Effect of copper variation in yeast hydrolysate on C‐terminal lysine levels of a monoclonal antibody

Abstract

The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This article addresses the control of C-terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large-scale production of a murine cell line exhibited variation in the C-terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture. Small-scale studies demonstrated sensitivity of the cells to copper, where a single low dose of copper to the culture impacted cell lactate metabolism and C-terminal lysine processing. Subsequent analytical tests indicated that the yeast hydrolysate powder, added to the basal media and nutrient feed in the process, contained varying levels of trace copper across lots. The measured copper concentrations in yeast hydrolysate lots correlated well with the variation in lactate and pH trends and C-terminal lysine levels of the batches in manufacturing. Small-scale studies further demonstrated that copper supplementation to yeast hydrolysate lots with low concentrations of copper can shift the metabolic performance and C-terminal lysine levels of these cultures to match the control, high copper cultures. Hence, a strategy of monitoring, and if necessary supplementing, copper in yeast-hydrolysate powders resulted in the ability to control and ensure product quality consistency. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:463-468, 2017.

Online control of cell culture redox potential prevents antibody interchain disulfide bond reduction

“…In the previously published work where increased Cu in the bioreactor lead to increased C-terminal lysine, increased lactate consumption was also observed (Mitchelson et al, 2017). In our case, we did not observe a significant change in lactate consumption across conditions (Figure 2c) despite very different amounts of Cu added to the bioreactor (Figure 3d).…”

“…Increased Cu in the bioreactor has been previously demonstrated to increase the amount of C‐terminal lysine present on a mAb (Mitchelson, Mondia, & Hughes, 2017). We thus quantified the percent C‐terminal lysine.…”

Elemental metal variance in cell culture raw materials for process risk profiling

“…As shown in Figure 4, increasing copper concentrations gave an increasing titer trend with decreased lactate production. Although not shown here, increased copper concentrations can also lead to increased oxidative culture stress and, through the action of the copper essential cofactor peptidylglycine α‐amidating mono‐oxygenase, an increase in basic protein isoforms 9,14 . To mitigate the risk of these two negative effects and to saturate the impact of potential impurity shifts, intermediate basal and feed copper concentrations were chosen for future development work.…”

“…Raw material variability in cell culture media has been shown to cause an abundance of problems which can affect a wide range of cell culture characteristics including cell growth, product quality, and drug efficacy. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] There are multiple sources for raw material nonuniformity including material misformulation, impurities and contaminants, and chemical degradation. 5 The most common impurities are trace elements from numerous raw material sources.…”

Investigating trace metal precipitation in highly concentrated cell culture media with Pourbaix diagrams

“…Today, there exists an entire industry centered around the formulation of cell culture media to suit specific cell lines (Jayme et al, 1997) and medium development is a major topic of study under the cell culture umbrella (Ritacco et al, 2018) as it has been demonstrated that improvements in medium formulation (and the subsequent optimization of the process feed strategy) can result in higher productivity and lower cost of goods in protein manufacturing processes (Hakkinen et al, 2018; Y. M. Huang et al, 2010; Xu et al, 2017). A prominent topic in the development of media is the availability of trace metals, such as copper, iron, and manganese, which are critical to maintain product titer and quality (Ehret et al, 2019; Mitchelson et al, 2017; Vijayasankaran et al, 2013).…”