PEGylation techniques MCQs With Answer

Introduction

PEGylation—the covalent attachment of polyethylene glycol (PEG) chains to therapeutic proteins—is a cornerstone technique in modern protein formulation to improve pharmacokinetics, stability, and reduce immunogenicity. For M.Pharm students, understanding PEGylation requires grasping diverse chemistries (NHS esters, maleimides, aldehydes, click reagents), how PEG size and architecture alter hydrodynamic radius and clearance, and how reaction conditions, purification and analytical methods determine product quality. This quiz set focuses on mechanistic details, practical considerations in conjugation and formulation, and analytical/regulatory challenges. These MCQs aim to deepen comprehension of PEGylation techniques with focused, exam-relevant scenarios and clear answers to support advanced coursework and research applications.

Q1. What is the primary pharmacokinetic advantage conferred by PEGylation of a therapeutic protein?

• Increased renal clearance due to reduced size
• Enhanced receptor-mediated uptake in the liver
• Decreased immunogenicity but unchanged half-life
• Increased hydrodynamic radius leading to reduced renal filtration and prolonged circulation

Correct Answer: Increased hydrodynamic radius leading to reduced renal filtration and prolonged circulation

Q2. Which PEGylation chemistry is most commonly used to selectively modify free thiol groups on cysteine residues?

• NHS ester-mediated acylation
• Maleimide-thiol Michael addition
• Reductive amination with aldehyde-PEG
• Carbodiimide coupling to carboxyl groups

Correct Answer: Maleimide-thiol Michael addition

Q3. NHS-activated PEG reacts primarily with which functional group on proteins under typical reaction conditions?

• Carboxylate side chains (Asp/Glu)
• Thiols (Cys)
• Primary amines (lysine ε-amino and N-terminal α-amino)
• Phenolic hydroxyls (Tyr)

Correct Answer: Primary amines (lysine ε-amino and N-terminal α-amino)

Q4. Reductive amination using aldehyde-PEG is often used for N-terminal targeting because:

• Lysine side chains are more nucleophilic than the N-terminus at low pH
• The N-terminal α-amino group has a lower pKa and is more reactive at mildly acidic pH
• It forms permanent thioether bonds with cysteines
• It requires copper catalysis to proceed

Correct Answer: The N-terminal α-amino group has a lower pKa and is more reactive at mildly acidic pH

Q5. Which analytical technique provides the best direct measurement of the intact mass increase after PEGylation?

• Size-exclusion chromatography (SEC) retention time only
• SDS-PAGE band shift without mass measurement
• MALDI-TOF or ESI mass spectrometry with deconvolution
• Dynamic light scattering (DLS) hydrodynamic diameter

Correct Answer: MALDI-TOF or ESI mass spectrometry with deconvolution

Q6. Site-specific PEGylation strategies are preferred over random lysine PEGylation because they:

• Always produce higher molecular weight conjugates
• Guarantee no change in biological activity
• Yield homogeneous products with predictable activity and easier characterization
• Require no purification after reaction

Correct Answer: Yield homogeneous products with predictable activity and easier characterization

Q7. Which factor most strongly influences the renal clearance threshold of PEGylated proteins?

• PEG chemical end-group identity (e.g., methoxy vs hydroxyl)
• Hydrodynamic radius conferred by PEG molecular weight and architecture
• The buffer ionic strength during conjugation
• Presence of residual catalysts from coupling reactions

Correct Answer: Hydrodynamic radius conferred by PEG molecular weight and architecture

Q8. A common drawback of PEGylation that has emerged clinically is:

• Complete elimination of immunogenicity for all proteins
• Generation of anti-PEG antibodies that can accelerate clearance or cause hypersensitivity
• Unrestricted tissue penetration of large PEGylated proteins
• Infinite stability in all storage conditions

Correct Answer: Generation of anti-PEG antibodies that can accelerate clearance or cause hypersensitivity

Q9. Maleimide-thiol conjugates can be unstable in vivo due to which chemical process?

• Hydrolysis of amide bonds formed with lysines
• Retro-Michael reaction or thiol exchange leading to loss or transfer of PEG
• Oxidation of PEG backbone to shorter chains
• Spontaneous cleavage of the peptide backbone adjacent to PEG

Correct Answer: Retro-Michael reaction or thiol exchange leading to loss or transfer of PEG

Q10. Click chemistry (CuAAC) used for PEGylation offers which main advantage?

• Requires no catalyst and proceeds in all buffers
• Provides bioorthogonal, high-yield, and often site-specific coupling between azide and alkyne pairs
• Reacts selectively with native lysine residues without modification
• Produces reversible linkages that are cleaved in plasma

Correct Answer: Provides bioorthogonal, high-yield, and often site-specific coupling between azide and alkyne pairs

Q11. When using NHS-PEG for lysine modification, what condition helps minimize over-PEGylation and preserve activity?

• Use very high molar excess of NHS-PEG and long reaction times
• Carry out reaction at extremely alkaline pH (>10)
• Control molar ratio of PEG:protein, short reaction time, and lower temperature
• Include reducing agents like DTT to keep disulfides reduced

Correct Answer: Control molar ratio of PEG:protein, short reaction time, and lower temperature

Q12. Which purification method is most commonly used to separate unreacted PEG from PEGylated protein in early development?

• Dialysis against organic solvents
• Size-exclusion chromatography (SEC) or ultrafiltration/diafiltration based on size differences
• Affinity chromatography to unmodified PEG
• Reverse-phase HPLC with strong denaturants only

Correct Answer: Size-exclusion chromatography (SEC) or ultrafiltration/diafiltration based on size differences

Q13. Which PEG architecture generally provides a larger increase in hydrodynamic size per unit mass compared with linear PEG?

• Short linear PEG (2 kDa)
• Branched or multi-arm PEGs
• Single methoxy-terminated linear PEG only
• PEGylated lipids instead of PEG polymers

Correct Answer: Branched or multi-arm PEGs

Q14. A common analytical indicator of heterogeneous PEGylation on SDS-PAGE is:

• A single sharp band at the native molecular weight
• A smear or multiple shifted bands indicating a distribution of PEG attachments
• Complete disappearance of protein staining
• Increased electrophoretic mobility to much lower apparent mass

Correct Answer: A smear or multiple shifted bands indicating a distribution of PEG attachments

Q15. How does PEGylation generally affect proteolytic degradation of a protein drug?

• It increases susceptibility to all proteases
• It reduces proteolytic degradation by sterically shielding cleavage sites
• It converts the protein into a protease substrate for specific peptidases only
• It causes immediate proteolysis during storage

Correct Answer: It reduces proteolytic degradation by sterically shielding cleavage sites

Q16. For maleimide-PEG conjugation to be selective for free cysteine, which preparatory step is often required?

• Oxidize all cysteines to form disulfides
• Reduce disulfide bonds to generate free thiols and protect essential disulfides if necessary
• Acetylate lysines to create more reactive cysteines
• Add copper catalysts to promote thiol reactivity

Correct Answer: Reduce disulfide bonds to generate free thiols and protect essential disulfides if necessary

Q17. Which parameter is most useful for quantifying average PEG-to-protein ratio (degree of PEGylation) in a product mixture?

• Visual inspection of color change after reaction
• UV absorbance at 280 nm without further analysis
• LC-MS deconvolution combined with chromatographic peak integration
• Measuring solution viscosity only

Correct Answer: LC-MS deconvolution combined with chromatographic peak integration

Q18. Cleavable PEG linkers are designed to:

• Make the PEG irreversibly bound to the protein for lifetime stability
• Enable release of native protein in response to a biological trigger (e.g., pH, enzymes, redox)
• Promote permanent aggregation of the protein
• Increase immunogenicity intentionally

Correct Answer: Enable release of native protein in response to a biological trigger (e.g., pH, enzymes, redox)

Q19. Which of the following is an approved therapeutic example of a PEGylated protein used clinically?

• Unmodified insulin
• Pegfilgrastim (PEGylated filgrastim)
• Native erythropoietin without modification
• Non-PEGylated monoclonal antibody

Correct Answer: Pegfilgrastim (PEGylated filgrastim)

Q20. During formulation, which concern is specific to PEGylated protein therapeutics compared with non-PEGylated ones?

• PEGylated proteins never aggregate under any conditions
• Potential formation of anti-PEG antibodies and need to monitor anti-PEG responses during clinical development
• PEG eliminates the need for buffer optimization
• PEGylated proteins are incompatible with lyophilization

Correct Answer: Potential formation of anti-PEG antibodies and need to monitor anti-PEG responses during clinical development

Download