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

Abstract

The objective of this study was to identify critical formulation and processing variables affecting aggregation of bovine IgG during freeze-drying when no lyoprotective solute is used. Parameters examined were phosphate buffer concentration and counterion (Na versus K phosphate), added salts, cooling rate, IgG concentration, residual moisture level, and presence of a surfactant. 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 of aggregate formation involving denaturation of IgG at the ice/freeze-concentrate interface which is reversible upon freeze-thawing, but becomes irreversible after freeze-drying and reconstitution. Rapid cooling (by quenching in liquid nitrogen) results in more and larger aggregates than slow cooling on the shelf of the freeze-dryer. This observation is consistent with surface 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 reconstituted freeze-dried solids than in nonannealed controls, with a corresponding decrease in specific surface area of the freeze-dried, annealed system. Increasing the concentration of IgG significantly improves the stability of IgG against freeze-drying-induced aggregation, which may be explained by a smaller percentage of the protein residing at the ice/freeze-concentrate interface as IgG concentration is increased. A sodium phosphate buffer system consistently results in more turbid reconstituted solids than a potassium phosphate buffer system at the same concentration, but this effect is not attributable to a pH shift during freezing. Added salts such as NaCl or KCl contribute markedly to insoluble aggregate formation. Both sodium and potassium chloride contribute more to turbidity of the reconstituted solid than either sodium or potassium phosphate buffers at similar ionic strength, with sodium chloride resulting in a substantially higher level of aggregates than potassium chloride. At a given cooling rate, the specific surface area of dried solids is approximately a factor of 2 higher for the formulation containing sodium chloride than the formulation containing potassium chloride. Turbidity is also influenced by the extent of secondary drying, which underscores the importance of minimizing secondary drying of this system. Including a surfactant such as polysorbate 80, either in the formulation or in the water used for reconstitution, decreased, but did not eliminate, insoluble aggregates. There was no correlation between pharmaceutically acceptability of the freeze-dried cake and insoluble aggregate levels in the reconstituted product.

Detection and prevention of protein aggregation before, during, and after purification

“…Alternately, the turbidity, or light scattered by precipitates at a nonabsorbing wavelength, can be used to rapidly detect insoluble protein aggregates. Turbidity measurements require large volumes of at least 10 lM final protein concentration [3]. The yields of many protein preparations are too low to allow screening by buffer dilution.…”

“…Many proteins are also potential pharmaceutical agents [1][2][3][4][5]. One significant impediment to the study and utilization of proteins is their extreme sensitivity to solution conditions.…”

“…Alternately, sample turbidity can be measured to assay for protein aggregation. However, turbidity measurements require high concentrations of protein and cannot detect low concentrations of aggregates or small, soluble aggregates [3,32]. Further, turbidity requires purified samples, prohibiting its use for proteins that aggregate during expression or purification.…”

Scientific Considerations for Generic Synthetic Salmon Calcitonin Nasal Spray Products

“…Peptide aggregates may be generated during the manufacture of a drug substance and drug product, as well as during storage (28)(29)(30)(31). Salmon calcitonin has been shown to be susceptible to dimerization and further aggregation (32).…”

Stabilization of Protein Structure in Freeze-Dried Amorphous Organic Acid Buffer Salts

“…For example, freezing of certain buffer systems (e.g., sodium phosphate) often induces crystallization of a component salt and resulting shift of the local pH surrounding the proteins. [7][8][9][10][11] Freeze-drying from some buffer systems (e.g., L-histidine, citrate, or Tris) often leads to higher activity retention of proteins (e.g., coagulation factor VIII, recombinant human interleukin-1 receptor antagonist) relative to those from other buffers. [12][13][14][15] Conformation of the proteins lyophilized in these buffer systems is of particular interest.…”