Elucidation of cell culture impacts on hydroxylysine levels in monoclonal antibodies using high-throughput analytical quantification and media components.

Hydroxylysine (Hyl) is a post-translational hydroxyl modification of lysine that is not commonly observed at very high levels and thus is not usually considered a product quality attribute (PQA). Post-translation modifications (PTMs) are considered potential PQAs when elevated levels are observed - requiring monitoring and investigation. In a recent monoclonal antibody expression using Media A, Hyl levels were observed at ~20%-35%. At such elevated percentage levels, Hyl was considered a PQA - triggering a root-cause investigation in the upstream activities like cell culture conditions and media components. Initial detection of the Hyl modification originated from non-quantitative, intact mass analysis with confirmation of site-location determined by peptide mapping. Through the root-cause investigation, it was determined that levels of Hyl were underestimated by ~10-fold using tryptic peptide mapping analysis without inclusion of miscleaved peptides. The analytical procedure was revised from trypsin-digestion to IdeS-digestion, a reduced mass analysis, to accurately and rapidly quantify Hyl levels of investigational samples. Proprietary Media B was utilized to reduce the Hyl level by 2-fold to ~10%-15%. Further investigation into the media and feed components determined that increasing concentration of Fe(III) content decreased Hyl levels. Supplementation of Fe(III) served as a robust mitigation strategy of Hyl reduction in upstream process. Media B was used to scale up to a 500 L bioreactor while maintaining the lower Hyl level. The analytical and cell culture methods developed in this study can be leveraged to detect and tune Hyl levels.