BIOT7018 S1, 2024

BIOT7018 S1, 2024

Question 1

Dr. Patel is investigating a signaling pathway in models of rheumatoid arthritis, a painful chronic
autoimmune disease characterized by swelling and deformity. Dr Patel has found that Cytokine X
reduces inflammation, while Cytokine Y increases inflammation associated with the disease. Based on
this knowledge, how might you use this information to help patients suffering from rheumatoid
arthritis?
• Cytokine X-Based Therapy: Develop a treatment that enhances the activity or levels of
Cytokine X in patients with rheumatoid arthritis, as it reduces inflammation. This could
involve administering recombinant Cytokine X or developing drugs that stimulate the body's
production of Cytokine X.
• Cytokine Y Inhibition: Create therapies that inhibit the activity or production of Cytokine Y,
as it increases inflammation. This could involve using monoclonal antibodies that specifically
target and neutralize Cytokine Y, or small molecule inhibitors that block the signaling
pathway activated by Cytokine Y.
• Combination Therapy: Consider a combination approach that simultaneously increases
Cytokine X and inhibits Cytokine Y to synergistically reduce inflammation in rheumatoid
arthritis patients.
• Personalized Medicine: Investigate the possibility of personalized medicine approaches,
where patients are screened for their levels of Cytokine X and Cytokine Y, and therapies are
tailored accordingly to optimize the balance between these cytokines.
• Safety and Efficacy Testing: Conduct preclinical and clinical trials to test the safety and
efficacy of these therapies in reducing inflammation and improving symptoms in rheumatoid
arthritis patients. It's important to consider the potential risks associated with reducing an
inflammatory pathway. Suppressing the natural function of cytokines could lead to side
effects, such as increased susceptibility to infections or other immune-related disorders.
BIOT7018 S1, 2024

Question 2
The table below shows a list of insulin products currently in the market, some of which are innovator
drugs and some are biosimilars.
1. Considering the requirements for a product to be a biosimilar, identify in this list the pair of
products that constitute a reference product and its biosimilar. (0.25 marks)
2. Describe the aspects of a biosimilar that must be identical to the reference and the aspects
that can be different? (0.25 marks)
• Biosimilars must be identical molecules to the reference,
• this requires that the amino acid sequence and modifications are identical.
• Aspects of biosimilars that can be different include the expression system and the
formulation.

Amino
Expression
Company Insulin acid Modifications Formulations
system
changes

P>K Clear solution, glycerin,

E. coli (B28) sodium phosphate,
Eli Lilly HUMALOG none
(bacteria) K>P metacresol, zinc oxide, pH 7.0
(B29) to 7.8
Clear solution, glycerin,
S.
phenol, metacresol, zinc
cerevisiae P>D
Novo Nordisk NovoLog none oxide, sodium phosphate,
(yeast) (B28)
sodium chloride, pH of 7.2 to
7.6
S.
T Clear solution, glycerin,
cerevisiae glutamic acid
Novo Nordisk TRESIBA deletion metacresol, phenol, zinc, pH
(yeast) C16 fatty acid
(B30) 7.6

N>G
(A21) Clear solution, glycerol,
E. coli
Sanofi-Aventis LANTUS RR none metacresol, zinc, polysorbate
(bacteria)
added 20, pH 4
(B31-32)
N>G
(A21) Clear solution, glycerol ,
Biocon P. pastoris
SEMGLEE RR none metacresol, polysorbate 20,
Biologics (yeast)
added zinc chloride, pH 4
(B31-32)
BIOT7018 S1, 2024

Quiz 4
Company PepGenix wants to develop a peptide inhibitor that blocks protein DIM-1 from binding
protein DIM-L1. They have elucidated the X-ray crystal structure of protein DIM-1 bound to protein
DIM-L1. You are part of the protein design team.
a) Consider what information the X-ray structure provides in terms of how DIM-1 binds
to DIM-L1, and which residues are important for function and which for structure.
How will you go about designing a peptide inhibitor? Describe some of the key
considerations and experiments. (0.5 marks)
• Identify the contact surface from the crystal structure. (this a rationale drug-
based design we identify, Hotspots? Where key amino acids bind to the
target. Mimic that structure using in silico design approach and utilizing
scaffold to make a mimetics. Check the binding affinity of the naïve and
compare it with the one you synthesize( the optimized one). Then check the
binding using crystallography. Also check activity in vitro binding assays.
Or/And in vivo cytotoxicity )
• Check if interaction surface can be represented by a small peptide.
• Synthesize and check activity of peptide fragment.

b) You have successfully designed a highly potent inhibitor called SuperPep-A. You would
like to attach a radioactive metal for imaging biodistribution in small animals. How
should you decide on the conjugation site? (0.5 marks)
• Identify regions of the peptide not involved in the interaction site.
Preference those most distal from the interaction surface.
 BIOT7018 S1, 2024

Quiz 5
Question 1
Your collaborator has produced a targeted chemotherapeutic nanomedicine for cancer. It’s comprised
of doxorubicin attached to a novel polymer through an acid degradable linker. The polymer is further
functionalized with a fluorescent dye and an antibody that binds to Prostate Specific Membrane
Antigen. Briefly define which component is. and how they might each function when administered
into the body. (0.5 marks)
a. Targeting ligand Prostate-specific-membrane-Antigen binding antibody functions by
enhancing delivery of nanomedicine to the tumor site through binding of cell surface
antigens and increasing retention
b. Carrier/scaffold Functions as macromolecular carrier which is low fouling and soluble
to allow circulation and tumour delivery of payload
c. Prodrug: (doxorubicin attached through an linker) reducing systemic distribution of
the chemotherapeutic and then cleaving under acidic conditions in the tumour to
locally deliver the active chemotherapeutic (doxorubicin)
d. Imaging agent Fluorescent Dye Allows fluorescent imaging of the nanomedicine and
how it behaves. Fluorescent dyes primarily used for in vitro assays and preliminary
preclinical assessment (limited human applications due to depth penetration
limitations)
Question 2
You and your collaborator have received broad exposure from the success of your targeted
nanomedicine. You are now approached by a radiopharmaceutical company to repurpose your
successful nanomedicine carrier for a new application which they could then develop for the clinic.
They would like the new nanomedicine to:
a. Be targeted to breast cancer : Substitute anti-PSMA antibody for a targeting ligand specific for
breast cancer (antibody, antibody fragment, aptamer, small molecule ligand etc.). Common target
would be EGFR, HER2, HER3 (not a required answer)
b. Deliver siRNA as the treatment payload: Substitute doxorubicin payload for siRNA with appropriate
linker chemistries. These are commonly disulfide linkages (but not necessarily a required answer)
c. Be able to be imaged using Positron Emission Tomography (PET; nuclear imaging): Substitute the
fluorescent dye for a radiometal chelator appropriate for PET. This is a metal binding macrocycle
able to specifically bind a radioisotope for PET imaging such as 64Cu (discussed in lecture).
Describe what substitutions would need to be made to your original nanomedicine as described in
Question 1. What development and optimization processes and tests might you need to do during in
vitro and preclinical development in terms of deciding (0.5 marks):
d. how many targeting ligands to attach: Generate a series of nanomedicines with
varied targeting ligand densities on the surface. Assess both cellular targeting (in vitro
and in vivo behaviours) in terms of binding and MPS clearance to find an optimal level
where tumour association is enhanced without rapid clearance of targeted construct
from circulation.
e. what your product shelf-life might be: Undertake stability and efficacy testing over
time, primarily in terms of therapeutic payload as siRNA is likely the least stable
component of this construct. Assays could be in terms of cellular transfection efficacy
in vitro, direct measurements of siRNA identity (DNA electrophoresis etc) or
preclinical efficacy studies over time
f. how your material behaves in the body: Undertake PET imaging of nanomedicine in
preclinical models (mouse models of breast cancer) or clinical patients to assess
pharmacokinetics and biodistribution of the new optimized construct. Understanding
these factors will provide an understanding of how long your nanomedicine may
remain in the body (clearance profile), optimization of dosing regimens (are multiple
doses required?) and where the material may be deposited (predicting potential off-
target effects)
BIOT7018 S1, 2024

Quiz 6
As you conduct a presentation on public health strategies, a group of medical students eagerly seeks
clarification on vaccine types and their respective advantages and disadvantages. They are particularly
interested in comprehending the differences between whole pathogen vaccines, molecular-based
vaccines, and viral vector vaccines to evaluate their effectiveness and limitations against infectious
diseases. How would you expand upon the advantages and disadvantages of these distinct vaccine
types to address their inquiries effectively?

Question 2.You are approached by a curious individual who is keen to understand the complexities
surrounding the development of inactivated influenza vaccines and the strategies employed to tackle
them. As an expert in the field, how would you explain the hurdles faced during the development of
this vaccine and the current initiatives aimed at over-coming these obstacles?
• Strain matching from viral/antigenic diversity
• Inactivated vaccine using chemical agents/detergents
• Egg allergies, Production time
• Vaccine efficacy
• Cell-based vaccine technology
BIOT7018 S1, 2024

Quiz7

Question 1
The 2023 Nobel Prize in Physiology or Medicine was jointly to Katalin Karikó and Drew Weissman for
their discoveries concerning nucleotide base modifications that enabled the development of effective
mRNA vaccines. Can you please discuss what these nucleotides modifications are and how they impact
the performance of mRNA vaccines and therapies?

A major challenge for mRNA therapies is the activation of the innate immune response in recipient
cells, which suppresses translation and reduces the performance of mRNA therapies. The TOLL
receptors of the innate immune response can detect features of the mRNA, including double-
stranded RNA and uridine residues. However, the receptors are less sensitive at detecting modified
nucleotides resulting in lower activation of the innate immune response. Furthermore, modified
nucleotides can also increase the stability and translation of the mRNA
N1-methylpseudouridine is a commonly used modified nucleoside used in the COVID-19 mRNA
vaccines to improve stability and reduce immunogenicity. It is a derivative of pseudouridine, which is
a naturally occurring isomer of the nucleoside uridine found in RNA. In N1-methylpseudouridine, a
methyl group is attached to the nitrogen atom at position 1 of the pseudouridine ring.

Question 2
Please discuss the advantages and disadvantages of mRNA vaccines and therapies compared to other
biologics (antibodies, recombinant proteins etc.)? Use illustrative examples where possible.

Rapid design: mRNA vaccines can be developed and manufactured more quickly than traditional
biologics. This was exemplified during the COVID-19 pandemic when mRNA vaccines were developed
and approved within a year. However, the biotechnology industry has extensive experience in
producing and regulating biologics, leading to well-established manufacturing processes and
regulatory pathways.

Manufacture: mRNA production is based on in vitro transcription, which is a rapid and scalable
process. The same manufacturing process can be used for different mRNA sequences, making it
easier to produce vaccines for new variants or different diseases. However, each biologic often
requires customized manufacturing processes, including complex cell culture systems and
purification processes, which are time-consuming and expensive, and which can complicate scale-up
and production.

Intracellular Targets: mRNA is uptake into the cells, where it is translated within the cytoplasm,
allowing the target intracellular proteins. Antibodies are generally limited to extracellular targets and
cannot easily access intracellular proteins, limiting their range of potential therapeutic targets

Stability: mRNA is unstable and is degraded quickly within cells, leading to transient expression of
the encoded protein for 1-2 weeks. This can necessitate multiple doses or booster shots. Antibodies
typically have long half-lives in the body, often lasting for weeks to months, providing sustained
therapeutic effects.

Storage: mRNA vaccines often require very low temperatures for storage and transportation,
complicating logistics, especially in regions without advanced cold chain infrastructure. Many
biologics are stable at refrigerated temperatures, simplifying storage and distribution.
 BIOT7018 S1, 2024

Quiz 8

Question 1
What factors determine the effectiveness of natural killer cells in combating cancer, and how can we
use this knowledge to develop better immunotherapies for cancer treatment?
The activity of NK cells is balanced between signals of activating (e.g. NKG2D, NKp46, NKp30, NKp44)
and inhibitory receptors (e.g. KIR (for humans) or Ly49 (for mouse), where overall inhibitory receptors
are always dominant.
All health cells express class I MHC (the cellular window, which constantly expose neo peptides and
normal peptides to signal to T cell receptors if they are altered or not via their TCRs) which is a main
ligand for KIRs or Ly49, ensuring NK cell inhibition against normal cells.
Cancers, often stop expressing MHC I to avoid sensing by T cells, but by doing that they automatically
become highly immunogenic and susceptible for NK cell surveillance and killing.
Current immunotherapy developing aims to enhance NK cell activation by providing ligands for
activating receptors, and also boosting their function with other factors like cytokine or activating
antibodies to enhance their recognition of tumour cells and activation through Fc receptors.

Question 2
What are the advantages and limitations of using immunotherapy as a cancer treatment? Name and
explain at least 2 advantages and 2 limitations.
Advantages of Immunotherapy:
1. Targeted approach: Unlike traditional chemotherapy or radiation, immunotherapy works by
targeting specific cancer cells and does not harm healthy cells.
2. Long-term benefits: Immunotherapy has been shown to provide long-term benefits, with some
patients experiencing a complete and durable response.
3. Fewer side effects: Immunotherapy causes fewer side effects than traditional cancer treatments
because it does not damage healthy cells.
4. Increased survival rates: Immunotherapy has been shown to increase survival rates in some types of
cancer, including melanoma and lung cancer.
Limitations of Immunotherapy:
1. Limited effectiveness: Not all patients respond to immunotherapy, and even among those who do,
the response may be temporary.
2. High cost: Immunotherapy is expensive, which can be a barrier for many patients.
3. Potential side effects: While immunotherapy causes fewer side effects than traditional cancer
treatments, it can still cause immune-related side effects that can be severe.
4. Resistance: Some cancers can develop resistance to immunotherapy, making it less effective over
time.
To overcome these limitations and develop more effective and personalized immunotherapy
treatments, several strategies are being explored:
1. Combination therapy: Researchers are studying the use of combination therapies, such as
combining immunotherapy with chemotherapy or radiation therapy, to increase the effectiveness of
treatment.
2. Identifying predictive biomarkers: Researchers are identifying biomarkers that can predict which
patients are likely to respond to immunotherapy. This can help personalize treatment and improve
outcomes.
BIOT7018 S1, 2024

Quiz 9

Question 1

Angela works for a Biotech company. Her supervisor provides her with the sequence below, which
comes from a bacterial organism, and tells her that she needs to express it in mammalian cells as a
secreted peptide.

MAPKSTKCTGPNPTATSKNSTGKCE*

Look carefully at the peptide sequence. Can you identify any potential issue(s) in the strategy
suggested by her supervisor? Justify your answer. Answer should include a description of the potential
issue(s) and an explanation of why there is/are potential issue(s).

1. Presence of 2xC that can make disulfide bonds.

2. Presence of 1xsequon, and another one that looks like a sequon but it is not.
3. Needs a signal sequence.

Question 2
BIOT7018 S1, 2024

Angela's company now wishes to produce their monoclonal antibody as a treatment against Japanese
encephalitis virus. Based on the picture above, Angela decides that the protein should be expressed
in a eukaryotic organism.

• Explain how she arrived at that conclusion and why bacteria would not be a suitable host.
• PTMs such as N-linked glycans, and disulfide bonds. The Ab is large quaternary
structure.
• What changes could she make to the protein that could make it suitable for expression in
bacteria? Would you suggest Angela to make these changes?
• remove the glycan sites as a minimum, which is problematic because these are
necessary for the interaction of the mAb with the immune system (for the Fc
mediated function of the mAb). The disulfide bonds could potentially be handled
with specific bacterial strains, and if you mutate them you would not be able to
assemble the full molecule. Nevertheless bacteria is not a good system for a full
length mAb, the mA just too big
 BIOT7018 S1, 2024

Quiz 10

Question 1
Your mAb-producing CHO cell line has a doubling time of 24 hours (i.e. 1 day). You have estimated your
passaging schedule for your expansion protocol, where you’re aiming to inoculate a 50L bioreactor.
For each passage, estimate the time required to reach the desired cell densities.
a) You inoculate a flask of 30 mL of media. Your density is 0.3 million cells per mL (i.e. 0.3 x 10 6
cells/mL) at the time of inoculation. How long will it take for your cells to reach 9.6 x 10 6
cells/mL?
5 days

b) You use these cells to passage. Your next flask is passaged to 0.4 x 106 cells/mL. How long will
it take for your cells to reach 6.4 x 106 cells/mL?
4 days
c) After some number of passages, you inoculate your 50 L batch-phase bioreactor at a density
of 3.5 x 106 cells/mL. Estimate the cell density after 3 days.
28*10^6 cells/mL
d) After 3 days, you notice that your cells have only reached a density of 16 x 106 cells/mL with a
high viability. Given your knowledge of batch cells dynamics, discuss two reasons which would
contribute to a lower-than-expected cell density. Suggest which of the two you discussed is
the most likely contributor.
• Extended lag phase: This typically occurs when the cells used for inoculation are in
a stationary phase. When this happens, cells require time to become prolific again
and divide at a maximum growth rate
• high-cell density effect: large number of cells produce substantial amounts of
growth-inhibiting metabolites which attenuate growth (triggering proliferation
arrest).
growth rate for 5 days (part a), it infers that if the cells you used to inoculate (part
b; grown for 4 days to a relatively low density) are still in exponential phase. This
means a lag phase is unlikely. This suggests that your current density of 16 x 106
cells/mL has reached (or is about to reach) a stationary phase.
Question 2
When you monitored your batch, you found that excessive base addition was required to your 50 L
CHO cell culture.
a) Describe why this would occur.
Mammalian cell cultures can produce excess amounts of lactic acid – with some clones some clones
producing more than others. This leads to growth inhibition.
 BIOT7018 S1, 2024

You also took samples of the supernatant at 24 hours and 72 hours. You ran an SDS-PAGE gel of your
purified monoclonal antibody (IgG) samples and observed different results for each sample. The first
sample (24 hr) gave a clear dominant band for your antibody, while the second (72 hr) yielded multiple
bands.
b) What do the multiple bands in your second sample indicate?

Your sample has degraded in the second sample and might not be considered a suitable product.
Multiple bands can indicate irreversible separation of the heavy chain from the light chain.

c) What does this suggest about the effect of your batch process on your product?

The conditions toward the end of the batch cause product instability. This might be due metabolites
produced by the cell lines or product residence times in the bioreactor which are not suitable for the
mAb being produced. A combination of the two reasons is also plausible.

d) How would you change your 50 L culture to address this change in product you observed?

The bioreactor configuration should change. A number of possibilities should be considered: reducing
the seeding density to reduce the amount of ‘toxic’ metabolite carry-over, changing to a fed batch to
dilute ‘toxic’ metabolites, changing to perfusion for continuous harvest of mAb (reducing residence
times) and removal of growth inhibiting metabolites.

Quiz 11
Question 1:

Dr. Cooper is researching a novel protein with potential therapeutic applications. She has successfully
expressed the protein in E. coli using an inducible expression system. However, her initial purification
approach using gel filtration has encountered challenges. Consider the following details: Initial
Purification Step:. Dr. Cooper’s gel filtration purification approach failed to effectively remove
impurities, resulting in a protein sample with low purity. She needs to revise her purification strategy
to obtain a highly pure protein for further characterization and functional studies.

What alternative purification strategy would you recommend to Dr. Cooper? Design a multi-step
purification process that ensures high purity. Keep in mind that the protein does not carry a
purification tag. Scalable purification techniques are preferred as the process needs to be
commercially viable.
1. Cation Exchange Capture: This is optional in this case. Hydrophobic interaction chromatography
or an optimized non-denaturing precipitation method can also be used as the first purification step.
Run will be in Bind and Elute mode.
2. Hydrophobic Interaction Polishing: High salt elution in IEX initial step provides excellent
conditions for using HIC as the following step as high salt conditions facilitate protein binding to HIC
resins.
3. Final Anion Exchange Polishing: Seamlessly connects to HIC as low salt condition is used for HIC
elution and as a starting point for IEX. This will be done in Flow through mode to allow viruses and
endotoxins to bind to the column.
4. Ultrafiltration/diafiltration by TFF: If the buffer needs to be exchanged at any point (definitely
before fill and finish but likely in preparation for the initial capture step).

Also consider:
• If the expression system is changed to mammalian, more effective methods need to be
introduced to remove viruses. This can combine viral inactivation by chemical (e.g. low pH)
means and final viral filtration
BIOT7018 S1, 2024

Question 2:

Consider the following purification methods commonly used in bioprocessing. For each method,
describe critical parameters that should be carefully considered when implementing them in a scalable
operation. Your response should highlight both the advantages and challenges associated with
scalability.

1. Chromatography (Protein A affinity, ion exchange, hydrophobic interaction)

Describe the principles behind each type of chromatography. Identify critical parameters during
binding and elution of target proteins.
Highlight any limitations or bottlenecks that may arise during large-scale purification.
• Protein A chromatography is an affinity-based separation technique.
• It relies on the reversible interaction between a protein (in this case, antibodies)
and a specific ligand immobilised on a chromatographic matrix.
• The ligand, protein A in this case, selectively binds to the Fc region of
immunoglobulins (IgG) with high specificity1.
• The immobilised protein A acts as an affinity ligand, capturing antibodies from a
complex mixture (cell culture supernatants are an acceptable matrix).
Applications:
• Protein A chromatography is commonly used for capturing recombinant
monoclonal antibodies.
• o It is the standard technique in biomanufacturing for antibody purification.
Critical Parameters:
1.Binding Conditions:
• Protein A binds to the Fc region of IgG through electrostatic and hydrophobic
interactions.
• Optimal pH and salt concentration influence binding strength.
• High binding capacity is essential for efficient capture.
2.Elution Conditions:
• Elution involves disrupting the protein A-IgG interaction.
• Harsh elution conditions (e.g., low pH or high salt) may affect protein integrity.
• Gradual elution with milder conditions is preferred.
3.Sample Loading:
• Proper sample loading ensures maximum binding.
• Overloading can lead to poor resolution and reduced yield.

2. Ion Exchange Chromatography:

• In ion exchange chromatography, the stationary phase contains charged groups (e.g.,
resins with positively or negatively charged functional groups).
• Components interact with these charges based on their own charge properties.
• Binding affinity can be adjusted by moving the pH away from the isoelectric point of the
protein to increase surface charges and by lowering the ionic strength of the feed
material.
• By adjusting pH or ionic strength, we can selectively elute components from the column.
• IEX can be run in flow-through (collect whatever doesn’t bind to the column) or bind and
elute mode (specifically collect the eluate).
3. Hydrophobic Interaction Chromatography (HIC):
• HIC exploits differences in hydrophobicity.
• The stationary phase contains hydrophobic groups (e.g., phenyl or butyl groups).
• Binding affinity can be adjusted by increasing the salt (ionic strength) concentration of
the feed solution.
• Components interact with these hydrophobic groups, and elution occurs by decreasing
the salt concentration or increasing the organic solvent concentration.
• HIC can be run in flow-through (collect whatever doesn’t bind to the column) or bind and
elute mode (specifically collect the eluate).
 BIOT7018 S1, 2024

Factors Impacting Scalability:

• Column Size:
• Larger columns allow for higher sample loading and better resolution.
• However, larger columns require more material and longer run times.
• Flow Rates:
• Faster flow rates reduce separation time but may compromise resolution.
• Slower flow rates improve resolution but increase overall run time.
• Resin Compatibility:
• The choice of resin affects selectivity and binding capacity. o Compatibility with the
sample matrix and elution conditions is crucial. o Resin stability and reusability
impact scalability.
Limitations and Bottlenecks:
• Sample Complexity:
o Complex mixtures may lead to overlapping peaks, reducing resolution.
• Column Packing:
o Uniform packing is essential for reproducibility. o Variability in packing can affect
scalability.
• Pressure Limits:
o High flow rates and large columns increase pressure. o Systems must handle
pressure without compromising performance.
• Sample Loading:
o Overloading the column can lead to peak distortion and poor resolution. o
Balancing sample size and column capacity is critical.

4. Precipitation (e.g., ammonium sulfate, polyethylene glycol):

Explain how protein precipitation works.
Identify critical parameters like protein concentration, pH, and temperature.
Address potential issues related to solubility, yield, and downstream processing.
• Hydrophobic Aggregation:
o Protein precipitation primarily occurs through hydrophobic
aggregation.
o When proteins are dissolved in a solution, their hydrophobic
regions become exposed.
o These hydrophobic patches on the protein surface can interact
with each other, leading to aggregation.
o Properly folded proteins have water molecules forming a shell
around them, but precipitation disrupts this shell.
• Dehydration of Hydrophobic Patches:
o Another mechanism involves dehydrating the water molecules
around hydrophobic patches.
o By removing the water barrier, hydrophobic parts of proteins
come into contact, leading to aggregation and precipitation.
Critical Parameters:
• Protein Concentration:
o Higher protein concentrations favor precipitation.
o However, excessive concentration can lead to over-aggregation
and poor solubility.
• pH:
o pH affects protein charge and solubility.
Adjusting pH can enhance or inhibit precipitation.
• Temperature:
o Lower temperatures promote protein aggregation
o .Optimal temperature depends on the specific protein.
Precipitating Agent:
o Common agents include ammonium sulphate and
 BIOT7018 S1, 2024

polyethylene glycol (PEG).

o The choice depends on the protein’s properties and
downstream requirements.

Potential Issues:
• Solubility Challenges:
• Some proteins may not precipitate efficiently due to high
solubility.
• Finding the right precipitant and conditions is crucial.
• Yield Loss:
• Precipitation can lead to loss of protein yield.
• Aggregated proteins may not be fully recoverable.
• • Downstream Processing:
• Precipitated proteins often require further purification
steps.
• Removing contaminants and optimizing recovery are
essential.
• Aggregation and Denaturation:
• Aggregates can form irreversible structures.
• Denaturation during precipitation affects protein
functionality.

5. Ultrafiltration and Diafiltration:

Define ultrafiltration and diafiltration.
Discuss membrane pore size, flux rates, and buffer exchange considerations.
Consider the impact of membrane fouling and scalability challenges.
Ultrafiltration (UF):
• UF is a membrane-based separation technique that selectively
separates solutes based on their molecular size.
• It uses a semi-permeable membrane with specific pore sizes to
retain larger molecules (such as proteins) while allowing smaller
molecules (such as salts and water) to pass through.
• UF is commonly used for concentrating and desalting protein
solutions.
Diafiltration (DF):
• DF is a variant of UF that involves continuous buffer exchange.
• During DF, a protein solution is repeatedly diluted with fresh buffer
(usually the same buffer used during UF) to remove impurities and
exchange the buffer.
• DF is essential for adjusting the final buffer composition and
ensuring product stability.

Membrane Pore Size and Flux Rates:

• Membrane Pore Size: o

• UF membranes have defined pore sizes (usually expressed in molecular weight cutoff, MWCO).
• Common MWCO ranges for UF membranes are 1 kDa to 100 kDa. O
• The choice of membrane depends on the target molecule’s size and the desired separation.
• Smaller MWCO membranes retain smaller molecules, while larger MWCO membranes allow
larger molecules to pass through.
 BIOT7018 S1, 2024

Flux Rates:
• Flux refers to the rate at which solvent (usually water) passes through the membrane.
• High flux rates are desirable for efficient processing.
• Factors affecting flux include:
o Transmembrane pressure (TMP): Higher TMP increases flux.
o Membrane properties (pore size, material, surface charge).
o Fouling (discussed below).

Buffer Exchange Considerations:

• Buffer Composition:
o The choice of buffer affects protein stability, solubility, and interactions.
o Buffer components (salts, pH, additives) impact UF/DF performance.
• Buffer Exchange Efficiency:
o DF aims to achieve complete buffer exchange.
o The number of diafiltration steps and diavolumes determine efficiency.
o Properly designed DF ensures minimal residual impurities.
• Buffer Compatibility:
o Ensure compatibility between the initial buffer and the diafiltration buffer.
o Sudden changes in buffer conditions can affect protein stability.

Impact of Membrane Fouling and Scalability Challenges:

• Membrane Fouling:
o Fouling occurs when particles (e.g., proteins, aggregates) accumulate on the
membrane surface.
o Fouling reduces flux and requires cleaning or replacement.
o Strategies to minimise fouling:
▪ Pre-filtration to remove large particles.
▪ Use appropriate membrane materials (hydrophilic vs. hydrophobic).
▪ Optimize operating conditions.
• Scalability Challenges:
o UF/DF processes must be scalable from lab-scale to production.
o Challenges include:
▪ Maintaining consistent flux rates.
▪ Ensuring uniform membrane packing.
▪ Addressing pressure limitations in large-scale systems.
BIOT7018 S1, 2024
BIOT7018 S1, 2024

Quiz 12.

Figure 1: monoclonal antibody schematic (mAb).

Question 1.
Imagine you work in a company that manufactures Rapid Antigen Tests (RATs) for COVID-19. The
company is designing a RAT that can not only measure the presence of an infection, but also identify
the strain of SARS-CoV-2. As part of this process, you are responsible for quality control of production
of a monoclonal antibody (mAb) which specifically binds to the spike protein from the omicron variant
of SARS-CoV-2 but which does not bind to the spike protein of the alpha variant of SARS-CoV-2.

a) As part of the quality control you will run SDS-PAGE of the mAb at reducing and non-reducing
conditions, which results do you expect to see (hint: see figure 1 above)?
For the reducing SDS-PAGE it is expected that the disulfide bonds in the protein structure will break,
resulting in two different bands corresponding to the heavy chain (~50 kDa) and the light chain (~25
kDa). For the non-reduced SDS-PAGE it is expected that the mAb will stay intact and to see a band
corresponding to the full length protein (~150 kDa).

b) Design an assay you could use to ensure the binding specificity of this mAb.
Enzyme-linked immunosorbent assay (ELISA) is an easy method to detect binding specificity. An assay
can be designed where an 96-well ELISA plate is coated with the antigen (e.g. Spike protein) and
different concentrations mAb is added to different wells on the plate for a short incubation. The plate
is then washed several times with buffer before incubated with a secondary antibody (e.g. anti-Fc),
typically conjugated to horseradish peroxidase (HRP). The plate is again washed several times and HRP
detected using a colorimetric substrate. The colour intensity of a well reflects the amount of mAb
bound to the antigen. For quality control you want to ensure the mAb consistently binds the antigen
at the different concentrations.