title: Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability.
abstract: Immunoglobulin G (IgG), the most common class of commercial monoclonal antibodies (mAbs), exists as multimers of two identical light chains (LC) and two identical heavy chains (HC) assembled together by disulfide bridges. Due to the kinetics of mAb assembly, it is suggested that expression of LC and HC in equal amounts is not optimal for IgG production. We designed a set of vectors using internal ribosome entry site (IRES) elements to control LC and HC expression. The intracellular LC:HC ratio of stable IgG expressing Chinese hamster ovary (CHO) cell pools can be controlled effectively at four different ratios of 3.43, 1.24, 1.12, and 0.32. The stable pools were used to study the impact of LC:HC ratio on mAb expression and quality. Gene amplification was most effective for pools with excess LC and generated the highest mAb titers among the transfected pools. When LC:HC ratio was greater than one, more than 97% of the secreted products were IgG monomers. The products also have similar N-glycosylation profiles and conformational stabilities at those ratios. For pools presented a lower LC:HC ratio of 0.32, monomers only constituted half of the product with the other half being aggregates and mAb fragments. High mannose-type N-glycans increased while fucosylated and galactosylated glycans decreased significantly at the lowest LC:HC ratio. Product stability was also adversely affected. The results obtained provide insights to the impact of different LC:HC ratios on stable mAb production and useful information for vector design during generation of mAb producing cell lines.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
authors.forename: Steven C L
authors.initials: SC
authors.lastname: Ho
authors.forename: Esther Y C
authors.initials: EY
authors.lastname: Koh
authors.forename: Miranda
authors.initials: M
authors.lastname: van Beers
authors.forename: Monika
authors.initials: M
authors.lastname: Mueller
authors.forename: Corrine
authors.initials: C
authors.lastname: Wan
authors.forename: Gavin
authors.initials: G
authors.lastname: Teo
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Song
authors.forename: Yen Wah
authors.initials: YW
authors.lastname: Tong
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Yuansheng
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human