Introduction: This set of MCQs on the production of Hepatitis B vaccine is designed for M. Pharm students specializing in Advanced Pharmaceutical Biotechnology. The questions focus on recombinant HBsAg-based vaccine manufacturing, expression systems, upstream fermentation parameters, downstream purification and viral inactivation considerations, formulation with adjuvants, quality control assays, and regulatory requirements. Emphasis is placed on deeper technical concepts such as VLP assembly, host-cell related impurities, glycosylation impacts, and potency testing. Use these questions to test and strengthen your conceptual understanding of both practical production challenges and scientific rationale behind modern Hepatitis B vaccine development.
Q1. Which method is the most commonly used to produce the modern Hepatitis B vaccine antigen (HBsAg)?
- Plasma-derived purification from infected donors
- Recombinant HBsAg expressed in Saccharomyces cerevisiae forming virus-like particles
- Recombinant expression in Escherichia coli with chemical refolding
- Inactivated whole hepatitis B virions propagated in cell culture
Correct Answer: Recombinant HBsAg expressed in Saccharomyces cerevisiae forming virus-like particles
Q2. What is the characteristic size of the HBsAg virus-like particles (VLPs) that are the principal antigen in recombinant Hepatitis B vaccines?
- 5–8 nm
- 22 nm
- 100–200 nm
- 500–1000 nm
Correct Answer: 22 nm
Q3. Which viral gene encodes the small surface antigen (HBsAg) commonly used in vaccine production?
- C gene (core antigen)
- S gene (small surface antigen)
- P gene (polymerase)
- X gene (regulatory protein)
Correct Answer: S gene (small surface antigen)
Q4. Compared to bacterial systems, why is Saccharomyces cerevisiae often preferred for HBsAg expression?
- Yeast produce endotoxin which enhances immunogenicity
- Yeast is cheaper than bacteria for large-scale culture
- Yeast can perform eukaryotic folding and allow VLP assembly while lacking endotoxin
- Yeast glycosylation exactly matches human glycosylation
Correct Answer: Yeast can perform eukaryotic folding and allow VLP assembly while lacking endotoxin
Q5. A major downstream polishing step used in modern recombinant HBsAg purification to increase purity is:
- Cesium chloride density gradient ultracentrifugation only
- Ion-exchange chromatography
- Simple ammonium sulfate precipitation with no chromatography
- Heat denaturation to remove host proteins
Correct Answer: Ion-exchange chromatography
Q6. The most commonly used adjuvant in licensed Hepatitis B vaccines to enhance immune response is:
- Freund’s complete adjuvant
- Aluminum hydroxide (alum)
- Monophosphoryl lipid A (MPL) alone
- CpG oligonucleotide adjuvant
Correct Answer: Aluminum hydroxide (alum)
Q7. Which analytical test is routinely used to quantify HBsAg content during production and batch release?
- Endotoxin assay (LAL) only
- HPLC size-exclusion without antigen-specific detection
- HBsAg-specific ELISA using reference standards
- DNA gel electrophoresis
Correct Answer: HBsAg-specific ELISA using reference standards
Q8. What is the principal reason E. coli is not the preferred host for producing immunogenic HBsAg VLPs?
- E. coli secretes HBsAg too efficiently causing loss
- Inability to form properly folded VLPs and absence of eukaryotic processing/glycosylation
- E. coli over-glycosylates proteins interfering with antigenicity
- E. coli inherently contains high levels of viral contaminants
Correct Answer: Inability to form properly folded VLPs and absence of eukaryotic processing/glycosylation
Q9. Which impurity is of particular regulatory concern for recombinant yeast-derived Hepatitis B vaccines and must be quantified/controlled?
- Residual host-cell DNA
- Endotoxin from yeast
- Prion proteins
- Aluminum metal contamination
Correct Answer: Residual host-cell DNA
Q10. The inclusion of pre-S1 and pre-S2 regions in some Hepatitis B vaccine constructs primarily aims to:
- Reduce manufacturing cost by shortening fermentation
- Enhance immunogenicity and broaden the neutralizing antibody response
- Increase thermostability of the vaccine formulation
- Enable oral delivery without adjuvant
Correct Answer: Enhance immunogenicity and broaden the neutralizing antibody response
Q11. During scale-up of yeast fermentation for HBsAg, which parameter is most critical to control to maximize VLP yield?
- Light exposure of the fermenter
- pH and dissolved oxygen to maintain optimal metabolic state
- Magnetic field strength around the bioreactor
- Addition of plant extracts to the medium
Correct Answer: pH and dissolved oxygen to maintain optimal metabolic state
Q12. Which statement best describes the role of the ‘a’ determinant of HBsAg in vaccine efficacy?
- It is a non-immunogenic internal peptide irrelevant to protection
- The ‘a’ determinant is a conserved conformational epitope that induces neutralizing antibodies across genotypes
- It only affects T-cell responses and not antibody generation
- It is important for viral replication but not for vaccine design
Correct Answer: The ‘a’ determinant is a conserved conformational epitope that induces neutralizing antibodies across genotypes
Q13. Which quality control assay is typically used to detect residual host-cell proteins in a recombinant HBsAg vaccine batch?
- Host-cell protein (HCP) ELISA
- Bradford total protein assay alone
- UV absorbance at 260 nm
- Reverse transcriptase activity assay
Correct Answer: Host-cell protein (HCP) ELISA
Q14. Plasma-derived Hepatitis B vaccines historically required additional viral inactivation steps. Which production process specifically necessitates robust viral inactivation?
- Recombinant yeast-derived HBsAg production
- Synthetic peptide vaccine manufacture
- Plasma-derived HBsAg purification from human donors
- DNA vaccine plasmid production in bacteria
Correct Answer: Plasma-derived HBsAg purification from human donors
Q15. Regulatory guidance commonly sets limits for residual host-cell DNA in recombinant vaccines. A widely cited conservative limit is:
- ≤100 mg per dose
- ≤10 ng per dose
- ≤1 g per dose
- There is no limit for residual DNA
Correct Answer: ≤10 ng per dose
Q16. Co-expression of molecular chaperones or foldases in the yeast expression host is used to:
- Increase plasmid copy number only
- Improve correct folding, assembly of HBsAg and formation of VLPs
- Remove the need for downstream purification
- Increase glycosylation heterogeneity intentionally
Correct Answer: Improve correct folding, assembly of HBsAg and formation of VLPs
Q17. Adsorption of HBsAg to aluminum salts in the final formulation primarily provides which of the following benefits?
- Complete sterilization of the vaccine
- Depot effect for slow antigen release and enhanced antibody response
- Replacement for cold-chain storage
- Neutralization of host-cell DNA contamination
Correct Answer: Depot effect for slow antigen release and enhanced antibody response
Q18. Which assay is most appropriate to demonstrate in vivo potency of an HBsAg vaccine batch for regulatory release?
- In vitro SDS-PAGE alone
- Mouse immunogenicity test measuring anti-HBs seroconversion compared to reference standard
- Simple pH measurement of the formulation
- In vitro LAL endotoxin test alone
Correct Answer: Mouse immunogenicity test measuring anti-HBs seroconversion compared to reference standard
Q19. A manufacturing challenge unique to yeast-produced HBsAg compared with mammalian cells is:
- Yeast produce no proteins, causing low yield
- Yeast hypermannosylation of N-linked glycans that can alter antigen properties
- Yeast generate mammalian-like glycosylation making regulatory approval easier
- Yeast release infectious hepatitis B virions spontaneously
Correct Answer: Yeast hypermannosylation of N-linked glycans that can alter antigen properties
Q20. For Hepatitis B vaccine potency and standardization, the unit of biological activity typically referenced is:
- Colony forming units (CFU)
- International Units (IU) based on WHO reference preparations and in vivo immunogenicity
- Optical density at 600 nm
- Milliequivalents of aluminum
Correct Answer: International Units (IU) based on WHO reference preparations and in vivo immunogenicity
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