines of therapy, including T-DM1, was
enrolled to test the efficacy of T-Dxd. Interestingly, 60.9% of patients
showed an objective response, and the median duration and median
response were 16.4 months and 14.8 months, respectively, with a
median time response of 1.6 months (95% CI) (Modi et al., 2020;
2021). In addition, the efficacy of T-Dxd and T-DM1 was evaluated in
the DESTINY -Breast 03 trial (phase III), which enrolled 524 patients
with HER2+ metastatic BC previously treated with trastuzumab and
taxane. Randomization data showed superior efficacy of T-Dxd in
terms of ORR (79.9% vs. 34.2%), PFS (not reached vs. 6.8 months, p <
0.001), and OS (94.1% vs. 85.9%) (Cortés et al., 2021; 2022). In
general, hematotoxicity, nausea, and fatigue were the most common
grade ≥3 adverse effects observed in patients treated with T-Dxd. Of
note, treatment with T-Dxd was also associated with pulmonary
toxicity and, in particular, interstitial lung disease (ILD), a group of
respiratory diseases that require careful management and may lead to
treatment discontinuation (Diéras et al., 2017; Cardoso et al., 2020).
Finally, the DESTINY-Breast04 trial evaluated the use of T-Dxd
versus physician’s choice chemotherapy in 577 patients with low
HER2+ metastatic BC who had received prior chemotherapy in the
metastatic setting or developed a recurrence within 6 months of
completing adjuvant chemotherapy. The results of this study showed
that administration of T-Dxd resulted in significantly longer median
progression-free (9.9 months versus 5.1 months) and overall survival
(23.4 months versus 16.8 months) than pharmacologic therapy in
enrolled patients (Modi et al., 2022).
2.8.2.2 Enfortumab vedotin
®
Enfortumab vedotin (Padcev ; Astellas Pharma US,
Seagen) is a fully human IgG1 conjugated to the
microtubule inhibitor MMAE via a protease-cleavable linker
on cysteine residues and has a DAR of 3.8 (Joubert et al., 2020).
It targets nectin-4, a transmembrane protein involved in
multiple cellular signaling pathways, including cell adhesion,
proliferation, and migration, and is overexpressed in several
malignancies. in 2019, Padcev was approved by the FDA for
locally advanced or metastatic urothelial carcinoma following
Pt-containing therapy and a PD -1 or PD -L1 inhibitor (Alt
et al., 2020; Chang et al., 2021) and received EU-wide
marketing approval in 2022.
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2.8.2.3 Sacituzumab govitecan
®
Sacituzumab govitecan (Trodelvy ; Gilead Sciences) is an ADC
consisting of a humanized IgG1 mAb targeting Tumor-associated
calcium signal transducer 2 (TROP2), a transmembrane
glycoprotein involved in cell self-renewal, proliferation, invasion,
and survival, and plays an important role in intracellular calcium
signaling. It is generally overexpressed in most solid tumors,
including triple negative breast cancer (TNBC) (Furlanetto et al.,
2022). The mAb harnesses to malignant cells SN-38, a DNA
topoisomerase I inhibitor that causes DNA breaks and ultimately
cell death. IgG1 and payload are connected via an acid-cleavable
linker bound to cysteine residues with a DAR between 7 and 8 (Tong
et al., 2021). Trodelvy was approved by the FDA in 2020 and by the
EMA in 2021 for the treatment of locally advanced or metastatic
TNBC in patients who have received at least two prior therapies and
in locally advanced or metastatic urothelial carcinoma following Pt-
containing therapy and a PD -1 or PD -L1 inhibitor (Mehanna et al.,
2019; Bardia et al., 2021; Tong et al., 2021).
2.8.2.4 Tisotumab vedotin-tftv
®
Tisotumab vedotin-tftv (Tivdak ; Seagen) consists of a fully
human IgG1, an enzymatically cleavable linker, MMAE as a payload,
and a DAR equal to 4. It targets tissue factor (TF), a membrane
protein related to cancer metastasis and invasiveness that is highly
expressed in various solid tumors. Being the last ADC on the market,
it was approved by the FDA in 2021 for the treatment of relapsed/
refractory metastatic cervical cancer with disease progression during
or after chemotherapy (Liu et al., 2011; Alley et al., 2019; Heitz et al.,
2023).
2.8.2.5 Mirvetuximab soravtansine
®
Mirvetuximab soravtansine (ELAHERE ; ImmunoGen) was
approved by the FDA in 2022 for the treatment of adult patients
with Folate Receptor α (FRα)-positive, platinum-resistant epithelial
ovarian cancer who have previously received 1–3 systemic therapies.
It targets FRα, a member of the folate receptor family that is
overexpressed on several epithelial-derived cancer cells (Macor
et al., 2006). It consists of a chimeric mAb bound via a cleavable
linker to DM4, a potent tubulin targeting agent that belongs to the
maytansinoids. Like other ADC, the drug exerts its cytotoxic effect
after internalization into cancer cells, leading to their death by
blocking their mitotic fuse formation (Dilawari et al., 2023; Heo,
2023; Kaplon et al., 2023).
2.8.2.6 Disitamab vedotin
®
Disitamab vedotin (Aidixi ; RemeGen) was approved by the
NMPA (National Medical Products Administration of China) in
2021 as a second-line treatment for patients with HER2-expressing,
locally advanced or metastatic urothelial carcinoma (mUC) who
have previously received Pt-containing chemotherapy (Fu et al.,
2022), and approved for patients with HER2-overexpressing locally
advanced or metastatic gastric cancer who have received at least two
systemic chemotherapy regimens (Deeks, 2021). It delivers HER2+
cancer cell MMAE (DAR equals 4) via a cleavable linker bound to a
humanized mAb. Interestingly, Aidixi showed high specific
antigenic activity and stronger tumor activity compared to other
HER2+-targeted ADCs in preclinical experiments and in animal
models (Shi et al., 2022).
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2.8.2.7 Cetuximab sarotalocan
®
Cetuximab sarotalocan (Akalux ; Rakuten Medical) received
PMDA (Pharmaceuticals and Medical Devices Agency of Japan)
approval in 2020 (Gomes-da-Silva et al., 2020). It is comprised of a
chimeric IgG1 mAb specifically targeting epidermal growth factor
receptor (EGFR), the triggering of which is involved in cell
proliferation, angiogenesis, and invasion/metastasis. The
conjugation of Akalux is not with a small molecule, but with a
light-activatable near-infrared dye called 700DX (DAR 1.3–3.8) (Fu
et al., 2022). In this case, we would also like to consider it as ADC
given its mechanism of action against cancer cells. After interaction
with ADC-EGFR, this ADC inhibits EGFR signaling pathway and
achieves high anticancer effect by laser activation of 700DX dye. In
this way, malignant cells are targeted and rapidly eliminated while
healthy cells surrounding the tumor mass are spared. It has been
approved for the treatment of unresectable locally advanced or
recurrent head and neck squamous cell carcinoma (HNSCC)
(Kitamura et al., 2020; Omura et al., 2023).
2.9 The therapeutic strategy of ADC reveals
challenges and limitations and can be
combined in combinatorial regimens
2.9.1 Payloads are the main cause of ADCs toxicity
In 2 decades, ADCs have achieved remarkable results in the
treatment of hematologic malignancies and solid tumors and
represent a valid alternative in the field of clinical oncology.
Although they have been designed to release cytotoxic agents by
targeting selective cell populations, a striking number of clinical
trials have shown that ADCs are not free of adverse effects,
sometimes leading to toxicities commonly observed with
conventional chemotherapy (Dumontet et al., 2023; Tarantino
et al., 2023). In general, a significant proportion of patients
suffered various toxicities, sometimes so severe (or fatal) to
require dose reduction or interruption, treatment delays, and
supportive medications (Dumontet et al., 2023; Tarantino et al.,
2023). Each of the blocks that form an ADC can result in significant
side effects when these compounds are administered to the human
body. Even if the nature of mAbs is responsible for moderate/severe
immunogenic side effects, especially in ADCs preparations using
murine and chimeric mAb scaffolds (Kim and Kim, 2015), the
primary manifestation of a toxicity profile is highly dependent on
the type of payload (Zahavi and Weiner, 2020). Most ADCs used in
the clinic are loaded with tubulin inhibitors or DNA-interacting
agents that exert their cytotoxic effects in the range of nano- or
picomolar concentrations and are highly toxic when used in
unconjugated form (Mecklenburg, 2018). Unfortunately, because
only a very small percentage of ADCs reach their targets and a part
of the payload is released prematurely, a significant portion of the
dose is virtually free to interact with numerous non-target healthy
cells and cause unconventional systemic or local side effects
(Dahlgren and Lennernäs, 2020). The out-of-target toxicities are
closely related to the linker. Non-cleavable linkers exhibit greater
stability in plasma and the ADCs are better tolerated by the body.
However, in the majority of ADCs used in the clinic and foremost to
the ones use in solid tumors, cleavable linkers are preferred as they
have shown more benefits, probably due to the bystander side effect.
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This out-of-cell toxicity has two sides. On the one hand, it may
extend the efficacy of treatment to antigen-negative cells found in
the tumor niche, to cells that form the core of the tumor mass being
less accessible to the ADC (Jain, 2005; Mahadevan and Von Hoff,
2007; Xiao and Yu, 2021), and to cells that have low/heterogeneous
expression of the antigen. On the other hand, due to the non-specific
effect of the conventional small drugs, normal cells can also suffer
severe damages with potential unpredictable consequences
(Donaghy, 2016; Staudacher and Brown, 2017). Other limitations
relate to the pharmacokinetic properties of ADCs. Rapid clearance
and aggregation represent two important aspects that may
negatively impact the therapeutic activity of these compounds
(Lucas et al., 2018; Mahmood, 2021; Pettinato, 2021). To solve
complications and improve the body compatibility of ADC, the
mAb component and linker can be chemically modified by
glycosylation or PEGylation (Mahmood, 2021; Edwards et al.,
2022). While the former has not been studied in detail within
this platform, the latter allows overcoming some drawbacks
(Pettinato, 2021). PEGylation involves the addition of
polyethylene glycol (PEG) to specific amino acid residues. In
general, it has been shown that the use of PEG as a linker can
improve the solubility of ADCs and reduce aggregation, improving
their pharmacokinetic by enhancing stability and distribution in the
body (Verhoef and Anchordoquy, 2013; Mahmood, 2021).
2.9.2 Clinical manifestations of ADCs include major
toxicities
As ADCs are developed to limit their exposure to healthy tissues,
they are associated with quite manageable toxicities, with nausea,
vomiting, diarrhea and fatigue being among the most frequent ones.
Unfortunately, the large amount of data provided by several clinical
trials also highlights the presence of severe toxicities (grade 3 or
higher) that include peripheral neuropathy and hematotoxicity,
often dose-limiting (Masters et al., 2018; Professional Committee
on Clinical Research of Oncology Drugs et al., 2022). Peripheral
neuropathy includes tingling, pain in the extremities, numbness, and
rarely muscle weakness and is commonly associated with ADCs
carrying cleavable linkers bound to tubulin inhibitors (i.e., all ADCs
loaded with MMAE) and Mirvetuximab soravtansine, carrying DM4
(Nguyen et al., 2023). As cleavable linkers are associated to the
premature drug release and that these compounds block tubulin
polymerization, this common side effect is not unexpected as
microtubules are deeply involved in the axonal transport, an
essential process to the growth and maturation of neurons
(Yogev et al., 2016). Hematologic side effects include anemia,
neutropenia, thrombocytopenia, leukopenia and are mainly due
to the off-target Fc receptor-mediated uptake of ADCs into
immune cells, with neutropenia being the most prominent
toxicity in ADC-based monotherapy (Mahalingaiah et al., 2019).
Besides, major toxicities responsible for dose limitations are related
to drug classes and mainly include hepatotoxicity (for MMAF, DM1,
and calicheamicin), skin toxicity (for MMAE and PBD), and ocular
toxicity (for MMAF and DM4) (Zhao et al., 2020; Hurwitz et al.,
2023). Of note, the mechanism underlying corneal toxicity has not
been solved yet but occurred in a relevant percentage of patients
treated with Belantamab mafotodin, Trastuzumab emtansine and
Mirvetuximab soravtansine, all loaded with tubulin inhibitors
(Aschauer et al., 2022; Domínguez-Llamas et al., 2023). Other
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relevant clinical manifestations include gastrointestinal side effects
upon administration of Sacituzumab govitecan and Trastuzumab
deruxtecan, two ADC used in the treatment of breast cancer and
loaded with Topoisomerase inhibitors and serosal effusion for
duocarmycin and PBD, the latter responsible of nephrotic
toxicity in Loncastuzimab tesirine preparation (Nguyen et al.,
2023). In addition, Trastuzumab emtansine and Trastuzumab
deruxtecan, both targeting HER2+ breast cancer, are associated
with an increased risk of interstitial lung disease (ILD)/
pneumonitis, which must be carefully managed with dose
adjustment and supportive care recommendations to avoid fatal
outcomes (Ma et al., 2018; Hackshaw et al., 2020). Like other
strategies based on small molecules and immunotherapy, ADCs
show some challenges that need to be carefully addressed to improve
their efficacy and reduce systemic side effects. A variety of
approaches is being taken into consideration in clinics to deeply
counteract the manifestation of side effects, and many of them focus
on an individual basis. Extensive efforts are being made to identify
patients who have potentially life-threatening toxicities at an early
stage and offer them supportive measures, such as dose and schedule
adjustments. The investigation of patients’ history and data on
previous treatments, comorbidities, or genetic profiles, including
pharmacogenetic analysis to identify SNPs or potential mutations in
key genes, will undoubtedly play a critical role in the determination
of the best strategy to improve the tolerability and efficacy of ADCs
(Lambert and Morris, 2017; Dumontet et al., 2023; Tarantino et al.,
2023).
2.9.1 ADCs can be used in combination therapies
In addition to the use of ADCs as monotherapy, recent preclinical
and clinical studies have also focused on ADCs combinations with
chemo-immunotherapics. Ideally, concomitant administration of
ADCs with antiangiogenic agents, i.e., agents that damage neo-blood
vessels to facilitate ADC penetration into the tumor niche, or
immunomodulatory drugs that promote immune surveillance should
amplify the body’s anti-neoplastic response to the tumor mass and its
TME, without or with limited severe toxicities and safety issues
(Dumontet et al., 2023; Fuentes-Antrás et al., 2023). Regarding ADC
combination with chemotherapy, promising data include the
combination of Brentuximab vedotin and CHP (cyclophosphamide,
doxorubicin, and prednisone) and Polatuzumab vedotin and
Rituximab-CHP in CD30+ peripheral T cell lymphoma (Herrera
et al., 2021) and in DLBCL (Tilly et al., 2022), respectively.
Similarly, the combination therapy based on Trastuzumab emtansine
and the selective anti-HER2 tyrosine kinase inhibitor (TKI) tucatinib
achieved remarkable benefits in metastatic breast cancer (Nader-Marta
et al., 2022) and the administration of bevacizumab, the mAb targeting
the Vascular-Endothelial Growth Factor (VEGF), with Mirvetuximab
soravtansine obtained similar benefits in preclinal models of ovarian
cancer (Ponte et al., 2016). Combinatorial approaches using ADCs and
immunotherapeutic agents have been recently explored in several types
of cancers (Fuentes-Antrás et al., 2023). Immune checkpoint inhibitors
(ICIs) are immunotherapy that enhance antineoplastic immune
responses by converting exhausted T-cells into activated ones and
bypassing the pathways that cause the tumor escape from the
immune system (Shiravand et al., 2022; von Arx et al., 2023). ICIs
targeting Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) and the
Programmed Cell Death Protein 1/its ligand (PD-1/PD-L1) axis
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have demonstrated robust clinical activity in several malignancies
but only a fraction of patients experience long-term benefits with
monotherapy (Wojtukiewicz et al., 2021). From this perspective, an
effort could come from ADCs, capable to enhance antitumor immune
responses by inducing tumor-specific adaptive immunity through
increasing T cell infiltration into the TME, while ICIs revitalize
exhausted T cells (Nicolò et al., 2022). Accordingly, encouraging
results come from the combination of ICIs and HER2-targeted
trastuzumab emtansine in the treatment of PD-L1+, HER2+
advanced breast cancer (Emens et al., 2020).
2.10 ADC strategy shows application in non-
oncology indications
In recent years, research groups have investigated the use of ADCs
in non-oncologic contexts, mainly focusing on the treatment of bacterial
infections and autoimmune diseases. Tvilum et al. achieved
antimicrobial efficacy by developing an Antibody-Antibiotic
Conjugate (AAC), an antibody directed against bacteria conjugated
to the antimicrobial molecule mitomycin C, for the treatment of
implant-associated biofilm infections caused by Staphylococcus
aureus, the most common causative agent in prosthetic joint
infections (Tvilum et al., 2023). In another study, O-Leary et al.
proposed the development of an Antibody-Bactericide Conjugate
(ABC), an antibody conjugated to an antimicrobial peptide that
exerts its effect by binding to the cell surface of P. aeruginosa,
providing another interesting example of ADC activity against
bacterial infections (O’Leary et al., 2023). Regarding inflammatory
diseases, Yasunaga et al. developed an ADC targeting IL-7 receptor
(IL-7R) conjugated with MMAE and showed that ADC-mediated
immunoregulation of the IL-7R, the upregulation of which is a
common mechanism in the pathogenesis of autoimmunity,
specifically depleted IL-7R-positive cells in the inflammation site of a
mouse model of autoimmune arthritis and abrogated disease
progression (Yasunaga et al., 2017). In the same year, Lee et al.
demonstrated in a model of rheumatoid arthritis (RA) the
immunosuppressive efficacy of TCZ-ALD, an ADC that targets the
IL-6 receptor (IL-6R) and is conjugated to the small molecule
alendronate. TCZ-ALD blocks the IL-6R activity and macrophages
activity in the manifestation of RA symptoms and joint inflammation
(Lee et al., 2017). A few years later, Gillard et al. achieved immune reset
of disease-causing reactive T cells by a single administration of CD45-
ADC and bone marrow transplantation, resulting in significant disease-
modifying effect in mouse models of autoimmune disease (Gillard et al.,
2020). In addition, a few studies also addressed the use of ADCs in
cardiovascular (Lim et al., 2015) and renal diseases (Kvirkvelia et al.,
2015), highlighting the potential application of this therapeutic strategy
in other pathological conditions.
2.11 Challenges and solutions in the
manufacturing of ADCs
2.11.1 Process development consists of several
steps
The main goal in the manufacture of ADCs by companies is to
produce a pure and bioburden-free compound that is safe for the
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human body for clinical use. Since the preparations of ADC consist
of mixtures of mAbs with multiple conjugation sites and small
organic molecules, several analytical steps are required in the
development of these compounds, and several challenges must be
overcome. Overall, the production of ADCs can be divided into
three steps: production of the antibody, synthesis of the drug-linker
complex, and conjugation to form the final ADC (Hutchinson et al.,
2018; Bulger et al., 2023). The conjugation step is of fundamental
importance as it determines the therapeutic efficacy of the
biomolecule. Conventional conjugation methods based on lysine
side-chains or reduced cysteines result in heterogeneous ADC
preparations whose safety and therapeutic efficacy are difficult to
predict. To overcome this crucial limitation, site-selective
conjugation methods have recently been developed. In this way, a
known number of drug molecules are constantly bound to selected
sites on mAbs, resulting in a more homogeneous mixture with
improved batch-to-batch consistency and therapeutic efficacy (Cao
et al., 2019; Nejadmoghaddam et al., 2019). After the conjugation
step, ADC proceeds to purification and filling into aseptic vials, all
following the sterile pipeline of current Good Manufacturing
Practices (cGMP). Quality is controlled throughout the
development process. Production requires a biological
manufacturing environment that allows for the safe handling of
sensitive structures, such as various powders and chemicals, purified
mAbs, and high potency drugs, to minimize potential pollution and
losses that may occur at various stages of production. To ensure
product purity and sterility, synthesis and bioconjugation reactions
are performed in aseptic rooms and conditions, with regular
documentation of instrument accuracy in accordance with cGMP
standards. To reduce potential contamination and exposure from
the use of hazardous substances, each step of the experimental
process is achieved by providing in-depth expertise and specialized
equipment to personnel and operators (Hutchinson et al., 2018;
Schmidhalter et al., 2019). In addition, the production of ADC
usually involves several departments/services and, in this sense, an
extremely low temperature supply chain and secure packaging must
be ensured to reduce possible variations, damage and leakage during
long-term storage and transportation (Ducry, 2012). Considering
the complexity and number of processes involved, the production of
ADCs is quite laborious, time-consuming and economically
expensive. Large capital investments are required to cover a
multitude of steps ranging from the design of the experiment, the
invention and development of new drugs to product innovation,
differentiation, and safety monitoring, all at a cost that must make
the finished drug marketable (www.cbo.gov). To overcome these
barriers, contract development and manufacturing organizations
(CDMOs), companies that provide drug development and
manufacturing services to the pharmaceutical industry, are
turning to single-use technologies (SUTs) and automated end-to-
end systems (Schmidhalter et al., 2019). SUTs are sterile, single-use
items made of various plastics, most of which can be used in the
same way as their stainless-steel counterparts without the need for
sterilization and recycling after use. As a result, these technologies
offer significant advantages over traditional reusable systems by
reducing the risk of cross-contamination, better ensuring sterility,
allowing more flexibility throughout the process and, most
importantly, improving cost efficiency and reducing time to
market (Schmidhalter et al., 2019). In these terms, continuous
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improvement in technology and investments by major
biopharmaceutical companies in extensive research programs will
drive the growth of the ADC market (valued at approximately
$7 billion in June 2022 but expected to reach $22.4 billion in
2030 (www.researchandmarkets.com) and increase the number of
available ADC-based therapies in new medical areas.
2.11.2 Analytical characterization startegies
Another challenge in the production of ADCs is the evaluation
of their biochemical attributes to obtain a safe and effective product.
Critical quality attributes of ADCs that must be carefully controlled
during the manufacturing process include determination of DAR
and distribution of drug, residual non-conjugated species, especially
in terms of mAb and payload, and evaluation of size and charge
variants in the final preparations. Nowadays, various in vitro
instruments and techniques are used either alone or in
combination to perform comprehensive analytical
characterization of ADCs, including spectroscopic,
chromatographic, and mass spectrometric methods (Wagh et al.,
2018; Liu et al., 2022).
2.11.2.1 Determination of purity
Purity of compound is a fundamental goal in any
biopharmaceutical manufacturing process. One of the techniques
used to evaluate the purity of ADC preparations is size exclusion
chromatography followed by ultraviolet detection (SEC-UV), a
method that separates molecules by size and, in some cases,
molecular weight, and is used to fractionate large
macromolecules such as proteins (e.g., mAbs) or protein
complexes (de Mel et al., 2019). In ADC synthesis, separation of
macromolecules by size allows purification of the mAb from
potential fragments, aggregates, and particles, three examples of
undesirable species that affect the efficacy of the final ADC as well as
its safety when administered to patients (Jiang et al., 2019). In
general, coupling SEC with multi-angle laser light scattering (SEC-
MALS) offers some advantages for biopharmaceutical applications.
In MALS detection, a laser beam passes through a sample solution
containing the target molecule, and depending on the size of the
molecules, the intensity of the scattered light is measured at specific
angles (Some et al., 2019). Compared to SEC itself, SEC-MALS
requires high-purity columns but provides additional information
by increasing the sensitivity for detecting impurities in the
preparation (Beck et al., 2019). Other techniques for determining
the presence of aggregation or fragmentation include dynamic light
scattering (DLS), sedimentation velocity analytical
ultracentrifugation (SV-AUC) (Ducry, 2012), and capillary
electrophoresis followed by sodium dodecyl sulfate analysis (CE-
SDS), a valuable method commonly used in the biopharmaceutical
industry for mAbs and ADC preparations to determine batch
consistency and overall protein purity (Wagner et al., 2020). In
addition, various residual species in ADC preparations may pose a
potential safety risk to patients and are a critical quality attribute that
must be carefully evaluated. The levels of these unconjugated forms
can be monitored by measuring the charge on the molecules
(Wakanar, 2011). Since the bioconjugation reaction between the
mAb and the payload can significantly alter the electrostatic profile
of the newly formed ADC, charge-based separation techniques such
as ion exchange chromatography (IEC), isoelectric focusing gel
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electrophoresis (IEF), and capillary isoelectric focusing (cIEF) can

provide information on charge heterogeneity, drug distribution
pattern, and overall preparation quality (Ducry, 2012). To date,
imaged capillary isoelectric focusing (iCIEF) is considered a robust
method in biopharmaceutical quality control because it can
quantitatively separate samples based on the isoelectric point (pI)
of individual variants (Wagh et al., 2018; Abbood, 2023). Therefore,
iCIEF can be used to rapidly measure the content of free mAb in an
ADC mixture according to the different pI of the mAb and its
conjugated form. However, the drawbacks of this assay are that
conjugates cannot be distinguished from reaction intermediates and
other impurities, and it can only be used for conjugation chemistries
that result in significant changes in charge and net pI (Wagh et al.,
2018).

2.11.2.2 Determination of DAR and drug distribution

Probably the hottest topic in the ADC manufacturing
process is the ability to achieve bioconjugation reactions that
lead to the synthesis of a homogeneous mixture and thus a
controlled DAR and payload. Various analytical methods have
been used to determine these parameters, including ultraviolet/
visible spectroscopy (UV/Vis) (Andris et al., 2018), hydrophobic
interaction chromatography (HIC) (Andris and Hubbuch,
2020), reversed-phase liquid chromatography (RPLC) (Chen
et al., 2019), and mass spectrometry (MS). As for MS, its
extended use in characterization of ADCs is discussed in
other works (Debaene et al., 2014; Zmolek et al., 2016; Liu
and Chen, 2022; Barbero et al., 2023). UV/Vis spectrometry is
relatively easy to measure DAR compared to the other
techniques, although it requires sufficiently different
absorption profiles between the mAb and the payload and a
UV/Vis chromophore on the payload (Wagh et al., 2018).
Among liquid chromatographic methods, HIC and RPLC are
routinely used to measure average DAR and drug distribution
(Ouyang, 2013; D’atri et al., 2019). HIC is a method that allows
determination of species distribution based on differences in
their hydrophobic properties and uniquely preserves the native
structures and activity of ADCs. Because the analysis is
performed under mild, non-denaturing conditions, ADCs can
be studied in their native conformation, which is an advantage
because isolation of chromatographically pure species allows
their further characterization in subsequent analysis. This
method is commonly used to analyze cysteine-conjugated
ADCs and other site-specific conjugations but cannot be
applied to ADCs obtained by lysine conjugation because the
greater heterogeneity of these preparations complicates
chromatographic separation (D’atri et al., 2019; Liu and
Chen, 2022). Nevertheless, HIC requires a large amount of
starting material, shows low efficiency for randomly
conjugated ADCs, and cannot separate positional isomers
from cysteine-conjugated ADCs or determine the chain, H or
L, to which the drug was conjugated (Becker et al., 2020;
Fleming, 2020; Liu and Chen, 2022). The RPLC method also
separates the components of a mixture according to their
hydrophobicity, but this approach requires denaturation of
the proteins, resulting in the loss of some information about
the distribution of some ADCs and the loss of certain DAR
species (Liu and Chen, 2022).
FIGURE 3
Overview on the tumor-targeting ADCs. Taking advantage of
blood circulation, ADCs reach the tumor microenvironment (TME),
which is composed by cancer-associated fibroblasts (CAFs) and other
cell types and interact with malignant cells that exposed the
tumor associated/specific antigen on their surface. In addition to their
canonical mechanism of action, ADCs can potentiate their positive
response against the tumor mass. To this aim, ADCs hydrophobic
payloads may diffuse through the cell membrane inducing the killing
of neighborhood antigen-negative cells via bystander killing effect
(black arrows) and/or the ADCs antibody Fc fragment may elicit anti-
tumor immunity (ADCC, CDC, and ADCP) by engaging immune
effector mechanisms, such as complement system, macrophages and
NK cells. All together, these mechanisms aim to induce the death of
the cellular component of the tumor mass via apoptosis.
2.12 Conclusion and perspectives
Nowadays, ADC represents a solid strategy to treat different
types of malignancies. The design of ADCs provides an exceptional
opportunity to selectively deliver an effective anti-tumor chemical to
the target cell and eliminate it without severe toxic off-target effects.
The main advantage of ADC lies in its mechanism of action, as it
offers the potential to overcome some major limitations of
conventional small molecule-based therapies, such as low
therapeutic index and high off-tumor toxicity The possibility of
killing neighboring antigen-negative cells, through the bystander
effect, in the mass and in the TME and the potential activation of
direct and indirect anti-cancer mechanisms via mAb-antigen
interaction and immune cell activation, respectively, argue for
their use in the clinic (Figure 3). In this context, it is worth
noting that the benefits associated with the activity of ADCs
when administered as monotherapy may encounter consistent
clinical limitations due to the presence of tumor-derived
resistance mechanisms and several manageable and few severe
adverse effects mainly caused by the payload of the ADC
(Fuentes-Antrás et al., 2023). Despite the promising results
obtained so far, the ADC technology is still under investigation
and has some limitations in terms of its pharmacokinetic properties

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and biological efficacy in different tumor contexts. To develop a
more suitable generation of ADCs, future prospects in this field are
based on the optimization of available technologies and especially on
the discovery of new methodologies. Accordingly, profound
improvements in the validation of newly developed mAbs, in the
synthesis of less immunogenic and more stable linkers, and in the
discovery of more effective payloads are being actively explored by
scientists in this field, and similar advance are also focused on the
development of more appropriate formulations as well as on the
identification of new target antigens. Given the central role of the
TME in solid tumor progression and spread, targeting novel
candidates upregulated in stromal cells, blood vessels and most
importantly immune cells within the TME could lead to greater
inhibition of tumor escape mechanisms and metabolic dysfunctions
to achieve long-lasting therapeutic effects. Moreover, combinatorial
therapies with different drug classes have already shown synergistic
and promising results in the treatment of hematologic and solid
tumors by enhancing the anticancer efficacy and therapeutic index
of ADCs. Based on these observations, all these efforts are aimed at
developing a new generation of ADCs that will undoubtedly show
significant improvements in terms of pharmacological properties,
therapeutic efficacy and safety in the field of oncology and in other
pathological conditions.
Author contributions
FR: Writing–original draft. MDB: Writing–review and editing.
PM: Writing–review and editing. GT: Writing–review and editing.
Funding
The authors declare financial support was received for the
research, authorship, and/or publication of this article. The
research leading to these results has received funding from the
European Union—NextGenerationEU through the Italian Ministry
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00000019 “HEAL ITALIA” to MDB (Spoke 5) and GT (Spoke 6)
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Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
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The AAPS Journal, Vol. 16, No. 4, July 2014 ( # 2014)
DOI: 10.1208/s12248-014-9598-3

Review Article

Understanding Pharmaceutical Quality by Design

Lawrence X. Yu,1,6 Gregory Amidon,2 Mansoor A. Khan,1 Stephen W. Hoag,3 James Polli,3
G. K. Raju,4,5 and Janet Woodcock1

Received 17 November 2013; accepted 24 March 2014; published online 23 May 2014

Abstract. This review further clarifies the concept of pharmaceutical quality by design (QbD) and describes
its objectives. QbD elements include the following: (1) a quality target product profile (QTPP) that identifies
the critical quality attributes (CQAs) of the drug product; (2) product design and understanding including
identification of critical material attributes (CMAs); (3) process design and understanding including
identification of critical process parameters (CPPs), linking CMAs and CPPs to CQAs; (4) a control strategy
that includes specifications for the drug substance(s), excipient(s), and drug product as well as controls for
each step of the manufacturing process; and (5) process capability and continual improvement. QbD tools and
studies include prior knowledge, risk assessment, mechanistic models, design of experiments (DoE) and data
analysis, and process analytical technology (PAT). As the pharmaceutical industry moves toward the
implementation of pharmaceutical QbD, a common terminology, understanding of concepts and expectations
are necessary. This understanding will facilitate better communication between those involved in risk-based
drug development and drug application review.
KEY WORDS: control strategy; critical quality attributes; pharmaceutical quality by design; process
understanding; product understanding.

INTRODUCTION Consider document; and ICH Q11 (Development and Manu-

Quality by design (QbD) is a concept first developed by the
quality pioneer Dr. Joseph M. Juran (1). Dr. Juran believed that
quality should be designed into a product, and that most quality
crises and problems relate to the way in which a product was
designed in the first place. Woodcock (2) defined a high-quality
drug product as a product free of contamination and reliably
delivering the therapeutic benefit promised in the label to the
consumer. The US Food and Drug Administration (FDA)
encourages risk-based approaches and the adoption of QbD
principles in drug product development, manufacturing, and
regulation. FDA’s emphasis on QbD began with the recognition
that increased testing does not necessarily improve product
quality. Quality must be built into the product.
Over the years, pharmaceutical QbD has evolved with the
issuance of ICH Q8 (R2) (Pharmaceutical Development), ICH
Q9 (Quality Risk Management), and ICH Q10 (Pharmaceutical
Quality System) (3–5). In addition, the ICH Q1WG on Q8, Q9,
and Q10 Questions and Answers; the ICH Q8/Q9/Q10 Points to
facture of Drug Substance) have been issued, as have the
conclusions of FDA-EMA’s parallel assessment of Quality-By-
Design elements of marketing applications (6–9). These docu-
ments provide high level directions with respect to the scope and
definition of QbD as it applies to the pharmaceutical industry.
Nonetheless, many implementation details are not
discussed in these guidances or documents. There is confusion
among industry scientists, academicians, and regulators despite
recent publications (10–13). This paper is intended to describe
the objectives of pharmaceutical QbD, detail its concept and
elements, and explain implementation tools and studies.
PHARMACEUTICAL QUALITY BY DESIGN
OBJECTIVES
Pharmaceutical QbD is a systematic approach to devel-
opment that begins with predefined objectives and empha-
sizes product and process understanding and control based on
sound science and quality risk management (3). The goals of
pharmaceutical QbD may include the following:

1. To achieve meaningful product quality specifications

1
Center for Drug Evaluation and Research, Food and Drug that are based on clinical performance
Administration, Silver Spring, Maryland 20993, USA. 2. To increase process capability and reduce product
2
University of Michigan, Ann Arbor, Michigan 48109, USA.
3 variability and defects by enhancing product and
University of Maryland, Baltimore, Maryland 21201, USA.
4 process design, understanding, and control
Massachusetts Institute of Technology, Cambridge, Massachusetts
02139, USA. 3. To increase product development and manufacturing
5
Light Pharm Inc., Cambridge, Massachusetts 02142, USA. efficiencies
6
To whom correspondence should be addressed. (e-mail: 4. To enhance root cause analysis and postapproval
lawrence.yu@fda.hhs.gov) change management

771 1550-7416/14/0400-0771/0 # 2014 American Association of Pharmaceutical Scientists

772
Under QbD, these goals can often be achieved by
linking product quality to the desired clinical performance
and then designing a robust formulation and manufactur-
ing process to consistently deliver the desired product
quality.
Since the initiation of pharmaceutical QbD, the FDA
has made significant progress in achieving the first
objective: performance-based quality specifications. Some
examples of FDA policies include tablet scoring and bead
sizes in capsules labeled for sprinkle (14,15). The recent
FDA discussions on the assayed potency limits for narrow
therapeutic index drugs and physical attributes of generic
drug products reflect this trend (16). Nonetheless, it
should be recognized that ICH documents (3–9) did not
explicitly acknowledge clinical performance-based specifi-
cations as a QbD goal, although this was recognized in a
recent scientific paper (10).
The second objective of pharmaceutical QbD is to
increase process capability and reduce product variability
that often leads to product defects, rejections, and recalls.
Achieving this objective requires robustly designed prod-
uct and process. In addition, an improved product and
process understanding can facilitate the identification and
control of factors influencing the drug product quality.
After regulatory approval, effort should continue to
improve the process to reduce product variability, defects,
rejections, and recalls.
QbD uses a systematic approach to product design and
development. As such, it enhances development capability,
speed, and formulation design. Furthermore, it transfers
resources from a downstream corrective mode to an
upstream proactive mode. It enhances the manufacturer’s
ability to identify the root causes of manufacturing
failures. Hence, increasing product development and
manufacturing efficiencies is the third objective of phar-
maceutical QbD.
The final objective of QbD is to enhance root cause
analysis and postapproval change management. Without good
product and process understanding, the ability to efficiently
scale-up and conduct root cause analysis is limited and
requires the generation of additional data sets on the
proposed larger scale. FDA’s change guidances (17,18)
provide a framework for postapproval changes. Recently,
the FDA issued a guidance intended to reduce the regulatory
filing requirements for specific low-risk chemistry,
manufacturing, and control (CMC) postapproval manufactur-
ing changes (19).
ELEMENTS OF PHARMACEUTICAL QUALITY
BY DESIGN
In a pharmaceutical QbD approach to product develop-
ment, an applicant identifies characteristics that are critical to
quality from the patient’s perspective, translates them into the
drug product critical quality attributes (CQAs), and estab-
lishes the relationship between formulation/manufacturing
variables and CQAs to consistently deliver a drug product
with such CQAs to the patient. QbD consists of the following
elements:
Yu et al.
1. A quality target product profile (QTPP) that identifies
the critical quality attributes (CQAs) of the drug
product
2. Product design and understanding including the
identification of critical material attributes (CMAs)
3. Process design and understanding including the iden-
tification of critical process parameters (CPPs) and a
thorough understanding of scale-up principles, linking
CMAs and CPPs to CQAs
4. A control strategy that includes specifications for the
drug substance(s), excipient(s), and drug product as
well as controls for each step of the manufacturing
process
5. Process capability and continual improvement
Quality Target Product Profile that Identifies the Critical
Quality Attributes of the Drug Product
QTPP is a prospective summary of the quality charac-
teristics of a drug product that ideally will be achieved to
ensure the desired quality, taking into account safety and
efficacy of the drug product. QTPP forms the basis of design
for the development of the product. Considerations for
inclusion in the QTPP could include the following (3):
& Intended use in a clinical setting, route of adminis-
tration, dosage form, and delivery system(s)
& Dosage strength(s)
& Container closure system
& Therapeutic moiety release or delivery and attributes
affecting pharmacokinetic characteristics (e.g., disso-
lution and aerodynamic performance) appropriate to
the drug product dosage form being developed
& Drug product quality criteria (e.g., sterility, purity,
stability, and drug release) appropriate for the
intended marketed product
Identification of the CQAs of the drug product is the
next step in drug product development. A CQA is a
physical, chemical, biological, or microbiological property
or characteristic of an output material including finished
drug product that should be within an appropriate limit,
range, or distribution to ensure the desired product
quality (3). The quality attributes of a drug product may
include identity, assay, content uniformity, degradation
products, residual solvents, drug release or dissolution,
moisture content, microbial limits, and physical attributes
such as color, shape, size, odor, score configuration, and
friability. These attributes can be critical or not critical.
Criticality of an attribute is primarily based upon the
severity of harm to the patient should the product fall
outside the acceptable range for that attribute. Probability
of occurrence, detectability, or controllability does not
impact criticality of an attribute.
It seems obvious that a new product should be ade-
quately defined before any development work commences.
However, over the years, the value of predefining the target
characteristics of the drug product is often underestimated.
Consequently, the lack of a well-defined QTPP has resulted in
wasted time and valuable resources. A recent paper by Raw
Understanding Pharmaceutical Quality by Design
et al. (12) illustrates the significance of defining the correct
QTPP before conducting any development. Also, QbD exam-
ples exemplify the identification and use of QTPPs (20–22).
Product Design and Understanding
Over the years, QbD’s focus has been on the process
design, understanding, and control, as discussed in the ICH
Q8 (R2) guidance (3). It should be emphasized that product
design, understanding, and control are equally important.
Product design determines whether the product is able to
meet patients’ needs, which is confirmed with clinical studies.
Product design also determines whether the product is able to
maintain its performance through its shelf life, which is
confirmed with stability studies. This type of product under-
standing could have prevented some historical stability
failures.
The key objective of product design and understanding is
to develop a robust product that can deliver the desired
QTPP over the product shelf life. Product design is open-
ended and may allow for many design pathways. Key
elements of product design and understanding include the
following:
& Physical, chemical, and biological characterization of
the drug substance(s)
& Identification and selection of excipient type and
grade, and knowledge of intrinsic excipient variability
& Interactions of drug and excipients
& Optimization of formulation and identification of
CMAs of both excipients and drug substance
To design and develop a robust drug product that has the
intended CQAs, a product development scientist must give
serious consideration to the physical, chemical, and biological
properties of the drug substance. Physical properties include
physical description (particle size distribution and particle
morphology), polymorphism and form transformation, aqueous
solubility as a function of pH, intrinsic dissolution rate,
hygroscopicity, and melting point(s). Pharmaceutical solid
polymorphism, for example, has received much attention
recently since it can impact solubility, dissolution, stability, and
manufacturability. Chemical properties include pKa, chemical
stability in solid state and in solution, as well as photolytic and
oxidative stability. Biological properties include partition coef-
ficient, membrane permeability, and bioavailability.
Pharmaceutical excipients are components of a drug
product other than the active pharmaceutical ingredient.
Excipients can (1) aid in the processing of the dosage
form during its manufacture; (2) protect, support, or
enhance stability, bioavailability, or patient acceptability;
(3) assist in product identification; or (4) enhance any
other attribute of the overall safety, effectiveness, or
delivery of the drug during storage or use (23). They
are classified by the functions they perform in a pharma-
ceutical dosage form. Among 42 functional excipient
categories listed in USP/NF (24), commonly used excipi-
ents include binders, disintegrants, fillers (diluents), lubri-
cants, glidants (flow enhancers), compression aids, colors,
sweeteners, preservatives, suspending/dispersing agents,
pH modifiers/buffers, tonicity agents, film formers/coatings,
flavors, and printing inks. The FDA’s inactive ingredients
773
database (25) lists the safety limits of excipients based on
prior use in FDA-approved drug products.
It is well recognized that excipients can be a major
source of variability. Despite the fact that excipients can alter
the stability, manufacturability, and bioavailability of drug
products, the general principles of excipient selection are not
well-defined, and excipients are often selected ad hoc without
systematic drug-excipient compatibility testing. To avoid
costly material wastage and time delays, ICH Q8 (R2)
recommends drug-excipient compatibility studies to facilitate
the early prediction of compatibility (3). Systematic drug-
excipient compatibility studies offer several advantages as
follows: minimizing unexpected stability failures which usual-
ly lead to increased development time and cost, maximizing
the stability of a formulation and hence the shelf life of the
drug product, and enhancing the understanding of drug-
excipient interactions that can help with root cause analysis
should stability problems occur.
Formulation optimization studies are essential in developing a
robust formulation that is not on the edge of failure. Without
optimization studies, a formulation is more likely to be high risk
because it is unknown whether any changes in the formulation itself
or in the raw material properties would significantly impact the
quality and performance of the drug product, as shown in recent
examples (26,27). Formulation optimization studies provide impor-
tant information on the following:
& Robustness of the formulation including establishing
functional relationships between CQAs and CMAs
& Identification of CMAs of drug substance, excipients,
and in-process materials
& Development of control strategies for drug substance
and excipients
In a QbD approach, it is not the number of optimization
studies conducted but rather the relevance of the studies and
the utility of the knowledge gained for designing a quality
drug product that is paramount. As such, the QbD does not
equal design of experiments (DoE), but the latter could be an
important component of QbD.
Drug substance, excipients, and in-process materials may
have many CMAs. A CMA is a physical, chemical, biological,
or microbiological property or characteristic of an input
material that should be within an appropriate limit, range,
or distribution to ensure the desired quality of that drug
substance, excipient, or in-process material. For the purpose
of this paper, CMAs are considered different from CQAs in
that CQAs are for output materials including product
intermediates and finished drug product while CMAs are for
input materials including drug substance and excipients. The
CQA of an intermediate may become a CMA of that same
intermediate for a downstream manufacturing step.
Since there are many attributes of the drug substance
and excipients that could potentially impact the CQAs of the
intermediates and finished drug product, it is unrealistic that a
formulation scientist investigate all the identified material
attributes during the formulation optimization studies. There-
fore, a risk assessment would be valuable in prioritizing which
material attributes warrant further study. The assessment
should leverage common scientific knowledge and the
formulator’s expertise. A material attribute is critical when a
realistic change in that material attribute can have a
774
Table I. Typical Input Material Attributes, Process Parameters, and Quality Attributes of Pharmaceutical Unit Operations
Pharmaceutical unit operation
Input material attributes
• Particle size
• Particle size distribution
• Fines/oversize
• Particle shape
• Bulk/tapped/true density
• Cohesive/adhesive properties
• Electrostatic properties
• Moisture content
• Particle/granule size
• Particle/granule size
distribution
• Fines
• Particle/granule shape
• Bulk/tapped/true density
• Adhesive properties
• Electrostatic properties
• Hardness/plasticity
• Viscoelasticity
• Brittleness
• Elasticity
• Solid form/polymorph
• Moisture content
• Granule porosity/density
• Particle size distribution
• Fines/Oversize
• Particle shape
• Bulk/tapped/true density
• Cohesive/adhesive properties
• Electrostatic properties
• Hardness/plasticity
• Viscoelasticity
• Brittleness
• Elasticity
• Solid form/polymorph
• Moisture content
Yu et al.
Process parameters Quality attributes
Blending/mixing
• Type and geometry of mixer • Blend uniformity
• Mixer load level • Potency
• Order of addition • Particle size
• Number of revolutions (time and speed) • Particle size distribution
• Agitating bar (on/off pattern) • Bulk/tapped/true density
• Discharge method • Moisture content
• Holding time • Flow properties
• Environment temperature and RH • Cohesive/adhesive properties
• Powder segregation
• Electrostatic properties
Size reduction/comminution
Ribbon milling
• Ribbon dimensions
• Ribbon density
• Ribbon porosity/solid fraction
Impact/cutting/screening mills • Particle/granule size
• Mill type • Particle/granule size distribution
• Speed • Particle/granule shape
• Blade configuration, type, orientation • Particle/granule shape factor
• Screen size and type (e.g., aspect ratio)
• Feeding rate • Particle/granule density/Porosity
• Bulk/tapped/true density
Fluid energy mill • Flow properties
• Number of grinding nozzles • API polymorphic form
• Feed rate • API crystalline morphology
• Nozzle pressure • Cohesive/adhesive properties
• Classifier • Electrostatic properties
• Hardness/Plasticity
Granule/ribbon milling • Viscoelasticity
• Mill type • Brittleness
• Speed • Elasticity
• Blade configuration, type, orientation
• Screen size and type
• Feeding rate
Wet granulation
High/low shear granulation • Endpoint measurement
• Type of granulator (High/low shear, top/bottom drive) (e.g., power consumption, torque,
• Fill level etc.)
• Pregranulation mix time • Blend uniformity
• Granulating liquid or solvent quantity • Potency
• Impeller speed, tip speed, configuration, location, power • Flow
consumption/torque • Moisture content
• Chopper speed, configuration, location, power consumption • Particle size and distribution
• Spray nozzle type and location • Granule size and distribution
• Method of binder excipient addition (dry/wet) • Granule strength and uniformity
• Method of granulating liquid addition (spray or pump) • Bulk/tapped/true density
• granulating liquid temperature • API polymorphic form
• granulating liquid addition rate and time • Cohesive/adhesive properties
• Wet massing time (post-granulation mix time) • Electrostatic properties
• Bowl temperature(jacket temperature) • Granule brittleness
• Product temperature • Granule elasticity
• Post mixing time • Solid form/polymorph
• Pump Type: Peristaltic, Gear type
• Granulating liquid vessel (e.g., pressurized, heated)
Fluid bed granulation
• Type of fluid bed
• Inlet air distribution plate
• Spray nozzle (tip size, type/quantity/ pattern/configuration/position)
• Filter type and orifice size
Understanding Pharmaceutical Quality by Design
Pharmaceutical unit operation
Input material attributes
• Particle size, distribution
• Fines/oversize
• Particle shape
• Cohesive/adhesive properties
• Electrostatic properties
• Hardness/plasticity
• Viscoelasticity
• Brittleness
• Elasticity
• Solid form/polymorph
• Moisture content
• Particle size, distribution
• Fines/oversize
• Particle shape
• Cohesive/adhesiveproperties
• Electrostatic properties
• Hardness/plasticity
• Bulk/tapped/true density
• Viscoelasticity
• Brittleness
• Elasticity
775
Table I. (continued)
Process parameters Quality attributes
• Fill level
• Bottom screen size and type
• Preheating temperature/time
• Method of binder excipient addition (dry/wet)
• Granulating liquid temperature
• Granulating liquid quantity
• Granulating liquid concentration/viscosity
• Granulating liquid holding time
• Granulating liquid delivery method
• Granulating liquid spray rate
• Inlet air, volume, temperature, dew point
• Atomization air pressure
• Product and filter pressure differentials
• Product temperature
• Exhaust air temperature, flow
• Filter shaking interval and duration
Drying
Fluidized bed • Granule size and distribution
• Inlet air volume, temperature, dew point • Granule strength, uniformity
• Product temperature • Flow
• Exhaust air temperature, flow • Bulk/tapped/true density
• Filter type and orifice size • Moisture content
• Shaking interval and duration • Residual solvents
• Total drying time • API polymorphic form or transition
• Purity profile
Tray • Moisture profile (e.g. product
• Type of tray dryer temperature vs. LOD)
• Bed thickness/tray depth (depth of product per tray) • Potency
• Type of drying tray liner (e.g., paper, plastic, • Cohesive/adhesive properties
synthetic fiber, etc.) • Electrostatic properties
• Quantity carts and trays per chamber
• Quantity of product per tray
• Drying time and temperature
• Air flow
• Inlet dew point
Vacuum/microwave
• Jacket temperature
• Condenser temperature
• Impeller speed
• Bleed air volume
• Vacuum pressure
• Microwave power
• Electric field
• Energy supplied
• Product temperature
• Bowl and lid temperature
• Total drying time
Roller compaction/chilsonation
• Type of roller compactor • Ribbon appearance (edge attrition,
• Auger (feed screw) type/design (horizontal, splitting, lamination, color, etc.)
vertical or angular) • Ribbon thickness
• Deaeration (e.g., vacuum) • Ribbon density (e.g., envelop
• Auger (feed screw) speed density)
• Roll shape (cylindrical or interlocking). • Ribbon porosity/solid fraction
• Roll surface design (smooth, knurled, serrated, • Ribbon tensile strength/breaking
or pocketed) force
• Roll gap width (e.g., flexible or fixed) • Throughput rate
• Roll speed • API polymorphic form and transition
• Roll pressure
776
Pharmaceutical unit operation
Input material attributes
• Solid form/polymorph
• Particle size, distribution
• Fines/oversize
• Particle shape
• Cohesive/adhesiveproperties
• Electrostatic properties
• Hardness/plasticity
• Bulk/tapped/true density
• Viscoelasticity
• Brittleness
• Elasticity
• Solid form/polymorph
• Particle size, distribution
• Fines/oversize
• Particle shape
• Melting point
• Density
• Solid form/polymorph
• Moisture content
• Particle/granule size
and distribution
• Fines/oversize
• Particle/granule shape
• Cohesive/adhesive
properties
• Electrostatic properties
• Hardness/plasticity
• Bulk/tapped/true density
• Viscoelasticity
• Brittleness
• Elasticity
• Solid form/polymorph
• Moisture
• Particle/granule size and
distribution
• Fines/oversize
• Particle/granule shape
• Cohesive/adhesive properties
• Electrostatic properties
• Hardness/plasticity
• Bulk/tapped/true density
• Viscoelasticity
• Brittleness
Yu et al.
Table I. (continued)
Process parameters Quality attributes
• Roller temperature
• Fines recycled (yes or no, # of cycles)
Extrusion–Spheronization
• Type of extruder (screw or basket) • Extrudate
• Screw length, pitch, and diameter • Density
• Screw channel depth • Length/thickness/diameter
• Screw blade configuration • Moisture content
• Number of screws (single/dual) • API polymorphic form and transition
• Die or screen configuration (e.g., radial or axial) • Content uniformity
• Die length/diameter ratio • Throughput
• Roll diameter (mm)
• Screen opening diameter (mm) • Pellets after spheronization
• Screw speed (rpm) • Pellets size and distribution
• Feeding rate (g/min) • Pellets shape factor (e.g. aspect
• Type and scale of spheronizer ratio)
• Spheronizer load level • Bulk/Tapped density
• Plate geometry and speed • Flow properties
• Plate groove design (spacing and pattern) • Brittleness
• Air flow • Elasticity
• Residence time • Mechanical strength
• Friability
Hot melt extrusion
• Screw design (twin/single) • Extrudate density
• Screw speed • Length/thickness/diameter
• Screw opening diameter (mm) • Polymorphic form and transition
• Solid and liquid feed rates • Content uniformity
• Feeder type/design • Throughput
• Feed rate
• No. of zones
• Zone temperatures
• Chilling rate
Tabletting
• Type of press (model, geometry, number of stations) • Tablet appearance
• Hopper design, height, angle, vibration • Tablet weight
• Feeder mechanism (gravity/forced feed, shape of wheels, • Weight uniformity
direction of rotation, number of bars) • Content uniformity
• Feed frame type and speed • Hardness/tablet breaking force/
• Feeder fill depth tensile strength
• Tooling design (e.g., dimension, score configuration, • Thickness/dimensions
quality of the metal) • Tablet porosity/density/solid fraction
• Maximum punch load • Friability
• Press speed/dwell time • Tablet defects
• Precompression force • Moisture content
• Main compression force • Disintegration
• Punch penetration depth • Dissolution
• Ejection force
• Dwell Time
Encapsulation
• Machine type • Capsule appearance
• Machine fill speed • Weight
• Tamping Force • Weight uniformity
• No. of tamps • Content uniformity
• Auger screw design/speed • Moisture content
• Powder bed height • Slug tensile strength
• Disintegration
• Dissolution
Understanding Pharmaceutical Quality by Design
Pharmaceutical unit operation
Input material attributes
• Elasticity
• Solid form/polymorph
• Moisture
• Tablet dimensions
• Tablet defects
• Hardness/plasticity
• Density
• Porosity
• Moisture content
• Tablet dimensions
• Tablet defects
• Hardness/plasticity
• Density/porosity
moisture content
• Size/dimensions
• Polymer type
membrane thickness
777
Table I. (continued)
Process parameters Quality attributes
Pan coating
• Type of pan coater (conventional or side-vented) • Coating efficiency
• Pan (fully perforated or partial perforated) • Core tablet weight before and after
• Baffle (design, number, location) preheating
• Pan load level • Moisture (gain/loss) during
• Pan rotation speed preheating
• Spray nozzle (type, quantity, pattern, configuration, • Environmental equivalency factor
spray pattern) • Coated drug product (e.g., tablet or
• Nozzle to bed distance capsule) appearance
• Distance between nozzles • % weight gain
• Nozzle orientation • Film thickness
• Total preheating time • Coating (polymer and /or color)
• Inlet air flow rate, volume, temperature, dew point uniformity
• Product temperature • Hardness/breaking force/Tensile
• Individual nozzle spray rate strength
• Total spray rate • Friability
• Atomization air pressure • Moisture (gain/loss) during overall
• Pattern air pressure process
• Exhaust air temperature, air flow • Residual solvent(s)
• Total coating, curing time and drying time • Disintegration
• Dissolution
• Tablet defects
• Visual attributes
Fluid bed coating
• Type of fluid bed coater • Coating efficiency
• Fluid bed load level • Core tablet weight before and after
• Partition column diameter preheating
• Partition column height • Moisture (gain/loss) during
• Number of partition columns preheating
• Air distribution plate type and size • Environmental equivalency factor
• Filter type and orifice size • Coated drug product (e.g., tablet or
• Filter differential pressure capsule) appearance
• Filter shaking interval and duration • % weight gain
• Spray nozzle (type, quantity, pattern, configuration) • Film thickness
• Nozzle port size • Coating (polymer and /or color)
• Total preheating time uniformity
• Spray rate per nozzle • Hardness/breaking force/tensile
• Total spray rate strength
• Atomization air pressure • Friability
• Inlet air flow rate, volume, temperature, dew point • Moisture (gain/loss) during overall
• Product temperature process
• Exhaust air temperature, air flow • Residual solvent(s)
• Total coating, curing and drying time • Disintegration
• Dissolution
• Tablet defects
• Visual attributes
Laser drilling
• Conveyor type • Opening diameter (internal and
• Conveyor speed external)
• Laser power • Depth
• Number of pulses • Shape of the opening
• Type(s) of lens(es)
• One or two sided
• Number of holes
778
significant impact on the quality of the output material.
Product understanding includes the ability to link input
CMAs to output CQAs. The steps taken to gain product
understanding may include the following:
1. Identify all possible known input material attributes
that could impact the performance of the product
2. Use risk assessment and scientific knowledge to
identify potentially high risk attributes
3. Establish levels or ranges of these potentially high-risk
material attributes
4. Design and conduct experiments, using DoE when
appropriate
5. Analyze the experimental data and, when possible, apply
first principle models to determine if an attribute is critical
6. Develop a control strategy. For critical material
attributes, define acceptable ranges. For noncritical
material attributes, the acceptable range is the range
investigated. When more than one excipient is in-
volved, these defined acceptable ranges may be
termed formulation design space
Process Design and Understanding
A pharmaceutical manufacturing process usually consists
of a series of unit operations to produce the desired quality
product. Unit operations may be executed in batch mode or in a
continuous manufacturing process. A unit operation is a discrete
activity that involves physical or chemical changes, such as
mixing, milling, granulation, drying, compression, and coating.
A process is generally considered well-understood when (1) all
critical sources of variability are identified and explained, (2)
variability is managed by the process, and (3) product quality
attributes can be accurately and reliably predicted (28).
Process parameters are referred to as the input operating
parameters (e.g., speed and flow rate) or process state variables
(e.g., temperature and pressure) of a process step or unit operation.
A process parameter is critical when its variability has an impact on
a critical quality attribute and therefore should be monitored or
controlled to ensure the process produces the desired quality.
Under this definition, the state of a process depends on its CPPs
and the CMAs of the input materials. Table I lists the typical
manufacturing unit operations, material attributes, process param-
eters, and quality attributes for solid oral dosage forms.
Process robustness is the ability of a process to deliver
acceptable drug product quality and performance while tolerating
variability in the process and material inputs (29). The effects of
variations in process parameters and material attributes are
investigated in process robustness studies. The analysis of these
experiments identifies CPPs that could affect drug product quality
and establishes limits for these CPPs (and CMAs) within which
the quality of drug product is assured. The relationship between
input CMAs and CPPs and output CQAs is shown in Fig. 1.
Steps to establish process understanding are very similar
to those of product understanding and include the following:
1. Identify all possible known process parameters that
could impact the performance of the process
2. Use risk assessment and scientific knowledge to
identify potentially high-risk parameters
Yu et al.
3. Establish levels or ranges of these potentially high-risk
parameters
4. Design and conduct experiments, using DoE when
appropriate
5. Analyze the experimental data and, when possible,
determine scalability and apply first principle models
to determine if a process parameter is critical. Link
CMAs and CPPs to CQAs when possible.
6. Develop a control strategy. For critical parameters,
define acceptable ranges. For noncritical parameters,
the acceptable range is the range investigated. When
more than one process parameter or material attribute
is involved, these defined acceptable ranges may be
termed process design space
While developing a strategy for investigating both
product design and understanding and process design and
understanding, studies can be designed in such a way that
both the objectives of product and process understanding are
achieved simultaneously. In addition, an interactive (or
interdependent) relationship among material attributes, pro-
cess parameters, and product attributes can be more easily
developed when such analyses are performed in carefully
planned and designed experimental studies.
ICH Q8 (R2) defines design space as the multidimen-
sional combination and interaction of input variables (e.g.,
material attributes) and process parameters that have been
demonstrated to provide assurance of quality (3). Parameter
movements that occur within the design space are not
subjected to regulatory notification. However, movement
out of the design space is considered to be a change and
would normally initiate a regulatory postapproval change
process. Design space is proposed by the applicant and is
subject to regulatory assessment and approval. Thus, design
space is the direct outcome of analysis of the DoE data or
validated models such as first-principle models.
Design space may be scale and equipment dependent.
Therefore, the design space determined at laboratory scale
may need to be justified for use at commercial scale.
Approaches for justification may include geometric consider-
ations, kinematic considerations, heat and mass transfer, or
dimensionless numbers as well as continual verification
during commercial manufacturing. Justification is needed
because the mechanistic understanding of pharmaceutical
unit operations may be limited and scale-up is largely based
on general rule of thumb and trial-and-error approaches;
however, when mechanistic understanding or reliable
CPPs
Pharmaceutical
CMAs CQAs
Unit
Input Operation Output
Materials Materials or
Product
CQAs = f (CPP1, CPP2 , CPP3 …CMA1, CMA2, CMA3…)
Fig. 1. Link input critical material attributes (CMAs) and critical
process parameters (CPPs) to output critical quality attributes
(CQAs) for a unit operation
Understanding Pharmaceutical Quality by Design 779

empirical models (i.e., extensive process understanding) release testing (e.g., the use of predictive models as a
exists, then the design space can be translated across scale. surrogate for traditional release test, where the model may
Pharmaceutical products are frequently manufactured by be defined in terms of traditional in-process measurements).
a combination of unit operations. For example, tablets Level 2 consists of pharmaceutical control with reduced
prepared by direct compression may simply involve blending end-product testing and flexible material attributes and process
and compression. However, when tablets are prepared by wet parameters within the established design space. QbD fosters
granulation, unit operations may involve blending, granula- product and process understanding and facilitates identification
tion, wet milling, drying, dry milling, blending for lubrication, of the sources of variability that impact product quality.
compression, coating, and packaging. In such cases, the Understanding the impact that variability has on in-process
output of the first unit operation becomes an input of materials, downstream processing, and drug product quality
subsequent unit operations. Process understanding could be provides an opportunity to shift controls upstream and to reduce
conducted on each unit operation or a combination of unit
the reliance on end-product testing (3).
operations to determine CMAs, CPPs, and CQAs. Figure 2
Level 3 is the level of control traditionally used in the
shows an example how the CMAs and CPPs were deter-
pharmaceutical industry. This control strategy relies on extensive
mined, using an example of an immediate release dosage
end-product testing and tightly constrained material attributes
form (20).
and process parameters. Due to limited characterization of the
Control Strategy sources of variability and inadequate understanding of the
impact that CMAs and CPPs have on the drug product CQAs,
The knowledge gained through appropriately designed any significant change in these requires regulatory oversight.
development studies culminates in the establishment of a Significant industry and regulatory resources are spent debating
control strategy. As shown in Fig. 3, control strategy could issues related to acceptable variability, the need for additional
include three levels of controls as follows: controls, and the establishment of acceptance criteria.
In reality, a hybrid approach combining levels 1 and 2
Level 1 utilizes automatic engineering control to can be used. ICH Q8 (R2) (3) defines a control strategy as a
monitor the CQAs of the output materials in real time. This planned set of controls, derived from current product and
level of control is the most adaptive. Input material attributes process understanding that ensures process performance and
are monitored and process parameters are automatically product quality. The controls can include parameters and
adjusted to assure that CQAs consistently conform to the attributes related to drug substance and drug product
established acceptance criteria. Level 1 control can enable materials and components, facility and equipment operating
real-time release testing and provides an increased level of conditions, in-process controls, finished product specifica-
quality assurance compared to traditional end-product test- tions, and the associated methods and frequency of monitor-
ing. It should be noted that adoption of process analytical ing and control. A control strategy can include, but is not
technology (PAT) is not the only way to implement real-time limited to, the following (3):

Material Attributes Process Parameters

Acetriptan [solid state form, solubility, morphology, particle size distribution (PSD), bulk,
density, flowability, cohesiveness, moisture content, hygroscopicity, chemical stability,
process impurities, residual solvents, etc…]
Lactose [type, grade, source, amount, polymorphism, PSD, morphology, aspect ratio, bulk,
density, moisture content, flowability, compressibility, lot-to-lot variability, etc…]
Microcrystalline Cellulose (MCC) [type, grade, source, amount, PSD, bulk, density,
morphology, flowability, moisture, content, compressibility, lot-to-lot variability, etc…]
Croscarmellose [type, grade, source, amount, degree of substitution, PSD, moisture
content, lot-to-lot variability, etc…]
Talc [type, grade, amount, PSD, density, specific surface area, moisture content, lot-to-lot
variability, etc…]
Mg St [type, grade, source, amount, PSD, specific surface area, moisture content, lot-to-lot
variability, etc…]
Pre-roller compaction blending and lubrication [blender type, order of addition, fill
level, rotation speed, time, number of revolutions, intensifier bar (on/off),
environment (temp and RH), etc…]
Roller compaction [compactor type, feed screw speed, deaeration, roller surface
design, roller pressure, roller speed, roller gap, environment (temp and RH), etc…]
Milling [Mill type, blade configuration, speed, screen type, mesh size, # of recycles,
environment (temp and RH), etc…]
Final blending and Lubrication [blender type, order of addition, fill level, rotation
speed, time, number of revolutions, intensifier bar (on/off), environment (temp and
RH), etc…]
Compression [Press type, number of stations, tooling design, feed frame paddle speed,
feeder fill depth, pre-compression force, main compression force, press speed
(dwell time), hopper design, hopper fill level, drop height of finished tablets, run
time, environment (temp and RH), etc…]

Risk Assessment
Prior Knowledge
First Principle
Critical Material Attributes* DoEs Critical Process Parameters*
Acetriptan: PSD, polymorphic form
Pre-roller compaction blending and lubrication:
Lactose and MCC: amount and ratio
number of revolutions
Croscarmellose: amount
Roller compaction: roller pressure and roller gap
Talc: amount
Milling: mill screen orifice size
Mg St: amount
Final blending and Lubrication: none within the ranges studied
(Note: Excipients type, grade, and source are fixed and its quality is
Compression: main compression force
controlled per compendial/in-house specifications.)
Drug Product
Critical Quality Attributes
(Part of QTPP)

DoEs & Scale-up Physicochemical DoEs & Scale-up

-With interactions and Assay -With interactions
quadratic responses Content Uniformity and quadratic
Dissolution
-Univariate OK if responses
proven that there are Safety -Univariate OK if
Stability
no interactions proven that there are
Degradation Products
no interactions
Efficacy
Bioavailability

*Conclusion is drawn based upon the ranges studied and the control strategy for other variables (fixed or controlled within the ranges studied)

Fig. 2. Product and process understanding: an example for immediate release dosage forms
780
Fig. 3. Control strategy implementation options
& Control of input material attributes (e.g., drug substance,
excipient, in process material, and primary packaging
material) based on an understanding of their impact on
processability or product quality
& Product specification(s)
& Controls for unit operations that have an impact on
downstream processing or product quality (e.g., the impact
of drying on degradation and particle size distribution of
the granulate on dissolution)
& In-process or real-time release testing in lieu of end-product
testing (e.g., measurement and control of CQAs during
processing)
& A monitoring program (e.g., full product testing at regular
intervals) for verifying multivariate prediction models
Process Capability and Continual Improvement
Process capability measures the inherent variability of a
stable process that is in a state of statistical control in
relation to the established acceptance criteria. Table II
shows the definition, calculation formula, and description of
process capability indices (30) that are useful for monitoring
the performance of pharmaceutical manufacturing process-
es. Calculations based on the inherent variability due to
common cause of a stable process (i.e., in a state of
statistical control) result in process capability (Cp and Cpk)
indices. When the process has not been demonstrated to be
in a state of statistical control, the calculation needs to be
Table II. Process Capability Indices and Their Measures
Index Description
ðUSL−LSLÞ
Cp ¼ Estimates process capability when the data mean is centered between upper and lower specification limits.
6b
σ
Cpkl ¼ ðMean−LSLÞ Estimates process capability when the data mean is not centered between upper and lower specification limits or when
3b
σ
specifications consist of a lower limit only.
Cpku ¼ ðUSL−MeanÞ Estimates process capability when the data mean is not centered between upper and lower specification limits or when
3b
σ
specifications consist of an upper limit only.
USL upper specification limit, LSL lower specification limit, σ
b (sigma hat) inherent variability due to common cause of a stable process
Yu et al.
based on sample standard deviation of all individual
(observed) samples taken over a longer period of time; the
result is a process performance index (Pp and Ppk). A state
of statistical control is achieved when the process exhibits
no detectable patterns or trends, such that the variation
seen in the data is believed to be random and inherent to
the process (31).
When a process is not in a state of statistical control, it is
because the process is subject to special cause (source of
intermittent variation in a process). Special causes can give rise
to short-term variability of the process or can cause long-term
shifts or drifts of the process mean. Special causes can also create
transient shifts or spikes in the process mean. On the other hand,
common cause is a source of inherent variation that is random,
always present, and affects every outcome of the process. In a
QbD development process, the product and process under-
standing gained during pharmaceutical development should
result in early identification and mitigation of potential sources
of common cause variation via the control strategy. The
manufacturing process will move toward a state of statistical
control, and, once there, the manufacturer will continue to
improve process capability by reducing or removing some of the
random causes present and/or adjusting the process mean
towards the preferred target value to the benefit of the patient.
In a non-QbD approach, common cause variation is more likely
to be discovered during commercial production and may
interrupt commercial production and cause drug shortage when
it will require a root cause analysis.
Process capability can be used to measure process
improvement through continuous improvement efforts that
focus on removing sources of inherent variability from the
process operation conditions and raw material quality.
Ongoing monitoring of process data for Cpk and other
measures of statistical process control will also identify when
special variations occur that need to be identified and
corrective and preventive actions implemented.
Continuous improvement is a set of activities that the
applicant carries out in order to enhance its ability to meet
requirements. Continual improvements typically have five
phases as follows (32):
& Define the problem and the project goals, specifically
& Measure key aspects of the current process and
collect relevant data
& Analyze the data to investigate and verify cause-and-
effect relationships. Determine what the relationships
are, and attempt to ensure that all factors have been
considered. Seek out root cause of the defect if any.
Understanding Pharmaceutical Quality by Design
& Improve or optimize the current process based upon
data analysis using techniques such as design of
experiments to create a new, future state process.
Set up pilot runs to establish process capability.
& Control the future state process to ensure that any
deviations from target are corrected before they
result in defects. Implement control systems such as
statistical process control, production boards, visual
workplaces, and continuously monitor the process.
In addition, continuous improvement can apply to legacy
products. Legacy products usually have a large amount of
historical manufacturing data. Using multivariate analysis to
examine the data could uncover major disturbances in the form
of variability in raw materials and process parameters. Contin-
uous improvement could be achieved by reducing and control-
ling this variability. Newer processes associated with a design
space facilitate continuous process improvement since appli-
cants will have regulatory flexibility to move within the design
space (ICH Q8).
PHARMACEUTICAL QUALITY BY DESIGN TOOLS
Prior Knowledge
Although not officially defined, the term “prior knowl-
edge” has been extensively used in workshops, seminars,
and presentations. In regulatory submissions, applicants
often attempt to use prior knowledge as a “legitimate”
reason for substitution of scientific justifications or
conducting necessary scientific studies.
Knowledge may be defined as a familiarity with someone
or something, which can include information, facts, descrip-
tions, and/or skills acquired through experience or education.
The word “prior” in the term “prior knowledge” not only
means “previous,” but also associates with ownership and
confidentiality, not available to the public. Thus, for the
purpose of this paper, prior knowledge can only be obtained
through experience, not education. Knowledge gained
through education or public literature may be termed public
knowledge. Prior knowledge in the QbD framework general-
ly refers to knowledge that stems from previous experience
that is not in publically available literature. Prior knowledge
may be the proprietary information, understanding, or skill
that applicants acquire through previous studies.
Risk Assessment
ICH Q9 quality risk management indicates that “the
manufacturing and use of a drug product, including its
components, necessarily entail some degree of risk.… The
evaluation of the risk to quality should be based on scientific
knowledge and ultimately link to the protection of the patient
and the level of effort, formality, and documentation of the
quality risk management process should be commensurate with
the level of risk (4).” The purpose of ICH Q9 is to offer a
systematic approach to quality risk management and does not
specifically address risk assessment in product development.
However, the risk assessment tools identified in ICH Q9 are
applicable to risk assessment in product development also.
781
The purpose of risk assessment prior to development
studies is to identify potentially high-risk formulation and
process variables that could impact the quality of the drug
product. It helps to prioritize which studies need to be conducted
and is often driven by knowledge gaps or uncertainty. Study
results determine which variables are critical and which are not,
which facilitates the establishment of a control strategy. The
outcome of the risk assessment is to identify the variables to be
experimentally investigated. ICH Q9 (4) provides a
nonexhaustive list of common risk assessment tools as follows:
& Basic risk management facilitation methods (flow-
charts, check sheets, etc.)
& Fault tree analysis
& Risk ranking and filtering
& Preliminary hazard analysis
& Hazard analysis and critical control points
& Failure mode effects analysis
& Failure mode, effects, and criticality analysis
& Hazard operability analysis
& Supporting statistical tools
It might be appropriate to adapt these tools for use in specific
areas pertaining to drug substance and drug product quality.
Mechanistic Model, Design of Experiments, and Data
Analysis
Product and process understanding is a key element of
QbD. To best achieve these objectives, in addition to mechanis-
tic models, DoE is an excellent tool that allows pharmaceutical
scientists to systematically manipulate factors according to a
prespecified design. The DoE also reveals relationships between
input factors and output responses. A series of structured tests
are designed in which planned changes are made to the input
variables of a process or system. The effects of these changes on
a predefined output are then assessed. The strength of DoE over
the traditional univariate approach to development studies is the
ability to properly uncover how factors jointly affect the output
responses. DoE also allows us to quantify the interaction terms
of the variables. DoE is important as a formal way of
maximizing information gained while minimizing the resources
required. DoE studies may be integrated with mechanism-based
studies to maximize product and process understanding.
When DoE is applied to formulation or process devel-
opment, input variables include the material attributes (e.g.,
particle size) of raw material or excipients and process
parameters (e.g., press speed or spray rate), while outputs
are the critical quality attributes of the in-process materials or
final drug product (e.g., blend uniformity, particle size or
particle size distribution of the granules, tablet assay, content
uniformity, or drug release). DoE can help identify optimal
conditions, CMAs, CPPs, and, ultimately, the design space.
FDA scientists have shown the use of DoE in product and
process design in recent publications (33–39).
Process Analytical Technology
The application of PAT may be part of the control
strategy (28). ICH Q8 (R2) identifies the use of PAT to
782
ensure that the process remains within an established design
space (3). PAT can provide continuous monitoring of CPPs,
CMAs, or CQAs to make go/no go decisions and to
demonstrate that the process is maintained in the design
space. In-process testing, CMAs, or CQAs can also be
measured online or inline with PAT. Both of these applica-
tions of PAT are more effective at detecting failures than end-
product testing alone. In a more robust process, PAT can
enable active control of CMAs and/or CPPs, and timely
adjustment of the operating parameters if a variation in the
environment or input materials that would adversely impact
the drug product quality is detected.
Application of PAT involves four key components as
follows (40):
& Multivariate data acquisition and analysis
& Process analytical chemistry tools
& Process monitoring and control
& Continuous process optimization and knowledge
management
Multivariate data acquisition and analysis requires building
scientific understanding about a process and identifying critical
material attributes and process parameters that affect product
quality and integrating this knowledge into the process control,
which is essentially the same as the process understanding in the
context of QbD. Process analytical chemistry tools provide real-
time and in situ data about the status of the process. Multivariate
data analysis takes the raw information from the PAT tools and
connects it to CQAs. Based on the outcome of the data analysis,
process controls adjust critical variables to assure that CQAs are
met. The information collected about the process provides a basis
for further process optimization. Studies in FDA laboratories
indicated the promise of several PAT tools and chemometric
approaches (41–44).
CONCLUSION
The goals of implementing pharmaceutical QbD are to
reduce product variability and defects, thereby enhancing
product development and manufacturing efficiencies and
postapproval change management. It is achieved by designing
a robust formulation and manufacturing process and establish-
ing clinically relevant specifications. The key elements of
pharmaceutical QbD can include the QTPP, product design
and understanding, process design and understanding, and scale
up, control strategy, and continual improvement. Prior knowl-
edge, risk assessment, DoE, and PAT are tools to facilitate
QbD implementation. Finally, product and process capability is
assessed and continually improved postapproval during product
lifecycle management.
ACKNOWLEDGMENT
The authors would like to thank Lane V. Christensen,
Devinder Gill, Frank Holcombe Jr, Robert Iser, Khalid Khan,
Robert Lionberger, Jennifer Maguire, Christine Moore, Yingxu
(Daniel) Peng, Andre Raw, Bhagwant Rege, Susan
Rosencrance, Vilayat Sayeed, Paul Schwartz, Glen Smith, Yue
Yu et al.
(Helen) Teng, Youmin Wang, Huiquan Wu, Abhay Gupta,
Ziyaur Rahman, and Naiqi Ya for their valuable suggestions.
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