(716b) Critical Factors That Affect the Hydroxylysine Levels in Monoclonal Antibody Production

Hydroxylysine (Hyl) is an enzyme-catalyzed post translational modification (PTM) that arises from hydroxyl modification of lysine. Hyl is a well-known component of collagen but is usually not observed in cell cultures at high levels (>10%). Monitoring of PTMs is critical as presence of PTMs such as Hyl in cell culture may lead to challenges in process robustness and product consistency. For our study, the quantification of the Hyl level was done using IdeS digestion, a reduced mass analysis technique. At elevated Hyl level of 20-35% observed in the bioreactor, Hyl was considered as a product quality attribute and required further investigation. Detecting and modulating the levels of Hyl present in cell culture remains a major challenge. Bioreactor parameters such as pH and temperature downshift had little impact on the Hyl levels. With use of optimized media/feeds, it was observed that the Hyl levels could be reduced by 50%. Media additives such as Fe (III) were found to be critical in modulating Hyl levels. The results identified from this study can support other biologic projects to enable proper modulation of levels of Hyl in cell culture.