Visualisation of intracellular production bottlenecks in suspension-adapted CHO cells producing complex biopharmaceuticals using fluorescence microscopy

Mathias, Sven; Fischer, Simon; Handrick, René; Fieder, Jürgen; Schulz, Patrick; Bradl, Harald; Gorr, Ingo H.; Gamer, Martin; Otte, Kerstin

doi:10.1016/j.jbiotec.2018.02.009

Cited by 29 publications

(18 citation statements)

References 26 publications

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“…This revealed a positive correlation with the titer, similar to that seen at the population level with qRT‐PCR and western blotting assays (Figure E). IFC images revealed differential cytoplasmic localization of the HC polypeptide and HC mRNA, consistent with the previous findings of HC polypeptide residing in the endoplasmic reticulum (ER) and Golgi, and mRNA localized with ribosomes on the ER surface (Figure F). As both the HC mRNA and polypeptide show a positive correlation with the final titer early in culture and with each other, HC polypeptide was selected as the marker of productivity at the single‐cell level as the assay is more time‐ and cost‐efficient than that for mRNA.…”

Section: Resultssupporting

confidence: 90%

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

Pekle

Smith²,

Rosignoli³

et al. 2019

Biotechnology Journal

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Biopharmaceutical manufacturing using Chinese hamster ovary (CHO) cells requires the generation of high‐producing clonal cell lines. During cell line development, cell cloning using fluorescence‐activated cell sorting (FACS) has the potential to combine isolation of single cells with sorting based on specific cellular attributes that correlate with productivity and/or growth, identifying cell lines with desirable phenotypes for manufacturing. This study describes the application of imaging flow cytometry (IFC) to characterize recombinant cell lines at the single‐cell level to identify cell attributes predictive of productivity. IFC assays are developed to quantify the organelle content and recombinant heavy‐chain (HC) and light‐chain (LC) polypeptide as well as messenger RNA (mRNA) amounts in single cells. The assays are then validated against orthogonal standard flow cytometry, western blot, and quantitative reverse transcription polymerase chain reaction (qRT‐PCR) methods. The authors describe how these IFC assays may be used in cell line development and show how cellular properties can be correlated with productivity at the single‐cell level, allowing the isolation of such cells during the cloning process. From the analysis, HC polypeptide and mRNA are found to be predictive of productivity early in the culture; however, specific organelle content did not show any correlation with productivity.

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Section: Resultssupporting

confidence: 90%

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

Pekle

Smith²,

Rosignoli³

et al. 2019

Biotechnology Journal

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“…The HMW increase could be induced by 60% higher LC1 and HC1 mRNA levels that potentially led to saturating endoplasmic reticulum (ER) folding capacity and increased aggregation as described in other reports (Gomez et al, ; Schroder, Schafer, & Friedl, ; Zhou, Raju, Alves, & Gilbert, ). A recent study pointed to the ER as a bottleneck in particular for the folding and secretion of B‐Abs (Mathias et al, ).…”

Section: Resultsmentioning

confidence: 99%

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody

Gómez

Wieczorek

et al. 2018

Biotech & Bioengineering

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Therapeutic bispecific antibodies are formed by assembly of multichain polypeptides. In general, a bispecific antibody has two different light chains and two different heavy chains that fold and correctly pair via engineered interchain interactions. Because of some incorrect assembly, product-related impurities can be prevalent (e.g., half molecules, mispaired light chains, homodimers), requiring its removal during subsequent purification. In this study, we investigated the modulation of impurity levels in a stable Chinese hamster ovary cell line X expressing a bispecific antibody A formed by two light chains (LC1 and LC2) and two heavy chains (HC1 and HC2) that assembled intracellularly into a heterodimer (LC1-HC1 + LC2-HC2) via engineered charged residues. Cell line X exhibited the best volumetric productivity, growth, and viability in culture compared with other clones but also showed higher levels of half antibody species (>10%); therefore, to minimize process yield loss, better understanding, and control of impurity formation was pursued. We found this cell line decreased half antibody levels from 16% to 1% when temperature changed from 36°C to 32.5°C or 31.5°C. However, lower temperature also increased high-molecular-weight (HMW) species from 4% to 12%. To determine the impurity species composition, we characterized enriched fractions with half antibody or HMW. Intact mass spectrometry analysis revealed half antibody was LC2-HC2, whereas HMW was a mixture with ~50% as LC1-HC1 homodimer. Results suggested LC2-HC2 was easily folded and could be secreted as half antibody, especially at 36°C. On the contrary, LC1-HC1 was more susceptible to misfold or aggregate, a phenomenon more acute for cell line X at lower culture temperature because of 60% increased LC1 and HC1 messenger RNA levels. Although temperature modulation was cell line X-specific, the propensity of LC2-HC2 to form half antibodies and LC1-HC1 to aggregate appeared in other cell lines also expressing bispecific antibody A, suggesting an amino-acid sequence-dependent mechanism. In summary, impurity formation in cell line X was temperature-dependent and was influenced by different molecule characteristics between the LC1-HC1 and LC2-HC2 parts. Ultimately, we selected a biphasic cell culture process with a growth phase followed by a lower temperature phase to improve product quality and purification yield.

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“…In light of the challenges encountered in trying to control all aspects of large scale bioreactor processes, the approaches consisting in making the cells and the culture process more resistant to potential sources of oxidative stress have to be considered. Although, increased energy metabolism and a consequent activity of the folding/secretion machinery have been observed or modeled in high producer cell lines, it remains possible to limit ROS byproducts of these cellular processes (Borth et al, ; Ghorbaniaghdam, Chen, Henry, & Jolicoeur, ; Mathias et al, ; Prashad & Mehra, ; Templeton et al, ). Two of the main strategies considered to date consist in (a) the supplementation of the cell culture media with antioxidant molecules, and (b) the engineering of metabolic pathways associated with oxidative stress.…”

Section: Discussionmentioning

confidence: 99%

Oxidative stress‐alleviating strategies to improve recombinant protein production in CHO cells

Chevallier

Andersen

Malphettes

2019

Biotech & Bioengineering

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Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high‐level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.

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Visualisation of intracellular production bottlenecks in suspension-adapted CHO cells producing complex biopharmaceuticals using fluorescence microscopy

Cited by 29 publications

References 26 publications

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

Culture temperature modulates half antibody and aggregate formation in a Chinese hamster ovary cell line expressing a bispecific antibody

Oxidative stress‐alleviating strategies to improve recombinant protein production in CHO cells

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