(15d) Designing in Process Robustness: Minimizing Proteolysis Through Analytical and Chromatographic Tools

Host cell protein removal is a primary goal during purification process development for vaccines and therapeutic proteins, however the subset of protease impurities is a particular threat to product yield, quality, and product/process consistency.  The presence of such enzymes in the process can lead to proteolytic clipping of the product, resulting in a heterogeneous mixture of degraded product fragments and isoforms.  Resolution of the desired product from these closely-related degradates represents a significant separation challenge and can result in project delays and elevated developmental cost (and cost of goods) for vaccines and therapeutics.  Due to the complexity and constraints of genetically removing proteases from the host cell system, the purification process is often tasked with solving these problems by minimizing degradation and eliminating the offending protease activity.  The exact solution to the problem, however, is highly dependent on the particular host cell, product, and process conditions.  In this work we outline a strategic methodology that has been consistently utilized to rapidly identify and minimize protease activity.  Examples comprising unique experimental designs and statistics will be used to illustrate chromatographic and filtration development aimed at minimizing residual proteases while maintaining high product recovery.  The resolving power of mixed-mode resins combined with tailored buffer/wash matrixes was instrumental in identifying conditions yielding the lower proteolytic activity.  Rapid development methods, such as high-throughput screening, were exploited to shorten the development life cycle and map the chromatography design space for optimal removal of protease activity and product degradates.  Novel fluorescent linkers enabling a low level of detection and real time analytics were used to detect protease and inform development decisions.  By understanding the operating conditions that minimize protease activity, we have been able to design multiple tiers of robustness into several high value biological processes.