SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield.

This study investigates sec-hplc analysis of column load and flow-through provides critical understanding of low protein a step yield.. The research utilizes high-performance liquid chromatography (HPLC) as the primary analytical technique to address specific challenges in biopharmaceutical analysis. For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the... Research Background and Significance Protein A affinity chromatography is a cornerstone purification step in biopharmaceutical manufacturing, especially for monoclonal antibodies (mAbs), bispecific antibodies (bsAbs), and Fc-fusion proteins. Despite its widespread utilization, challenges such as unexpectedly low yield at the Protein A capture step can arise, significantly impacting overall process efficiency and product recovery. One notable issue is the presence of non-binding aggregates formed during cell culture, which can affect binding capacity and lead to yield losses. This study addresses the critical need for robust analytical techniques that provide insight into these yield issues by employing Size-Exclusion Chromatography coupled with High-Performance Liquid Chromatography (SEC-HPLC). By analyzing both the column load and the flow-through fractions, the researchers seek to elucidate the root causes of low Protein A step yield and propose strategies for process optimization. Experimental Design and Methodology The study employed SEC-HPLC as a primary analytical tool to characterize both the Protein A column load and the flow-through fractions from several biopharmac