Evaluation of production of recombinant human interleukin-2 in fluidized bed bioreactor

The production of recombinant human interleukin-2 in a fluidized bed bioreactor containing porous glass carriers is described. Cultivations were carried out with different medium formulations over 80 days. Maximal cell densities and product yield could be maintained even when protein free medium was perfused, with less than 10% cell washout. Due to this effective immobilization of the cells in the reactor, continuous operation was easy to perform. Final cell densities on the order of 3.8 x 10(8) mL(-1) intrasphere volume were reached while the interleukin-2 production rate was 0.75 mg L(-1) d(-1). The production rate showed a maximum of a 1.9 fold decrease compared with a homogeneous stirred bubble-free aerated system. This result was in contrast to that achieved with hybridoma cell lines, where better performance was obtained with the fluidized bed bioreactor. The situation may reflect the problems caused by the dense cell culture with adherent cells, as previously shown in a hollow-fiber bioreactor with the same cell line.     

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.     

Growth kinetics of Chinese hamster ovary cells in a compact loop bioreactor. 3. Selection and characterization of an anchorage-independent subline and medium improvement.

Four sublines of Chinese hamster ovary (CHO) cells were selected or cloned on a 10% fetal calf serum supplemented MEM-alpha medium. Three of them were monolayer cultures and could proliferate by 2000 times a week (mu = 1.1 d 1) in T-flasks. The other subline, S1, could grow in suspension even in static T-flask cultures. The stability in chromosome number of these cell lines was investigated. By evaluating the kinetic growth parameters, i.e. the specific rates of growth, glucose consumption and lactic acid production, and the yields of cells and lactic acid from glucose, the S1 cells were considered to be the most suitable subline for the bioreactor suspension culture. The S1 cells reached the greatest maximum of cell concentration among all cell lines tested because of their efficient glucose utilization. Observed nutrient limitations in the S1 cell culture was overcome by modification of the medium composition, that is addition of 10 mg l-1 hypoxanthine, 1 mg l-1 FeSO4.7H2O, and 0.1 mg l-1 sodium putrescine, elimination of glutamine, supplementation of 6 mM asparagine and double amount of isoleucine, leucine, methionine and vitamins other than ascorbic acid, cyanocobalamin and biotin, increase of NaHCO3 concentration from 26 to 40 mM, and finally decrease of NaCl concentration from 122 to 100 mM. With this modified medium, 7.2 X 10(6) ml-1 of the maximum cell concentration was observed in a glucose fed-batch culture, the cell concentration which was twice as much as in batch cultures with the original medium.     

Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.

An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids     

Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor

Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.     

Evaluation of a system producing the hemopoietic factor. WEHI-3B cell line function, when encapsulated in a polypropylene hollow fibre

The purpose of our study was evaluation of functioning of WEHI-3B (an mouse cell line producing IL-3) cells encapsulated in hollow fibers (HF). In vitro: the WEHI-3B cells were encapsulated in HF of polypropylene K600 silikonized, and cultured over two weeks. In vivo: the encapsulated WEHI-3B after weeks culture, were implanted subcutaneously into mice for 1 week. After explantation encapsulated WEHI 3-B were cultured again in culture medium for one week. The production of IL-3 by encapsulated WEHI-3B cells was assessed by evaluation of IL-3 dependent, BaF3 cells viability. The percent number of one day survival of BaF3 cells in the culture medium supplemented with 15% of encapsulated WEHI-3B in vitro or encapsulated WEHI-3B after in vivo conditioned medium was comparable with positive control. Possible replacement of recombinant cytokines with HF encapsulated cytokine-producing cells may be a chance for continous supplementation of the factors for hematopoietic stem cells differentiation.     

Induction of LAK cells by high density dialyzing culture device and its cytotoxicity

Lymphokine activated killer (LAK) cells are generated by culture of lymphocytes with interleukin 2 (IL-2) in short term culture (3 to 5 days) and are used for adoptive immunotherapy for advanced cancer patients. The culture condition hitherto reported are essentially based on the rotating culture system, in which the maximum cell density was at 2 X 10(6) cell/ml and the cell recovery was usually less than 100%. The inability to induce LAK cells efficiently in vitro made the culturing of cells for therapy rather difficult and costly work because the mean infusion dose of LAK cells of one patient requires more than 1 X 10(10)/ml. We have therefore attempted to culture lymphocytes in 10 times higher concentration comparing with conventional methods. By using a new dialyzing culture system under continuous regulation of the amount of infused IL-2, nutrition medium, and pO2 and pCO2, we could culture cells at 2 X 10(7)/ml for more than 21 days and the resulted LAK cells showed a 100 times increase of activity on a per cell basis. By limiting dilution procedure, these killer cells mostly express T cell markers such as CD3 and CD8 but dose not express CD16.     

Optimal nitrogen supply as a key to increased and sustained production of a monoclonal full‐size antibody in BY‐2 suspension culture

Plant cell cultures have been used as expression hosts for recombinant proteins for over two decades. The quality of plant cell culture‐produced proteins such as full‐size monoclonal antibodies has been shown to be excellent in terms of protein folding and binding activity, but the productivity and yield fell short of what was achieved using mammalian cell culture, in which the key to gram‐per‐liter expression levels was strain selection and medium/process optimization. We carried out an extensive media analysis and optimization for the production of the full‐size human anti‐HIV antibody 2G12 in N. tabacum cv. BY‐2. Nitrogen source and availability was found to be one key factor for the volumetric productivity of plant cell cultures. Increased amounts of nitrate in the culture medium had a dramatic impact on protein yields, resulting in a 10–20‐fold increase in product accumulation through a combination of enhanced secretion and higher stability. The results were scalable from shake flasks to stirred‐tank bioreactors, where the maximum yield per cultivation volume was 8 mg L^?1 over 7 days. During the stationary phase, antibody levels were 150‐fold higher in nitrogen‐enriched medium compared to standard medium. The enhanced medium appeared not to affect antibody quality and activity, as determined by Western blots, surface plasmon resonance binding assays and N‐glycan analysis. Biotechnol. Bioeng. 2010;107: 278–289. © 2010 Wiley Periodicals, Inc.     

Insect cell growth evaluation during serum-free adaptation in stirred suspension cultures

Sf-9 insect cells were adapted to three different serum-free media (SF900II, EXCELL 401 and IPL/41 supplemented) in 125 ml stirred vessels by gradually reducing serum concentration from 10 to 0% (v/v). TC100 medium sup-plemented with 10% fetal bovine serum was used as control. With this procedure it was possible to obtain cells fully adapted to SF900II and EXCELL 401 in 5 weeks. The adapted cells could be frozen in serum-free medium and thawed without any decrease in specific growth rate or maximum cell concentration. Even after 4 months of culture in stirred vessels at 170 rpm the specific growth rate and maximum cell concentration (0.031 h and 4.8 × 10 cells/ml, respectively) remained constant.     

Improved paclitaxel and baccatin III production in suspension cultures of Taxus media

A cell suspension culture of Taxus media was established from a stable callus line of this species. The growth rate and production of paclitaxel and baccatin III of this cell suspension were significantly increased during the shake flask culture in its respective optimum media for cell growth and product formation, which were selected after assaying 24 different culture media. The highest yields of paclitaxel (2.09 mg L(-1)) and baccatin III (2.56 mg L(-1)) in the production medium rose (factors of 7.0 and 3.0, respectively) in the presence of methyljasmonate (220 microg g(-1) FW). When the elicitor was added together with mevalonate (0.38 mM) and N-benzoylglycine (0.2 mM), the increase in the yields of paclitaxel and baccatin III was even higher (factors of 8.3 and 4.0, respectively). Thereafter, a two-stage culture for cell suspension was carried out using a 5-l stirred bioreactor running for 36 days, the first stage being in the cell growth medium until cells entered their stationary growth phase (12 days) and the second stage being in the production medium supplemented with the elicitor and two putative precursors in the concentrations indicated above. Under these conditions, 21.12 mg L(-1) of paclitaxel and 56.03 mg L(-1) of baccatin III were obtained after 8 days of culture in the production medium.     

深山含笑的组织培养和快速繁殖

组织培养深山含笑的实验结果表明：利用休眠芽和种子萌发时实生苗的上胚轴及下胚轴为外植体均能诱导出愈伤组织和不定芽,其中无菌实生苗的上胚轴最易诱导出不定芽,无菌实生苗的下胚轴最易诱导出愈伤组织。外体植体在ＭＳ＋１．０－２．０ｍｇ Ｌ＾－１ ２,４－Ｄ培养基上只产生愈伤组织;在ＭＳ＋３．０ｍｇ Ｌ＾－１ ＢＡ＋０．２ｍｇ Ｌ＾－１ ＮＡＡ培养基上产生较多的不定芽和较多的愈伤组织;在ＭＳ＋２．０ｍｇ Ｌ＾     

Glycosylation of an immunoglobulin produced from a murine hybridoma cell line: the effect of culture mode and the anti-apoptotic gene, bcl-2

The impact of bcl-2 over-expression on the glycosylation pattern of an antibody produced by a bcl-2 transfected hybridoma cell line (TB/C3.bcl-2) was investigated in suspension batch, continuous and high cell density culture (Flat hollow fibre, Tecnomouse system). In all culture modes bcl-2 over-expression resulted in higher cell viability. Analysis of the glycans from the IgG of batch cultures showed that >95% of the structures were neutral core fucosylated asialo biantennary oligosaccharides with variable terminal galactosylation (G0f, G1f and G2f) consistent with previous analysis of glycans from the conserved site at Asn-297 of the IgG protein. The galactosylation index (GI) was determined as an indicator of the glycan profile (=(G2 + 0.5* G1)/(G0 + G1 + G2)). GI values in control cultures were comparable to bcl-2 cultures during exponential growth (0.53) but declined toward the end of the culture when there was a loss in cell viability. Low dilution rates in chemostat culture were associated with reduced galactosylation of the IgG glycans in both cell lines. However, at the higher dilution rates the GI for IgG was consistently higher in the TB/C3.bcl-2 cultures. In the hollow fibre bioreactor the galactosylation of the IgG glycans was considerably lower than in suspension batch or continuous cultures with GI values averaging 0.38. Similar low galactosylation values have been found previously for high density cell cultures and these are consistent with the low values obtained when the dissolved oxygen level is maintained at a low value (10%) in controlled suspension cultures of hybridomas.     

High-density tissue-like cultivation of JAR choriocarcinoma cells for the in vitro production of human xylosyltransferase

Human xylosyltransferase is the chain-initiating enzyme involved in the biosynthesis of glycosaminoglycans. Large amounts of xylosyltransferase are required to study the biochemical properties of the native enzyme. To achieve this goal a scale-up of animal cell culture systems was inevitable due to the small amounts of the enzyme present in tissues, e.g. only 0.5 microg XT can be obtained from a chick embryo. JAR choriocarcinoma cells cultured with 10% fetal calf serum were found to secrete xylosyltransferase with relatively high activities (1.10 mU l(-1)). To reduce contaminating proteins JAR cells were adapted to serum-free conditions. Xylosyltransferase activities up to 0.22 mU l(-1) were determined in the harvested cell culture supernatant. Scaling-up of JAR cell culture in the hybrid hollow fiber bioreactor Tecnomouse resulted in the production of 15.8 mU or 270 microg XT in 0.5 l of XT-enriched cell culture supernatant using 57 l of serum-free cell culture medium. The XT activity per ml harvest solution was 200-280-fold higher in this cell culture supernatant than in cell culture flasks. In addition, the specific XT activity of the bioreactor product was 6 microU mg(-1) of total protein, which is 2-fold higher than that obtained under static culture conditions. This study clearly demonstrates the successful high-density, tissue-like cultivation of JAR choriocarcinoma cells in a hollow fiber bioreactor resulting in an effective production of native human xylosyltransferase.     

用改装的Ｓuper—Spinner搅拌瓶连续灌流培养产凝血酶原ＣＨＯ工程细胞

在动物细胞培养过程中对培养体系实施培基连续灌流能及时地补充细胞生长所需的营养物质、控制细胞代谢产物对细胞的影响 ,实现细胞的高密度长期培养 ,提高目的产品的生产效率[1,2 ] 。细胞连续灌流培养的前提是在实施培基连续灌流的同时培养体系能有效地截留细胞[3] 。这一前提增加了细胞培养装置的复杂程度 ,使之特化为价格昂贵的生物反应器 ,限制了细胞连续灌流培养的应用。如能通过对普通的细胞搅拌培养瓶进行改进 ,使之能用于细胞的连续灌流培养 ,则有利于细胞连续灌流培养的推广应用。1　材料和方法1 1　细胞产人重组凝血酶原ＣＨＯ工…     

Ex vivo expansion of bone marrow mesenchymal stem cells using microcarrier beads in a stirred bioreactor

Bone marrow mesenchymal stem cells (bmMSCs) have recently gained attention as a useful resource in the fields of regenerative medicine and tissue engineering. However, the number of bmMSCs obtained from available donors is very low. Here we developed a culture strategy for in vitro expansion of bmMSCs in a 1.5 L stirred bioreactor with microcarrier beads. First, the microcarriers (Cytodex 3) were equilibrated in culture medium containing 3% fetal bovine serum (FBS) for at least 30 min prior to cell addition. After inoculation, the FBS concentration of the medium was maintained at 3% (v/v) in the first 24 h and thereafter maintained at 1% (v/v) and a developed feeding regimen was applied over 5 days. The maximum cell density of 2.6 × 10⁶ cells/mL was achieved at day 5, corresponding to a 10.4 ± 0.8 fold increases in total cell number. Among the harvested cells, 98.95% expressed CD29 and 84.48% expressed CD90, suggesting that the majority of expanded bmMSCs still retained their differentiation potential. Therefore, the developed microcarrier-based stirred bioreactor culture system is an effective method to generate significant numbers of bmMSCs for potential applications and research studies.     

Suspended aggregates as an immobilization mode for high-density perfusion culture of HEK 293 cells in a stirred tank bioreactor

Cells of the human embryonic kidney cell line (HEK 293) grown in repeated suspension and perfusion systems were characterized and described. Cell aggregates that formed immediately after the HEK 293 cells were inoculated in stirred vessels in serum-containing Dulbecco’s modified Eagle’s medium (D-MEM)/F-12 medium. The mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 63 to 239 μm after 1 and 8 days of culture in spinner flasks, respectively. No significant differences in cell performance were observed between HEK 293 cell populations grown as suspended aggregates and those grown as anchored monolayers. Replacing the D-MEM/F-12 with CD 293 medium caused the compact spherical cell aggregates to dissociate into single cells and small irregular aggregates without any apparent effect on cell performance. Moreover, the spherical cell aggregates could reform from individual cells and small aggregates when exposed to the serum-containing D-MEM/F-12 dominant medium. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5-l stirred tank bioreactor for 17 days resulted in a maximum viable cell density of 1.2×10⁷ cells ml⁻¹. These results demonstrate the feasibility and proof-of-concept for using aggregates as an immobilization system in large-scale stirred bioreactors because a small-scale culture can be used as easily as the inoculum for larger bioreactors.The first two authors contributed equally to this work.     

Scale-up of mouse embryonic stem cell expansion in stirred bioreactors

The aim of this study was to develop a robust, quality controlled and reproducible large-scale culture system using serum-free (SF) medium to obtain vast numbers of embryonic stem (ES) cells as a starting source for potential applications in tissue regeneration, as well as for drug screening studies. Mouse ES (mES) cells were firstly cultured on microcarriers in spinner flasks to investigate the effect of different parameters such as the agitation rate and the feeding regimen. Cells were successfully expanded at agitation rates up to 60 rpm using the SF medium and no significant differences in terms of growth kinetics or metabolic profiles were found between the two feeding regimens evaluated: 50% medium renewal every 24 h or 25% every 12 h. Overall, cells reached maximum concentrations of (4.2 ± 0.4) and (5.6 ± 0.8) ×10(6) cells/mL at Day 8 for cells fed once or twice per day; which corresponds to an increase in total cell number of 85 ± 7 and 108 ± 16, respectively. To have a more precise control over culture conditions and to yield a higher number of cells, the scale-up of the spinner flask culture system was successfully accomplished by using a fully controlled stirred tank bioreactor. In this case, the concentration of mES cells cultured on microcarriers increased 85 ± 15-fold over 11 days. Importantly, mES cells expanded under stirred conditions, in both spinner flask and fully controlled stirred tank bioreactor, using SF medium, retained the expression of pluripotency markers such as Oct-4, Nanog, and SSEA-1 and their differentiation potential into cells of the three embryonic germ layers.     

Effect of bench-scale culture conditions on murine IgG heterogeneity

A stable murine hybridoma cell line, secreting IgG1 antibodies (7H3) against the soluble type I receptor for Tumor Necrosis Factor (sTNF-R1), was cultivated in two different bioreactor systems, a hollow fiber and a stirred tank fermentor, in order to evaluate the effect of culture conditions on antibody structural and functional heterogeneity. Conventional serum-supplemented and serum-free media were chosen for fermentation in stirred tank bioreactor, whereas only serum-supplemented media were used for hollow fiber cultivation. Extent of glycosylation, determined by lectin binding assays, and charge heterogeneity of murine monoclonal antibodies displayed relevant variations according to the fermentation system used. After complete sugars removal by N-glycosidase F treatment, charge heterogeneity were still observed suggesting the occurrence of additional modifications at the protein level. In vitro culture in serum-supplemented media carried out with the hollow fibre system led to higher productivity but greater antibody charge heterogeneity and differences in lectin-binding profile than cultivation in the stirred tank bioreactor.Results cumulatively indicated that hybridoma cultivation methods, but also cultivation time, influence antibody heterogeneity, both in the protein and sugar moieties. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 17-25, 1997.     

Continuous production of diatom Entomoneis sp. in mechanically stirred tank and flat-panel airlift photobioreactors

Continuous production of diatom Entomonies sp. was performed in mechanically stirred tank and flat-panel airlift photobioreactors (FPAP). The maximum specific growth rate of diatom from the batch experiment was 0.98 d^?1. A series of dilution rate and macronutrient concentration adjustments were performed in a stirred tank photobioreactor and found that the dilution rate ranged from 0.7 to 0.8 d^?1 and modified F/2 growth media containing nitrate at 3.09?mg N/L, phosphate at 2.24?mg P/L, and silicate at 11.91?mg Si/L yielded the maximum cell number density. Finally, the continuous cultivation of Entomonies sp. was conducted in FPAP using the optimal conditions determined earlier, resulting in the maximum cell number density of 19.69?×?10⁴ cells/mL, which was approximately 47 and 73% increase from the result using the stirred tank photobioreactor fed with modified and standard F/2 growth media, respectively.