Combination of temperature shift and hydrolysate addition regulates anti-IgE monoclonal antibody charge heterogeneity in Chinese hamster ovary cell fed-batch culture

Abstract

Charge heterogeneity has been broadly studied as a critical quality attribute during monoclonal antibody (mAb) production that may subsequently affect product stability and biopotency. However, the charge variation distribution is poorly controlled, so methods of more effective control need to be explored. In this study, the combined effects of temperature shift (37-34, 37-32, or 37-30 °C) and hydrolysate addition (0.100 g/L) to culture feed on the charge heterogeneity of anti-IgE mAb were investigated. The results showed that the distribution of charge variation was significantly regulated by the combination of hydrolysate addition with a highly sub-physiological temperature (34 °C). In addition, under this condition, the main peak content significantly increased, and the acidic peak content significantly decreased. Furthermore, we explored Lys variant content, which is the major basic variant content, as well as its relationship with temperature shift and hydrolysate addition. Lys variant levels were positively related to the Lys and Arg concentrations in the medium and negatively related to carboxypeptidase B and carboxypeptidase H transcript levels. The combination of temperature shift and hydrolysate addition can thus effectively improve anti-IgE mAb charge heterogeneity and significantly increase main variant levels and decrease acidic variant levels.

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants

“…Control strategies can be established based on the relationship of these parameters with charge variants. Overall, longer duration [ 136 , 137 , 138 ], higher temperature [ 137 , 138 , 139 , 140 , 141 , 142 ], and exposure to light [ 143 ] increased acidic species. N-terminal Gln cyclization mainly occurs during the cell culture stage and is driven by pH, temperature, and duration [ 144 ].…”

“…Typically, C-terminal Lys increases with cell culture duration, while C-terminal amidation decreases with the cell culture duration due to the dynamic changes between the amount of mAb products and the respective specific enzymes [ 115 , 145 ]. Lower temperature results in increased levels of basic species caused by incomplete removal of C-terminal Lys due to decreased carboxypeptidase activity [ 140 , 141 , 146 ]. In one study, trisulfide levels were low on days 12–13, but higher on earlier and later days and varied significantly with bioreactor operating parameters [ 97 ].…”

“…An aged medium that has been stored for 10 weeks caused increased acidic species [ 137 ]. The addition of hydrolysate caused decreased levels of acidic species and increased levels of basic species compared to controls operated at the same temperatures [ 141 ].…”