Development of hyper osmotic resistant CHO host cells

We have developed a cell culture platform for monoclonal antibody (IgG) production by Chinese Hamster Ovary (CHO) cells. The platform feed used the continuous feeding method. This platform can maintain high cell density and produce high antibody titer. However because operation of continuous feed is complex, contract manufacturing organization (CMO) that can perform continuous feed is limited. Therefore, we tried to change the feeding method from continuous feed to bolus feed. However the previous studies showed that the rapid changes of osmolality by bolus feed and the hyper osmolality repressed the cell culture growth and the final titer. In this study, we developed hyper osmotic resistant CHO-S host cell A (resistant to 450mOsm). To establish osmotic resistant CHO-S host cells, original CHO-S cells were passaged on a hyper osmotic basal media with repetition for about 100 days. We demonstrated that there were obviously differences in the cell growth under osmotic pressure of iso- (328 mOsm) and hyper- (450 mOsm) osmolality between the two host cells. Metabolic analysis of cell culture supernatant on CHO-S host cell A and CHO-S host cells with/ without osmotic stress performed. Compared to original CHO-S host cells, the osmotic resistant CHO-S host cell A has a greater capacity to generate osmolytes (sorbitol and erthritol) and decreased level of oxidized glutathione (GSSG), which suggests the osmotic resistant CHO-S host cells A handles osmotic stress better. Moreover, the characteristic of osmotic resistant on hyper osmotic resistant CHO-S host cell A was maintained even after 7 passages on a basal medium (330 mOsm). We will establish hyper osmotic resistant antibody production CHO cell line by using the CHO-S host cell A

Genotype of CHO host cell line has higher impact on mAb production and quality than process strategy or cell culture medium

Chinese hamster ovary (CHO) cells comprise a variety of lineages, including CHO-DXB11, CHO-K1, CHO-DG44 and CHO-S. Despite the fact that CHO cell lines share a common ancestor, extensive mutagenesis and clonal selection have resulted in substantial genetic heterogeneity among them. Data from sequencing shows that different genes are lacking from individual CHO cell lines and that each cell line harbors a unique set of mutations that are relevant to the bioprocess. However, literature outlining how the observed genetic differences affect CHO cell performance during bioprocess operations remains scarce. In this study, we examined 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 in all cell lines by using bacterial artificial chromosomes (BACs) as transfer vector. Cell-specific growth, product formation and heavy and light chain mRNA levels were studied in batch, fed-batch and perfusion cultures. Furthermore, two different cell culture media were investigated. Product quality was studied through glycoprofiling, and the thermal denaturation was analyzed using differential scanning calorimetry (DSC). We found CHO cell line-specific preferences for mAb production or biomass synthesis that were determined by the host cell line rather than product-specific mRNA levels. Additionally, quality attributes of the expressed mAb were influenced by the host cell line and medium used

Lipidomic analysis to enhance the understanding of Chinese Hamster ovary cells

Chinese Hamster Ovary (CHO) cell lines are common hosts for the production of biotherapeutic proteins. Achieving high level of specific protein production by CHO cell lines remains a challenge. In order to address this issue, we are incorporating lipidomic analyses to study the role of lipids played in CHO-S cells. In our study, we have applied chromatography (TLC) methods for lipid analysis in terms of lipid polarity. For polar lipids, 2-D HPTLC (2-dimensional high performance TLC) was used instead of conventional 1D- TLC by virtue of its high separation capacity. The eluting solvent system was optimized for the 1st and 2nd dimension, respectively. Neutral lipids were separated on 1-D HPTLC with the optimal elution solvent of hexane-diethyl ether-acetic acid. The lipid spots on the TLC plates were stained by 0.2% of 2,7-dichlorofluorescein dissolved in ethanol solution and illuminated with UV. Multiple lipid standards were also run to correctly identify the lipid spots and the fluorescence of lipid spots was semi-quantitatively measured with ImageJ. By optimization of TLC conditions, the lipids of CHO-S cell line were separated successfully and the lipid contents were semi-quantified. From neutral lipids result, we observed high level of certain lipids in CHO-S cell lines. We will further investigate which lipid play a key role in various cell processes

Cre-loxP-controlled cell-cycle checkpoint engineering in Chinese Hamster ovary cells

The gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the productive cell line construction of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering1). Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system2). A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by Cre recombinase protein expression. Overexpression of the floxed mutant CDC25B significantly enhanced the efficiency of transgene amplification and productivity. Moreover, the specific production rate of the isolated clone CHO Cre-1 and Cre-2 were approximately 11-fold and 15-fold higher than that of mock-transfected clone CHO Mock-S. Chromosomal aneuploidy was increased by mutant CDC25B overexpression, but Cre-1 and Cre-2 did not show any changes in chromosome number during long-term cultivation, as is the case with CHO Mock-S. Our results suggest that high-producing and stable cells can be constructed by conditionally controlling a cell-cycle checkpoint integrated in conventional gene amplification systems

Nature sẽ ủng hộ kế hoạch Plan S

Tại thị trường Châu Âu, Springer Nature cho biết có đến 4 quốc gia có hơn 70% tác giả đang lựa chọn công bố mở. Tuy nhiên, để đạt được 30% còn lại thì các nhà xuất bản như Springer Nature cần đưa ra các lựa chọn hấp dẫn và thuyết phục hơn là ép buộc các tác giả phải lựa chọn Open Access như yêu cầu hiện nay

Engineering CHO cells for the production of Hard-To-Produce proteins

Over the past decades, the CHO cell has become increasingly popular as the favorite host cell line for the production of protein based therapeutic drugs. In comparison with the popularity of the CHO cells and the frequent use of these cells to produce a large part of the bestselling blockbuster drugs, less intensive efforts have been done to understand the machinery used by the CHO cells during growth and production. The main approach has (broadly speaking) been to approach the CHO cell as a “black box” where one could insert the gene of interest, perform a number of amplifying steps, like gene amplification, selection for stable clones, intense screening for stably expressing high producers, and massive efforts to optimize a specific bioprocess for the selected cell line(s). Since 2013, the Novo Nordisk Foundation Center for Biosustainablity at the Technical University of Denmark has embarked on a large CHO program to open up the “black box”, to get a deeper understanding of the available machinery inside the protein producing “cell factory” that is CHO cells. We are using this understanding to engineer new CHO cell lines having significantly improved features for the production of therapeutic proteins. We are not only doing this by improving the titer, quality, downstream processing and speed of development for already well-known proteins (e.g. Ab), but also for the production of therapeutic proteins that cannot be produced in CHO cells today, due low titer, wrong post translational modifications, and/or low activity. By combining the competences embedded in the CHO program, we are able to exploit the combination of genome scale modelling, high throughput protein expression, deep understanding of both the glycosylation machinery as well as the secretory and metabolic pathways involved in the expression of secreted proteins. This knowledge is being used as input to a high throughput CHO cell line engineering pipeline, able to engineer up to 10 cell lines and 25 gene targets in parallel. This has resulted in a large number of new CHO cell lines enabling the production of proteins with specific tailor-made glycoprofiles, higher quality, less degradation, improved bioprocess, higher viable cell density and better cell viability. We have made a cell lines where we have removed a number of naturally expressed host cell proteins (HCP) from CHO, which has resulted in higher titer and higher VCD, cell lines showing increased resistance to viral infections, cell lines displaying homogenous glycoprofiles, reduced degradation, and drastically changed cell lines that does not produce lactate. These features are currently being combined to engineer CHO cells able to produce proteins that have not been possible to produce with adequate product quality and titer using CHO cells to date

A community genome-scale model of Chinese Hamster ovary cell metabolism identifies differences in the efficiency of resource utilization for various bioprocesses

Genome-scale models of metabolism have successfully been employed in many microbial and eukaryotic metabolic engineering efforts by guiding pathway engineering and media optimization. They have also been used to explore the genotype-phenotype relationship in mammalian cells. The publication of the genomic sequence for Chinese hamster ovary (CHO) cells has allowed generation of genome-scale metabolic models (GeMs) for this organism. Here we have developed a high-quality community CHO GeM via careful reconciliation and manual curation of three independently developed CHO GeMs. This metabolic model, consisting of over 4000 metabolites and 6000 reactions, is capable of integrating proteomic, transcriptomic, and metabolomic data and can accurately simulate experimentally measured growth rates. Integration of transcriptomic and proteomic data from CHO-K1 and CHO-S shed light on the enzymatic basis for various amino acid auxotrophies characteristic of the cell lines. We show that experimental arginine and cysteine auxotrophies are recapitulated by model predictions (via reaction inactivation) while the characteristic proline auxotrophy is not, due to detectable levels of expression in biosynthetic pathways for this amino acid. We additionally used the model to assess the metabolic limitations on recombinant protein producing lines subject to different cell line and process modifications and found that some alterations result in specific productivities up to 20-fold lower than computational predictions of metabolically feasible production rates. The results indicate a possible secretory bottleneck and implicate engineering the secretory pathway as a lucrative target to pursue in future CHO cell line engineering

A Note on the Hybrid Equilibrium in the Besley-Smart Model

This note shows that there is always a non-empty set of parameter values for which the hybrid equilibrium in the Besley and Smart(2003) model is unstable in the sense of Cho and Kreps. This set may include all the parameter values for which a hybrid equilibrium exists. For these parameter values, it is shown that a fully separating equilibrium always exists, which is Cho-Kreps stable. In this equilibrium, the good incumbent distorts ?scal policy to signal his type. An implication is that equilibrium in their model is not (generically) unique.

Symbols and Rituals on the Grounds of Queer Culture Festivals

The Queer Culture Festivals (QCF) in South Korea have been rapidly growing as a social movement that promotes visibility and pride of the LGBTQ population. This study explores rituals and symbols at QCFs: territorialization of the festivals grounds; booth activities; staged speeches and slogans; queerthemed artefacts; and participants bodily expressions. These various activities question and mock the hegemonic notions of heteronormativity and gender binaries, the ideology of the normal family, Confucian puritanism, and the antiqueer rhetoric of Evangelical Christians. QCFs also deploy playful symbols to subvert the stereotypes of LGBTQ people as abnormal, amoral, and sinful; instead they depict LGBTQ as proud and worthy. The article argues that, in comparison with secularized, individualized, and commercialized festivals of contemporary South Korea, QCFs have retained the ritualism, communality, and subversiveness of traditional festivals—and this difference is due to queer participants realizing their yearning for a utopian world via their participation in QCF

Growth characterization of CHO DP-12 cell lines with different high passage histories

Heinrich C, Timo W, Christina K, Northoff S, Noll T. Growth characterization of CHO DP-12 cell lines with different high passage histories. In: Hansjörg H, ed. BMC Proceedings. BMC Proceedings. Vol 5. BioMed Central; 2011