ABSTRACT
Upon exposure to shaking stress, an IgG1 mAb formulation in both the liquid and lyophilized state formed subvisible particles. Because freeze-drying was expected to minimize protein physical instability under these conditions, the extent and nature of aggregate formation in the lyophilized preparation were examined using a variety of particle characterization techniques. The effects of formulation variables such as residual moisture content, reconstitution rate, and reconstitution medium were also examined. Upon reconstitution of shake-stressed lyophilized mAb, differences in protein particle size and number were observed by microflow digital imaging, with the reconstitution medium having the largest impact. Shake stress had minor effects on the structure of protein within the particles as shown by SDS-PAGE and FTIR analysis. The lyophilized mAb was shake stressed to different extents and stored for 3 months at different temperatures. Both extent of cake collapse and storage temperature affected the physical stability of the shake-stressed lyophilized mAb upon subsequent storage. These findings demonstrate that physical degradation upon shaking of a lyophilized IgG1 mAb formulation includes not only cake breakage, but also results in an increase in subvisible particles and turbidity upon reconstitution. The shake-induced cake breakage of the lyophilized IgG1 mAb formulation also resulted in decreased physical stability upon storage.
Subject(s)
Antibodies, Monoclonal/chemistry , Chemistry, Pharmaceutical/methods , Immunoglobulin G/chemistry , Stress, Mechanical , Drug Stability , Drug Storage/methods , Freeze Drying/methodsABSTRACT
Silicone oil used as a lubricant in prefilled syringes has the potential to induce formation of particles in protein formulations. In the current study, we used a therapeutic fusion protein, albinterferon α2b , to evaluate protein aggregation and particle formation in the presence of silicone oil microdroplets or immobilized silicone interfaces. Tertiary structure of albinterferon α2b adsorbed on silicone oil microdroplets was perturbed in a formulation containing only buffer. In contrast, native-like tertiary structure was retained for albinterferon α2b adsorbed on silicone oil microdroplets in 300 mM sodium chloride or 300 mM sucrose formulations. Agitation of albinterferon α2b samples in the presence of silicone oil droplets or siliconized beads, respectively, caused albinterferon α2b aggregation and subvisible particle formation in formulations containing buffer or 300 mM sucrose. Adsorption of albinterferon α2b onto silicone oil was inhibited by addition of 0.01% (w/v) polysorbate 80, and this excipient prevented aggregation during agitation in the presence of silicone oil microdroplets. Aggregation was also reduced in the presence of 300 mM sodium chloride during agitation at least in part because of the increased conformational stability of the protein.
Subject(s)
Albumins/chemistry , Interferon-alpha/chemistry , Silicone Oils/chemistry , Water/chemistry , Acrylamides/chemistry , Adsorption , Algorithms , Buffers , Chemistry, Pharmaceutical , Chromatography, High Pressure Liquid , Electrochemistry , Emulsions , Fluorescence , Particle Size , Particulate Matter , Protein Conformation , Sodium Chloride/chemistry , Tryptophan/chemistryABSTRACT
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.
