Biophysical Characterization for Product Comparability - Spectroscopic methods such as circular dichroism can detect subtle differences in higher order structure before and after changes in process

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Biophysical Characterization for Product Comparability
Spectroscopic methods such as circular dichroism can detect subtle differences in higher order structure before and after changes in process and formulation.


BioPharm International Supplements



Figure 4. Gravitational sweep AUC analysis for a VLP conjugate formulated with low and high salt concentrations. The centrifugation speed was varied from 3,000 to 20,000 rpm. The size distribution profiles were obtained using SedAnal.
Another example of applying gravitational sweep AUC is in formulation development for a Qβ virus-like-particle (VLP) conjugated with a peptide at multiple sites. In a formulation matrix with higher salt concentration, SEC analysis suffered significant loss in total peak area, and AF4 suggested the presence of very large aggregates (data not shown). To confirm, the VLP conjugate in low and high salt formulations were analyzed with gravitational sweep AUC (Figure 4). The distribution profiles are markedly different. The protein formed very large and heterogeneous aggregates in the high salt formulation that peaked at ~665 S. The minimum radius and the minimum molecular weight for the equivalent anhydrous sphere are ~32 nm and ~111 MDa, respectively. Particles of this size may not be recoverable by the matrix used in SEC chromatography.


Figure 5. Dynamic light scattering for drug substance batches produced at different manufacturing sites or by different manufacturing processes. The scattered light intensity was measured at the scattering angle of 173. The size distributions were analyzed using the CONTIN algorithm, and averaged over multiple measurements.
DLS is another powerful biophysical technique that offers different advantages in characterizing aggregates. DLS measurement is simpler and offers higher throughput than AUC, and requires less substantial sample dilution, and is more sensitive to trace amounts of very large particles than SEC and AUC, which makes DLS very useful in detecting large aggregates. Figure 5 presents a DLS analysis of the bacterial Qβ VLP samples to evaluate a downstream process change. The process change did not generate any large aggregates. However, despite the fact that the size distribution is monomodal, the peak has shifted significantly to a larger size, suggesting the presence of small aggregates that are not resolvable by DLS. AUC–SV analysis showed similar results (Figure 6). On the other hand, no substantial difference was detected by either DLS or AUC in samples produced at different manufacturing sites using the original process, indicating that the original process is robust with regard to site change.


Figure 6. AUC–SV for drug substance batches produced at different manufacturing sites or by different manufacturing processes. The samples were analyzed at a centrifugation speed of 20,000 rpm and data were analyzed using Sedfit. Each profile is the average of two replicates.


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