Pilot production of u-PA with porous microcarrier cell culture

A recombinant DNA CHO cell line secretingurokinase-type plasminogen activator (u-PA) wascultivated with Cytopore cellulose porousmicrocarriers in a 30l Biostat UC stirred tankreactor. After 26 days of culture, using a spinfilter toretain cells in bioreactor, the cell density couldreach 1.33 × 10⁷ ml^-1. The maximal u-PAactivity in supernatant was 7335 IU·ml^-1, and204l supernatant containing 7.1 g u-PA was harvested.After 100 days of culture with 0.1% fetal bovineserum medium, a modified cell retention system whichcan be washed-out backward, substituted thespinfilter to prevent filter clogging. The maximalcell density was over 10⁷ ml^-1, the maximalu-PA activity in supernatant reached 6250IU·ml^-1, and 1604l supernatant containing about51 g u-PA was harvested. Compared to perfusionculture, batch medium-replaced culture could raiseutilizing efficiency of the medium, increase cell specificproductivity and improve the quality of the product which wasnot steady in a 37 °C environment. Cells can movefrom seed porous microcarriers occupied by cells tovacant microcarriers spontaneously, withouttrypsinization, and continue to grow until all microcarriers contained cells. It shows that Cytoporeporous microcarriers are very useful and convenient toscale up cultivation step by step.     

用多孔微载体Cytopore高密度培养CHO工程细胞生产rHEPO

在２．２Ｌｃｅｌｉｇｅｎ灌注培养系统中采用Ｃｙｔｏｐｏｒｅ多孔微载体培养产重组人促红细胞生成素（ｒＨＥＰＯ）的细胞Ｃ２。细胞密度达到１×１０７／ｍＬ,ｒＨＥＰＯ最高可达９．７ｍｇ／Ｌ。该载体具有表面积高,使用浓度低,便于扩大培养的特点,便于进行大规模培养     

用天冬酰胺代替谷氨酰胺培养产尿激酶原CHO工程细胞

以产尿激酶原CHO工程细胞CL－11G的细胞生长和目的产物的表达为观察指标,对用天冬酰胺替代培养基中谷氨酰胺的可行性进行研究。结果表明,溶液中游离态的天冬酰胺的自发分解速度明显低于谷氨酰胺,用天冬酰胺替代培基中的谷氨酰胺培养11G细胞不影响细胞的生长和目的产物的表达,有助于克服因谷氨酰胺的自行分解而造成的培养基氨的过度积聚对细胞代谢的不利影响。     

Optimization of parameters affecting on CHO cell culture producing recombinant erythropoietin

Abstract

Several factors may affect erythropoietin (EPO) sugar structures including designing cell culture procedure, pH, concentration of additives, dissolved oxygen, and other physicochemical parameters. In this study, we investigated the influence of changes in effective parameters and compounds on the growth rate of Chinese hamster ovary cell (CHO) cells producing recombinant EPO. Cell culture was performed at different temperature, buffering conditions, and varied concentrations of additives such as pyruvic acid, insulin, GlutaMAX, and sodium butyrate. Results indicated that the optimal temperature and pH were 37?°C and 7.2, respectively. Also, optimal concentrations for pyruvic acid, butyrate, glutamate, and insulin were obtained to be 20?mM, 1?mM, 2?mM, and 40?μg/mL, respectively. Then, cell culture was performed in microcarrier-coated spinner flasks under the optimized condition. The results showed recombinant human EPO (rhEPO) production with adequate purity. Optimization of physicochemical conditions and culture media are important factors to improve the quantity and quality of protein products. This study showed that cell growth and recombinant EPO protein production significantly increased under the optimized conditions. The results of this research can also be used in scale-up to increase the efficiency of EPO production.

Abbreviations: EPO: erythropoietin; CHO cell: Chinese hamster ovary cell; rhEPO: recombinant human EPO; DMEM: modified eagle’s medium; FBS: fetal bovine serum; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; IGF-1: insulin-like growth factor 1     

Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: Improving the efficiency of cell line generation

Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide‐ranging transfectant population. This identification process is on the critical path for first‐in‐human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One‐hundred and seventy‐five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Glycolipid membrane anchored recombinant protein production from CHO cells cultGred on porous microcarriers

Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano-transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl-phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T-flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate-buffered saline (PBS) containing 10 mU/mL phosphatidylinositol-phospholipase C (PI-PLC). The GPI anchor was selectively cleaved by PI-PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 mug/mL due to difficulties in maintaining stable confluent T-flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 10(7) cell/mL. The p97 was harvested at up to 100 mug/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum. (c) 1994 John Wiley & Sons, Inc.     

A low-cost chemically defined protein free medium for a recombinant CHO cell line producing prothrombin

A chemically defined protein free medium, DF6S, was developed for the cultivation of a recombinant Chinese hamster ovary cell line (CHO2DS) producing human prothrombin in suspension batch culture. DF6S was formulated by optimizing DME/F12 with amino acids and supplementing the optimized DME/F12 with aurintricarboxylic acid, ethanolamine, ferric sulfate, Pluronic F68, putrescine and sodium pyruvate. From a seeding density of 2.3 × 10⁵ cells ml^–1, CHO2DS cells grown in suspension in DF6S medium reached a maximal cell density of 1.92 × 10⁶ cells ml^–1 with an accumulated prothrombin concentration of 16.7 mg l^–1 after 6 days in culture.     

免疫亲和层析法纯化单链尿激酶型纤溶酶原激活剂

尿激酶原 (Ｐｒｏ ｕｒｏｋｉｎａｓｅ ,ｐｒｏ ＵＫ) ,也称单链尿激酶型纤溶酶原激活剂 (Ｓｉｎｇｌｅ ｃｈａｉｎｕｒｏｋｉｎａｓｅ ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ｓｃｕ ＰＡ) ,与ｔ ＰＡ一样是第二代溶栓药物。给药时 ,保持无活性的酶原状态 ,只激活被纤维蛋白吸附的纤溶酶原 ,而对游离的纤溶酶原没有作用 ,即只在血栓表面才能活化转变为双链尿激酶 (Ｔｗｏ ｃｈａｉｎｕｒｏｋｉｎａｓｅ ｔｙｐｅｐｌａｓｍｉｎｏｇｅｎａｃｔｉｖａｔｏｒ,ｔｃｕ ＰＡ或ＵＫ) ,因而具有较高的特异性溶血栓作用[1 ] 。尿激酶原…     

High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures

A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 10⁷ cells per ml were obtained. The described system is scaleable.     

Mapping and partial characterization of proteases expressed by a CHO production cell line

The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12. Several species with metalloproteinase activity were characterized and found to be related to each other. The results indicate that an MMP pro-enzyme of >/=200 kDa was released from the CHO cells during the production phase. The enzyme expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a number of smaller, less specific MMPs were formed with the smallest form, a 19.4 kDa protein, being the most active. These results may be of particular relevance for other production processes using CHO cells for the expression of recombinant proteins.     

Production of recombinant Von Willebrand factor by CHO cells cultured in macroporous microcarriers

Recombinant Chinese hamster ovary cells producing Von Willebrand factor have been successfully grown in gelatin macroporous microcarriers (Cultispher-G). Serum-free cultures were maintained in 1, 4, and 10 liter fermentors for more than two months. Comparative studies with Cytodex-3 microcarriers have been performed in 1 liter fermentors. The lower specific Von Willebrand factor productivity of CHO cells cultivated on Cultispher-G were offset by higher cell densities (10⁷–2×10⁷ cells/ml). Volumetric Von Willebrand factor productivity was influenced by oxygen concentration, and remained stable during scale-up from 1 to 10 liter fermentors.     

Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line

The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day. This revised version was published online in August 2006 with corrections to the Cover Date.     

Functional heterogeneity and heritability in CHO cell populations

In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing—new parental cell lines which are inherently more “fit for purpose.” One‐hundred and ninety‐nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell‐specific proliferation rate during extended deep‐well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell‐specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub‐culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N‐glycan processing were identified. Finally, for clonal populations most “evolved” by extended sub‐culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N‐glycosylation. Rapid‐specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone‐specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and isolate CHO cell variants with improved functional properties in vitro. Biotechnol. Bioeng. 2013; 110: 260–274. © 2012 Wiley Periodicals, Inc.     

Long-term stable production of monocyte-colony inhibition factor (M-CIF) from CHO microcarrier perfusion cultures

Monocyte-colony inhibition factor (M-CIF) was produced in microcarrier perfusion cultures from engineered Chinese hamster ovary (CHO) cells. Three and fifteen liter microcarrier perfusion bioreactors equipped with internal spin filters were operated for over two months. Approximately 60 L and 300 L of culture filtrate were harvested from the 3L and 15L microcarrier perfusion bioreactors respectively. During the perfusion operation, cell density reached 2–6 × 10⁶ cells/ml. Importantly, stable expression of M-CIF from the CHO cells under non-selection condition was maintained at a level of 4–10 mg/L. Specific productivity was maintained at 1.8–3.4 mg/billion cells/day. The ability of the recombinant CHO cells to migrate from microcarrier to microcarrier under our proprietary HGS-CHO-3 medium greatly facilitated microcarrier culture scale-up and microcarrier replenishment. Future directions for microcarrier perfusion system scale-up and process development are highlighted. This revised version was published online in August 2006 with corrections to the Cover Date.     

Batch, fed-batch, and microcarrier cultures with CHO cell lines in a pressure-cycle driven miniaturized bioreactor

Miniaturized bioreactors for suspension cultures of animal cells, such as Chinese Hamster Ovary (CHO) cells, could improve bioprocess development through the ability to cheaply explore a wide range of bioprocess operating conditions. A miniaturized pressure-cycled bioreactor for animal cell cultures, described previously (Diao et al., 2008), was tested with a suspension CHO cell line producing commercially relevant quantities of human IgG. Results from the suspended CHO cell line showed that the cell growth was comparable to conventional flask controls and the target protein production was enhanced in the minibioreactor, which may be due to the relatively high oxygen transfer rate and the moderate shear stress, measured and simulated previously. Microcarrier culture using an anchorage-dependent CHO cell line and Cytodex 3 also showed a similar result: comparable growth and enhanced production of a model protein (secreted alkaline phosphatase or SEAP). Various fed-batch schemes were applied to the CHO cells producing human IgG, yielding cell numbers (1.1 × 10(7) /mL) at day 8 and titers of human IgG (2.3 g/L) at day 14 that are typical industrial values for CHO cell fed-batch cultures. The alteration of the volumetric oxygen transfer coefficient is a key parameter for viability of the CHO cell line producing human IgG. We conclude that the minibioreactor can provide favorable cell culture environments; oxygen transfer coefficient and mixing time can be altered to mimic values in a larger scale system allowing for potential prediction of response during scale-up.     

Two-stage depth filter perfusion culture for recombinant antibody production by recombinant Chinese hamster ovary cell

A rCHO cell line of DUKX origin 26*-320, producing recombinant antibody against the human platelet, was cultivated in a two-stage depth filter perfusion system (DFPS) for 20 days in order to attain high recombinant antibody concentration. The productivity of the first stage DFPS bioreactor reached 53 times that of the batch culture in a controlled stirred tank reactor and was showed 12.1 mg/L antibody concentration at a perfusion rate of 6.0 d⁻¹. Glucose concentration in the first DFPS was maintained at 1.5 g/L to avoid cell damage in the perfusion culture. A second stage DFPS system was attached to the first DFPS, which resulted in a low glucose concentration of 0.02 g/L and a high antibody concentration of 23.9 mg/L. The two-stage depth filter perfusion culture yielded 60% higher product concentration than the batch and 49-fold higher productivity of 69.3 mg/L/d in comparison with that (1.4 mg/L/d) in a batch system. Furthermore, antibody concentration of the second stage was 97% higher than that of the first stage, and the antibody productivities were comparable to that of the first stage. This two-stage DFPS system also showed potential for higher titer production of recombinant antibody and high volumetric productivity for long-term culture of bio-pharmaceutical substances.