Evaluation of heavy chain C‐terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells

Hu, Zhilan; Tang, Danming; Misaghi, Shahram; Jiang, Guoying; Yu, Christopher; Yim, Mandy; Shaw, David; Snedecor, Brad; Laird, Michael W.; Shen, Amy

doi:10.1002/btpr.2444

Cited by 17 publications

(15 citation statements)

References 18 publications

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“…In addition to the CpB treatment method, other mitigation strategies that affect C‐terminal lysine levels, for example, cell line engineering and cell culture medium and process optimization, have been reported in literature. Hu et al (2017) reported that the CHO cells with C‐terminal lysine deletion in the cDNA sequence indeed produced less C‐terminal lysine‐related basic variants, but resulted in a lower titer and higher total basic species due to a more prevalent proline amidation than the wild type CHO cells. Furthermore, it was found that the CHO cells with C‐terminal lysine and glycine deletion in the cDNA sequence produced lower basic species and comparable titers to the wide type CHO cells.…”

Section: Discussionmentioning

confidence: 99%

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

Zheng

Dawood

et al. 2021

Biotech & Bioengineering

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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.

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Section: Discussionmentioning

confidence: 99%

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

Zheng

Dawood

et al. 2021

Biotech & Bioengineering

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“…C-terminal Lys does not impact mAb structure, stability, or biological functions including PK [2,9,13,180,181,182], though, one study demonstrated that the removal of C-terminal Lys is required for optimal CDC [183]. Interestingly, inclusion of the C-terminal Lys codon may impact mAb titer of cell culture [184]. C-terminal Lys can be rapidly removed from mAbs during circulation with a half-life of 62 minutes [185].…”

Section: C-terminal Modificationsmentioning

confidence: 99%

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

Beck

Liu²

2019

Antibodies

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Recombinant monoclonal antibodies (mAbs) intended for therapeutic usage are required to be thoroughly characterized, which has promoted an extensive effort towards the understanding of the structures and heterogeneity of this major class of molecules. Batch consistency and comparability are highly relevant to the successful pharmaceutical development of mAbs and related products. Small structural modifications that contribute to molecule variants (or proteoforms) differing in size, charge or hydrophobicity have been identified. These modifications may impact (or not) the stability, pharmacokinetics, and efficacy of mAbs. The presence of the same type of modifications as found in endogenous immunoglobulin G (IgG) can substantially lower the safety risks of mAbs. The knowledge of modifications is also critical to the ranking of critical quality attributes (CQAs) of the drug and define the Quality Target Product Profile (QTPP). This review provides a summary of the current understanding of post-translational and physico-chemical modifications identified in recombinant mAbs and endogenous IgGs at physiological conditions.

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“…Cell line development has also been particularly effective at controlling C‐terminal Lys. Investigations into the carboxypeptidase activity, the enzyme that cleaves the C‐terminal Lys, allows for targeted modulation by cell line engineering (Dick, Qiu, Mahon, Adamo, & Cheng, ; Hu et al, ), and deletion of C‐terminal amino residues has also successfully controlled the basic charge variants (Hu et al, ; Jiang et al, ). While cell line development may effectively control protein modifications caused by endogenous mechanisms, many charge variant modifications occur due to extracellular interactions in a molecule‐dependent manner that may be difficult to predict and control.…”

Section: Upstream Process Strategies For Charge Variants Controlmentioning

confidence: 99%

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

Chung

Tian

Tan

et al. 2018

Biotech & Bioengineering

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Controlling the charge profile of therapeutic protein is a critical challenge in the current quality-by-design (QbD) paradigm, throughout all phases of biologics process development (PD): cell line development, upstream cell culture, recovery process, downstream purification, and analytical characterization. Charge variant profiles may influence efficacy and/or lead to unintended side-effects. Thus, maintaining a consistent charge profile is of tremendous importance, and increasingly, researchers have focused efforts toward developing strategies to mitigate variability during cell culture and to improve separation and detection of charge variants. Current understanding of factors affecting charge variant formation during manufacturing remains inadequate, and sometimes, even substantial commitment of resources may still not fully achieve the desired or consistent profiles. As such, this review attempts to provide a comprehensive resource for the biologics community by summarizing the impact of charge variants and CQA management, analytical methods for charge variant detection, as well as strategies in downstream and upstream PD for controlling charge variant profiles.

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Evaluation of heavy chain C‐terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells

Cited by 17 publications

References 18 publications

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

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

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

Industrial bioprocessing perspectives on managing therapeutic protein charge variant profiles

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