Nutrient Optimization Reduces Phosphorylation and Hydroxylation Level on an Fc-Fusion Protein in a CHO Fed-Batch Process

Abstract

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.

Progress in fed-batch culture for recombinant protein production in CHO cells

“…Gangwar et al also indicated that vitamins can inhibit the acidic variants while it enhances the basic variants (Gangwar et al 2021 ). Hou et al found that increasing rate of vitamin C and nicotinamide feeding can reduce the phosphorylation level in CHO cells to 3% and attenuated the hydroxylation level to 9.4% (Hou et al 2019 ). Adding B vitamins including 0.005 g/L vitamin B2, 0.0035 g/L pyridoxine (vitamin B6), 0.03 g/L pyridoxal (vitamin B6), 0.0985 g/L folic acid and 0.024 g/L vitamin B12 can improve packed cell volume (PCV) and antibody titer (Vijayasankaran et al 2013 ).…”

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics

“…7 Together, these observations and the data presented here demonstrate that CHO cells are generally capable of introducing high levels of hydroxyproline and sulfotyrosine in therapeutic proteins, which thereby significantly increase the heterogeneity of the molecules. In a very recent study by Hou et al, 12 the impact of specific nutrients on phosphoserine (~20%) and hydroxylysine (~25%) of a CHO platform, fed batch-produced Fc-fusion protein was reported. Although the exact influence of the nutrients is unknown, increased vitamin C, ferric citrate, and niacinamide feeding rates and a decreased cysteine feeding rate reduced the phosphorylation level to~3%.…”

“…6,7 Further uncommon enzyme-catalyzed PTMs (based on the experience and knowledge from IgGs and monoclonal antibodies (mAbs)) have been reported in non-IgG domains and linkers of antibody-fusion proteins. [8][9][10][11][12] In therapeutic proteins, PTMs are especially critical if they negatively influence drug potency or drug safety. Even if PTMs just increase the product heterogeneity of the desired molecules, when identified they need to be diminished or removed by manufacturing process optimizations or further protein engineering, or they at least have to be monitored and controlled to demonstrate batch consistency or comparability of manufactured clinical material.…”

Potent Bispecific Neutralizing Antibody Targeting Glycoprotein B and the gH/gL/pUL128/130/131 Complex of Human Cytomegalovirus

“…The density and viability of cells were determined before each feeding. Supernatant harvest and antibody purification followed procedures described previously (54).…”