Melissa D. Perkins

Comparative Effects of pH and Ionic Strength on Protein–Protein Interactions, Unfolding, and Aggregation for IgG1 Antibodies

Changes in protein-protein interactions, protein unfolding, and nonnative aggregation were assessed for a series of human IgG1 antibodies as a function of pH and solution ionic strength (I). Unfolding transitions were characterized with differential scanning calorimetry. Protein-protein interactions were characterized with the apparent second virial coefficient (A(2)) from light scattering. Aggregation pathways were assessed using size-exclusion chromatography and multi-angle laser light scattering, aggregation kinetics, and structural changes monitored by circular dichroism spectroscopy and thioflavine T (ThT) binding. Ionic strength had relatively minor qualitative effects on unfolding, while pH had large effects for all four antibodies. A(2) was sensitive to both pH and I, and indicated that electrostatic interactions and nonuniform surface-charge distributions were important near neutral pH. Depending on solution pH and I, distinct aggregation pathways were found for each antibody, and these shared similar patterns versus pH, I, and A(2). Main differences observed across different antibodies included thermal unfolding transitions in DSC and the effects of pH and I on aggregation kinetics and pathways. These correlated strongly with whether aggregates of a given antibody bound ThT, suggesting possible differences with respect to conformational changes and/or regions of the proteins that are structurally involved in stabilizing the aggregates.

Characterization of subvisible particle formation during the filling pump operation of a monoclonal antibody solution.

Characterization and control of aggregate and subvisible particle formation during fill-finish process steps are important for biopharmaceutical products. The filling step is of key importance as there is no further filtration of the drug product beyond sterile filtration. Filling processes can impact product quality by introducing physical stresses such as shear, friction, and cavitation. Other detrimental factors include temperature generated in the process of filling, foaming, and contact with filling system materials, including processing aids such as silicone oil. Certain pumps may shed extrinsic particles that may lead to heterogeneous nucleation-induced aggregation. In this work, microflow imaging, size-exclusion chromatography (SEC), and turbidimetry were utilized to quantify subvisible particles, aggregation, and opalescence, respectively. The filling process was performed using several commonly used filling systems, including rotary piston pump, rolling diaphragm pump, peristaltic pump, and time-pressure filler. The rolling diaphragm pump, peristaltic pump, and time-pressure filler generated notably less protein subvisible particles than the rotary piston pump, although no change in aggregate content by SEC was observed by any pump. An extreme increase in subvisible particles was also reflected in an increase in turbidity.

Demonstrating the Stability of Albinterferon Alfa-2b in the Presence of Silicone Oil

Silicone oil is often used to decrease glide forces in prefilled syringes and cartridges, common primary container closures for biopharmaceutical products. Silicone oil has been linked to inducing protein aggregation (Diabet Med 1989;6:278; Diabet Care 1987;10:786-790), leading to patient safety and immunogenicity concerns. Because of the silicone oil application process (Biotech Adv 2007;25:318-324), silicone oil levels tend to vary between individual container closures. Various silicone oil levels were applied to a container closure prior to filling and lyophilization of an albumin and interferon alfa-2b fusion protein (albinterferon alfa-2b). Data demonstrated that high silicone oil levels in combination with intended and stress storage conditions had no impact on protein purity, higher order structure, stability trajectory, or biological activity. Subvisible particulate analysis (1-10 µm range) from active and placebo samples from siliconized glass barrels showed similar particle counts. Increases in solution turbidity readings for both active and placebo samples correlated well with increases in silicone oil levels, suggesting that the particles in solution are related to the presence of silicone oil and not large protein aggregates. Results from this study demonstrate that silicone oil is not always detrimental to proteins; nevertheless, assessing the impact of silicone oil on a product case-by-case basis is still recommended.