Assessing the risk of leachables from single-use bioprocess containers through protein quality characterization

Abstract

Leachables from single-use bioprocess containers (BPCs) are a source of process-related impurities that have the potential to alter product quality of biotherapeutics and impact patient health. Leachables often exist at very low concentrations making it difficult to detect their presence and challenging to assess their impact on protein quality. A small-scale stress model based on assessing protein stability was developed to evaluate the potential risks associated with storing biotherapeutics in disposable bags caused by the presence of leachables. Small-scale BPCs were filled with protein solution at high surface area-to-volume ratios (≥ 3x the surface area-to-volume ratio of manufacturing scale BPCs) and incubated at stress temperatures (e.g. 25°C or 30°C for up to 12 weeks) along with an appropriate storage vessel (e.g., glass vial or stainless steel) as a control for side-by-side comparison. Changes in protein size variants measured by size exclusion chromatography (SEC), capillary electrophoresis, and particle formation for two monoclonal antibodies (mAbs) using both the small scale stress model and a control revealed a detrimental effect of gamma irradiated BPC on protein aggregation and significant BPC batch-to-batch differences. It was found that preincubation of the empty BPCs prior to protein storage improved protein stability suggesting the presence of volatile or heat sensitive leachables. In addition, increasing the polysorbate 20 concentration lowered, but did not completely mitigate, the leachable-protein interactions indicating the presence of a hydrophobic leachable. Overall, this model can inform the risk of BPC leachables on bio-therapeutics during routine manufacturing and assist in making decisions on the selection of a suitable BPC for the manufacturing process via assessing changes in product quality.