Biophysical parameters in preformulation MCQs With Answer

Introduction: This blog provides a focused set of MCQs on biophysical parameters used during preformulation of protein therapeutics, tailored for M.Pharm students. It covers experimental techniques (DLS, SEC, DSC, CD, FTIR, MS), descriptors (hydrodynamic radius, zeta potential, second virial coefficient B22, Tm), and mechanistic concepts (colloidal vs conformational stability, preferential hydration, ionic strength effects). Questions emphasize interpretation of data, selection of appropriate analytical methods, and implications for formulation design such as aggregation risk, viscosity, and high‑concentration behavior. Use these items to test and deepen understanding of how biophysical characterization guides development of stable, manufacturable protein formulations.

Q1. Which biophysical parameter directly reports the effective size of a protein particle as it diffuses in solution?

• Hydrodynamic radius (Rh)
• Isoelectric point (pI)
• Zeta potential
• Melting temperature (Tm)

Correct Answer: Hydrodynamic radius (Rh)

Q2. Which technique is most commonly used to measure the hydrodynamic radius and size distribution of proteins in solution non‑invasively?

• Dynamic light scattering (DLS)
• Size‑exclusion chromatography (SEC)
• SDS‑PAGE
• Analytical ultracentrifugation (AUC)

Correct Answer: Dynamic light scattering (DLS)

Q3. A positive value of the second virial coefficient (B22) is generally interpreted as which of the following?

• Net repulsive protein–protein interactions indicating improved colloidal stability
• Net attractive interactions promoting aggregation
• No interactions between protein molecules
• Irreversible aggregation tendency

Correct Answer: Net repulsive protein–protein interactions indicating improved colloidal stability

Q4. Which analytical technique provides a direct calorimetric measurement of a protein’s thermal unfolding transition and yields the melting temperature (Tm)?

• Differential scanning calorimetry (DSC)
• Circular dichroism (CD)
• Dynamic light scattering (DLS)
• Fourier‑transform infrared spectroscopy (FTIR)

Correct Answer: Differential scanning calorimetry (DSC)

Q5. Which method is most sensitive to changes in protein secondary structure (alpha‑helix vs beta‑sheet) in solution?

• Circular dichroism (CD) spectroscopy
• Intrinsic fluorescence of tryptophan
• Size‑exclusion chromatography (SEC)
• Zeta potential measurement

Correct Answer: Circular dichroism (CD) spectroscopy

Q6. Which measurement reports the electrophoretic mobility that is converted to surface charge information and stability estimates for colloidal proteins?

• Electrophoretic light scattering (zeta potential measurement)
• Dynamic light scattering (DLS)
• Analytical ultracentrifugation (AUC)
• Mass spectrometry (MS)

Correct Answer: Electrophoretic light scattering (zeta potential measurement)

Q7. Which technique separates monomeric protein from soluble oligomeric species and provides quantitative distribution for preformulation assessment?

• Size‑exclusion chromatography (SEC)
• Dynamic light scattering (DLS)
• Intrinsic fluorescence
• Circular dichroism (CD)

Correct Answer: Size‑exclusion chromatography (SEC)

Q8. Intrinsic fluorescence, mainly from tryptophan residues, is most useful in monitoring which aspect of protein behavior?

• Tertiary structure changes and local environment of aromatic residues
• Overall molecular weight
• Colloidal charge distribution
• Secondary structure content

Correct Answer: Tertiary structure changes and local environment of aromatic residues

Q9. Which assay specifically reports increased exposure of hydrophobic patches on a protein surface during unfolding or aggregation?

• ANS (8‑anilino‑1‑naphthalenesulfonate) binding fluorescence assay
• Circular dichroism (CD)
• Dynamic light scattering (DLS)
• Differential scanning calorimetry (DSC)

Correct Answer: ANS (8‑anilino‑1‑naphthalenesulfonate) binding fluorescence assay

Q10. What is the expected effect on protein solubility when the formulation pH is close to the protein’s isoelectric point (pI)?

• Decreased solubility and increased propensity to aggregate
• Maximum solubility and minimized aggregation
• No change in solubility
• Increased net charge and electrostatic repulsion

Correct Answer: Decreased solubility and increased propensity to aggregate

Q11. For high‑concentration subcutaneous formulations, which preformulation parameter is most critical to ensure syringeability and patient acceptability?

• Viscosity of the protein solution
• Isoelectric point (pI)
• Intrinsic fluorescence intensity
• Hydrodynamic radius at infinite dilution

Correct Answer: Viscosity of the protein solution

Q12. In a thermal unfolding experiment, the melting temperature (Tm) is defined as which of the following?

• The midpoint temperature where native and unfolded populations are equal
• The temperature where aggregation begins irreversibly
• The temperature at which enzymatic activity is zero
• The onset temperature of minor conformational changes

Correct Answer: The midpoint temperature where native and unfolded populations are equal

Q13. Which chemical denaturant is frequently used for reversible equilibrium unfolding experiments to derive free energy of unfolding by extrapolation?

• Urea
• Sodium dodecyl sulfate (SDS)
• Formaldehyde
• Glutaraldehyde

Correct Answer: Urea

Q14. Hydrogen–deuterium exchange coupled to mass spectrometry (HDX‑MS) is most useful in preformulation for mapping what property of a protein?

• Conformational dynamics and solvent accessibility of backbone amides
• Overall molecular weight
• Primary amino acid sequence
• Surface charge distribution

Correct Answer: Conformational dynamics and solvent accessibility of backbone amides

Q15. How does increasing ionic strength typically affect electrostatic interactions between protein molecules in solution?

• It screens electrostatic repulsion, often reducing long‑range repulsive forces
• It increases net electrostatic repulsion indefinitely
• It converts repulsion into permanent covalent crosslinks
• It has no effect on electrostatic interactions

Correct Answer: It screens electrostatic repulsion, often reducing long‑range repulsive forces

Q16. The stabilization mechanism where excipients (e.g., sugars) are excluded from the protein surface, promoting preferential hydration of the protein, is known as:

• Preferential exclusion (preferential hydration)
• Direct binding stabilization
• Hydrophobic shielding
• Electrostatic complexation

Correct Answer: Preferential exclusion (preferential hydration)

Q17. Which analytical method is most appropriate to resolve charge variants and accurately determine a protein’s isoelectric point in preformulation?

• Capillary isoelectric focusing (cIEF)
• Size‑exclusion chromatography (SEC)
• Dynamic light scattering (DLS)
• Differential scanning calorimetry (DSC)

Correct Answer: Capillary isoelectric focusing (cIEF)

Q18. Which spectroscopic technique analyzes the amide I vibrational band and is widely used to characterize protein secondary structure in both solution and solid formulations?

• Fourier‑transform infrared spectroscopy (FTIR)
• Intrinsic fluorescence spectroscopy
• Analytical ultracentrifugation (AUC)
• Size‑exclusion chromatography (SEC)

Correct Answer: Fourier‑transform infrared spectroscopy (FTIR)

Q19. A measured zeta potential with a magnitude greater than approximately ±30 mV typically indicates what about a protein colloidal system?

• Good electrostatic stabilization against aggregation
• Immediate precipitation is inevitable
• No charge on the protein surface
• High propensity for covalent cross‑linking

Correct Answer: Good electrostatic stabilization against aggregation

Q20. Which pair of biophysical parameters is most frequently combined in preformulation to predict aggregation propensity and guide formulation choice?

• Second virial coefficient (B22) and melting temperature (Tm)
• Isoelectric point (pI) and intrinsic fluorescence intensity
• Zeta potential and amide I absorbance maximum
• Hydrodynamic radius at infinite dilution and SDS‑PAGE band intensity

Correct Answer: Second virial coefficient (B22) and melting temperature (Tm)

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