Modulating cell culture oxidative stress reduces protein glycation and acidic charge variant formation

Abstract

Controlling acidic charge variants is critical for an industrial bioprocess due to the potential impact on therapeutic efficacy and safety. Achieving a consistent charge variant profile at manufacturing scale remains challenging and may require substantial resources to investigate effective control strategies. This is partially due to incomplete understanding of the underlying causes for charge variant formation during the cell culture process. To address this gap, we examined the effects of four process input factors (temperature, iron concentration, feed media age, and antioxidant (rosmarinic acid) concentration) on charge variant profile. These factors were found to affect the charge profile by modulating the cell culture oxidative state. Process conditions with higher acidic peaks corresponded to elevated supernatant peroxide concentration, intracellular reactive oxygen species (ROS) levels, or both. Changes in glycation level were the primary cause of the charge heterogeneity, and for the first time, supernatant peroxide was found to positively correlate with glycation levels. Based on these findings, a novel mathematical model was developed to demonstrate that the rate of acidic species formation was exponentially proportional to the concentrations of supernatant peroxide and protein product. This work provides critical insights into charge variant formation during the cell culture process and highlights the importance of modulating of cell culture oxidative stress for charge variant control.

Antibody Structure and Function: The Basis for Engineering Therapeutics

“…Upstream consideration of developability metrics should reduce the frequency of failures in later downstream development stages. Optimization of stability in the early stages of discovery can reduce complications in upstream and downstream process optimization as well as increase the potential for successful drug product formulations [218,219]. Nonetheless, downstream processing can minimize some levels of oxidation via the presence of free radical scavengers, elimination of redox metal ions, addition of chelation agents, protection from light, decrease in storage temperature, and reduction of exposure to oxygen.…”

Productivity improvement and charge variant modulation for intensified cell culture processes by adding a carboxypeptidase B (CpB) treatment step

“…From the regulatory point of view, higher basic species derived from C‐terminal lysine are of minimal concern in terms of safety and efficacy because this lysine will be rapidly cleaved in vivo by endogenous serum CpB after intravenous injection to patients (Brorson & Jia, 2014). Nevertheless, charge variants still need to be monitored to ensure manufacturing consistency and product comparability (Chung et al, 2019). The percentage of proteins that comprise the main peak of this charge variants profile is commonly specified in the release specification for most mAb products.…”

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line

“…In addition, it was reported that iron forms ROS that tend to increase acidic mAb species. [17][18][19][20][21][22] Consequently, our data indicate that it is advisable to identify the source of iron variation in soy protein hydrolysates. Two main sources can be envisioned: the soy grit itself or the manufacturing process.…”

“…The importance of iron content in cell culture media and its impacts on cell growth, mAb production and acidic charge variants was described in several publications. [15][16][17][18][19][20][21][22] Cellular iron deficiency can be associated with a lower cell growth, while higher level of iron has shown to be potentially cytotoxic due to Fenton reaction. 15,16 Impact on cell growth due to transitional metals variations, here e.g., iron, in cell culture media is a mechanism described by Dixon et al in 2012 as ferroptosis.…”