A control strategy to investigate the relationship between specific productivity and high-mannose glycoforms in CHO cells

Zalai, Dénes; Hevér, Helga; Lovász, Krisztina; Molnár, Dóra; Wechselberger, Patrick; Hofer, Alexandra; Párta, László; Putics, Ákos; Herwig, Christoph

doi:10.1007/s00253-016-7380-4

Cited by 13 publications

(9 citation statements)

References 44 publications

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“…Since these experiments were performed in rich medium within a short time frame, these changes most likely were not caused by media depletion, but rather by bottlenecks within the cellular protein synthesis and processing machinery in the ER and Golgi, or possibly by limited precursor availability within the cells. These results correspond well to changes in glycan quality observed in high producers of mAbs that had been shifted to higher productivity . The elucidation of the relationship of qP and N‐glycan processing in these studies was limited by the range of qP achievable through bioprocess‐modification alone.…”

Section: Discussionsupporting

confidence: 73%

“…This multifactorial process is readily influenced by both subclone‐specific phenotypes and bioprocess conditions, posing a substantial challenge to establishing suitable clones and maintaining a desirable glycan profile during production . With the boundaries of productivity being pushed further and further, several reports have demonstrated significant shifts toward reduced N‐glycan maturation in mAb‐production processes steered toward higher specific productivity (qP) by reduction in temperature, or alteration of feeding profile …”

Section: Introductionmentioning

confidence: 99%

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mRNA Transfection into CHO‐Cells Reveals Production Bottlenecks

Coats

Bydlinski

Maresch

et al. 2019

Biotechnology Journal

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Obtaining highly productive Chinese hamster ovary (CHO)-cell clones for the production of therapeutic proteins relies on multiple time-consuming selection steps. Several CHO-cell strains with high degrees of genomic and epigenetic variation are available. Each harbor potential advantages and disadvantages for any given product, particularly those considered difficult to express. A simple test system to quickly assess compatibility of cell line and product may therefore prove useful. Transient plasmid transfection falls short of the specific productivities of stable producer cells, making it unsuitable for the elucidation of high specific productivity bottlenecks. The aim of the study is to reach specific productivities approaching those of industrial production cell lines by transfection of in vitro transcribed mRNA. The system is characterized with respect to transfection efficacy (by quantitative PCR) and protein production (by flow cytometry and biolayer interferometry). Fluorescence of intracellular eGFP saturates at higher amounts of mRNA per cell, while the amount of secreted and intracellular EPO-Fc remain linearly correlated to the amount of mRNA taken up. Nevertheless, MS shows a severe reduction in N-glycosylation quality. This method allows for rapid elucidation of bottlenecks that would otherwise remain undetected until later during cell line development, giving insight into suitable strategies for preemptive targeted metabolic engineering and host cell line optimization.The ORCID identification number(s) for the author(s) of this article can be found under https://doi.

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Section: Discussionsupporting

confidence: 73%

Section: Introductionmentioning

confidence: 99%

mRNA Transfection into CHO‐Cells Reveals Production Bottlenecks

Coats

Bydlinski

Maresch

et al. 2019

Biotechnology Journal

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“…The increased mannose in the SDM could be related to the higher specific productivity in the ambr® 250 batches for mAb2, as demonstrated by the higher titer in the ambr® 250 in combination with the lower VCD shown previously. Work by Zalai et al demonstrated the association of high specific productivity and high mannose formation. Additionally, Pacis et al linked increases in high mannose to increasing culture osmolality, similar to the effect seen in the mAb2 process, which showed an increase in mannose in the SDM and higher osmolality.…”

Section: Resultsmentioning

confidence: 99%

Scale‐down model qualification of ambr® 250 high‐throughput mini‐bioreactor system for two commercial‐scale mAb processes

Manahan

Nelson

Cacciatore

et al. 2019

Biotechnology Progress

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Recent advances in high‐throughput (HTP) automated mini‐bioreactor systems have significantly improved development timelines for early‐stage biologic programs. Automated platforms such as the ambr® 250 have demonstrated the ability, using appropriate scale‐down approaches, to provide reliable estimates of process performance and product quality from bench to pilot scale, but data sets comparing to large‐scale commercial processes (>10,000 L) are limited. As development moves toward late stages, specifically process characterization (PC), a qualified scale‐down model (SDM) of the commercial process is a regulatory requirement as part of Biologics License Application (BLA)‐enabling activities. This work demonstrates the qualification of the ambr® 250 as a representative SDM for two monoclonal antibody (mAb) commercial processes at scales >10,000 L. Representative process performance and product quality associated with each mAb were achieved using appropriate scale‐down approaches, and special attention was paid to pCO2 to ensure consistent performance and product quality. Principal component analysis (PCA) and univariate equivalence testing were utilized in the qualification of the SDM, along with a statistical evaluation of process performance and product‐quality attributes for comparability. The ambr® 250 can predict these two commercial‐scale processes (at center‐point condition) for cell‐culture performance and product quality. The time savings and resource advantages to performing PC studies in a small‐scale HTP system improves the potential for the biopharmaceutical industry to get products to patients more quickly.

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“…6,7 Multiple cell culture parameters have been implicated to modulate the high-mannose glycan content. Low pH (6.9), 8 low glutamine (<0.1 mM) or glucose (<0.70 mM), 9 elevated manganese concentrations, 10 osmolality over 400 mOsm/kg, 11 higher specific productivity, 12 and advancing cell age 13 have been linked to increased high-mannose glycans. In addition, using mannose as the major carbon source for cells resulted in 26% more high-mannose species, 14 and gene knockout of the glycoenzyme mannosidase I resulted in highmannose glycans as the most abundant species.…”

Section: Introductionmentioning

confidence: 99%

A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

Brantley

Mitchelson

Khattak

2020

Biotechnology Progress

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N-linked glycosylation of therapeutic monoclonal antibodies is an important product quality attribute for drug safety and efficacy. An increase in the percent of high mannose N-linked glycosylation may be required for drug efficacy or to match the glycosylation profile of the innovator drug during the development of a biosimilar. In this study, the addition of several chemical additives to a cell culture process resulted in high mannose N-glycans on monoclonal antibodies produced by Chinese hamster ovary (CHO) cells without impacting cell culture performance. The additives, which include known mannosidase inhibitors (kifunensine and deoxymannojirimycin) as well as novel inhibitors (tris, bis-tris, and 1-amino-1-methyl-1,3-propanediol), contain one similar molecular structure: 2-amino-1,3-propanediol, commonly referred to as serinol. The shared chemical structure provides insight into the binding and inhibition of mannosidase in CHO cells. One of the novel inhibitors, tris, is safer compared to kifunensine, 35x as cost-effective, and stable at room temperature. In addition, tris and bis-tris provide multiple low-cost alternatives to kifunensine for manipulating glycosylation in monoclonal antibody production in a cell culture process with minimal impact to productivity or cell health.

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A control strategy to investigate the relationship between specific productivity and high-mannose glycoforms in CHO cells

Cited by 13 publications

References 44 publications

mRNA Transfection into CHO‐Cells Reveals Production Bottlenecks

mRNA Transfection into CHO‐Cells Reveals Production Bottlenecks

Scale‐down model qualification of ambr® 250 high‐throughput mini‐bioreactor system for two commercial‐scale mAb processes

A class of low‐cost alternatives to kifunensine for increasing high mannose N‐linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells

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