Andreas Castan

Influence of cell culture media and feed supplements on cell metabolism and quality of IgG produced in CHO-K1, CHO-S, and CHO-DG44

Background Chinese hamster ovary (CHO) cells have become the preferred expression system for the production of complex recombinant proteins. In this study, chemically defined CHO cell culture media and feed substrates from different vendors were investigated regarding their influence on cell metabolism, antibody titer and quality. Special emphasis was put on elucidating how these attributes change with the use of different CHO host cell lines. For this purpose, CHO-K1, CHO-DG44, and CHO-S each producing the same IgG antibody were adapted to ActiCHOTM P and CD CHO medium. All three cell lines were grown both in batch and in fedbatch cultures using the manufacturer’s specific concentrated feed supplements. The impact of the different media and feeds on antibody production, cell growth, cell-specific nutrient consumption, by-product formation and IgG quality was analyzed throughout the process.

Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression

In biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess.

Upstream Processing Equipment

Abstract Modern bioprocesses have become quite diverse with highly specialized designs serving a broad spectrum of customers and their products. With the specialization, there has been a growth in platform technologies, starting with the cell line or organism and extending into equipment design. Despite the breadth and diversity of equipment designs, all equipment must be scalable and address the needs of the related stakeholders, from the scientist/engineer to the maintenance personnel. In this chapter, the requirements of upstream bioprocessing equipment are introduced and discussed in detail from an engineering standpoint, with the bioreactor in focus, and including examples of available bioreactor technology.

Cell Line Development

Abstract Cell lines suitable for stable production of recombinant proteins must be possible to scale up for commercial GMP production. In particular, the cell line should: (i) be adapted to serum-free and suspension growth conditions, (ii) be able to grow at a high rate, and (iii) have a high secretory capacity. It must also exhibit an efficient energy metabolism without excess secretion of by-products and should have well-tuned protein secretion and glycosylation machinery. Furthermore, it must be stable with respect to product quality and productivity over time, and the timelines and population doublings from gene transfection to production cell line should be as short as possible. In this chapter, we describe how such stable CHO cell lines can be developed, propose options for an optimized cell line development process and discuss host cell engineering strategies and biosafety analysis.

Differential gene expression of a feed-spiked super-producing CHO cell line

Feed supplements are concentrated cell culture media that contain a variety of nutrients, which can be added during a bioprocess. During fed-batch cultivation, feed media are typically added to a growing cell culture to maximize cell and product concentrations. In this study, only a single shot of feed medium was added on day 0 to a basal cell culture medium and compared to non-supplemented basal medium (feed-spiked at day 0 versus control experiments) by cultivation of a recombinant mAb expressing CHO cell line in batch mode under controlled conditions in a bioreactor. Since the feed-spike at day 0 was based on existing medium components without introducing additional supplements, a desirable process with decreased complexity was generated. Unlike cells in basal medium, feed-spiked cultures reached almost 2× higher peak cell concentrations (10 × 106 c/mL vs. 18 × 106 c/mL) and 3× higher antibody concentrations (0.8 g/L vs. 2.4 g/L). Batch process time and the integral over the viable cell count were similar for both process types. Constantly high cell-specific production rates in feed-spiked cultures (70 pg/cell/day) compared to continuously declining rates in basal medium (from 70 to 10 pg/cell/day) were responsible for an overall 70% higher cell-specific production rate and the higher product concentrations. To associate gene expression patterns to different process proceedings, transcriptome analysis was performed using microarrays. Several transcripts that are involved with glutamine de novo synthesis and citric acid cycle were significantly upregulated on several days in feed-spiked cultures. The top identified gene ontology (GO) terms related well to cell cycle and primary metabolism, cellular division as well as nucleobase formation or regulation, which indicated a more active proliferative state for feed-spiked cultures. KEGG biochemical pathway analysis and Gene set enrichment analysis (GSEA) further confirmed these findings from a complementary perspective. Moreover, several interesting gene targets, which have not yet been associated with recombinant protein expression, were identified that related to a higher proliferative state, growth, protein synthesis, cell-size control, metabolism, cell survival as well as genes that are associated with the control of the mammalian target of rapamycin (mTOR) in feed-spiked cultures. Analysis of critical product quality attributes (i.e. glycosylation, charge variants and size distribution) showed that feed-spiking did not change antibody quality.

Bioprocessing of Recombinant CHO-K1, CHO-DG44, and CHO-S: CHO Expression Hosts Favor Either mAb Production or Biomass Synthesis

Chinese hamster ovary (CHO) cells comprise a variety of lineages including CHO-DXB11, CHO-K1, CHO-DG44, and CHO-S. Despite all CHO cell lines sharing a common ancestor, extensive mutagenesis, and clonal selection has resulted in substantial genetic heterogeneity among them. Data from sequencing show that different genes are missing in individual CHO cell lines and each cell line harbors a unique set of mutations with relevance to the bioprocess. However, not much literature is available about the influence of genetic differences of CHO on the performance of bioprocess operations. In this study, the host cell-specific differences among three widely used CHO cell lines (CHO-K1, CHO-S, and CHO-DG44) and recombinantly expressed the same monoclonal antibody (mAb) in an isogenic format by using bacterial artificial chromosomes (BACs) as transfer vector in all cell lines is examined. Cell-specific growth and product formation are studied in batch, fed-batch, and semi-continuous perfusion cultures. Further, two different cell culture media are used to investigate their effects. The authors find CHO cell line-specific preferences for mAb production or biomass synthesis that are determined by the host cell line. Additionally, quality attributes of the expressed mAb are influenced by the host cell line and media.