Bioprocessing of cyclic AMP MCQs With Answer

Introduction: This quiz collection on Bioprocessing of cyclic AMP (cAMP) is designed for M.Pharm students preparing for advanced topics in Bioprocess Engineering and Technology. It covers biochemical formation of cAMP, enzymatic and microbial production strategies, reactor design considerations, immobilized enzyme systems, analytical assays, downstream recovery, and quality control aspects relevant to pharmaceutical bioprocesses. Questions emphasize understanding of pathway regulation, kinetic and mass-transfer limitations, choice of analytical methods (HPLC, ELISA, radioassay), and practical challenges in scale-up and purification. The set balances conceptual depth and application-oriented problems to sharpen problem-solving skills needed for research or industrial bioprocess development involving cAMP and related cyclic nucleotides.

Q1. Which enzyme catalyzes the conversion of ATP to cyclic AMP in biological systems?

• Adenylate cyclase
• Guanylate cyclase
• Adenosine deaminase
• Phosphodiesterase

Correct Answer: Adenylate cyclase

Q2. In an immobilized enzyme reactor designed for enzymatic synthesis of cAMP from ATP, which factor most directly limits conversion at high substrate concentration?

• Enzyme intrinsic turnover number (kcat)
• External mass transfer of ATP to the particle surface
• Substrate inhibition by ATP
• pH of the bulk solution

Correct Answer: External mass transfer of ATP to the particle surface

Q3. Which analytical method provides the highest specificity and quantitation for separating and measuring cAMP in complex biological matrices?

• Enzyme-linked immunosorbent assay (ELISA)
• High-performance liquid chromatography coupled with mass spectrometry (HPLC-MS)
• Colorimetric phosphate assay
• Turbidimetric assay

Correct Answer: High-performance liquid chromatography coupled with mass spectrometry (HPLC-MS)

Q4. When designing a fed-batch fermentation to produce cAMP via recombinant microbial expression of adenylate cyclase, which feed strategy helps avoid accumulation of acetate and maintains high ATP availability?

• Exponential feeding to match specific growth rate
• Bolus glucose feeding every hour
• No feeding; batch culture only
• Continuous high-rate glucose feeding

Correct Answer: Exponential feeding to match specific growth rate

Q5. Which of the following downstream steps is most appropriate for initial recovery of cAMP from a fermented cell broth where cAMP is predominantly intracellular?

• Direct ultrafiltration of whole broth
• Cell lysis followed by centrifugation to remove debris
• Adsorption to nonpolar resin without lysis
• Salt-induced precipitation of cAMP

Correct Answer: Cell lysis followed by centrifugation to remove debris

Q6. Phosphodiesterases (PDEs) affect cAMP levels in a bioprocess. Which strategy is best to preserve produced cAMP during downstream processing?

• Add PDE inhibitors or adjust pH to inactivate PDEs immediately after lysis
• Increase incubation temperature to accelerate release of cAMP
• Introduce divalent cations to stabilize PDE activity
• Delay downstream processing to allow equilibration

Correct Answer: Add PDE inhibitors or adjust pH to inactivate PDEs immediately after lysis

Q7. In a kinetic model of adenylate cyclase converting ATP to cAMP, which parameter most directly determines the maximal enzymatic rate in Michaelis-Menten kinetics?

• Km (Michaelis constant)
• kcat (turnover number)
• Substrate concentration
• Inhibitor concentration

Correct Answer: kcat (turnover number)

Q8. Which solvent system is most suitable for extracting cAMP from aqueous lysate prior to chromatographic purification, considering cAMP polarity?

• Nonpolar organic solvent like hexane
• Water-miscible polar organic solvent such as methanol followed by solid-phase extraction
• Chloroform:methanol (2:1) biphasic extraction
• Supercritical CO2 extraction

Correct Answer: Water-miscible polar organic solvent such as methanol followed by solid-phase extraction

Q9. For routine monitoring of intracellular cAMP in process development, which assay balances throughput and sensitivity most effectively?

• Radioimmunoassay (RIA)
• HPLC with UV detection
• Commercial ELISA kit designed for cAMP
• NMR spectroscopy

Correct Answer: Commercial ELISA kit designed for cAMP

Q10. Which immobilization support characteristic is most important to retain adenylate cyclase activity when used for repeated cAMP production cycles?

• High hydrophobicity to increase enzyme adsorption
• Appropriate pore size allowing substrate diffusion but limiting enzyme leaching
• Strong oxidizing surface groups
• Extremely rigid, nonporous beads

Correct Answer: Appropriate pore size allowing substrate diffusion but limiting enzyme leaching

Q11. Which regulatory mechanism reduces adenylate cyclase activity in cells and therefore would lower cAMP biosynthesis?

• Activation by Gs protein signaling
• Phosphorylation by protein kinase A causing feedback inhibition
• Upregulation of ATP synthesis
• Allosteric activation by forskolin

Correct Answer: Phosphorylation by protein kinase A causing feedback inhibition

Q12. While scaling up a bioreactor for cAMP production, which dimensionless number is most critical to assess mixing and oxygen transfer similarity between scales?

• Péclet number
• Reynolds number
• Damköhler number
• Oxygen mass transfer coefficient times area (kLa)

Correct Answer: Oxygen mass transfer coefficient times area (kLa)

Q13. During chromatographic purification, which stationary phase is most effective to separate cAMP from structurally similar nucleotides based on charge properties?

• Reverse-phase C18
• Anion-exchange resin
• Size-exclusion gel
• Hydrophobic interaction resin

Correct Answer: Anion-exchange resin

Q14. Which storage condition best preserves purified cAMP stability for long-term storage in a pharmaceutical lab?

• Room temperature in aqueous solution
• Prepared as lyophilized powder and stored at −20°C or lower, protected from moisture
• Stored in acidic aqueous buffer at 37°C
• Stored in direct sunlight to avoid microbial growth

Correct Answer: Prepared as lyophilized powder and stored at −20°C or lower, protected from moisture

Q15. When comparing chemical synthesis versus enzymatic production of cAMP, which advantage is specific to enzymatic routes?

• Lower catalyst cost per mole synthesized
• Greater stereospecificity and milder reaction conditions
• Ability to use extreme pH and temperatures
• Less requirement for purification

Correct Answer: Greater stereospecificity and milder reaction conditions

Q16. In an experiment to maximize intracellular cAMP, inhibition of which cellular process would most directly increase steady-state cAMP levels?

• ATP synthesis by oxidative phosphorylation
• Phosphodiesterase-mediated cAMP degradation
• Protein translation by ribosomes
• Glycolytic flux

Correct Answer: Phosphodiesterase-mediated cAMP degradation

Q17. During HPLC method development for cAMP, which mobile phase modification improves peak shape for a polar anionic nucleotide on ion-exchange or reverse-phase columns?

• Use pure water with no buffer
• Add volatile buffer like ammonium acetate and adjust pH to suppress ionization
• Increase organic solvent to 90% acetonitrile
• Use nonvolatile salt at very high concentration

Correct Answer: Add volatile buffer like ammonium acetate and adjust pH to suppress ionization

Q18. Which process analytical technology (PAT) tool can provide near real-time monitoring of intracellular cAMP trends during a fermentation?

• Offline ELISA sampled every 24 hours
• Online mass spectrometry sampling combined with rapid quench
• End-point UV absorbance of whole broth
• Colony forming unit counts

Correct Answer: Online mass spectrometry sampling combined with rapid quench

Q19. Which impurity class is most challenging to remove from cAMP preparations because of very similar chemical structure and charge?

• Inorganic salts such as NaCl
• Other cyclic nucleotides like cGMP
• Large proteins and cell debris
• Hydrophobic lipid contaminants

Correct Answer: Other cyclic nucleotides like cGMP

Q20. For regulatory quality control of pharmaceutical-grade cAMP intended as a research reagent, which specification is essential to demonstrate reproducible potency and safety?

• Only pH measurement of final solution
• Identity, purity by HPLC, endotoxin level, and stability data
• Visual color inspection only
• Microbial count without specification limits

Correct Answer: Identity, purity by HPLC, endotoxin level, and stability data

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