Overexpression of the regulatory subunit of glutamate-cysteine ligase enhances monoclonal antibody production in CHO cells

Orellana, Camila A., Marcellin, Esteban, Gray, Peter P. and Nielsen, Lars K. (2017) Overexpression of the regulatory subunit of glutamate-cysteine ligase enhances monoclonal antibody production in CHO cells. Biotechnology and Bioengineering, 114 8: 1825-1836. doi:10.1002/bit.26316


Author Orellana, Camila A.
Marcellin, Esteban
Gray, Peter P.
Nielsen, Lars K.
Title Overexpression of the regulatory subunit of glutamate-cysteine ligase enhances monoclonal antibody production in CHO cells
Journal name Biotechnology and Bioengineering   Check publisher's open access policy
ISSN 1097-0290
0006-3592
Publication date 2017-08-01
Year available 2017
Sub-type Article (original research)
DOI 10.1002/bit.26316
Open Access Status Not yet assessed
Volume 114
Issue 8
Start page 1825
End page 1836
Total pages 12
Place of publication Hoboken, NJ, United States
Publisher John Wiley & Sons
Language eng
Subject 1305 Biotechnology
1502 Bioengineering
2402 Applied Microbiology and Biotechnology
Abstract For decades, Chinese hamster ovary (CHO) cells have been the preferred host for therapeutic monoclonal antibody (mAb) production; however, increasing mAb titer by rational engineering remains a challenge. Our previous proteomic analysis in CHO cells suggested that a higher content of glutathione (GSH) might be related to higher productivity. GSH is an important antioxidant, cell detoxifier, and is required to ensure the formation of native disulfide bonds in proteins. To investigate the involvement of GSH in mAb production, we generated stable CHO cell lines overexpressing genes involved in the first step of GSH synthesis; namely the glutamate-cysteine ligase catalytic subunit (Gclc) and the glutamate-cysteine ligase modifier subunit (Gclm). The two genes were reconstructed from our RNA-Seq de novo assembly and then were functionally annotated. Once the sequences of the genes were confirmed using proteogenomics, a transiently expressed mAb was introduced into cell lines overexpressing either Gclc or Gclm. The new cell lines were compared for mAb production to the parental cell line and changes at the proteome level were measured using SWATH. As per our previous proteomics observations, overexpressing Gclm improved productivity, titer, and the frequency of high producer clones by 70%. In contrast, overexpressing Gclc, which produced a higher amount of GSH, did not increase mAb production. We show that GSH cannot be linked to higher productivity and that Gclm may be controlling other cellular processes involved in mAb production yet to be elucidated. Biotechnol. Bioeng. 2017;114: 1825–1836.
Formatted abstract
For decades, Chinese hamster ovary (CHO) cells have been the preferred host for therapeutic monoclonal antibody (mAb) production; however, increasing mAb titer by rational engineering remains a challenge. Our previous proteomic analysis in CHO cells suggested that a higher content of glutathione (GSH) might be related to higher productivity. GSH is an important antioxidant, cell detoxifier, and is required to ensure the formation of native disulfide bonds in proteins. To investigate the involvement of GSH in mAb production, we generated stable CHO cell lines overexpressing genes involved in the first step of GSH synthesis; namely the glutamate-cysteine ligase catalytic subunit (Gclc) and the glutamate-cysteine ligase modifier subunit (Gclm). The two genes were reconstructed from our RNA-Seq de novo assembly and then were functionally annotated. Once the sequences of the genes were confirmed using proteogenomics, a transiently expressed mAb was introduced into cell lines overexpressing either Gclc or Gclm. The new cell lines were compared for mAb production to the parental cell line and changes at the proteome level were measured using SWATH. As per our previous proteomics observations, overexpressing Gclm improved productivity, titer, and the frequency of high producer clones by 70%. In contrast, overexpressing Gclc, which produced a higher amount of GSH, did not increase mAb production. We show that GSH cannot be linked to higher productivity and that Gclm may be controlling other cellular processes involved in mAb production yet to be elucidated.
Keyword CHO
Gclc
Gclm
Glutamate-cysteine ligase
Glutathione
Monoclonal antibodies
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
Australian Institute for Bioengineering and Nanotechnology Publications
 
Versions
Version Filter Type
Citation counts: TR Web of Science Citation Count  Cited 0 times in Thomson Reuters Web of Science Article
Scopus Citation Count Cited 0 times in Scopus Article
Google Scholar Search Google Scholar
Created: Sun, 09 Jul 2017, 01:00:20 EST by System User on behalf of Learning and Research Services (UQ Library)