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.

Development of a rapid and reliable analytical method for screening poloxamer 188 for use in cell culture process.

Poloxamer P188 is a common nonionic surfactant additive used in cell culture media as a cellular protectant from the hydrodynamic forces and shear stress during bioprocessing. Presence of a hydrophobic high molecular weight impurity contaminant has been shown to compromise its protective properties and lead to batch failure. In this work we present, a reliable, sensitive, and rapid analytical method to detect and quantify the contaminant impurity in poloxamer 188. This method replaces a laborious and time-consuming functional test in the form of a shake flask assay. The method is based upon reversed-phase liquid chromatography with charged aerosol detection, simple mobile phase compositions, and a three-step gradient. The method was optimized to resolve the impurity from the main P188 fraction in less than 10 min. Analytical method qualification and functional test comparison demonstrate equivalent or better high throughput impurity screening performance. Attempts to identify the impurity and establish suitable method positive control standards are also discussed.

Endotoxin inactivation via steam-heat treatment in dilute simethicone emulsions used in biopharmaceutical processes.

Simethicone emulsion is used to regulate foaming in cell culture operations in biopharmaceutical processes. It is also a potential source of endotoxin contamination. The inactivation of endotoxins in dilute simethicone emulsions was assessed as a function of time at different steam temperatures using a Limulus amebocyte lysate kinetic chromogenic technique. Endotoxin inactivation from steam-heat treatment was fit to a four-parameter double exponential decay model, which indicated that endotoxin inactivation was biphasic, consisting of fast and slow regimes. In the fast regime, temperature-related effects were dominant. Transitioning into the slow regime, the observed temperature dependence diminished, and concentration-related effects became increasingly significant. The change in the Gibbs free energy moving through the transition state indicated that a large energy barrier must be overcome for endotoxin inactivation to occur. The corresponding Arrhenius pre-exponential factor was >>10(12) s(-1) suggesting that endotoxins in aqueous solution exist as aggregates. The disorder associated with the endotoxin inactivation reaction pathway was assessed via the change in entropy moving through the transition state. This quantity was positive indicating that endotoxin inactivation may result from hydrolysis of individual endotoxin molecules, which perturbs the conformation of endotoxin aggregates, thereby modulating the biological activity observed. Steam-heat treatment decreased endotoxin levels by 1-2 logarithm (log) reduction (LRV), which may be practically relevant depending on incoming raw material endotoxin levels. Antifoam efficiency and cell culture performance were negligibly impacted following steam-heat treatment. The results from this study show that steam-heat treatment is a viable endotoxin control strategy that can be implemented to support large-scale biopharmaceutical manufacturing.