(1233-B) Automating the CE-SDS and cIEF workflows for downstream analysis on the BioPhase 8800 system

Abstract: Protein therapeutics is a rapidly growing industry for treating serious illnesses like cancer, autoimmune diseases, etc. Monoclonal antibodies, antibody-drug conjugates, fusion and multi-specific antibodies are a few examples of the different protein therapeutic modalities on the market today. These molecules may undergo post-translational modifications (PTM), producing functional variants directly affecting the overall protein charge. Capillary isoelectric focusing (cIEF) is a robust, reproducible, and high-resolution technique to characterize therapeutic recombinant proteins' charge variant heterogeneity. CE-SDS protein analysis kit is used to resolve reduced and non-reduced proteins by size, and quantify the purity and heterogeneity, by electrophoretic means. The BioPhase 8800 system is a multi-capillary system that analyzes 8 samples simultaneously, significantly decreasing high-throughput bottlenecks. However, sample preparation includes multiple steps and is laborious.
In this poster, we describe the automation of sample preparation for CE-SDS Protein analysis and charge heterogeneity assays on the Biomek i5 multichannel workstation for downstream separation on the BioPhase 8800 system, which significantly reduces hands-on time for users, minimizes exposure to hazardous chemicals and improves sample preparation reproducibility.
The entire automated workflow, for sample preparation, preparation of reagent inlet and outlet trays and the sample inlet and outlet trays for the BioPhase 8800, can be done in 3 hours (CE-SDS kit), and in 2 hours 20 mins (cIEF kit) for 96 samples on the Biomek i5 multichannel workstation. A plate with USP IgG reference standard was prepared with each of the automated methods. The robustness of the automated sample preparation for CE-SDS method was assessed by evaluating the inter- and intra-capillary consistency of corrected peak area (CPA) %, relative migration time (RMT) for heavy chain (HC), non-glycosylated heavy chain (ng-HC) and light chain (LC) of the reduced IgG. The robustness of the automated sample preparation for cIEF method was assessed by evaluating the inter- and intra-capillary consistency of corrected peak area (CPA) %, Detection Time (DT) for the acidic, basic and main isoforms of the IgG.
For the sample run, no cross-contamination or reagent carryover artifacts were observed. The intra-capillary reproducibility for CPA% was less than 0.47, 2.24 and 0.38 for LC, ng-HC and HC respectively. The intra-capillary reproducibility for CPA% was less than 0.47, 2.24 and 0.38 for LC, ng-HC and HC respectively.
Inter-capillary reproducibility values for % RSD for CPA% were less than 0.64, 0.24 and 0.53 for basic, main, and acidic groups, respectively.

Overall, these results indicate that the automated sample preparation methods are robust, reproducible and reduce hands-on time.