Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments

An efficient method of formulating serum-free medium (SFM) for production of therapeutic antibodies by recombinant CHO (rCHO) cells was developed using two rCHO cell lines producing a therapeutic antibody. In this method, ten kinds of SFM were prepared by supplementing the basal SFM with statistically designed mixtures (total 5 g L^?1) of three non-animal-derived hydrolysates: yeastolate, soy hydrolysate, and wheat gluten hydrolysate. When the two rCHO cell lines were cultivated, the mixtures of soy hydrolysate and wheat gluten hydrolysate showed a positive effect on cell growth. On the other hand, the mixtures including a high portion of yeastolate significantly enhanced specific antibody productivity. To reconstitute the mixture ratios of the three hydrolysates for high growth and antibody production, the effect of each medium was analyzed by the statistical program Design-Expert®. The resulting medium gave a 1.9–3.3-fold increase in the maximum antibody concentration, compared to the basal SFM. Taken together, the supplementation of hydrolysates to the basal SFM with the help of statistical analysis is an efficient means of developing SFM for therapeutic antibody production by rCHO cells.     

Development of a serum-free medium for the production of humanized antibody from chinese hamster ovary cells using a statistical design

Summary To develop serum-free (SF) media for the production of humanized antibody from recombinant Chinese hamster ovary (rCHO) cells, a statistical optimization approach based on a Plackett-Burman design was adopted. A basal medium was prepared by supplementing α-minimal essential medium (α-MEM) with Fe(NO₃)₃·9H₂O, CuCl₂, ZnSO₄·7H₂O, and Na₂SeO₃ which are generally contained in SF medium formulations. Insulin, transferrin, and ethanolamine were also supplemented to the basal medium to determine their optimal concentrations. From this statistical analysis, serine, phenylalanine, and tyrosine were identified as important determinants for cell growth. Also, putrescine, linoleic acid, and hydrocortisone were shown to be important for both cell growth and antibody production. The SF medium was formulated by supplementing the basal medium with components showing positive effects on cell growth and/or antibody production. Cell growth and antibody production in this SF medium were comparable to those in α-MEM supplemented with 5% dialyzed fetal bovine serum. Taken together, the results obtained here show that a Plackett-Burman design facilitates the development of SF media for rCHO cells aimed at producing a humanized antibody.     

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product, particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and non-adapted CHO cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when cells were adapted to SFX-CHO^TM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in the enriched SFM^TM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells.     

The optimization of serum-free medium for the production of the scu-PA by the addition of algal extracts

The addition of 2.8 g/ml algal extracts enhanced both scu-PA production and cell growth in a serum-free medium, compared to a conventional serum-free medium for the cultivation of recombinant CHO cells. The growth rate and scu-PA production were relatively lower in the serum-free medium than 5% serum containing medium: however, specific scu-PA production rate was higher in the serum-free medium due to the long-term period of cultivation (3.66×10^–4 vs. 2.48×10^–4 IU/cell/day). Overall scu-PA production rate was also greater in an enforced serum-free medium as 25,000 IU/day over 50 d of perfusion cultivation. The conversion ratio of scu-PA to tcu-PA was greatly reduced in the serum-free medium during perfusion cultivation (10% compared to 20% conversion in a serum containing medium).     

表达抗-CD25单克隆抗体的GS-NS0骨髓瘤细胞无血清培养及代谢特性

抗-CD25单克隆抗体作为免疫抑制剂拥有广阔的市场前景和巨大的经济价值。本实验以表达抗?CD25单克隆抗体的GS-NS0细胞为研究对象,开发了支持其大规模培养和抗体表达的无血清低蛋白培养基,批培养最大活细胞密度和最大抗体浓度分别达3×106cells/mL和300mg/L以上,比商业无血清培养基(Excell 620+0.2% primatone)分别提高了100%和46%。通过批培养实验,研究了细胞的生长、葡萄糖和氨基酸代谢、以及产物表达特点,并揭示了批培养过程中初始葡萄糖浓度对GS-NS0细胞生长与代谢的影响规律。为优化GS-NS0细胞培养过程和抗CD25单抗成功迈向产业化提供了重要的科学依据。     

Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells

To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g l^–1 YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 g ml^–1, which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced q_hTPO (the specific rate of hTPO production). The supplementation of YH in SFM increased q_hTPO by 294% and extended culture longevity by >2 days if the culture was terminated at a cell viability of 50%. Furthermore, cell viability throughout the culture using YH-supplemented SFM was higher than that using any other hydrolysate-supplemented SFM tested, thereby minimizing degradation of hTPO susceptible to proteolytic degradation. In addition, YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells.     

Evaluation of recombinant human transferrin (DeltaFerrinTM) as an iron chelator in serum-free media for mammalian cell culture

DeltaFerrin^TM, a yeast-derived recombinant human transferrin produced by Delta Biotechnology Ltd. (Nottingham UK), was found to be a suitable replacement for holo human transferrin in serum-free culture media of the MDCK cell line (chosen because of its transferrin dependence) in short-term screening assays. Long-term subculture was achieved with DeltaFerrin^TM supporting growth equivalent to that of holo human transferrin. DeltaFerrin^TM and a selection of chemical iron chelators were found in short-term assays to be equivalent to holo human transferrin in supporting growth of MDCK, BHK-21-PPI-C16 and Vero-PPI. In long-term subcultures, however, only DeltaFerrin^TM was found to support cell growth in a manner essentially equivalent to holo human transferrin in all three cell lines. For both BHK and Vero variants tested, recombinant preproinsulin production was unaltered by replacing holo human transferrin with DeltaFerrin^TM. As such, this is the first report of a recombinant human transferrin produced under animal-free conditions that can act as a universal iron chelator for cells grown in serum-free media (SFM).     

Proteomic understanding of intracellular responses of recombinant Chinese hamster ovary cells cultivated in serum-free medium supplemented with hydrolysates

In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (μ, SFM#S1) or the highest specific antibody productivity (q _Ab, SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.     

Stimulation of monoclonal antibody production by human-human hybridoma cells with an elevated concentration of potassium or sodium phosphate in serum-free medium

Potassium or sodium phosphate was found to stimulate the production of human monoclonal antibody by human-human hybridoma HB4C5. The addition of 15 mM Na-phosphate (pH 7.4) into serum-free culture medium increased the antibody production up to 4-fold, when seeded at cell density of 1×10⁵ cells/ml in dishes. At the higher cell density of 5×10⁵ cells/ml, K-phosphate was more effective than Na-phosphate, at the same concentration. In large-scale continuous culture, the addition of 10 mM Na-phosphate into serum-free culture medium stimulated antibody production by HB4C5 cells 6-fold.     

Autophagy and apoptosis of recombinant Chinese hamster ovary cells during fed-batch culture: effect of nutrient supplementation

Upon nutrient depletion during recombinant Chinese hamster ovary (rCHO) cell batch culture, cells are subjected to apoptosis, type I programmed cell death (PCD), and autophagy which can be type II PCD or a cell survival mechanism. To investigate the effect of nutrient supplementation on the two PCDs and protein production in rCHO cells, an antibody-producing rCHO cell line was cultivated in batch and fed-batch modes. The feed medium containing glucose, amino acids, and vitamins was determined through flask culture tests and used in bioreactor cultures. In the bioreactor cultures, the nutrient feedings extended the culture longevity and enhanced antibody production. In addition, cells in the fed-batch culture showed delayed onset of both apoptosis and autophagy, compared with those in the batch culture. The inhibition of apoptosis was demonstrated by a decreased amount of cleaved caspase-7 protein and less fragmentation of chromosomal DNA. Concurrently, reduced LC3 conversion, from LC3-I to LC3-II, was observed in cells that received the feeds. Cultivation with pharmacological autophagy inducer (rapamycin) or inhibitor (bafilomycin A1) indicated that autophagy is necessary for the cells to survive under nutrient depletion. Taken together, the delayed and relieved cell death by nutrient supplementation could improve antibody production.     

LONGTMR3IGF-I as a more potent alternative to insulin in serum-free culture of HEK293 cells

LONG^TMR³IGF-I, an analogue of insulin-like growth factor (IGF)-I, was specifically engineered for use in biopharmaceutical protein production in mammalian cells. LONG^TMR³IGF-I is capable of supporting the growth and survival of Chinese hamster ovary cells in serum-free media at concentrations at least 200-fold lower than required for insulin. LONG^TMR³IGF-I also acts as a more potent growth and survival factor than either insulin or native IGF-I in SF culture of human embryonic kidney (HEK293) cells. To investigate the basis of the enhanced potency of LONG^TMR³IGF-I we have examined the mechanism of action of these mitogens in HEK293 cells. All mitogens tested were found to activate the TypeI IGF receptor (IGF-IR) and insulin receptor (IR) in a dose-responsive manner. However, the level of activation of both receptors after stimulation with LONG^TMR³IGF-I, at lower concentrations, was greater than with either insulin or IGF-I. The greater potency of LONG^TMR³IGF-I in activating the IR, despite having a low affinity for IRs, suggests the presence of heterotetrameric IGF-IR/IR dimers. Interestingly, the decrease in IGF-IR activation at higher concentrations of LONG^TMR³IGF-I suggests that the dose-response curve may be bell-shaped.     

Interleukin-6 is antiproliferative to a mouse hybridoma cell line and promotive for its antibody productivity

Monoclonal antibody production by hybridoma cells at moderately slowed growth states would be favorable for commercial scale production since cells can devote their resources to performing the differentiated function, immunoglobulin production. We found that a purified recombinant human interleukin-6, which had been reported to support or stimulate proliferation of B cell hybridoma/plasmacytoma cells, suppressed growth of a hybridoma cell line in serum-free medium. In the presence of the interleukin, the growth-suppressed cells were viable for remarkably long periods in batch culture, and after removal of the interleukin from the culture medium, they started to proliferate at their normal growth rate. As the concentration of the interleukin increased in the culture, the growth rate decreased and the specific antibody productivity (antibody production rate per cell) increased to 5-fold of control at 10 U ml^–1 (2 ng ml^–1) of the interleukin.Abbreviations IL-1,2, and 6 interleukin-1, 2 and 6 - rhIL-6 recombinant human interleukin-6 - MCAb monoclonal antibody - TNP trinitrophenyl - unit (U) of interleukin-6 A unit (U) is equivalent to the amounts of IL-6 which gives one-half maximal IgM secretion by SKW6-CL4 cells (1U ml^–1=200 pg ml^–1)     

Enhanced human thrombopoietin production by sodium butyrate addition to serum-free suspension culture of bcl-2-overexpressing CHO cells

When sodium butyrate (NaBu) was added to serum-free suspension culture of recombinant CHO (rCHO) cells for enhanced expression of human thrombopoietin (hTPO), apoptotic cell death of rCHO cells was induced in a dose-dependent manner and hTPO quality was deteriorated in regard to sialic acid and acidic isoform contents. To overcome these problems, we overexpressed Bcl-2 protein, an antiapoptotic protein, in rCHO cells producing hTPO. Compared to serum-free suspension culture of control cells without Bcl-2 overexpression (R-neo cells) and NaBu addition, a more than 10-fold increase in the maximum hTPO concentration was obtained in serum-free suspension culture of cells with Bcl-2 overexpression (R-bc12-14 cells) and 3 mM NaBu addition. Both the enhanced specific productivity endowed by NaBu and the extended culture longevity provided by the antiapoptotic effect of Bcl-2 overexpression contributed to the enhancement of maximum hTPO concentration. The problem of quality reduction of hTPO induced by NaBu was not solved by Bcl-2 overexpression, but it was not that significant. Compared to the culture in the absence of NaBu, the percentage of hTPO isoforms in pI 3-5 with high in vivo biological activity produced by R-bc12-14 cells was decreased by approximately 18% in the presence of 3 mM. As a result, a more than 6-fold increase in the production of hTPO isoforms in pI 3-5 was achieved in R-bcl2-14 cell culture with 3 mM NaBu addition. Taken together, the data obtained suggest that Bcl-2 overexpression in rCHO cells and NaBu addition in serum-free suspension culture can be an effective means to enhance the production of highly glycosylated protein such as hTPO.     

Application of a cell-once-through perfusion strategy for production of recombinant antibody from rCHO cells in a Centritech Lab II centrifuge system

Based upon the results of scale-down intermittent perfusion processes, a cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred to the centrifuge and a fixed percentage was removed. Approximately 99% of the viable cells are transferred to another bioreactor filled with fresh medium by single operation of the Centritech Lab II centrifuge system for about 30 min. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress, oxygen limitation, nutrient limitation, and low temperature outside the bioreactor. The effects of culture temperature shift and fortified medium on cell growth and recombinant antibody production in the COT perfusion process were investigated. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33 degrees C was 157.8 mg/L, approximately 2.4-fold higher than that at 37 degrees C. By the use of a fortified medium at 37 degrees C, rCHO cells were maintained at high density above 1.2 x 10(7) cells/mL, and antibody was produced continuously in a range of 260-280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration. It will be an attractive choice for minimization of cell damage and stable long-term antibody production with high cell density.     

抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究

用无血清培养基或化学成分明确的培养基生产治疗用重组蛋白已成为趋势。然而,在此条件下凝血因子、糖蛋白激素等微量糖蛋白的表达十分困难,其主要原因之一是在细胞培养过程中工程细胞大量凋亡造成的细胞密度低和生存期短。通过将早期抗凋亡基因导入工程细胞并进行过表达可改善工程细胞的活细胞密度积分（integral viable cell concentration,IVCC）,提高表达量。该研究将bcl-xl基因导入工程细胞,筛选其高表达细胞株,并验证工程细胞的抗凋亡能力,获得了稳定表达抗凋亡蛋白和目的蛋白的工程细胞株。与母细胞相比,稳定表达Bcl－xL的工程细胞的IVCC提高了50％,最终目的蛋白表达增加超过200％,显示抗凋亡基因bcl-xl的过表达可改善工程细胞在无血清悬浮培养过程中的细胞凋亡,提高表达量,为表达人凝血因子、糖蛋白激素等微量糖蛋白奠定了基础。     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H^* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10⁶ cells ml^–1 producing 76.6 mg l^–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C cholesterol - CD cyclodextrin - dhfr dihydrofolate reductase - F68 Pluronic F68 - GS glutamine synthetase - MSX methionine sulphoximine - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - ADCC Antibody-dependant cellular cytotoxicity     

Monitoring of autophagy in Chinese hamster ovary cells using flow cytometry

Recently, autophagy, which is a degradative process, has drawn attention as an anti-cell death engineering target in addition to apoptosis in recombinant Chinese hamster ovary (rCHO) cell cultures for enhanced production of therapeutic proteins. Appropriate autophagy monitoring methods, that are suitable for long term CHO cell cultures, are necessary in order to investigate the culture conditions that affect the autophagy pathway and to select appropriate engineering targets for autophagy control. Herein, detailed protocols for autophagy monitoring methods based on flow cytometry are provided using the GFP-LC3-overexpressing CHO DG44 host cell line or MDC-like molecules in rCHO cells grown as an adherent culture with serum-containing medium or suspension culture with serum-free medium. Furthermore, combined with the apoptosis detection based on the Annexin V-PS interaction, the simultaneous detection of autophagy and apoptosis is also described. It is anticipated that the protocols described herein will assist in the fast, high throughput monitoring of autophagy that can support other existing autophagy assays.     

Continuous production of large amounts of monoclonal immunoglobulins in hollow fibers using protein-free medium

The performance of a protein-free medium was compared in culture flasks with a serum-supplemented medium and with a serum free medium in terms of cell growth and monoclonal antibody production by a murine hybridoma. We present results of continuous production in hollow fiber culture systems using serum-free medium and protein-free medium. In protein-free medium, it has been possible to produce large quantities of monoclonal antibody with a productivity similar to that obtained in serum-free medium. After a two steps purification process, monoclonal antibodies were characterized by SDS-PAGE, High Performance Size Exclusion Chromatography and Free Solution Capillary Electrophoresis. SDS-PAGE and high performance chromatography analysis have showed that purified monoclonal antibodies produced in serum-free medium or protein-free medium were similar. Furthermore, Capillary Electrophoresis characterization revealed that both MAbs were constituted by three isoforms with equivalent electrophoretic mobilities.Abbreviations CHES 2-(N-Cyclohexylamino)ethane-sulfonic acid - ECS Extracapillary Space - FSCE Free Solution Capillary Electrophoresis - HPSEC High Performance Size Exclusion Chromatography - ICS Intracapillary Space - MAb Monoclonal Antibody - PFM Protein-Free Medium - SFM Serum-Free Medium - SSM Serum-Supplemented Medium     

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using balb/c 3T3 mouse embryo fibroblasts

Summary A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-d-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1∶1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 μg/ml human transferrin, 100 μg/ml ovalbumin, and 1.0 μM dexamethanose. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-d-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of bFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED₅₀), ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED₅₀, of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in cultures. This work was supported by a contract from IMCERA Bioproducts, Inc.