title: Engineering toward a bacterial "endoplasmic reticulum" for the rapid expression of immunoglobulin proteins.
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.
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Dan
authors: D
authors: Groff
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Stephanie
authors: S
authors: Armstrong
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Patrick J
authors: PJ
authors: Rivers
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Juan
authors: J
authors: Zhang
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Junhao
authors: J
authors: Yang
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Evan
authors: E
authors: Green
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: James
authors: J
authors: Rozzelle
authors: Molecular Technologies Laboratories LLC; Ohio University Innovation Center; Athens, OH USA.
authors: Shengwen
authors: S
authors: Liang
authors: Molecular Technologies Laboratories LLC; Ohio University Innovation Center; Athens, OH USA.
authors: Joseph D
authors: JD
authors: Kittle
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Alexander R
authors: AR
authors: Steiner
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Ramesh
authors: R
authors: Baliga
authors: Halozyme Therapeutics Inc.; San Diego, CA USA.
authors: Christopher D
authors: CD
authors: Thanos
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Trevor J
authors: TJ
authors: Hallam
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Aaron K
authors: AK
authors: Sato
authors: Sutro Biopharma; South San Francisco, CA USA.
authors: Alice Y
authors: AY
authors: Yam
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001419:Bacteria / Q000235:genetics* / Q000378:metabolism*; D001709:Biotechnology; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007136:Immunoglobulins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D029968:Escherichia coli Proteins; D007074:Immunoglobulin G; D007136:Immunoglobulins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D019704:Protein Disulfide-Isomerases; C526649:dsbC protein, E coli; D000068878:Trastuzumab
keywords: DsbC; FkpA; IgG; OCFS; antibody; cell free; chaperone; high titer; rapid; strain engineering