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

Abstract

The goal of cell culture process intensification is to improve productivity while maintaining acceptable quality attributes. In this report, four processes, namely a conventional manufacturing Process A, and processes intensified by enriched N‐1 seed (Process B), by perfusion N‐1 seed (Process C), and by perfusion production (Process D) were developed for the production of a monoclonal antibody. The three intensified processes substantially improved productivity, however, the product either failed to meet the specification for charge variant species (main peak) for Process D or the production process required early harvest to meet the specification for charge variant species, Day 10 or Day 6 for Processes B and C, respectively. The lower main peak for the intensified processes was due to higher basic species resulting from higher C‐terminal lysine. To resolve this product quality issue, we developed an enzyme treatment method by introducing carboxypeptidase B (CpB) to clip the C‐terminal lysine, leading to significantly increased main peak and an acceptable and more homogenous product quality for all the intensified processes. Additionally, Processes B and C with CpB treatment extended bioreactor durations to Day 14 increasing titer by 38% and 108%, respectively. This simple yet effective enzyme treatment strategy could be applicable to other processes that have similar product quality issues.

Antibody charge variant modulation by in vitro enzymatic treatment in different Chinese hamster ovary cell cultures

“…In some cases, cell cultures would be terminated earlier to meet quality requirements, resulting in a loss of titer 9 . As discussed in detail in our previous study, 9 this in vitro CpB treatment protocol represents a simple and cost‐effective strategy to improve main peak without impacting other critical quality attributes. Alternative approaches depend on generating new cell lines or cell culture processes.…”

“…Therefore, it is safe to use an in vitro enzymatic treatment in the biopharmaceutic manufacturing. However, only one mAb product produced from a single 5‐L bioreactor was tested in this previous report (n = 1) 9 …”

“…[2][3][4][5] From the 1980s to the 2010s, manufacturing titers improved from tens of mg/L to ≥3 g/L, [6][7][8] while higher titers have often been reported in literature within the last 10 years (e.g., around 10 g/L titer or above in 10-18 days). [9][10][11][12][13] Although high titers can be achieved using platform fed-batch cell culture processes, critical product quality attributes, including charge variant species, size exclusion chromatography (SEC) impurities, and N-glycans, remain challenging to control and maintain throughout process development and the commercial biomanufacturing life cycle management. [14][15][16][17][18] Charge variants are modified mAb species with different net charge properties.…”

“…Although in vitro enzymatic treatments have been widely used in the food, environmental, and pharmaceutical industries, such as synthesis of small molecule drugs, [25][26][27] the biopharmaceutical industry has rarely adopted in vitro enzymatic treatments, for example, to increase titer or product quality control in cell culture process development. In a previous study, 9 we developed an in vitro treatment wherein CpB at a CpB:mAb ratio of only 1:10,000 (w/w) was added to the whole cell culture immediately prior to harvest for 2 h, which achieved the same enzymatic efficiency for C-terminal lysine removal at a much higher CpB:mAb ratio of 1:100 (w/w) reported by others. 28,29 The in vitro enzymatic treatment resulted in improved homogeneity of product quality with respect to the charge variant profile, thereby improving manufacturing consistency, and subsequently significantly improved cell culture titers by prolonging cell culture duration.…”

A novel twin‐column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants

“…The mAb mass weight is around 148 kDa and the pI is about 8.7. The CPB treatment was used to remove the C-terminal Lys [7,[31][32]. 15 μL CPB were added into 1 mL mAb sample with 1 g/L and kept for 30 min under 37°C.…”