Kinetic Analysis Enhances Monitoring of Product Quality During Process Development

Wesley McGinn-Straub, Product Manager, ForteBio

Researching a molecular interaction using only equilibrium data is like watching a story with still photographs rather than a motion picture. While some elements of the plot can be understood, missing information from between those still shots and character movements can lead to mis-interpretation of the events. And yet, full kinetic analyses of protein-protein interactions remain a relatively infrequent assay; most researchers have been trained in the theory of molecular interactions but have rarely measured an association and dissociation constant due to the technical nature of previous label-free instrumentation and the laborious nature of the assays. ForteBio has dramatically changed this landscape by introducing Dip and Read™ kinetic interaction assays on the Octet platform. The Dip and Read™ workflow enables biosensors to be dipped into samples in standard microplates to read molecular interactions, enabling rapid measurement of ka, kd and KD in a user-friendly, microfluidics-free environment.

With this improvement in format, the Octet platform brings label-free kinetic measurements out of the core lab and onto the research benchtop, having significant impact on the information available to scientists as they navigate the meandering paths of their research projects. Monitoring product quality of a potential biological is a good example of the impact kinetic data can have on a project. During process development, it is typically unreasonable to measure the bioactivity of every sample as the media type, feed type, feed strategy, temperature shift and harvest date, as well as purification strategy are altered. The common analytics of SDS-PAGE, HPLC and SEC track degradation and aggregation but it is often assumed de facto that the affinity of the interaction between the protein product and its target remains unaltered. One reason for this assumption is the lack of an analytical tool that can perform kinetic analyses on a routine basis yet requires no greater expertise or ideally less time than SDS-PAGE or SEC.

The process development group at Aragen Bioscience, a CRO based in California's Bay Area, encountered such a problem when they attempted to convert a research/academic  cell line to a manufacturing cell line during upstream development of a biological entity. Clone and media were selected based on titer and all routine analytics (SDS-PAGE, SEC, and HPLC) indicated that Lot B generated using the newly developed process was biophysically equivalent to a Lot A, derived from the original cell line and used as a "gold standard". However, the material generated by the new process possessed 50% less bioactivity than the gold standard.

The disparity was explained when the kinetics of Lot A and Lot B were measured on the Octet platform. The dissociation rate of Lot A was 10-fold higher than that of Lot B, and consequently the affinity was 12-fold lower. Dr. Oren Beske, vice-president of in-vitro services at Aragen Bioscience notes that "The Octet platform provided the biophysical basis for distinguishing Lot A from Lot B, a rationale for the loss in bioactivity for Lot B, and a method for monitoring future product quality on a daily basis. The assay is simple, does not require a dedicated user and therefore enables the group to routinely monitor the affinity of the biological and its target in a research environment where direction changes quickly and data needs to keep pace."

To learn more about the Octet platform, visit www.fortebio.com.