A platform for context-specific genetic engineering of recombinant protein production by CHO cells

Cartwright, Joseph F.; Arnall, Claire L.; Patel, Yash D.; Barber, Nicholas; Lovelady, Clare S.; Rosignoli, Guglielmo; Harris, Claire; Dunn, Sarah; Field, Ray; Dean, Greg; Daramola, Olalekan; Gibson, Suzanne J.; Peden, Andrew A.; Brown, Adam; Hatton, Diane; James, David C.

doi:10.1016/j.jbiotec.2020.02.012

Cited by 19 publications

(22 citation statements)

References 63 publications

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“…Greater enhancement was observed with CHO cells where mild hypothermia resulted in an 8.5-fold higher qP than that at 37°C, and a further increase in titer (10.9-fold, 5.4 mg/L) was obtained via increased cell accumulation. We anticipate that improved CHO systems (e.g., ExpiCHO-S cell line and ExpiCHO medium) as well as coexpression of genetic effectors and chemical chaperone addition (Cartwright et al, 2020;Johari et al, 2015) would significantly increase spike transient production in CHO cells. The CHO-derived spike exhibited a monomeric molecular mass of ~200 kDa by SDS-PAGE (Figure 1D) and a trimeric mass of ~670 kDa was measured using analytical size exclusion chromatography (Supplementary Figure S2).…”

Section: Accepted Articlementioning

confidence: 99%

Production of Trimeric SARS-CoV-2 Spike Protein by CHO Cells for Serological COVID-19 Testing

Johari

Jaffe²,

Scarrott

et al. 2020

Preprint

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We describe scalable and cost-efficient production of full length, His-tagged SARS-CoV-2 spike glycoprotein trimer by CHO cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both HEK and CHO cells mediated by PEI was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32ºC to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in ELISA format to detect IgG antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by PCR, including those who had displayed COVID-19 symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

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Section: Accepted Articlementioning

confidence: 99%

Production of Trimeric SARS-CoV-2 Spike Protein by CHO Cells for Serological COVID-19 Testing

Johari

Jaffe²,

Scarrott

et al. 2020

Preprint

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“…Finally, simply speeding up cell line development by new and more efficient selection mechanisms or by the above mentioned use of targeted integration and of pre-optimised cell lines, will reduce the number of divisions that a cell line will undergo until ready for manufacturing, thus also reducing the accumulation of variants or mutations. Finally, in all these approaches the impact of the product gene and its specific requirements for translation, processing and secretion will have to be taken into account [215,234]. Steps towards creating a Chinese hamster ovary (CHO) chassis cell line.…”

Section: What Is Missing Towards the Construction Of A Cho Chassis Cell?mentioning

confidence: 99%

Key Challenges in Designing CHO Chassis Platforms

et al. 2020

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Following the success of and the high demand for recombinant protein-based therapeutics during the last 25 years, the pharmaceutical industry has invested significantly in the development of novel treatments based on biologics. Mammalian cells are the major production systems for these complex biopharmaceuticals, with Chinese hamster ovary (CHO) cell lines as the most important players. Over the years, various engineering strategies and modeling approaches have been used to improve microbial production platforms, such as bacteria and yeasts, as well as to create pre-optimized chassis host strains. However, the complexity of mammalian cells curtailed the optimization of these host cells by metabolic engineering. Most of the improvements of titer and productivity were achieved by media optimization and large-scale screening of producer clones. The advances made in recent years now open the door to again consider the potential application of systems biology approaches and metabolic engineering also to CHO. The availability of a reference genome sequence, genome-scale metabolic models and the growing number of various “omics” datasets can help overcome the complexity of CHO cells and support design strategies to boost their production performance. Modular design approaches applied to engineer industrially relevant cell lines have evolved to reduce the time and effort needed for the generation of new producer cells and to allow the achievement of desired product titers and quality. Nevertheless, important steps to enable the design of a chassis platform similar to those in use in the microbial world are still missing. In this review, we highlight the importance of mammalian cellular platforms for the production of biopharmaceuticals and compare them to microbial platforms, with an emphasis on describing novel approaches and discussing still open questions that need to be resolved to reach the objective of designing enhanced modular chassis CHO cell lines.

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“…Greater enhancement was observed with CHO cells where mild hypothermia resulted in an 8.5-fold higher qP than that at 37°C, and a further increase in titer (10.9-fold, 5.4 mg/L) was obtained via increased cell accumulation. We anticipate that improved CHO systems (e.g., ExpiCHO-S cell line and ExpiCHO medium) as well as co-expression of genetic effectors and chemical chaperone addition (Cartwright et al, 2020;Johari et al, 2015) would significantly increase spike transient production in CHO cells.…”

Section: Introductionmentioning

confidence: 99%

“…Greater enhancement was observed with CHO cells where mild hypothermia resulted in an 8.5‐fold higher qP than that at 37°C, and a further increase in titer (10.9‐fold, 5.4 mg/L) was obtained via increased cell accumulation. We anticipate that improved CHO systems (e.g., ExpiCHO‐S cell line and ExpiCHO medium) as well as co‐expression of genetic effectors and chemical chaperone addition (Cartwright et al, 2020; Johari et al, 2015) would significantly increase spike transient production in CHO cells. The CHO‐derived spike exhibited a monomeric molecular mass of ~200 kDa by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) (Figure 1d) and a trimeric mass of ~670 kDa was measured using analytical size exclusion chromatography (Figure S2).…”

Section: Introductionmentioning

confidence: 99%

Production of trimeric SARS‐CoV‐2 spike protein by CHO cells for serological COVID‐19 testing

Johari

Jaffe

Scarrott

et al. 2020

Biotech & Bioengineering

Self Cite

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We describe scalable and cost‐efficient production of full length, His‐tagged severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS‐CoV‐2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9‐fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS‐CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9‐fold to 53 mg/L. Purification of recombinant spike by Ni‐chelate affinity chromatography initially yielded a variety of co‐eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme‐linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS‐CoV‐2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID‐19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS‐CoV‐2 trimer which can be used as antigen for mass serological testing.

show abstract

A platform for context-specific genetic engineering of recombinant protein production by CHO cells

Cited by 19 publications

References 63 publications

Production of Trimeric SARS-CoV-2 Spike Protein by CHO Cells for Serological COVID-19 Testing

Production of Trimeric SARS-CoV-2 Spike Protein by CHO Cells for Serological COVID-19 Testing

Key Challenges in Designing CHO Chassis Platforms

Production of trimeric SARS‐CoV‐2 spike protein by CHO cells for serological COVID‐19 testing

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