Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells
Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis...
Saved in:
Main Authors: | , , , , , , , , |
---|---|
Other Authors: | |
Format: | Article |
Language: | English |
Published: |
2016
|
Subjects: | |
Online Access: | https://hdl.handle.net/10356/81450 http://hdl.handle.net/10220/40785 |
Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
Institution: | Nanyang Technological University |
Language: | English |
id |
sg-ntu-dr.10356-81450 |
---|---|
record_format |
dspace |
spelling |
sg-ntu-dr.10356-814502023-12-29T06:48:39Z Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells Chng, Jake Wang, Tianhua Nian, Rui Lau, Ally Hoi, Kong Meng Yang, Yuansheng Ho, Steven C. L. Gagnon, Peter Bi, Xuezhi School of Chemical and Biomedical Engineering monoclonal antibody 2A peptide furin CHO cleavage efficiency GSG linker Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing. ASTAR (Agency for Sci., Tech. and Research, S’pore) Accepted version 2016-06-23T08:52:59Z 2019-12-06T14:31:15Z 2016-06-23T08:52:59Z 2019-12-06T14:31:15Z 2015 Journal Article Chng, J., Wang, T., Nian, R., Lau, A., Hoi, K. M., Ho, S. C. L., et al. (2015). Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells. mAbs, 7(2), 403-412. 1942-0862 https://hdl.handle.net/10356/81450 http://hdl.handle.net/10220/40785 10.1080/19420862.2015.1008351 25621616 en mAbs © 2014 Landes Bioscience. This is the author created version of a work that has been peer reviewed and accepted for publication in mAbs, published by Taylor & Francis on behalf of Landes Bioscience. It incorporates referee’s comments but changes resulting from the publishing process, such as copyediting, structural formatting, may not be reflected in this document. The published version is available at: [http://dx.doi.org/10.1080/19420862.2015.1008351]. 34 p. application/pdf |
institution |
Nanyang Technological University |
building |
NTU Library |
continent |
Asia |
country |
Singapore Singapore |
content_provider |
NTU Library |
collection |
DR-NTU |
language |
English |
topic |
monoclonal antibody 2A peptide furin CHO cleavage efficiency GSG linker |
spellingShingle |
monoclonal antibody 2A peptide furin CHO cleavage efficiency GSG linker Chng, Jake Wang, Tianhua Nian, Rui Lau, Ally Hoi, Kong Meng Yang, Yuansheng Ho, Steven C. L. Gagnon, Peter Bi, Xuezhi Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells |
description |
Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing. |
author2 |
School of Chemical and Biomedical Engineering |
author_facet |
School of Chemical and Biomedical Engineering Chng, Jake Wang, Tianhua Nian, Rui Lau, Ally Hoi, Kong Meng Yang, Yuansheng Ho, Steven C. L. Gagnon, Peter Bi, Xuezhi |
format |
Article |
author |
Chng, Jake Wang, Tianhua Nian, Rui Lau, Ally Hoi, Kong Meng Yang, Yuansheng Ho, Steven C. L. Gagnon, Peter Bi, Xuezhi |
author_sort |
Chng, Jake |
title |
Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells |
title_short |
Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells |
title_full |
Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells |
title_fullStr |
Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells |
title_full_unstemmed |
Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells |
title_sort |
cleavage efficient 2a peptides for high level monoclonal antibody expression in cho cells |
publishDate |
2016 |
url |
https://hdl.handle.net/10356/81450 http://hdl.handle.net/10220/40785 |
_version_ |
1787136595252477952 |