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MAbs. 2014 May-Jun;6(3):671-8. doi: 10.4161/mabs.28172. Epub 2014 Feb 11.

Engineering toward a bacterial "endoplasmic reticulum" for the rapid expression of immunoglobulin proteins.

Author information

1
Sutro Biopharma; South San Francisco, CA USA.
2
Molecular Technologies Laboratories LLC; Ohio University Innovation Center; Athens, OH USA.
3
Halozyme Therapeutics Inc.; San Diego, CA USA.

Abstract

Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies. However, given the ease and manipulability of bacteria, antibody discovery efforts often employ bacterial expression systems despite their limitations in generating high titers of functional antibody. Open-Cell Free Synthesis (OCFS) is a coupled transcription-translation system that has the advantages of prokaryotic systems while achieving high titers of antibody expression. Due to the open nature of OCFS, it is easily modified by chemical or protein additives to improve the folding of select proteins. As such, we undertook a protein additive screen to identify chaperone proteins that improve the folding and assembly of trastuzumab in OCFS. From the screen, we identified the disulfide isomerase DsbC and the prolyl isomerase FkpA as important positive effectors of IgG folding. These periplasmic chaperones function synergistically for the folding and assembly of IgG, and, when present in sufficient quantities, gram per liter IgG titers can be produced. This technological advancement allows the rapid development and manufacturing of immunoglobulin proteins and pushes OCFS to the forefront of production technologies for biologics.

KEYWORDS:

DsbC; FkpA; IgG; OCFS; antibody; cell free; chaperone; high titer; rapid; strain engineering

PMID:
24517929
PMCID:
PMC4011911
DOI:
10.4161/mabs.28172
[Indexed for MEDLINE]
Free PMC Article
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