Effects of buffer composition and processing conditions on aggregation of bovine IgG during freeze-drying

The objective of this study was to identify critical formulation and proces sing variables affecting aggregation of bovine IgG during freeze-drying whe n no lyoprotective solute is used. Parameters examined were phosphate buffe r concentration and counterion (Na versus K phosphate), added salts, coolin g rate, IgG concentration, residual moisture level, and presence of a surfa ctant. No soluble aggregates were detected in any formulation after either freezing/thawing or freeze-drying. No insoluble aggregates were detected in any formulation after freezing, but insoluble aggregate levels were always detectable after freeze-drying. The data are consistent with a mechanism o f aggregate formation involving denaturation of IgG at the ice/freeze-conce ntrate interface which is reversible upon freeze-thawing, but becomes irrev ersible after freeze-drying and reconstitution. Rapid cooling (by quenching in liquid nitrogen) results iri more and larger aggregates than slow cooli ng on the shelf of the freeze-dryer. This observation is consistent with su rface area measurements and environmental electron microscopic data showing a higher surface area of freeze-dried solids after fast cooling. Annealing of rapidly cooled solutions results in significantly less aggregation in r econstituted freeze-dried solids than in nonannealed controls, with a corre sponding decrease in specific surface area of the freeze-dried, annealed sy stem. Increasing the concentration of IgG significantly improves the stabil ity of IgG against freeze-drying-induced aggregation, which may be explaine d by a smaller percentage of the protein residing at the ice/freeze-concent rate interface as IgG concentration is increased. A sodium phosphate buffer system consistently results in more turbid reconstituted solids than a pot assium phosphate buffer system at the same concentration, but this effect i s not attributable to a pH shift during freezing. Added salts such as NaCl or KCI contribute markedly to insoluble aggregate formation. Both sodium an d potassium chloride contribute more to turbidity of the reconstituted soli d than either sodium or potassium phosphate buffers at similar ionic streng th, with sodium chloride resulting in a substantially higher level of aggre gates than potassium chloride. At a given cooling rate, the specific surfac e area of dried solids is approximately a factor of 2 higher for the formul ation containing sodium chloride than the formulation containing potassium chloride. Turbidity is also influenced by the extent of secondary drying, w hich underscores the importance of minimizing secondary drying of this syst em. Including a surfactant such as polysorbate 80, either in the formulatio n or In the water used for reconstitution, decreased, but did not eliminate , insoluble aggregates. There was no correlation between pharmaceutically a cceptability of the freeze-dried cake and insoluble aggregate levels in the reconstituted product.