Purification of monoclonal antibodies MCQs With Answer

Purification of monoclonal antibodies MCQs With Answer

Introduction: This quiz collection focuses on purification strategies for monoclonal antibodies (mAbs) tailored for M.Pharm students. It highlights key downstream unit operations: capture (Protein A affinity), intermediate purification (ion-exchange, hydrophobic interaction), polishing (SEC, polishing resins), and auxiliary steps such as viral inactivation/removal, filtration, and tangential flow filtration for concentration and buffer exchange. Questions emphasize mechanism, process parameters, troubleshooting (e.g., aggregation, ligand leaching), analytics for quality attributes (aggregates, charge variants, glycoforms), and regulatory considerations. The set is designed to reinforce conceptual understanding and practical decision-making required for designing robust mAb purification trains.

Q1. What is the primary molecular basis for using Protein A affinity chromatography in the capture step of IgG monoclonal antibodies?

• Protein A forms ionic bonds with the antigen-binding (Fab) region
• Protein A specifically binds the Fc region of IgG via high-affinity non-covalent interactions
• Protein A interacts with glycan moieties on the heavy chain
• Protein A selectively binds light chains of immunoglobulins

Correct Answer: Protein A specifically binds the Fc region of IgG via high-affinity non-covalent interactions

Q2. Which advantage best explains why Protein A capture is widely used as the first purification step for therapeutic IgG?

• It denatures host cell proteins, making them easier to remove
• It is highly selective for IgG Fc, yielding high purity in a single step
• It permanently removes aggregates by covalent trapping
• It provides strong hydrophobic interactions that eliminate charge variants

Correct Answer: It is highly selective for IgG Fc, yielding high purity in a single step

Q3. Why is low-pH elution from Protein A columns a common concern for monoclonal antibody quality?

• Low pH increases ionic strength causing more host cell protein co-elution
• Exposure to acidic pH can partially unfold mAbs, promoting aggregation and loss of potency
• Low pH prevents subsequent viral inactivation steps
• Low pH leads to irreversible cleavage of the Fc glycan

Correct Answer: Exposure to acidic pH can partially unfold mAbs, promoting aggregation and loss of potency

Q4. What is the main objective of the ‘polishing’ stage in a monoclonal antibody purification train?

• To capture the antibody from clarified harvest fluid
• To concentrate the product prior to fill-finish only
• To remove aggregates, remaining host cell proteins, DNA and charge variants to meet CQA limits
• To glycosylate the antibody uniformly

Correct Answer: To remove aggregates, remaining host cell proteins, DNA and charge variants to meet CQA limits

Q5. How does cation exchange (CEX) chromatography typically retain monoclonal antibodies?

• Antibodies bind to negatively charged resin at pH above their pI
• Antibodies bind to positively charged resin when pH is below their pI
• Antibodies are retained by hydrophobic patches in high-salt buffers
• Antibodies are separated based on molecular weight exclusion

Correct Answer: Antibodies bind to positively charged resin when pH is below their pI

Q6. Hydrophobic interaction chromatography (HIC) is often used in mAb purification. What is its separation principle?

• Separation based on isoelectric point differences in low salt
• Separation by hydrophobic interactions that increase at higher salt concentrations
• Size-based separation using porous beads
• Covalent trapping of aggregates to the ligand

Correct Answer: Separation by hydrophobic interactions that increase at higher salt concentrations

Q7. Which statement correctly describes the primary use of size exclusion chromatography (SEC) in mAb downstream processing?

• SEC is used as a high-capacity capture step for bulk removal of host cell proteins
• SEC separates species by size and is commonly used for aggregate analysis and final polishing
• SEC increases antibody binding to Protein A by removing glycans
• SEC denatures antibodies to facilitate viral inactivation

Correct Answer: SEC separates species by size and is commonly used for aggregate analysis and final polishing

Q8. What is the primary role of tangential flow filtration (TFF) in mAb processing?

• To fractionate proteins by charge
• To sterilize the product by removing viruses larger than 200 nm
• To concentrate the mAb and perform buffer exchange (diafiltration)
• To cleave Fc regions selectively

Correct Answer: To concentrate the mAb and perform buffer exchange (diafiltration)

Q9. Which common viral inactivation method is integrated into many mAb purification processes and why?

• Heat treatment at 80°C because antibodies remain fully stable
• Low-pH incubation (e.g., pH ~3.5 for defined hold time) because many enveloped viruses are inactivated
• High ionic strength incubation because it permanently removes viral genomes
• Protease digestion because it selectively degrades viral capsids without affecting mAb

Correct Answer: Low-pH incubation (e.g., pH ~3.5 for defined hold time) because many enveloped viruses are inactivated

Q10. Which technology is primarily used for effective removal of small, non-enveloped viruses during downstream processing?

• Protein A chromatography
• 20 nm nominal pore-size virus (nanofiltration)
• Hydrophobic interaction chromatography
• Size exclusion chromatography with >500 kDa cutoff

Correct Answer: 20 nm nominal pore-size virus (nanofiltration)

Q11. What is a commonly used approach to reduce host cell DNA levels in mAb downstream processing?

• High-pH elution in Protein A to precipitate DNA
• Treatment with nucleases (e.g., Benzonase) followed by anion exchange capture or depth filtration
• Passing product through SEC to specifically bind DNA
• Heating to 70°C to degrade DNA

Correct Answer: Treatment with nucleases (e.g., Benzonase) followed by anion exchange capture or depth filtration

Q12. Protein A ligand leaching into the product stream is a process concern. Which control strategy is most appropriate?

• Avoid using any column regeneration to reduce leaching
• Implement stringent wash and regeneration protocols, use engineered low-leach ligands, and monitor leachate levels analytically
• Use only high-pH elution to permanently inactivate leached ligand
• Rely solely on SEC at end of process to remove all leached ligands

Correct Answer: Implement stringent wash and regeneration protocols, use engineered low-leach ligands, and monitor leachate levels analytically

Q13. Which combination of operations is most effective for aggregate removal in a mAb process?

• Protein A capture followed by hydrophobic interaction or SEC polishing
• Direct sterile filtration only
• Low-pH viral inactivation only
• High-salt ultrafiltration without chromatography

Correct Answer: Protein A capture followed by hydrophobic interaction or SEC polishing

Q14. During elution and polishing, which buffer strategy helps minimize mAb aggregation and maintain stability?

• Use of chaotropic agents such as 6 M guanidine HCl in final buffer
• Inclusion of stabilizers (e.g., arginine, low ionic strength, appropriate pH) to reduce unfolding and aggregation
• Maintain very high ionic strength (>2 M NaCl) through final formulation
• Omit buffers and keep product in water to avoid salt effects

Correct Answer: Inclusion of stabilizers (e.g., arginine, low ionic strength, appropriate pH) to reduce unfolding and aggregation

Q15. What is an accepted cleaning-in-place (CIP) agent for sanitizing Protein A chromatography columns while maintaining ligand lifetime?

• 1–2% sodium hypochlorite for routine CIP of all columns
• 0.1–0.5 M NaOH for periodic sanitization and cleaning of most affinity resins
• 6 M guanidine HCl applied daily
• Pure ethanol flush as sole sanitant

Correct Answer: 0.1–0.5 M NaOH for periodic sanitization and cleaning of most affinity resins

Q16. Which process parameter most directly affects dynamic binding capacity (DBC) of a chromatography resin during mAb capture?

• Column color and material of column housing
• Residence time (flow rate) and ligand density on the resin
• Feed pH only, independent of flow rate or ligand density
• Downstream sterile filtration pore size

Correct Answer: Residence time (flow rate) and ligand density on the resin

Q17. Which analytical technique is most appropriate for resolving charge variants of monoclonal antibodies?

• Size exclusion chromatography (SEC)
• Capillary isoelectric focusing (cIEF) or ion-exchange chromatography
• Reverse phase HPLC only
• Dynamic light scattering (DLS)

Correct Answer: Capillary isoelectric focusing (cIEF) or ion-exchange chromatography

Q18. For quantitative measurement of soluble aggregates in a purified mAb product, which method is considered standard?

• SEC-HPLC with UV or light scattering detection
• SDS-PAGE under reducing conditions only
• pH titration curve analysis
• Endotoxin assay (LAL)

Correct Answer: SEC-HPLC with UV or light scattering detection

Q19. Which of the following is a primary advantage of single-use (disposable) technologies in mAb downstream processing?

• They eliminate the need for viral inactivation
• They reduce cleaning validation burden and cross-contamination risk, increasing flexibility
• They allow reuse of Protein A resin indefinitely
• They guarantee zero extractables and leachables

Correct Answer: They reduce cleaning validation burden and cross-contamination risk, increasing flexibility

Q20. Which list best represents critical quality attributes (CQAs) that downstream purification must control for therapeutic monoclonal antibodies?

• Color, taste, and boiling point
• Aggregate content, glycosylation pattern, charge variants, host cell protein and DNA levels
• Ligand molecular weight only
• Viscosity at 1000 rpm in presence of detergents

Correct Answer: Aggregate content, glycosylation pattern, charge variants, host cell protein and DNA levels

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