Investigating trace metal precipitation in highly concentrated cell culture media with Pourbaix diagrams.

Commercial production of therapeutic proteins using mammalian cells requires complex process solutions, and consistency of these process solutions is critical to maintaining product titer and quality between batches. Inconsistencies between process solutions prepared at bench and commercial scale may be due to differences in mixing time, temperature, and pH which can lead to precipitation and subsequent removal via filtration of critical solution components such as trace metals. Pourbaix diagrams provide a useful tool to model the solubility of trace metals and were applied to troubleshoot the scale-up of nutrient feed preparation after inconsistencies in product titer were observed between bench- and manufacturing-scale batches. Pourbaix diagrams modeled the solubility of key metals in solution at various stages of the nutrient feed preparation and identified copper precipitation as the likely root cause of inconsistent medium stability at commercial scale. Copper precipitation increased proportionally with temperature in bench-scale preparations of nutrient feed and temperature was identified as the root cause of copper precipitation at the commercial scale. Additionally, cell culture copper titration studies performed in bench-scale bioreactors linked copper-deficient mammalian cell culture to inconsistent titers at the commercial scale. Pourbaix diagrams can predict when trace metals are at risk of precipitating and can be used to mitigate risk during the scale-up of complex medium preparations.

Methods for identifying precipitates and improving stability of chemically defined highly concentrated cell culture media.

Currently, within the biopharmaceutical industry, media development is a key area of development as the ratios and concentrations of media components such as amino acids, metals, vitamins, sugars, salts, and buffering agents play arguably the largest role in cellular productivity and product quality. However, optimizing media for these targets often conflicts with solubility limitations and slow-rate chemical reactions that result in precipitation formation. Here we present methods such as inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence (XRF), colorimetry, and turbidity to identify multiple likely components of a complex precipitate that was observed in preparations of a custom nutrient feed medium across all storage conditions evaluated. Using these analytical methods, as well as adjustments to the formulation pH, increasing the pyruvate concentration, and removing sodium bicarbonate, we were able to extend the media shelf life from approximately 10 days to over 28 days. Alternatively, copper, selenium, and magnesium sources were removed from the media and no precipitation was observed until 32 days after prep, pointing to key metals as the probable root cause of precipitation. By analytically quantifying the precipitate using the methods above, instead of visual inspection, which is the current industry standard for media precipitation observation, we were better able to compare conditions to one another and relate them to the onset of precipitation. Cell culture performance and product quality remained comparable to the historical process despite the media formulation changes.

The Bcl-2 family antagonist ABT-737 significantly inhibits multiple animal models of autoimmunity.

The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-x(L), and Bcl-w protein function. There is evidence that Bcl-2-associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.

Elemental metal variance in cell culture raw materials for process risk profiling.

Elemental metals are critical raw material attributes which can impact cell culture performance and associated therapeutic protein product quality profiles. Metals such as copper and manganese act as cofactors and reagents for numerous metabolic pathways which govern cell growth, protein expression, and glycosylation, thus mandating elemental monitoring. The growing complexity of modern cell culture media formulations adds additional opportunities for elemental variance and its associated impact risks. This article describes an analytical technique applying inductively coupled plasma mass spectrometry to characterize a list of common raw materials and media powders used in mammalian cell culture and therapeutic protein production. We aim to describe a method qualification approach suitable for biopharmaceutical raw materials. Furthermore, we present detailed profiles of many common raw materials and discuss trends in raw material subtypes. Finally, a case study demonstrating the impact of an unexpected source of raw material variation is presented along with recommendations for raw material elemental risk profiling and control.