Process Removal of Impurities in

Biotech Products
CASSS Midwest Regional Forum
October 5, 2017

Warren R. Emery
Sr. Research Scientist
Bioproduct R&D, Eli Lilly and Company
 Pharmaceutical Process Development
PHARMACEUTICAL DEVELOPMENT

The aim of pharmaceutical development is to design a quality product and its

manufacturing process to consistently deliver the intended performance of the
product. The information and knowledge gained from pharmaceutical development
studies and manufacturing experience provide scientific understanding to support
the establishment of the design space*, specifications, and manufacturing controls.

ICH Q8 R2

• The downstream purification process must be designed

to control a wide variety of critical quality attributes,
including impurities and contaminants
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 Impurities
Process-related impurities encompass those that are derived from the
manufacturing process, i.e., cell substrates (e.g., host cell proteins, host cell DNA),
cell culture (e.g., inducers, antibiotics, or media components), or downstream
processing.
 HCP, DNA
 Detergent, Flocculant, Leached Protein A, processing enzymes, PEG reagents

Product-related impurities (e.g., precursors, certain degradation products) are

molecular variants arising during manufacture and/or storage that do not have
properties comparable to those of the desired product with respect to activity, efficacy,
and safety.
• Aggregates
• Fragments
• Post translational modifications, sequence variants ICH Q6B
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 Contaminants
Contaminants in a product include all adventitiously introduced materials not
intended to be part of the manufacturing process, such as chemical and biochemical
materials (e.g., microbial proteases) and/or microbial species.

POTENTIAL SOURCES OF VIRUS CONTAMINATION

Viral contamination of biotechnology products may arise from the original source of the cell lines
or from adventitious introduction of virus during production processes.
A. Viruses That Could Occur in the Master Cell Bank (MCB)
Cells may have latent or persistent virus infection (e.g., herpesvirus) or endogenous retrovirus which may be
transmitted vertically from one cell generation to the next, since the viral genome persists within the cell.
B. Adventitious Viruses That Could Be Introduced during Production
Adventitious viruses can be introduced into the final product by several routes including, but not limited to, the
following: 1) the use of contaminated biological reagents such as animal serum components; 2) the use of a
virus for the induction of expression of specific genes encoding a desired protein; 3) the use of a contaminated
reagent, such as a monoclonal antibody affinity column; 4) the use of a contaminated excipient during
formulation; 5) contamination during cell and medium handling.
ICH Q6B, ICH Q5A
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 Downstream Purification - Overview
Cell Culture Primary Recovery Purification

0.2 micron

Typical
Monoclonal Clarification
Centrifugation and Depth Filtration
Production Viral Capture Low pH Viral
Antibody Bioreactor Inactivation Chromatography Inactivation

Purification Purification

Process
R
P

Intermediate Polishing Virus Tangential DS DS

Chromatography Chromatography Filtration Flow UF/DF Dispensing Storage

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 Control Points Matrix
Unit Operations Influencing the Attributes

Dispensing/ Freezing
Primary Recovery

Protein A Capture
Chromatography

Chromatography

Chromatography

Drug Substance
Inactivation and

Tangential Flow
Detergent Viral

Viral Filtration
Low pH Viral

Intermediate
Clarification
Inactivation
Production
Bioreactor

Polishing

Drug Substance Critical
Quality Attributes
Product Related Impurities
Aggregates
Fragments
Post Translational Mods.
Process Related Impurities
Residual DNA
Residual Host Cell Proteins
Residual Protein A
Residual Detergent
Media Components
Contaminants
Microbial Safety
Viral Safety
UF/DF
  
O 
O 
O 
O  
O    
O  
O 
O 

O*     

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 Purification – Platform Toolbox Approach

Chromatography Bulk Operations Filtration Operations

R
P

Protein A Affinity Flocculation Viral Filtration*

Cation Exchange Detergent Viral Inactivation* Depth Filtration
Anion Exchange Low pH Viral Inactivation* Tangential Flow UF/DF
Hydrophobic Interaction Heat Inactivation Single-pass TFF
Mixed-mode PEGylation Membrane Adsorbers
Hydroxyapatite Enzymatic Reactions
Dye Affinity
* = dedicated viral clearance
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 Viral Safety
Mission: To design/develop downstream purification processes with robust and consistent
viral clearance capacity in support of clinical trials and commercialization

= Dedicated
viral clearance
unit operations

Detergent Protein A Low pH Intermediate Polishing Virus

Inactivation Chromatography Inactivation Chromatography Chromatography Filtration

• Dedicated viral clearance unit operations ensure orthogonal and robust safety
margins for retrovirus – broad platform applicability

• Other unit operations may provide additional clearance capacity – these are
more process specific than dedicated operations
AEX, Protein A, Heat Inactivation - CEX, HIC, Mixed Mode

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 Capture Chromatography - Protein A Affinity
Protein A: 42 kDa protein found in the cell wall of the bacteria Staphylococcus
aureus. It binds the heavy chain within the Fc region of most immunoglobulins
and also within the Fab region of human VH3 family.

A wide variety of Protein A resins are available, including engineered forms with Protein A
improved selectivity and increased cleanability (base stability). Chromatography

PROS
• Expensive resin, but highly selective affinity mode of chromatography (contributes to ROI)
• Robust (multi-log) reduction of DNA, HCP, media components, detergent, etc.
• Moderate viral clearance capability

CONS
• Protein A leaching that must be controlled downstream

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 Intermediate Chromatography
Goal: To reduce and control multiple process and product related impurities.

Cation Exchange (typically bind/elute)

• Manufacturing friendly – high load ratio, simple buffers
• Strong HCP, Aggregate, DNA reduction, possible product-related impurities Intermediate
Chromatography
• Possible virus clearance

Anion Exchange (b/e, flowthrough, membrane)

• Manufacturing friendly – (very) high load ratio, simple buffers
• Predictable and generally robust virus clearance
• Strong DNA and modest HCP and Aggregate reduction , possible product-related impurities

Mixed Mode (IEX/hydrophobic) (b/e, flowthrough)

• Manufacturing friendly – (very) high load ratio, simple buffers
• Alternative selectivity to straight CEX or AEX – opportunities for optimization
• Possible virus clearance
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 Polishing Chromatography
Goal: To provide an orthogonal mode of separation and serve as the final
control point for multiple process and product related impurities.
Hydrophobic Interaction
• Less manufacturing friendly – lower load ratio, heavy usage of kosmotropic salts
Polishing
• Very strong HCP and Aggregate reduction Chromatography
• Separation potential for truncated and misfolded product-related impurities
• Possible virus clearance (works best with more hydrophilic proteins)
Hydroxyapatite
• Resin beads composed of crystalline Ca5(PO4)3(OH)
• Strong Aggregate separation – but limited resin lifetime
Dye Affinity (Cibacron blue, etc.)
• Textile dyes – dye structure consists of a chromophore, linked to a reactive group, with
sulfonic acid groups – tend to interact with binding sites on proteins
• Unique selectivity for many proteins – especially enzymes
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 Case Study – Up-Front HCP Control
Problem: DNA and HCP reduction across Protein A are good, but can the
affinity column perform even better?
• Flocculation at the end of cell culture (chitosan, pDADMAC) can significantly
improve performance of primary recovery.
• Newer flocculation techniques can also play a significant role in impurity Flocculation
removal (DNA, HCP), and can lead to a simplified downstream process. A
cleaner feedstream can allow Protein A to perform at higher level
• Proprietary flocculation technique by Gagnon group at Bioprocess Institute, Singapore
• New stimulus reactive polymer from Merck Millipore (evaluated at Eli Lilly)

Nian, R., et. al., 2016, “Advance Chromatin Extraction Improves Capture Performance of Protein A Affinity
Chromatography”. Journal of Chromatography A

Kang, Y., et. al., 2013, “Development of a Novel and Efficient Cell Culture Flocculation Process Using a Stimulus
Responsive Polymer to Streamline Antibody Purification Processes”. Biotechnology and Bioengineering
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 Case Study – 2-for-1 Inactivation
Problem: Some product molecules may be susceptible to enzymatic
degradation by proteases that are expressed by mammalian cell culture
• HCPs with enzymatic activity can impact the stability of some bioproducts –
if so, they must be controlled by the purification process
• Enzymatic activity may be present at HCP levels below our ability to detect Heat Inactivation

• Heat inactivation of enzymes takes advantage of differences in thermal stability

between the product molecule and the enzymatic HCP
• Some viruses are also susceptible to heat treatment to achieve inactivation
• May be performed in batch at lower temp/longer time – or continuously by HTST
Lambooy, P., et. al., 2008, “Heat Inactivation of Protease During Downstream Processing of a Fusion Protein Enables
Purification of a Stable Bulk Drug Substance”. Recovery Conference

Bailey, M., et. al., 2007, “Evaluation of Microfluidics Reactor Technology on the Kinetics of Virus Inactivation”.
Biotechnology and Bioengineering
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 Downstream Purification - Overview
Cell Culture Primary Recovery Purification

0.2 micron

Typical
Monoclonal Clarification
Centrifugation and Depth Filtration
Production Viral Capture Low pH Viral
Antibody Bioreactor Inactivation Chromatography Inactivation

Purification Purification

Process
R
P

Intermediate Polishing Virus Tangential DS DS

Chromatography Chromatography Filtration Flow UF/DF Dispensing Storage

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