Mark Dysinger, Senior Scientist, Pfizer Global R&D

Amy Bass, Sr. Research Associate II, Takeda San Francisco, Inc

For cell line development, it is ideal to have a quick and robust method to accurately determine antibody titer from crude supernatant. We have incorporated ForteBio's antibody quantitation assay, using Protein A biosensors with the Octet RED machine, into Takeda San Francisco's Cell Line Development Platform. The quantitation assay has been characterized in-house for specificity, precision and accuracy. Results show that this assay is very specific for human antibodies showing no matrix effects from our various cell culture media. In addition, we see high precision when comparing numerous variability parameters. Lastly, titer values obtained from the Octet RED technology show high correlation to Protein A HPLC values. The antibody quantitation assay is used in our cell line development platform at the 96 well, fed-batch shake flask and bioreactor stages to streamline the evaluation of clones.

Islay Campbell, Field Application Scientist, ForteBio

Kinetics analysis of protein-protein and protein-particle interactions is a key application for real time, label free systems such as the Octet family of instruments. In this talk, tips and tricks for obtaining the best possible data will be presented. This will include optimization of target immobilization and analyte binding, along with regeneration strategies (if required). The Octet analysis software options will also be discussed, giving details of the analysis models and their applicability to data interpretation.

Jiamin Yu, Ph. D., Senior Scientist, diaDexus

Identification of constructs suitable for the recombinant protein production pipeline is a bottleneck for structural genomics efforts. A novel high-throughput approach, Binding Rate Screen, that can alleviate this bottleneck by screening crude lysates has been developed using the hexahistidine (His(6)) tag as a reporter. The constructs with the highest binding rates also exhibit high expression of soluble monomeric protein. Constructs expressing variations of the target protein can be prioritized on a time scale of minutes, providing a times savings of 10-100 fold.

Oren Beske, Ph. D., Vice President Aragen Biosciences

Monitoring both the physical characteristics and the bioactivity of a potential therapeutic protein are essential during the transition from a research-driven, bench scale production manufacturing process to a more robust industrialized process prior to initiating Phase I studies. This case study presents an example of a protein therapeutic expressed in mammalian cells that lost significant bioactivity when adapted to an industrial CHO production platform. Further investigation using various bioassays revealed that the reduction in specific activity was due to a fraction of the protein being expressed by the cells in an inactive form. Alternative purification processes were developed to identify and remove the inactive protein species in the preparation. Using the ForteBio Octet, an in vitro binding assay was rapidly developed and a kinetic analysis of the interaction revealed that the inactive form of the product possessed a faster dissociation constant from the target, resulting in a reduced affinity constant. With the source of the inactivity understood, a cell culture process was developed to increase specific activity of the expressed protein and identify clones producing product with high specific activity. Both the bioactivity assay and the ForteBio kinetic assay were used to track product quality during process development.