Applications of affinity chromatography in biopharmaceutical analysis MCQs With Answer

Introduction

Affinity chromatography is a powerful, selective technique widely used in biopharmaceutical analysis for purification, characterization, and quality control of proteins, antibodies, enzymes and biotherapeutics. B. Pharm students should understand ligand–target interactions, resin selection, affinity tags (e.g., His‑tag), Protein A/G, lectins, immobilization chemistry, elution strategies, dynamic binding capacity, and common problems like ligand leaching and nonspecific binding. Applications include capture steps in downstream processing, analytical assays for identity and purity, and enrichment prior to mass spectrometry or bioassays. Mastery of affinity chromatography principles improves competency in bioprocess development, validation, and regulatory expectations. Now let’s test your knowledge with 30 MCQs on this topic.

Q1. What is the primary basis for separation in affinity chromatography?

• Size of the molecule
• Hydrophobic interactions
• Specific reversible binding between ligand and target
• Electric charge of the molecule

Correct Answer: Specific reversible binding between ligand and target

Q2. Which ligand is most commonly used to purify monoclonal IgG antibodies?

• Avidin
• Protein A
• Ni-NTA
• Lectin ConA

Correct Answer: Protein A

Q3. Immobilized metal affinity chromatography (IMAC) primarily binds which tag?

• GST tag
• His tag
• FLAG tag
• Strep tag

Correct Answer: His tag

Q4. Which elution method is commonly used to release bound proteins in affinity chromatography?

• Increasing flow rate only
• Changing pH or using a competitive ligand
• Adding organic solvent to precipitate protein
• Heating the column to high temperature

Correct Answer: Changing pH or using a competitive ligand

Q5. Dynamic binding capacity (DBC) refers to:

• The total volume of resin in the column
• Amount of target bound under flow conditions at a specified breakthrough
• The static capacity measured without flow
• The maximum pressure the column can withstand

Correct Answer: Amount of target bound under flow conditions at a specified breakthrough

Q6. A common problem in affinity chromatography causing contamination of product is:

• Ligand leaching from the resin
• Excessive buffer viscosity
• Column overheating
• Too low sample concentration

Correct Answer: Ligand leaching from the resin

Q7. Lectin affinity chromatography is most useful for:

• Purifying nucleic acids
• Selective binding of glycoproteins based on sugar moieties
• Separating proteins by molecular weight
• Removing endotoxin from formulations

Correct Answer: Selective binding of glycoproteins based on sugar moieties

Q8. Which immobilization chemistry commonly links ligand to agarose beads via primary amines?

• Epoxy coupling
• CNBr activation
• EDC/NHS carbodiimide chemistry
• Maleimide-thiol coupling

Correct Answer: EDC/NHS carbodiimide chemistry

Q9. In affinity HPLC used for analysis, a major advantage is:

• Lowest cost compared to other HPLC modes
• High selectivity enabling direct analysis of complex samples
• No need for column regeneration
• Universal affinity for all proteins

Correct Answer: High selectivity enabling direct analysis of complex samples

Q10. What is the role of a spacer arm between ligand and resin?

• To reduce column pressure
• To increase ligand flexibility and accessibility for target binding
• To prevent elution
• To decrease ligand stability

Correct Answer: To increase ligand flexibility and accessibility for target binding

Q11. Competitive elution in affinity chromatography involves:

• Using a small molecule that displaces the target from the ligand
• Lowering the column temperature
• Diluting the sample with water
• Increasing salt concentration only

Correct Answer: Using a small molecule that displaces the target from the ligand

Q12. Which parameter is critical for validating an affinity purification method for biopharmaceuticals?

• UV absorbance only
• Specificity, recovery, reproducibility and robustness
• Color of the resin
• Sample container material only

Correct Answer: Specificity, recovery, reproducibility and robustness

Q13. Protein A binds to which region of IgG?

• Fab variable region
• Light chain constant region
• Fc region
• Hinge region only

Correct Answer: Fc region

Q14. For removal of host cell proteins using affinity steps, an important analytical application is:

• Determining protein tertiary structure only
• Quantifying residual host cell protein levels post‑purification
• Measuring pH of formulation
• Assessing color of the final product

Correct Answer: Quantifying residual host cell protein levels post‑purification

Q15. Which buffer condition commonly decreases nonspecific ionic interactions during affinity purification?

• Very low ionic strength buffer
• High salt concentration buffer
• Buffer without pH control
• Pure water

Correct Answer: High salt concentration buffer

Q16. What is the typical reason to use an affinity tag like GST or Strep-tag during development?

• To denature the protein for analysis
• To enable specific capture and simplify purification and detection
• To increase protein molecular weight only
• To prevent protein folding

Correct Answer: To enable specific capture and simplify purification and detection

Q17. A common analytical use of affinity chromatography before mass spectrometry is:

• Concentrating and enriching low‑abundance proteins of interest
• Running size exclusion separation
• Measuring residual solvents
• Sequencing DNA

Correct Answer: Concentrating and enriching low‑abundance proteins of interest

Q18. Which resin property affects flow rate and pressure drop in an affinity column?

• Color of the resin
• Particle size and porosity
• Ligand molecular weight only
• Elution buffer pH only

Correct Answer: Particle size and porosity

Q19. What is a major regulatory concern when using Protein A resins in biopharmaceutical manufacturing?

• Protein A increases product potency
• Residual Protein A ligand leaching into product requires monitoring and removal
• Protein A prevents sterilization
• Protein A changes product color

Correct Answer: Residual Protein A ligand leaching into product requires monitoring and removal

Q20. Affinity chromatography is especially useful in downstream “capture” steps because it:

• Requires the largest resin volume
• Provides high selectivity and yield for the target molecule from crude feedstocks
• Relies solely on size exclusion principles
• Is the cheapest chromatography mode

Correct Answer: Provides high selectivity and yield for the target molecule from crude feedstocks

Q21. In affinity purification, reducing target dissociation during washing is best achieved by:

• Using harsh denaturants
• Optimizing wash buffer composition to maintain binding conditions
• Running the column empty
• Using extremely low temperatures only

Correct Answer: Optimizing wash buffer composition to maintain binding conditions

Q22. Which analytical parameter assesses how tightly ligand and target interact?

• Column length
• Equilibrium dissociation constant (Kd)
• Buffer color
• Particle diameter

Correct Answer: Equilibrium dissociation constant (Kd)

Q23. When scaling up an affinity process from lab to manufacturing, a key factor to evaluate is:

• Only the aesthetic design of the column
• Mass transfer effects and dynamic binding capacity at process flow rates
• Replacing ligand with random proteins
• Removing all wash steps

Correct Answer: Mass transfer effects and dynamic binding capacity at process flow rates

Q24. Which statement about covalent immobilization of ligands is correct?

• Covalent immobilization prevents any ligand leaching and always preserves ligand activity
• Covalent immobilization secures ligand to resin but requires careful chemistry to preserve ligand activity
• Covalent immobilization is only used for synthetic polymers
• Covalent immobilization is reversible under mild conditions

Correct Answer: Covalent immobilization secures ligand to resin but requires careful chemistry to preserve ligand activity

Q25. In affinity HPLC, detection of bound analyte after elution is commonly done by:

• UV absorbance, fluorescence, or MS depending on analyte properties
• Visual inspection only
• Measuring resin color change
• pH meter exclusively

Correct Answer: UV absorbance, fluorescence, or MS depending on analyte properties

Q26. A disadvantage of affinity chromatography in biopharmaceutical production is:

• Its absolute lack of selectivity
• High cost of specific ligands and potential ligand instability or leaching
• Inability to process dilute feedstocks
• It always denatures the target protein

Correct Answer: High cost of specific ligands and potential ligand instability or leaching

Q27. Which of the following is an affinity resin regeneration concern?

• Accidental permanent denaturation of ligand reducing capacity
• Improved ligand activity over cycles
• Constant increase in pore size
• Better selectivity after each cycle

Correct Answer: Accidental permanent denaturation of ligand reducing capacity

Q28. For analytical method development using affinity capture prior to bioassay, critical considerations include:

• Capture efficiency, specificity, recovery and compatibility with downstream assay
• Only the column color
• Ignoring buffer composition
• Only the temperature of storage

Correct Answer: Capture efficiency, specificity, recovery and compatibility with downstream assay

Q29. Which affinity ligand is ideal for purifying biotinylated molecules?

• Streptavidin or avidin
• Protein L
• Ni-NTA
• Concanavalin A

Correct Answer: Streptavidin or avidin

Q30. During affinity purification, monitoring breakthrough in the column helps to:

• Decide when to regenerate the entire chromatography system
• Identify the point when target begins to appear in the column effluent indicating capacity is reached
• Measure the resin color change only
• Increase ligand synthesis

Correct Answer: Identify the point when target begins to appear in the column effluent indicating capacity is reached

G S Sachin

I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.

Mail- Sachin@pharmacyfreak.com