A high throughput ultra performance size exclusion chromatography assay for the analysis of aggregates and fragments of monoclonal antibodies

Abstract

Background: Size exclusion chromatography (SEC) has been employed as an essential assay for aggregate characterization of in-process intermediates, release testing and stability studies of biologics (Q6B-ICH). Ultra-performance SEC (UP-SEC) that enables improved separation of different size species within a shorter running time than HP-SEC is highly desired. Results: We developed a 5-min UP-SEC assay based on BEH200 column for analysis of monoclonal antibodies on UPLC systems following screening of 13 different SEC columns. This UP-SEC assay has been evaluated with multiple antibody stability and in-process samples. The performance parameters including the resolution have been studied. Conclusion: This new UP-SEC method with 80% shorter running time has demonstrated better or equivalent separation efficiency than the HP-SEC method. This UP-SEC has been successfully implemented in bioprocess development and analytical testing.