Site-specific protein delivery MCQs With Answer

Site-specific protein delivery MCQs With Answer

by

Introduction

Site-specific protein delivery is a rapidly evolving area in Advanced Pharmaceutical Biotechnology that focuses on delivering therapeutic proteins to precise biological locations with controlled orientation and stoichiometry. For M.Pharm students, mastering site-specific conjugation strategies, linker chemistry, targeting ligands, and bioorthogonal reactions is essential for designing safer and more effective biotherapeutics such as antibody-drug conjugates, enzyme therapies, and targeted cytokine delivery. This collection of MCQs explores enzymatic and chemical site-specific modification techniques, linker stability, payload release mechanisms, analytical characterization, and translational considerations like pharmacokinetics and immunogenicity. Use these questions to test and deepen your conceptual and practical understanding of modern targeted protein delivery approaches.

Q1. Which enzymatic method is commonly used for peptide bond formation at a specific C-terminal motif (LPXTG) to attach payloads to proteins?

  • Sortase A-mediated transpeptidation
  • Microbial transglutaminase crosslinking
  • Tyrosinase-mediated coupling
  • Pepsin proteolysis

Correct Answer: Sortase A-mediated transpeptidation

Q2. What is the main advantage of site-specific PEGylation over random PEGylation of therapeutic proteins?

  • Generates a heterogeneous mixture with variable bioactivity
  • Improves solubility while preserving defined activity and reduces immunogenicity
  • Makes purification more difficult and increases clearance
  • Always increases receptor binding affinity

Correct Answer: Improves solubility while preserving defined activity and reduces immunogenicity

Q3. Which amino acid residue is most frequently targeted for maleimide-based site-specific conjugation due to its nucleophilicity?

  • Lysine
  • Serine
  • Cysteine
  • Tyrosine

Correct Answer: Cysteine

Q4. In ADC design, what does DAR stand for and why is it important?

  • Drug-to-antibody ratio; it influences potency, pharmacokinetics, and toxicity
  • Drug activation rate; it determines linker cleavage speed
  • Delivery-associated response; it measures immune activation
  • Degradation and removal; it measures metabolic stability

Correct Answer: Drug-to-antibody ratio; it influences potency, pharmacokinetics, and toxicity

Q5. Which bioorthogonal reaction is characterized by a very fast rate and is especially useful for in vivo labeling: azide–alkyne cycloaddition or inverse electron-demand Diels–Alder (IEDDA)?

  • Classical copper-catalyzed azide–alkyne cycloaddition (CuAAC)
  • Strain-promoted azide–alkyne cycloaddition (SPAAC)
  • Inverse electron-demand Diels–Alder (IEDDA) between tetrazine and trans-cyclooctene
  • Staudinger ligation

Correct Answer: Inverse electron-demand Diels–Alder (IEDDA) between tetrazine and trans-cyclooctene

Q6. Which linker type is designed to be cleaved selectively in the reducing environment of the cytosol to release a payload?

  • Hydrazone linker
  • Disulfide linker
  • Non-cleavable thioether linker
  • PEO spacer

Correct Answer: Disulfide linker

Q7. Microbial transglutaminase (mTG) catalyzes site-specific conjugation by forming an isopeptide bond between which pair of residues or groups?

  • Lysine epsilon-amino group and glutamine side chain (γ-carboxamide)
  • Cysteine thiol and maleimide
  • Aspartate side chain and N-terminal amine
  • Tyrosine phenol and amine linker

Correct Answer: Lysine epsilon-amino group and glutamine side chain (γ-carboxamide)

Q8. Which analytical technique provides site-specific identification of conjugation sites and drug loading on proteins by peptide mapping?

  • Size-exclusion chromatography (SEC)
  • Bottom-up liquid chromatography–tandem mass spectrometry (LC-MS/MS)
  • Dynamic light scattering (DLS)
  • UV–visible spectrophotometry

Correct Answer: Bottom-up liquid chromatography–tandem mass spectrometry (LC-MS/MS)

Q9. Which strategy most effectively reduces heterogeneity when creating antibody–drug conjugates (ADCs)?

  • Random lysine conjugation using NHS-esters
  • Enzymatic or engineered cysteine conjugation at defined sites
  • Non-specific glycation reactions
  • High-temperature chemical activation of all residues

Correct Answer: Enzymatic or engineered cysteine conjugation at defined sites

Q10. Glycoengineering for site-specific conjugation typically targets which moiety on antibodies for homogeneous modification?

  • Peptide backbone amide bonds
  • Fc-region N-linked glycans
  • C-terminal carboxylate groups
  • Free N-terminal methionines

Correct Answer: Fc-region N-linked glycans

Q11. Which property is a major concern when using maleimide linkers for in vivo protein conjugates?

  • Excessive enzymatic stability and no release
  • Retro-Michael exchange leading to payload transfer and instability
  • Complete resistance to serum proteins
  • Inability to conjugate to thiols

Correct Answer: Retro-Michael exchange leading to payload transfer and instability

Q12. Site-specific incorporation of unnatural amino acids bearing bioorthogonal handles is typically achieved in cells using which technique?

  • Random chemical modification of methionines
  • Stop codon suppression with engineered tRNA/tRNA synthetase pairs
  • Thermal denaturation and refolding in the presence of labels
  • Edman degradation

Correct Answer: Stop codon suppression with engineered tRNA/tRNA synthetase pairs

Q13. Which linker cleavage mechanism is most appropriate for selective payload release within lysosomes of target cells?

  • Reduction-sensitive disulfide bond
  • Protease-cleavable peptide linker (e.g., valine–citrulline)
  • Hydrophobic non-cleavable thioether
  • pH-insensitive stable amide bond

Correct Answer: Protease-cleavable peptide linker (e.g., valine–citrulline)

Q14. Which targeting ligand is best suited for cell-specific internalization via receptor-mediated endocytosis in cancer therapy?

  • Non-specific albumin binding
  • An antibody or antibody fragment directed to an internalizing cell-surface receptor
  • Random polyethylene glycol chains
  • Hydrophobic fatty acids only

Correct Answer: An antibody or antibody fragment directed to an internalizing cell-surface receptor

Q15. What is the principal benefit of using a cleavable linker in a site-specific protein–drug conjugate?

  • Prevents any release of the drug at the target site
  • Allows controlled release of active payload in response to local triggers (pH, enzymes, redox)
  • Makes the conjugate permanently inactive
  • Guarantees no off-target exposure ever

Correct Answer: Allows controlled release of active payload in response to local triggers (pH, enzymes, redox)

Q16. Which approach yields the most homogeneous product when conjugating payloads to antibodies via engineered cysteines?

  • Reducing all interchain disulfides and random thiol modification
  • Introducing one or two engineered surface cysteines at predetermined locations and selective conjugation
  • Using non-specific lysine acylation
  • Attachment via non-selective oxidation

Correct Answer: Introducing one or two engineered surface cysteines at predetermined locations and selective conjugation

Q17. Which factor is most likely to increase immunogenicity of a site-specifically modified therapeutic protein?

  • Maintaining native glycosylation exactly
  • Introducing novel non-native chemical moieties or neo-epitopes
  • Reducing aggregation and impurities
  • Optimizing isoelectric point to native range

Correct Answer: Introducing novel non-native chemical moieties or neo-epitopes

Q18. For intracellular delivery of proteins, which addition often enhances endosomal escape after receptor-mediated endocytosis?

  • Incorporation of a protease-resistant spacer
  • Attachment of a fusogenic peptide or endosomolytic moiety
  • Increasing overall hydrophilicity only
  • Removal of all cationic residues

Correct Answer: Attachment of a fusogenic peptide or endosomolytic moiety

Q19. Which conjugation strategy is most likely to preserve antigen-binding activity of an antibody when attaching a cytotoxic payload?

  • Random modification of abundant surface lysines in Fab region
  • Site-specific conjugation at defined sites in Fc region or engineered sites away from CDRs
  • Crosslinking directly within complementarity-determining regions (CDRs)
  • High-temperature chemical labeling of all residues

Correct Answer: Site-specific conjugation at defined sites in Fc region or engineered sites away from CDRs

Q20. Which consideration is essential when selecting a site for engineering a conjugation handle on a therapeutic protein?

  • Proximity to active or binding sites that may disrupt function
  • Maximizing hydrophobic patches for aggregation
  • Ensuring the site is buried and inaccessible to solvent
  • Avoiding recombinant expression systems and using chemical synthesis only

Correct Answer: Proximity to active or binding sites that may disrupt function

Leave a Comment