Production of insulin MCQs With Answer

Introduction: This quiz collection on the production of insulin is designed specifically for M.Pharm students studying Advanced Pharmaceutical Biotechnology. It covers the molecular and bioprocessing aspects of insulin manufacture, including gene design, expression systems, folding and disulfide bond formation, downstream purification, analytical characterization, formulation and regulatory considerations. Questions focus on recombinant strategies in bacteria and yeast, inclusion body handling, enzymatic processing of proinsulin, chromatographic techniques, potency and impurity testing, and modern advances such as insulin analogs and biosimilars. The aim is to reinforce theoretical knowledge and practical understanding required for drug development, GMP production and quality control of therapeutic insulin preparations.

Q1. Which of the following describes the main advantage of expressing human proinsulin instead of separate A and B chains in Escherichia coli?

• Facilitates correct disulfide bond formation in the cytoplasm without refolding
• Simplifies downstream refolding because proinsulin folds intramolecularly and only requires enzymatic processing
• Reduces the risk of inclusion body formation and allows secretion into the medium
• Eliminates need for enzymatic cleavage to remove C-peptide

Correct Answer: Simplifies downstream refolding because proinsulin folds intramolecularly and only requires enzymatic processing

Q2. Which promoter is most commonly used for high-level expression of insulin precursors in E. coli BL21(DE3)?

• lac promoter
• T7 promoter
• AOX1 promoter
• GAL1 promoter

Correct Answer: T7 promoter

Q3. What is the primary role of carboxypeptidase B in insulin processing after trypsin cleavage of proinsulin-derived intermediates?

• To oxidize cysteine residues forming disulfide bonds
• To remove basic C-terminal residues (arginine/lysine) generated by trypsin so as to generate native termini
• To cleave peptide bonds adjacent to aromatic residues
• To glycosylate proinsulin for stability

Correct Answer: To remove basic C-terminal residues (arginine/lysine) generated by trypsin so as to generate native termini

Q4. When designing an expression construct for secretion of proinsulin in Saccharomyces cerevisiae, which sequence is most critical to include at the N-terminus?

• PelB signal peptide
• T7 leader sequence
• Alpha mating factor prepro signal peptide
• Trp attenuation sequence

Correct Answer: Alpha mating factor prepro signal peptide

Q5. Which of the following fermentation conditions most strongly promotes formation of inclusion bodies in E. coli expressing high levels of insulin precursors?

• Low induction temperature (15–20°C)
• High growth rate with strong induction at 37°C
• Use of glycerol as sole carbon source
• Expression under weak constitutive promoter

Correct Answer: High growth rate with strong induction at 37°C

Q6. During downstream purification of recombinant insulin, which chromatographic method is most effective for final polishing to achieve high purity and resolution of isoforms?

• Ion-exchange chromatography
• Size-exclusion chromatography
• Reverse-phase high-performance liquid chromatography (RP-HPLC)
• Affinity chromatography using His-tag resin

Correct Answer: Reverse-phase high-performance liquid chromatography (RP-HPLC)

Q7. Which analytical technique is most appropriate for confirming the intact molecular mass of recombinant insulin and detecting truncated isoforms?

• SDS-PAGE with Coomassie staining
• Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)
• UV-visible spectrophotometry at 280 nm
• Light scattering assay

Correct Answer: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Q8. Which modification in the insulin B-chain is characteristic of insulin lispro and accounts for its rapid-acting profile?

• Substitution of Lys B29 with Pro B28
• Exchange of Pro B28 and Lys B29
• Attachment of fatty acid at B29
• Glycosylation at B12

Correct Answer: Exchange of Pro B28 and Lys B29

Q9. What is the main reason yeasts (e.g., Pichia pastoris) are preferred over E. coli for secreted production of recombinant insulin in some processes?

• Yeast cannot form disulfide bonds, simplifying folding
• Yeast secrete proteins into the medium facilitating easier downstream purification and enable eukaryotic folding machinery
• Yeast growth rates are always faster than E. coli, increasing productivity
• Yeast expression eliminates requirement for codon optimization

Correct Answer: Yeast secrete proteins into the medium facilitating easier downstream purification and enable eukaryotic folding machinery

Q10. Which reagent system is commonly used to solubilize insulin inclusion bodies and promote oxidative refolding to form correct disulfide bonds?

• Urea with DTT (dithiothreitol) only
• Guanidine-HCl with redox pair glutathione oxidized/reduced (GSSG/GSH)
• Sodium dodecyl sulfate (SDS) and heat
• Formaldehyde crosslinking

Correct Answer: Guanidine-HCl with redox pair glutathione oxidized/reduced (GSSG/GSH)

Q11. In biosimilar insulin development, which critical quality attribute most directly assesses biological activity compared to an originator product?

• Host cell protein content
• In vitro receptor-binding potency and cell-based bioassay for metabolic response
• Residual DNA level
• Chromatographic purity percentage only

Correct Answer: In vitro receptor-binding potency and cell-based bioassay for metabolic response

Q12. Which regulatory guideline is most relevant for setting specifications and analytical procedures for biotechnology-derived peptides such as insulin?

• ICH Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products
• ICH Q1A: Stability Testing of New Drug Substances and Products
• ICH M4: Common Technical Document
• ICH Q3C: Residual Solvents

Correct Answer: ICH Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products

Q13. Which of the following is the best explanation for inclusion of zinc in commercial insulin formulations?

• Zinc catalyzes enzymatic degradation leading to rapid action
• Zinc stabilizes insulin hexamer formation, prolonging duration of action and improving storage stability
• Zinc prevents aggregation by chelating insulin monomers irreversibly
• Zinc masks immunogenic epitopes to reduce antibody formation

Correct Answer: Zinc stabilizes insulin hexamer formation, prolonging duration of action and improving storage stability

Q14. Which cell compartment in E. coli is commonly used to enhance disulfide bond formation of expressed proinsulin and reduce inclusion body formation?

• Cytoplasm without modifications
• Periplasmic space via signal peptide-directed secretion
• Inner membrane anchoring only
• Extracellular medium naturally without secretion signal

Correct Answer: Periplasmic space via signal peptide-directed secretion

Q15. Which impurity must be tested and limited specifically for products produced in Gram-negative bacteria such as E. coli?

• Host cell proteins (HCPs) only
• Endotoxin (lipopolysaccharide, LPS)
• Mycoplasma contamination
• Residual yeast-derived mannans

Correct Answer: Endotoxin (lipopolysaccharide, LPS)

Q16. Codon optimization of the insulin gene for expression in E. coli primarily improves which of the following?

• Folding specificity and disulfide bond formation
• Translation efficiency and overall protein yield by matching host tRNA abundance
• Secretion into the medium without signal peptide
• Proteolytic resistance of the expressed protein

Correct Answer: Translation efficiency and overall protein yield by matching host tRNA abundance

Q17. Which quality control method is most suitable to quantify residual host cell DNA in a recombinant insulin bulk drug substance?

• qPCR (quantitative polymerase chain reaction)
• Bradford protein assay
• ELISA for host cell proteins
• SDS-PAGE densitometry

Correct Answer: qPCR (quantitative polymerase chain reaction)

Q18. Which fermentation induction method is typically used with Pichia pastoris expression driven by the AOX1 promoter for insulin precursor production?

• IPTG induction
• Galactose induction
• Methanol induction
• Heat shock induction

Correct Answer: Methanol induction

Q19. What is the main analytical purpose of reversed-phase HPLC in insulin manufacturing quality control?

• To determine molecular weight precisely
• To separate and quantify insulin and its hydrophobic isoforms, oxidation products and degradants based on hydrophobicity
• To remove endotoxin from the bulk drug substance
• To measure the residual moisture content

Correct Answer: To separate and quantify insulin and its hydrophobic isoforms, oxidation products and degradants based on hydrophobicity

Q20. Which characteristic of insulin glargine provides its long-acting profile compared to human insulin?

• Single amino acid substitution that prevents hexamer formation
• Addition of two arginines at the C-terminus of the B-chain and substitution of Gly at A21, causing shift in isoelectric point and precipitation at physiological pH for prolonged release
• Conjugation with polyethylene glycol (PEG) to increase molecular weight
• Removal of C-peptide residues to increase receptor affinity

Correct Answer: Addition of two arginines at the C-terminus of the B-chain and substitution of Gly at A21, causing shift in isoelectric point and precipitation at physiological pH for prolonged release

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