Scale-up of suspension and anchorage-dependent animal cells

Alternative culture processes for laboratory scale-up (to 20 L) are described for both suspension and anchorage-dependent cells. Systems range from simple multiple culture units such as the roller bottle, through stirred suspension and microcarrier unit bioreactors, to highly sophisticated perfusion culture capable of maintaining cells at densities of about 10⁸/mL. Critical parameters in scale-up are discussed, and the advantages and disadvantages of each culture system are critically evaluated.     

A novel scale-up method for mammalian cell culture in packed-bed bioreactor

A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2×10⁷ cells ml^–1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.     

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…     

High-density culture of recombinant Chinese hamster ovary cells producing prothrombin in protein-free medium

A recombinant CHO cell line, CHO2DS, was immobilized on porous microcarrier Cytopore 1 and cultivated in 1 l modified Super-spinner and 2 l stirred tank bioreactor with the perfusion of a low-cost chemically defined protein-free medium DF6S. CHO2DS cells could enter into the inner space and grew both in the inner space and on the surface of Cytopore 1 in DF6S and produced prothrombin at 22 mg l^–1 after 10 days. From a seeding density of 5.7 × 10⁵ cells ml^–1, the highest viable cell density of CHO2DS was 1.12 × 10⁷ cells ml^–1.     

CHO细胞无血清结团灌流培养：超声-沉降柱二合一灌流系统

利用CHO细胞能在培养过程中自然结团的特性,采用超声—沉降柱二合一灌流系统能促进细胞结团和加强截留的特性,我们用无血清培养基连续灌流培养基因重组CHO细胞MK3-A2株,分泌表达rhTNK-tPA获得了成功。培养周期为77-110天,细胞结团率为90%左右,直径在285～570μm之间,细胞截留率保持在95%左右,成活率为85%以上,细胞密度达到2×107/ml左右,rhTNK-tPA生产率平均为89 mg/L/d,最高时达216mg/L/d。 结果表明,使用该灌流系统进行细胞结团培养可以取代微载体培养用于动物细胞制药的规模化生产。     

WAVETM生物反应器灌注培养生产种子细胞的开发和应用

近年来,国内中国仓鼠卵巢细胞(Chinese hamster ovary,CHO)生产罐的培养规模已达上千升,国外已达上万升,最终的生产罐前需要多级摇瓶、种子罐进行种子细胞扩增,扩增效率较低,严重影响了抗体、融合蛋白等生物制品的生产效率。文中利用WAVETM波浪式生物反应器,通过灌注培养的方法,成功地实现了种子细胞的高效扩增。WAVETM波浪式生物反应器灌注培养方法制备种子细胞,CHO细胞密度高达2.28×107cells/mL时仍处于指数生长期且细胞活力大于95%,以此细胞作为种子细胞,4×105cells/mL接种于另一个WAVETM生物反应器进行流加培养,最大活细胞密度仍可达1.73×107cells/mL。通过此种扩增方式,1台WAVETM20/50即可为1 000 L或2 000 L的生产罐提供种子细胞,种子细胞的扩增倍数(Split ratio)可以达到1∶50～1∶100倍,与传统不锈钢罐种子细胞扩增倍数1∶2～1∶10相比,可以显著减少2～3级种子罐,种子细胞的扩增时间减少7～9 d,极大地提高生产效率。     

A cell-detaching reactor for inoculation of anchorage-dependent CHD and Vero cells between stepwise-expanded bioreactors

A cell-detaching reactor was developed to collect cells growing on microcarriers for inoculation between stepwise-expanded bioreactors. It consisted of a trypsinization zone and a separation zone, which were separated by a 200-mesh stainless steel screen. The screen allowed the cells only to pass through to the next bioreactor, after the cells have been trypsinized and detached from microcarriers. The operating feasibility of the cell-detaching reactor was tested with anchorage-dependent recombinant Chinese hamster ovary (rCHO) and African green monkey kidney (Vero) cells. rCHO and Vero cells were first cultured in a small microcarrier bioreactor, and then inoculated via the cell-detaching reactor into either a packed-bed bioreactor (for rCHO cells) or a larger microcarrier bioreactor (for Vero cells). For rCHO cells, the cell density reached 1.3 × 10⁷ cells/ml in the perfusion culture, and Vero cells reached 1.3 × 10⁶ cells/ml in the batch culture.     

Evaluation of a microcarrier process for large-scale cultivation of attenuated hepatitis A

Microcarrier culture was investigated for the propagation of attenuated hepatitis A vaccine in the anchorage-dependent human fibroblast cell line, MRC-5. Cells were cultivated at 37°C for one to two weeks, while virus accumulation was performed at 32°C over 21 to 28 days. The major development focus for the microcarrier process was the difference between the cell and virus growth phases. Virus antigen yields, growth kinetics, and cell layer/bead morphology were each examined and compared for both the microcarrier and stationary T-flask cultures. Overall, cell densities of 4–5×10⁶ cells/ml at 5–10 g/l beads were readily attained and could be maintained in the absence of infection at either 37°C or 32°C. Upon virus inoculation, however, substantial cell density decreases were observed as well as 2.5 to 10-fold lower per cell and per unit surface area antigen yields as compared to stationary cultures. The advantages as well as the problems presented by the microcarrier approach will be discussed.     

A microcarrier culture method for the production of large quantities of viable Chlamydia pneumoniae

 We studied the propagation of Chlamydia pneumoniae strain TW-183 in HEp2 cells grown on microcarrier beads. Infection of the cells in microcarrier culture was optimized by addition of 7.5% polyethylene glycol 4000 (PEG4000) during adsorption. The yield in microcarrier culture was similar to that of microtitre-plate culture using centrifugation-assisted infection (120×10⁶ and 225×10⁶ bacteria/10⁶ HEp2 cells respectively), as was the burst size (505 and 449 bacteria produced/infecting bacterium respectively). However, up to 64% savings in labour time and 27% savings in culture medium were achieved if the microcarrier culture method was used instead of the microtitre-plate culture method. The optimal yield of viable bacteria could only be achieved at a narrow range of multiplicities of infection (0.24–1.14 inclusion-forming units/cell), independent of the mode of infection (centrifugation-assisted infection or PEG4000-facilitated infection by adsorption) and independent of incubation temperature (35°C or 37°C). The yield of microcarrier cultures was the same at an incubation temperature of 35°C or 37°C in contrast to an increased production at 35°C in the microtitre-plate culture method using centrifugation-assisted infection. In conclusion, the microcarrier culture method is useful to produce large quantities of viable Chlamydia pneumoniae economically. Received: 27 December 1995/Received revision: 4 April 1996/Accepted: 15 April 1996     

Process intensification in fed-batch production bioreactors using non-perfusion seed cultures

ABSTRACT

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.     

Defined Essential 8? Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm² of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x10⁶ cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.     

Microtuberization in potato (Solanum tuberosum L.)

Twenty-two genotypes of potato (Solanum tuberosum L.) were induced to form microtubers under six in vitro culture conditions. Cultures maintained under a short photoperiod (10 h of 6–12 μmol m^–2 s^–1) and low temperatures (day 20°±2°C and night 18°±2°C) had both a higher yield (255 mg/plantlet) and a greater number (2/plantlet) of microtubers than those maintained under long days (16 h of 38–50 μmol m^–2 s^–1) combined with high temperatures (day 28°±2°C and night 25°±2°C) (yield 207 mg/plantlet; microtuber number, 0.9/plantlet), over a wide range of genotypes. After the plantlets had been cultured under long days for an initial period of 60 days, continuous darkness advanced microtuberization by 2–3 months in various genotypes. Under short-day and low-temperature conditions the addition of 6-benzylaminopurine increased microtuber yield from 255 mg/plantlet to 645 mg/plantlet and average microtuber weight from 115 mg to 364 mg. A similar pattern was observed under conditions of long days and high temperature, and continuous darkness and low-temperature. Microtubers produced under light had a greater number of eyes (maximum average: 5.96/microtuber) than those produced in the dark (maximum average: 3.50/plantlet). The genotype × cultural conditions interactions were significant indicating the importance of developing genotype-specific protocols to maximize microtuberization. Received: 17 September 1997 / Revision received: 12 December 1997 / Accepted: 1 January 1998     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

The effect of calcium on flavanol production in cell suspension cultures of Polygonum hydropiper

Cultured Polygonum hydropiper cells maintained in Murashige and Skoog (MS) medium supplemented with 10^–6 m 2,4-D, 10^–6 m kinetin, 0.1% casamino acids and 3% sucrose were transferred to medium containing a higher concentration of calcium chloride (15 mm). The content of flavanols in the cells on the 6th day was approximately twice that of the control culture (31.9–60.7 mg/g dry wt). However, the contents of other secondary metabolites such as chlorogenic acid and gallic acid were not changed. The levels of flavanols in the culture medium remained unchanged throughout the 21-day culture period. Of the the inorganic components supplemented to the culture medium , only elevated levels of calcium chloride induced an increase in flavanol contents of the cells. The results indicated that the elevated concentration of calcium in the culture medium played an important role in activating the accumulation of flavanols. Received: 4 June 1998 / Revision received: 30 October 1998 / Accepted: 29 November 1998     

Differential stability of a human mini-chromosome in mouse cell lines

A 4 Mb human mini-chromosome, ΔΔ2, was transferred from Chinese hamster ovary (CHO) cells into a mouse L cell line. The mini-chromosome could be transferred intact into the L cells, with 112/119 clones maintaining a mini-chromosome of the same size as the original. Ten clones were grown for 30 days in continuous culture. The mini-chromosomes were maintained stably with or without selection at a copy number of 1–2 per cell and none experienced any size alterations, as determined by pulsed-field gel electrophoresis. Thus ΔΔ2 is structurally and mitotically stable in L cells. This contrasts with results in embryonic stem cells, in which ΔΔ2 is highly unstable. These findings indicate that established somatic cell lines, such as L cells and CHO cells, have less stringent controls over centromeric function than do normal embryonic cells. Received: 4 December 1997; in revised form: 25 February 1998 / Accepted: 14 April 1998     

High density culture of hybridoma cells in a dual hollow fiber bioreactor

Summary Hybridoma cells were cultured for two months in the dual hollow fiber bioreactor (DHFBR) which had been successfully used for high cell density cultures of various microbial cells. In batch suspension culture the concentration of monoclonal antibody (Mab) against human Chorionic Gonadotropin (hCG) and the cell density of Alps 25-3 hybridoma cells were obtained in 30 μg/mL and 2.35×10⁶ cells/mL, respectively. The continuous culture with DHFBR produced Mab of 100–130 μg/mL for 30 days and the estimated cell density in the extracapillary space of DHFBR was 1.87×10⁸ cells/mL based on the antibody production rate. The productivity of Mab was 205 mg/day per litre of the total reactor volume while that of the batch suspension culture was only 10 mg/L day.     

Enhanced Production of Human Recombinant Proteins from CHO cells Grown to High Densities in Macroporous Microcarriers

Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (β-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both β-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q _P) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q _P with increasing cell densities up to a threshold of >1 × 10⁸ cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q _P any further. A positive linear correlation (r ² = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 10⁶ to 3 × 10⁸ cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.     

Overexpression of survivin and cyclin D1 in CHO cells confers apoptosis resistance and enhances growth in serum-free suspension culture

To optimize Chinese Hamster Ovary (CHO) cell culture to recombinant protein therapeutic production, we stably overexpressed survivin and cyclin D1 in three CHO DG44-derived cell lines. The modifications conferred increases of 56–94% in S-phase fractions and decreases of 33–43% in early-stage apoptosis fractions. Clone 6.3, which expressed the highest levels of survivin and cyclin D1, reached significantly greater cell densities in suspension (2.7 × 10⁶ cells/ml) following serum deprivation. Nude mice inoculated with the modified cells showed no tumorigenesis suggesting that the CHO DG44-derived cell lines are viable candidates for biopharmaceutical production.     

Picophytoplankton abundance in the Velikaya Salma strait,White Sea

In July-August 2009, the abundance of picophytoplankton (Pico) in the Velikaya Salma strait varied from 3.4 × 10⁶ to 19.4 × 10⁶ cells/L, while its biomass (B) was 0.8–3.3 mg C/m³. In August 2010, Pico abundance was significantly higher (up to 216 × 10⁶ cells/L and 36.8 mg C/m³). Pico consisted mainly of cyanobacteria. It constituted 13 (2009) to 28% (2010) of the total phytoplankton biomass. In April 2010, Pico numbers varied from 0.1 × 10⁶ to 0.22 × 10⁶ cells/L and its biomass was 0.05–0.28 mg C/m³. Picoeukaryotes were predominant. Pico constituted not more than 2.7% of the phytoplankton biomass. In the ice column, the integrated Pico abundance was 430 × 10⁶ cells/m² and the integrated biomass was 365 μg C/m².     

The potential of hydrodynamic damage to animal cells of industrial relevance: current understanding

Suspension animal cell culture is now routinely scaled up to bioreactors on the order of 10,000 L, and greater, to meet commercial demand. However, the concern of the ‘shear sensitivity’ of animal cells still remains, not only within the bioreactor, but also in the downstream processing. As the productivities continue to increase, titer of ~10 g/L are now reported with cell densities greater than 2 × 10⁷ cells/mL. Such high, and potentially higher cell densities will inevitably translate to increased demand in mass transfer and mixing. In addition, achieving productivity gains in both the upstream stage and downstream processes can subject the cells to aggressive environments such as those involving hydrodynamic stresses. The perception of ‘shear sensitivity’ has historically put an arbitrary upper limit on agitation and aeration in bioreactor operation; however, as cell densities and productivities continue to increase, mass transfer requirements can exceed those imposed by these arbitrary low limits. Therefore, a better understanding of how animal cells, used to produce therapeutic products, respond to hydrodynamic forces in both qualitative and quantitative ways will allow an experimentally based, higher, “upper limit” to be created to guide the design and operation of future commercial, large scale bioreactors. With respect to downstream hydrodynamic conditions, situations have already been achieved in which practical limits with respect to hydrodynamic forces have been experienced. This review mainly focuses on publications from both the academy and industry regarding the effect of hydrodynamic forces on industrially relevant animal cells, and not on the actual scale-up of bioreactors. A summary of implications and remaining challenges will also be presented.