Transient CHO expression platform for robust antibody production and its enhanced N‐glycan sialylation on therapeutic glycoproteins

Abstract

Large‐scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO‐S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO‐S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO‐S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non‐Fc N‐linked glycans were expressed in transient ExpiCHO‐S™, the glycan pattern was unexpectedly found to have few sialylated N‐glycans, in contrast to glycans produced within a stable CHO expression system. To improve N‐glycan sialylation in transient ExpiCHO‐S™, we co‐transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co‐transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N‐glycans during transient ExpiCHO‐S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N‐glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019

Antibody Structure and Function: The Basis for Engineering Therapeutics

“…Glycan structure [67,69,[259][260][261][262][263]; High mannose and afucosylation affect stability [264]; Sialylation [265]; Fucosylation [266] Biological activity [267]; PK and PD [268]; Clearance [269] * Changes to critical amino acids linked to Fab-antigen or Fc-Fc receptor binding and functions.…”

Transfection of glycoprotein encoding mRNA for swift evaluation of N‐glycan engineering strategies

“…Their N-glycosylation capacity could be further optimized by combinatorial approaches targeting activated nucleotide sugar precursor synthesis, 17 transport into the Golgi 31 or by media supplementation. [32][33][34] We propose that mimicking high productivity by mRNA transfections could also be used to similarly evaluate engineering targets in other processing steps along the secretory pathway, such as folding, proteolytic cleavage, O-glycosylation or secretion rate. Since this mRNA system allows for testing numerous strategies upfront, at a very early stage of conception, the time-consuming process of generating stable cell lines for difficult-to-express proteins could be avoided during initial screening.…”

Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis

“…Considering that our main interest is related to glycoprotein hormones, we did not focus other important recombinant protein synthesis still based on CHO or HEK293 cell culture. We just recall some important works based on human cell lines in general (Fliedl et al 2015; Mortazavi et al 2019; Zhong et al 2019), on CHO cell (Bohm et al 2015; Croset et al 2012; Durocher and Butler 2009; Zhang et al 2010) and on HEK293 (Bohm et al 2015; Bouvette et al 2018; Croset et al 2012; Ding et al 2017; Gugliotta et al 2017; Hu et al 2018; Swiech et al 2012; Zhang et al 2010).…”