Optimized Selection Marker and CHO Host Cell Combinations for Generating High Monoclonal Antibody Producing Cell Lines

Abstract

There are several selection markers which are suitable for generating stably transfected Chinese hamster ovary (CHO) cell lines. Due to their different modes of action, each selection marker has its own optimal selection stringency in different host cells for obtaining high productivity. Using an internal ribosome entry site (IRES)-mediated tricistronic vector and a set of IRES variants with different strengths, the expression of five antibiotics resistance genes (ARGs) in CHO-K1 cells and dihydrofolate reductase (DHFR) in CHO DG44 cells is optimized to enhance the stringency of selection for high producing cells. There is an obvious optimal expression level for every selection marker, below or above which, the productivity is significantly lower. The enhanced productivity in ARG generated CHO K1 cells is due to selective integration of active site while the enhanced productivity in the amplified CHO DG44 cells results from increased gene copies. The high producing CHO K1 pools and clones generated using ARG exhibit better production stability than the amplified high producing CHO DG44 pools and clones. Loss of expression for the CHO K1 cell lines is due to loss of gene copies while for CHO DG44 is due to transcriptional silencing. mAb glycan profile also differed significantly between CHO K1 and CHO DG44 cell lines. These results would be helpful when developing optimized vectors for generating high mAb producing CHO cell lines.

Multiplexed engineering glycosyltransferase genes in CHO cells via targeted integration for producing antibodies with diverse complex-type N-glycans

“…Synthesis of IRES-GE1, IRES-GE2, other glycosyltransferase genes and inserting them into the control vector or the two basic vectors for expressing single or double glycosylation genes were all done by Genscript. The sequences of the wild-type IRES, IRESv18, FRT and FRT3 were described in previous studies 53 , 54 . The sequences of DsRed gene and pA were cloned from the pIRES2-DsRed vector (Clonetech) and the pcDNA3.1 (+) vector (Thermo Fisher Scientific), respectively.…”

“…Total RNA was isolated using RNeasy Mini Kit (Qiagen) from ten million cells collected from each fed-batch culture. Analysis of the mRNA levels for human glycosyltransferase genes (Table 1 ) and β-actin was done using either reverse transcription polymerase chain reaction (RT-PCR) or quantitative real-time PCR (qRT-PCR) as described previously 53 . Primer sequences used for mRNA analysis were listed in Supporting Table S2 .…”

“…The purified mAbs were analyzed for the N-glycosylation using hydrophilic interaction chromatography (HILIC) with fluorescence detection. The protocols for protein A purification and N-glycosylation analysis have been described in a previous study 53 . The data obtained from N-glycosylation analysis was analyzed with the UNIFI Biopharmaceutical software platform (version 1.8).…”

“…The N-glycan structures were assigned to peaks based on the alignment of observed and expected glucose unit (GU) values. The peak assignment followed the method described in a previous study 53 . The abundance of each structure was expressed as percentage of total peak area.…”

Enhanced Transgene Expression by Optimization of Poly A in Transfected CHO Cells

“…The expression of a heterologous gene in host cells depends on some components in the expression vector, such as an enhancer, promoter, intron, poly A, integration site, and regulatory sequences. Therefore, the proper selection of these elements can increase recombinant protein expression levels and stability ( Lee JS et al, 2018 ; Lee CP et al, 2018 ; Yeo et al, 2017 ). However, most studies have focused on the role of the enhancer, internal ribosome entry site (IRES), intron, promoters, and combinations of regulatory elements with promoters ( Hunter et al, 2019 ; Skipper et al, 2019 ; Yeo et al, 2017 ; Chai et al, 2018 ), few studies have been performed on the effects of poly A elements on transgene expression in CHO cells.…”