Abstract
An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50°C. A variety of excipients was tested for their ability to inhibit antibody aggregation. The amount of 4B9 aggregation, which was determined by analysis on a size-exclusion HPLC, was significantly reduced in the presence of low concentrations [between 0.1 and 1.0% (w/v)] of poly(vinylpyrrolidone) (PVP) molecules ranging in molecular weight from 10 to 40 kDa. When the PVP concentration was greater than 1.0%, antibody aggregation was enhanced, and with the highest molecular weight PVP, antibody precipitation occurred. HPLC was used to show that more PVP was associated with the 4B9 at 50°C than at 25°C. Differential scanning calorimetry revealed that PVP concentrations greater than 2.0% decreased the antibody thermal transition temperature. Enzyme-linked immunosorbent assays were used to assess the effects of PVP on the antigen binding capacity of 4B9 and on 4B9 quantitation. At 4°C, PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50°C, however, less 4B9 was detected in the 5.0% PVP solution. The heat stabilization of the 4B9 antibody by low concentrations of PVP can be explained by a weak binding of PVP to the native protein. The PVP may sterically interfere with protein–protein interactions, thus reducing aggregation. Higher concentrations of PVP lead to protein aggregation and precipitation, probably by a volume-exclusion mechanism. Low concentrations of less than 1.0% PVP can be used to stabilize proteins against heat-induced aggregation, but care should be exercised, since even slightly higher concentrations of PVP can also lead to protein destabilization.
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Gombotz, W.R., Pankey, S.C., Phan, D. et al. The Stabilization of a Human IgM Monoclonal Antibody with Poly(vinylpyrrolidone). Pharm Res 11, 624–632 (1994). https://doi.org/10.1023/A:1018903624373
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DOI: https://doi.org/10.1023/A:1018903624373