Novel engineered CHO DG44 host cell line demonstrates lowered UPR, increased titers and superior quality of recombinant vaccines

The Chinese Hamster Ovary (CHO) DG44 platform is routinely used at the Vaccine Production Program (VPP) for manufacturing Vaccine Research Center (VRC) pipeline therapeutic proteins. Clonal cell lines have been generated that express broadly neutralizing monoclonal antibodies (bNmAbs) against HIV-1 with titers ranging between 1 and 5 g/L. In the analysis of data from high and low productivity clones across a range of projects, high-producing clones were found to display increased viable cell density (VCD) and viability at later days in fed-batch culture. With an objective to understand the underlying mechanism for the observed differences in titer, we investigated the Unfolded Protein Response (UPR) pathway and found that multiple genes were differentially regulated among high- and low-producing clones. UPR-induced apoptosis was observed to be significantly higher in lower-producing clones and significantly lower in higher-producing clones at late days in culture. Our initial analysis emphasized a need to generate a platform host cell with lowered UPR and more efficient protein secretion capacity to achieve maximum yields. Through the course of developing and characterizing a panel of cell lines expressing varying levels of human furin for use in a trimeric viral antigen project, a CHO DG44 clone expressing low levels of human furin was identified that had high VCD and viability in later days of fed-batch culture. Reduced UPR at late days in these cells, particularly the lowered expression of apoptotic genes, was seen to correlate to the late day increases in VCD and viability. When the low furin expressing host CHO DG44 cells were used for recombinant protein expression, the increases in VCD and viabilities were maintained compared to platform CHO DG44 host and we achieved a 3 X increase in viral antigen titers without any change in the existing upstream process. It is our goal to characterize the quality of recombinant proteins expressed in DG44- furin engineered clones. The extent of unfolded protein response in viral antigen expressing clones, furin level, and the influence of these factors on the quality of protein is currently under investigation. Our work describes a successful effort to rationally develop a superior host cell for increased and efficient yield of difficult-to-synthesize recombinant targets and points to a potential path forward for generating higher-producing clones for bNmAbs as well

Evaluation of site-specific methylation of the CMV promoter and its role in CHO cell productivity of a recombinant monoclonal antibody

We previously demonstrated that increased monoclonal antibody productivity in dihydrofolate reductase (DHFR)-amplified CHO cells correlates with phosphorylated transcription factor-cytomegalovirus (CMV) promoter interactions. In this article, we extend the characterization to include CMV promoter methylation and its influence on NFκB and CREB1 transcription factor binding to the CMV promoter in two families of DHFR-amplified CHO cell lines. CMV promoter methylation was determined using bisulfite sequencing. To overcome Sanger-sequencing limitations due to high CG bias and multiple transgenes copies, pyrosequencing was used to determine the frequency of methylated cytosines in regions proximal to and containing the NFκB and CREB1 transcription-factor consensus binding sites. Chromatin immunoprecipitation was performed to interrogate transcription factor-DNA interactions. Antibodies to CREB1 and NFκB were used to immunoprecipitate formaldehyde-crosslinked protein-DNA fractions, followed by reverse transcription quantitative real-time polymerase chain reaction to quantitate the number of copies of CMV-promoter DNA bound to the various transcription factors. The relative unmethylated fraction at the CREB1 and NFκB consensus binding sites determined by pyrosequencing was correlated with transcription factor binding as determined by chromatin immunoprecipitation. Azacytidine treatment reduced methylation in all treated samples, though not at all methylation sites, while increasing transcription. Distinct promoter methylation patterns arise upon clonal selection in different families of cell lines. In both cell line families, increased methylation was observed upon amplification. In one family, the NFκB binding-site methylation was accompanied by increased CREB1 interaction with the promoter. In the other cell line family, lower methylation frequency at the NFκB consensus binding site was accompanied by more NFκB recruitment to the promoter region

Increased mAb production in amplified CHO cell lines is associated with increased interaction of CREB1 with transgene promoter

Most therapeutic monoclonal antibodies in biopharmaceutical processes are produced in Chinese hamster ovary (CHO) cells. Technological advances have rendered the selection procedure for higher producers a robust protocol. However, information on molecular mechanisms that impart the property of hyper-productivity in the final selected clones is currently lacking. In this study, an IgG-producing industrial cell line and its methotrexate (MTX)-amplified progeny cell line were analyzed using transcriptomic, proteomic, phosphoproteomic, and chromatin immunoprecipitation (ChIP) techniques. Computational prediction of transcription factor binding to the transgene cytomegalovirus (CMV) promoter by the Transcription Element Search System and upstream regulator analysis of the differential transcriptomic data suggested increased in vivo CMV promoter-cAMP response element binding protein (CREB1) interaction in the higher producing cell line. Differential nuclear proteomic analysis detected 1.3-fold less CREB1 in the nucleus of the high productivity cell line compared with the parental cell line. However, the differential abundance of multiple CREB1 phosphopeptides suggested an increase in CREB1 activity in the higher producing cell line, which was confirmed by increased association of the CMV promotor with CREB1 in the high producer cell line. Thus, we show here that the nuclear proteome and phosphoproteome have an important role in regulating final productivity of recombinant proteins from CHO cells, and that CREB1 may play a role in transcriptional enhancement. Moreover, CREB1 phosphosites may be potential targets for cell engineering for increased productivity

Inferring secretory and metabolic pathway activity from omic data with secCellFie

Understanding protein secretion has considerable importance in biotechnology and important implications in a broad range of normal and pathological conditions including development, immunology, and tissue function. While great progress has been made in studying individual proteins in the secretory pathway, measuring and quantifying mechanistic changes in the pathway's activity remains challenging due to the complexity of the biomolecular systems involved. Systems biology has begun to address this issue with the development of algorithmic tools for analyzing biological pathways; however most of these tools remain accessible only to experts in systems biology with extensive computational experience. Here, we expand upon the user-friendly CellFie tool which quantifies metabolic activity from omic data to include secretory pathway functions, allowing any scientist to infer properties of protein secretion from omic data. We demonstrate how the secretory expansion of CellFie (secCellFie) can help predict metabolic and secretory functions across diverse immune cells, hepatokine secretion in a cell model of NAFLD, and antibody production in Chinese Hamster Ovary cells