Glucocorticoid receptor‐mediated reduction of IgG‐fusion protein aggregation in Chinese hamster ovary cells

This study reports the effects of varying concentrations of copper sulfate on the metabolic and gene transcriptional profile of a recombinant Chinese hamster ovary (CHO) cell line producing an immunoglobulin G (IgG)-fusion protein (B0). Addition of 50 μM copper sulfate significantly decreased lactate accumulation in the cultures while increasing viable cell density and protein titer. These changes could be seen from day 6 and became increasingly evident with culture duration. Reducing the copper sulfate concentration to 5 μM retained all the above beneficial effects, but with the added benefit of reduced levels of the aggregated form of the B0 protein. To profile the cellular changes due to copper sulfate addition at the transcriptional level, Affymetrix® CHO microarrays were used to identify differentially expressed genes related to reduced cellular stresses and facilitated cell cycling. Based on the microarray results, down-regulation of the transferrin receptor and lactate dehydrogenase, and up-regulation of a cytochrome P450 family-2 polypeptide were then confirmed by Western blotting. These results showed that copper played a critical role in cell metabolism and productivity on recombinant CHO cells and highlighted the usefulness of microarray data for better understanding biological responses on medium modification.

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“…The proportions of monomer and high molecular weight species were quantified using HPLC‐Size Exclusion Chromatography as described previously. 6…”

Section: Methodsmentioning

confidence: 99%

Cell culture and gene transcription effects of copper sulfate on Chinese hamster ovary cells

Qian

Khattak

Xing

et al. 2011

Biotechnology Progress

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“…Furthermore, DEX has also been shown to rescue cytotoxicity and apoptosis induced in transfected HC92 rat cardiomyocytes (Kim et al, 2009), and reduced death of chinese hamster ovary (CHO) cells producing recombinant protein by modulating apoptosis-related gene expression (Jing et al, 2012). | 439 activity by modulating posttranslational events like folding or activesite stabilization, as Gc has been shown to improve CHO recombinant protein production by reducing protein aggregation through upregulation of genes that modulate redox conditions (Qian, Jing, & Li, 2010). | 439 activity by modulating posttranslational events like folding or activesite stabilization, as Gc has been shown to improve CHO recombinant protein production by reducing protein aggregation through upregulation of genes that modulate redox conditions (Qian, Jing, & Li, 2010).…”

Section: Dex Ameliorates Hmsc Protein Synthesis Inhibition Induced mentioning

confidence: 99%

Mechanisms of unprimed and dexamethasone‐primed nonviral gene delivery to human mesenchymal stem cells

Hamann

Broad

Nguyen

et al. 2018

Biotech & Bioengineering

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Human mesenchymal stem cells (hMSCs) are under intense study for applications of cell and gene therapeutics because of their unique immunomodulatory and regenerative properties. Safe and efficient genetic modification of hMSCs could increase their clinical potential by allowing functional expression of therapeutic transgenes or control over behavior and differentiation. Viral gene delivery is efficient, but suffers from safety issues, while nonviral methods are safe, but highly inefficient, especially in hMSCs. Our lab previously demonstrated that priming cells before delivery of DNA complexes with dexamethasone (DEX), an anti‐inflammatory glucocorticoid drug, significantly increases hMSC transfection success. This work systematically investigates the mechanisms of hMSC transfection and DEX‐mediated enhancement of transfection. Our results show that hMSC transfection and its enhancement by DEX are decreased by inhibiting classical intracellular transport and nuclear import pathways, but DEX transfection priming does not increase cellular or nuclear internalization of plasmid DNA (pDNA). We also show that hMSC transgene expression is largely affected by pDNA promoter and enhancer sequence changes, but DEX‐mediated enhancement of transfection is unaffected by any pDNA sequence changes. Furthermore, DEX‐mediated transfection enhancement is not the result of increased transgene messenger RNA transcription or stability. However, DEX‐priming increases total protein synthesis by preventing hMSC apoptosis induced by transfection, resulting in increased translation of transgenic protein. DEX may also promote further enhancement of transgenic reporter enzyme activity by other downstream mechanisms. Mechanistic studies of nonviral gene delivery will inform future rationally designed technologies for safe and efficient genetic modification of clinically relevant cell types.

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“…While this approach may be feasible with mAb processes, the high aggregate levels often encountered in expression of scFv‐IgGs present process robustness concerns, particularly when relying on a single column for the majority of aggregate clearance. Cell‐line engineering and cell culture condition optimization to reduce aggregate levels can be considered as potential solutions . However, the impact of the changes on titer and other impurity levels needs to be considered.…”

Section: Introductionmentioning

confidence: 99%

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform‐Like Purification Process

Andrade¹,

Arnold

Motabar³

et al. 2018

Biotechnology Progress

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Single chain variable fragment‐IgGs (scFv‐IgG) are a class of bispecific antibodies consisting of two single chain variable fragments (scFv) that are fused to an intact IgG molecule. A common trend observed for expression of scFv‐IgGs in mammalian cell culture is a higher level of aggregates (10%–30%) compared to mAbs, which results in lower purification yields in order to meet product quality targets. Furthermore, the high aggregate levels also pose robustness risks to a conventional mAb three column platform purification process which uses only the polishing steps (e.g., cation exchange chromatography [CEX]) for aggregate removal. Protein A chromatography with pH gradient elution, high performance tangential flow filtration (HP‐TFF) and calcium phosphate precipitation were evaluated at the bench scale as means of introducing orthogonal aggregate removal capabilities into other aspects of the purification process. The two most promising process variants, namely Protein A pH gradient elution followed by calcium phosphate precipitation were evaluated at pilot scale, demonstrating comparable performance. Implementing Protein A chromatography with gradient elution and/or calcium phosphate precipitation removed a sufficient portion of the aggregate burden prior to the CEX polishing step, enabling CEX to be operated robustly under conditions favoring higher monomer yield. From starting aggregate levels ranging from 15% to 23% in the condition media, levels were reduced to between 2% and 3% at the end of the CEX step. The overall yield for the optimal process was 71%. Results of this work suggest an improved three‐column mAb platform‐like purification process for purification of high aggregate scFv‐IgG bispecific antibodies is feasible. © 2018 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers. Biotechnol. Prog ., 35: e2720, 2019

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Glucocorticoid receptor‐mediated reduction of IgG‐fusion protein aggregation in Chinese hamster ovary cells

Cited by 14 publications

References 28 publications

Cell culture and gene transcription effects of copper sulfate on Chinese hamster ovary cells

Cell culture and gene transcription effects of copper sulfate on Chinese hamster ovary cells

Mechanisms of unprimed and dexamethasone‐primed nonviral gene delivery to human mesenchymal stem cells

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform‐Like Purification Process

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