THE STABILIZATION OF A HUMAN-IGM MONOCLONAL-ANTIBODY WITH POLY(VINYLPYRROLIDONE)

An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50 degrees C. A va riety of excipients was tested for their ability to inhibit antibody a ggregation. The amount of 4B9 aggregation, which was determined by ana lysis on a size-exclusion HPLC, was significantly reduced in the prese nce of low concentrations [between 0.1 and 1.0% (w/v)] of poly(vinylpy rrolidone) (PVP) molecules ranging in molecular weight from 10 to 40 k Da. When the PVP concentration was greater than 1.05%, antibody aggreg ation was enhanced, and with the highest molecular weight PVP, antibod y precipitation occurred. HPLC was used to show that more PVP was asso ciated with the 4B9 at 50 degrees C than at 25 degrees C. Differential scanning calorimetry revealed that PVP concentrations greater than 2. 0% decreased the antibody thermal transition temperature. Enzyme-linke d immunosorbent assays were used to assess the effects of PVP on the a ntigen binding capacity of 4B9 and on 4B9 quantitation. At 4 degrees C , PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50 degrees C, however, less 4B9 was detected i n 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 t o the native protein. The PVP may sterically interfere with protein-pr otein interactions, thus reducing aggregation. Higher concentrations o f PVP lead to protein aggregation and precipitation, probably by a vol ume-exclusion mechanism. Low concentrations of less than 1.0% PVP can be used to stabilize proteins against heat-induced aggregation, but ca re should be exercised, since even slightly higher concentrations of P VP can also lead to protein destabilization.