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Nutrient Optimization Reduces Phosphorylation and Hydroxylation Level on an Fc‐Fusion Protein in a CHO Fed‐Batch Process
Author(s) -
Hou Ye,
Su Hang,
Luo Zhiying,
Li Mingying,
Ma Xiaonan,
Ma Ningning
Publication year - 2019
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700706
Subject(s) - hydroxylation , phosphorylation , cysteine , chemistry , serine , biochemistry , enzyme
Phosphorylation and hydroxylation are post translational modifications (PTMs) rarely observed or reported in biopharmaceuticals. While developing a stable CHO cell line and a fed‐batch process to produce a biosimilar dulaglutide, a GLP1‐Fc fusion protein, the authors identified both serine phosphorylation and lysine hydroxylation. While the innovator dulaglutide contains less than 2% phosphorylated and only ≈6.5% hydroxylated GLP1‐Fc molecules, the clones that the authors obtained in the platform fed‐batch process have ≈20% phosphorylated and 25% hydroxylated GLP1‐Fc molecules. An optimization of the nutrient feed is carried out, which successfully reduces the phosphorylation level to ≈3% and the hydroxylation level to 9.4% using the lead clone. Four components, cysteine, vitamin C, ferric citrate, and niacinamide, are found to be important in reducing the phosphorylation level. An increase in vitamin C, ferric citrate, and niacinamide feeding rates and a decrease in the cysteine feeding rate helps to reduce the phosphorylation level. Niacinamide and cysteine are also found to be critical for hydroxylation. An increase in the niacinamide and cysteine feeding rate is beneficial in reducing the hydroxylation level. This study is the first to report the impact of nutrient components on serine phosphorylation and lysine hydroxylation in biopharmaceuticals.