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ABSTRACT In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary phase of the fed-batch culture. Higher degree of glucose starvation reduced intracellular concentrations of UDP-GlcNAc and UDP-GalNAc, but increased the levels of UDP-Glc and UDP-Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells was conducted to observe differences in protein abundance between early growth and early stationary phases. Generally higher expression of proteins involved in regulating cellular metabolism, extracellular matrix, apoptosis, protein secretion and glycosylation was found in early stationary phase. These analyses offered a systematic view of the intrinsic properties of these cells and allowed us to explore the root causes correlating culture duration with variations in the productivity and glycosylation quality of monoclonal antibodies produced with CHO cells. Biotechnol. Bioeng. 2015;112: 2172-2184. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4+ and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β- N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of [ABSTRACT FROM AUTHOR]

The human CMV promoter/enhancer is one of the strongest promoters for recombinant protein expression in mammalian cells, making the promoter very popular for production of recombinant antibodies. We used an antibody vector design where the antibody heavy and light chain genes were transcribed from a promoter complex consisting of two promoters arranged divergently with the 5′ ends of the promoters in close proximity. However, when two identical CMV promoters constituted this promoter complex, the antibody expression observed was lower than expected based on the strength of the individual promoters. To optimize expression we prepared truncated promoter complexes where only one CMV enhancer controlled the initiation of transcription from two divergent minimal CMV core promoters. Antibody expression from the truncated promoter complexes was analyzed both when transiently transfected and upon stable site-specific integration into a CHO DG44 derived cell line. The data showed that it was possible for one enhancer to drive the expression of two core promoters. However, efficient expression from both divergent core promoters was seen only when the unique region upstream of the CMV enhancer was removed. Notably, a 12-fold increase in expression was found from the best of the truncated promoter complexes after stable site-specific integration when compared to the full-length double CMV promoter complex. [ABSTRACT FROM AUTHOR]

We have previously described the development and implementation of a strategy for production of recombinant polyclonal antibodies (rpAb) in single batches employing CHO cells generated by site-specific integration, the SympressTM I technology. The SympressTM I technology is implemented at industrial scale, supporting a phase II clinical development program. Production of recombinant proteins by site-specific integration, which is based on incorporation of a single copy of the gene of interest, makes the SympressTM I technology best suited to support niche indications. To improve titers while maintaining a cost-efficient, highly reproducible single-batch manufacturing mode, we have evaluated a number of different approaches. The most successful results were obtained using random integration in a new producer cell termed ECHO, a CHO DG44 cell derivative engineered for improved productivity at Symphogen. This new expression process is termed the SympressTM II technology. Here we describe proof-of-principle data demonstrating the feasibility of the SympressTM II technology for single-batch rpAb manufacturing using two model systems each composed of six target-specific antibodies. The compositional stability and the batch-to-batch reproducibility of rpAb produced by the ECHO cells were at least as good as observed previously using site-specific integration technology. Furthermore, the new process had a significant titer increase. [ABSTRACT FROM AUTHOR]

Elk3 belongs to the Ets family of transcription factors, which are regulated by the Ras/mitogen-activated proteinkinase-signaling pathway. In the absence of Ras, this protein is a strong inhibitor of transcription andmay be directly involved in regulation of growth by downregulating the transcription of genes that are activatedduring entry into G. We have isolated theElk3 gene from the Chinese hamster ovary(CHO) cell line and investigated the transcriptional potential of this factor. Transient transfections revealedthat, in addition to its regulation of thepromoter, Elk3 from CHO cells seems to inhibit other promoterscontrolling expression of proteins involved in G/S phase progression; Cyclin D1 and DHFR. As has beendescribed for the Elk3 homologs Net (Mouse) and Sap-2 (Human), the results of the present study further indicatethat hamster Elk3 is a target of the Ras-Raf-MAPK pathway, and cotransfections with constitutivelyactive H-relieves its negative transcriptional activity. No cells stably expressing exogenous Elk3 could beobtained, possibly due to an unspecified toxic or growth retarding effect. These findings support a possiblerole for Elk3 in growth regulation and reveal a high degree of homology for this protein across species. [ABSTRACT FROM AUTHOR]

An abstract of the article "Recombinant antibody mixtures; optimization of cell line generation and single-batch manufacturing processes," by Lars S. Nielsen and colleagues is presented.