Modulation of high mannose levels in N‐linked glycosylation through cell culture process conditions to increase antibody‐dependent cell‐mediated cytotoxicity activity for an antibody biosimilar

Abstract

The regulatory approval of a biosimilar product is contingent on the favorable comparability of its safety and efficacy to that of the innovator product. As such, it is important to match the critical quality attributes of the biosimilar product to that of the innovator product. The N‐glycosylation profile of a monoclonal antibody (mAb) can influence effector function activities such as antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity. In this study, we describe efforts to modulate the high‐mannose (HM) levels of a biosimilar mAb produced in a Chinese hamster ovary cell fed‐batch process. Because the HM level of the mAb was observed to impact ADCC activity, it was desirable to match it to the innovator mAb's levels. Several cell culture process related factors known to modulate the HM content of N‐glycosylation were investigated, including osmolality, ammonium chloride (NH4Cl) addition, glutamine concentration, monensin addition, and the addition of alternate sugars and amino sugars to the feed medium. The process conditions evaluated varied in impact on HM levels, process performance and product quality. One condition, the addition of alternate sugars and amino sugars to feed medium, was identified as the preferred method for increasing HM levels with minimal disruptions to process performance or other product quality attributes. Interestingly, a secondary interaction between sugar and amino sugar supplemented feeds and osmolality was observed during process scale‐up. These studies demonstrate sugar and amino sugar concentrations and osmolality are critical variables to evaluate to match HM content in biosimilar and their innovator mAbs.

Insights into ultra-low affinity lipase-antibody noncovalent complex binding mechanisms

“…hile lipase expression during mAb production helps host cells survive, the results presented here indicate there is a possible opportunity to produce cell lines with partially deglycosylated lipases through asparagine-mutagenesis. Alternatively, feed-conditions 52 , 53 could be used to shift the production of glycans toward higher-molecular-weight glycans. Glycosylation-engineered lipases could minimize the formation of non-covalent mAb-lipase complexes and ultimately reduce the propensity for these types of enzymes to persist in formulation buffers, leading to visible particulates.…”

Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation

“…High mannose structures (Man5 and higher) have been reported to increase as a result of the addition of alternative sugars and amino sugars to the culture medium when producing a mAb using a CHO cell line, although upon scale‐up this supplementation caused a secondary interaction with osmolality. Mannose and glucosamine in combination with either fructose, galactosamine, or both fructose and galactosamine resulted in increased high‐mannose levels of the biosimilar compared with the innovator molecule in the region of 140%–160%, which correlated with an increased ADCC activity (>100%) compared with the innovator molecule (Rameez et al, 2021). As well as increasing ADCC activity, high mannose structures in the Fc region of IgG are also commonly associated with a faster serum clearance rate compared with other glycosylation structures in this region (Goetze et al, 2011; Yu et al, 2012), making mannosylation an important process to control during antibody production.…”