Mammalian cell-produced therapeutic proteins: heterogeneity derived from protein degradation

Dorai, Haimanti; Ganguly, Subinay

doi:10.1016/j.copbio.2014.07.007

Cited by 34 publications

(24 citation statements)

References 60 publications

(50 reference statements)

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“…Generally, lysine residues at the heavy chain C-terminus are removed in recombinant IgGs during cell culture process (Harris 1995), while incomplete cleavage of the lysine residues leads to the presence of lysine variants and further results in charge heterogeneity (Dorai and Ganguly 2014). Although the modification of C-terminal lysine has no substantial influence on antibody structure, stability, and functions (Ponniah et al 2014), the characterization of lysine variant level is essential to ensure the consistency of product quality.…”

Section: Introductionmentioning

confidence: 99%

Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures

Zhang

Tang

Sun

et al. 2015

Appl Microbiol Biotechnol

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C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.

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Section: Introductionmentioning

confidence: 99%

Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures

Zhang

Tang

Sun

et al. 2015

Appl Microbiol Biotechnol

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“…For example, chaperones may interact with unfolded and partially folded recombinant proteins to form large aggregates, which cause fouling of chromatography columns and exchanger filters (Lintern et al, ). Proteases and glycosidases may cause mAb fragmentation and clipping in serum‐free media, hence, reduce the overall product yield (Dorai & Ganguly, ; Gao et al, ). Therefore, efficient HCP removal is crucial to (i) ensure patient safety; (ii) reduce DSP cost; and (iii) maintain product integrity.…”

Section: Introductionmentioning

confidence: 99%

Cascading effect in bioprocessing—The impact of mild hypothermia on CHO cell behavior and host cell protein composition

Goey

Tsang

Bell

et al. 2017

Biotech & Bioengineering

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A major challenge in downstream purification of monoclonal antibodies (mAb) is the removal of host cell proteins (HCPs). Previous studies have shown that cell culture conditions significantly impact the HCP content at harvest. However, it is currently unclear how process conditions affect physiological changes in the host cell population, and how these changes, in turn, cascade down to change the HCP profile. We examined how temperature downshift (TDS) to mild hypothermia affects key upstream performance indicators, that is antibody titre, HCP concentration and HCP species, across the cell culture decline phase and at harvest through the lens of changes in cellular behavior. Mild hypothermic conditions introduced on day 5 of fed-batch Chinese hamster ovary (CHO) cell bioreactors resulted in a lower cell proliferation rate but larger percentages of healthier cells across the cell culture decline phase compared to bioreactors maintained at standard physiological temperature. Moreover, the onset of apoptosis was less evident in mild hypothermic cultures. Consequently, mild hypothermic cultures took an extra 5 days to reach an integral viable cell concentration (IVCC) and antibody yield similar to that of the control at standard physiological temperature. When cell viability dropped below 80%, mild hypothermic cell cultures had a reduced variety of HCP species by 36%, including approximately 44% and 27% lower proteases and chaperones, respectively, despite having similar HCP concentration. This study suggests that TDS may be a good strategy to provide cleaner downstream feedstocks by reducing the variety of HCPs and to maintain product integrity by reducing the number of proteases and chaperones.

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“…Cell death reduces product yield in cell culture (Arden & Betenbaugh, 2004), and can negatively affect product quality (Dorai & Ganguly, 2014). Cell death results in increased release of proteases, glycosylases, and sialidases, which degrade proteins and alter protein glycans structure, especially at the later stages of batch culture (Arden & Betenbaugh, 2004; Birch & Racher, 2006; Curling et al, 1990; Gramer & Goochee, 1993; Shuler & Kargi, 2002; Weikert et al, 1999; Wong, Wong, Nissom, Heng, & Yap, 2006).…”

Section: Introductionmentioning

confidence: 99%

Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production

Henry

MacDonald

Orellana

et al. 2020

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells are the predominant host cell line for the production of biopharmaceuticals, a growing industry currently worth more than $188 billion USD in global sales. CHO cells undergo programmed cell death (apoptosis) following different stresses encountered in cell culture, such as substrate limitation, accumulation of toxic by‐products, and mechanical shear, hindering production. Genetic engineering strategies to reduce apoptosis in CHO cells have been investigated with mixed results. In this review, a contemporary understanding of the real complexity of apoptotic mechanisms and signaling pathways is described; followed by an overview of antiapoptotic cell line engineering strategies tested so far in CHO cells.

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Mammalian cell-produced therapeutic proteins: heterogeneity derived from protein degradation

Cited by 34 publications

References 60 publications

Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures

Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures

Cascading effect in bioprocessing—The impact of mild hypothermia on CHO cell behavior and host cell protein composition

Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production

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