Equipment design considerations for large scale cell culture

Equipment design is frequently recognized as a key component in the success of GMP biologics manufacturing, but is not always implemented with full appreciation of the processing implications. In the case of mammalian cell culture, there are some recognized issues and risks that develop when transitioning to a large scale of operation. The developing demand for cell culture production capacity in the biopharmaceutical industry has led to a progressive increase in the scale of operation in the last decade. This review will provide a high level summary of the documented process difficulties unique to serum-free large scale (LS) cell culture, analyze the engineering constraints typical of these processes, and suggest some practical equipment design considerations to enhance the productivity, reliability and operability of such systems under GMP manufacturing conditions. A systems approach will be used to establish a good LS bioreactor design practice, providing a discussion on gas distribution, agitation, vessel design, SIP/CIP and control issues. This revised version was published online in July 2006 with corrections to the Cover Date.     

Media development for large scale Agrobacterium tumefaciens culture

A chemically defined media was developed for growing Agrobacterium tumefaciens at large scale for commercial production of recombinant proteins by transient expression in plants. Design of experiments was used to identify major and secondary effects of ten media components: sucrose, ammonium sulfate ((NH₄)₂SO₄), magnesium sulfate heptahydrate (MgSO₄*7H₂O), calcium chloride dihydrate (CaCl₂*2H₂O), iron (II) sulfate heptahydrate (FeSO₄*7H₂O), manganese (II) sulfate monohydrate (MnSO₄*H₂O), zinc sulfate heptahydrate (ZnSO₄*7H₂O), sodium chloride (NaCl), potassium chloride (KCl) and a sodium/potassium phosphate buffer (Na₂HPO₄/KH₂PO₄). Calcium and zinc were found to have no detectable impact on biomass concentration or transient expression level, and concentrations of the other components that maximized final biomass concentration were determined. The maximum specific growth rate of Agrobacterium strain C58C1 pTFS40 in this media was 0.33 ± 0.01 h^?1 and the final biomass concentration after 26 h of batch growth in shake flasks was 2.6 g dry cell weight/L. Transient expression levels of the reporter protein GUS following infiltration of a recombinant Agrobacterium strain C58C1 into N. benthamiana were comparable when the strain was grown in the defined media, Lysogeny Broth (LB) media, or yeast extract‐peptone (YEP) media. In LB and YEP media, free amino acid concentration was measured at three points over the course of batch growth of Agrobacterium strain C58C1 pTFS40; results indicated that l ‐serine and l ‐asparagine were depleted from the media first, followed by l ‐alanine and l ‐glutamic acid. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1218–1225, 2017     

Reactor design for large scale suspension animal cell culture

The scale of operation of freely suspended animal cell culture has been increasing and in order to meet the demand for recombinant therapeutic products, this increase is likely to continue. The most common reactor types used are stirred tanks. Air lift fermenters are also used, albeit less commonly. No specific guidelines have been published for large scale (≥10 000 L) animal cell culture and reactor designs are often based on those used for microbial systems. However, due to the large difference in energy inputs used for microbial and animal cell systems such designs may be far from optimal. In this review the importance of achieving a balance between mixing, mass transfer and shear effects is emphasised. The implications that meeting this balance has on design of vessels and operation, particularly in terms of strategies to ensure adequate mixing to achieve homogeneity in pH and dissolved gas concentrations are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

发热伴血小板减少综合征布尼亚病毒规模化生产培养工艺的建立

目的通过对发热伴血小板减少综合征布尼亚病毒（简称“新布尼亚病毒”）进行规模化生产培养工艺研究,为新布尼亚病毒规模化生产提供有力支持。方法采用细胞工厂培养Vero细胞,待其长成致密单层后,取工作种子批新布尼亚病毒毒种接种细胞,采用连续收获或细胞病变充分时收获培养液上清的方法收获病毒,并以病毒滴度、抗原含量作为评价指标选择基础培养基、培养基pH、人血白蛋白添加浓度、接种细胞日龄、接种病毒MOI以及病毒培养温度。结果按0．01-0．001MOI接种3-4日龄Vero细胞,病毒培养液选择含0．3％人血白蛋白pH7．6-7．8的DMEM溶液,35℃培养7d收获,病毒收获液病毒滴度7．87LgCCID50／mL、抗原含量170．1μ/mL。结论初步建立了新布尼亚病毒规模化生产培养工艺,为后续工业化生产提供了数据支持。     

Towards large scale fermentative production of succinic acid

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Segmented linear modeling of CHO fed‐batch culture and its application to large scale production

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed‐batch cultures. Using the model structure and parameter values from a small‐scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed‐batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785–797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

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     

Studies of some physical parameters for large scale protease production by SSF

For designing the large scale protease production system, through solid state fermentation, the important parameters are accumulation density, bed height, aeration etc. The optimum fermentation condition recorded were 0.3125 g/cc accumulation density and 1.0 cm bed height (without aeration). Aeration at 3 LPM and 2.0 cm bed height were found optimum for protease production by Rhizopus oryzae.     

Mathematical model for empirically optimizing large scale production of soluble protein domains

Background  

Efficient dissection of large proteins into their structural domains is critical for high throughput proteome analysis. So far, no study has focused on mathematically modeling a protein dissection protocol in terms of a production system. Here, we report a mathematical model for empirically optimizing the cost of large-scale domain production in proteomics research.     

Alcohol production from pineapple waste

Saccharomyces cerevisiae andZymomonas mobilis were grown on pineapple waste and their alcohol production characteristics compared. The pineapple waste consisted of 19% cellulose, 22% hemi-cellulose, 5% lignin and 53% cell soluble matters but concentration of soluble sugars, which included 5.2% sucrose, 3.1% glucose and 3.4% fructose, was relatively low and pretreatment of the substrate was needed. Pretreatment of pineapple waste with cellulase and hemi-cellulase and then fermantation withS. cerevisiae orZ. mobilis produced about 8% ethanol from pineapple waste in 48 h.

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Résumé On a fait croîtreSaccharomyces cerevisiae etZymomonas mobilis sur des déchets d'ananas, et on a comparé les caractéristiques de leur production d'alcool. Le déchet d'ananas consistait en 19% de cellulose, 22% d'hémicellulose, 5% de lignine et 53% de matières cellulaires solubles. Mais la concentration en sucres solubles qui comprenait 5.2% de sucrose, 3.1% de glucose et 3.4% de fructose, était relativement faible. Le prétraitement du substral s'avérait donc nécessaire. Le p_r étraitement ces déchets d'ananas avec la cellulase et l'hemicellulase, suivi de la fermentation parS. cerevisiae ouZ. mobilis ont produit environ 8% d'ethanol à partir de résidus d'ananas en 48 h.

     

Application of bioreactor systems for large scale production of horticultural and medicinal plants

Automation of micropropagation via organogenesis or somatic embryogenesis in a bioreactor has been advanced as a possible way of reducing costs. Micropropagation by conventional techniques is typically a labour-intensive means of clonal propagation. The paper describes lower cost and less labour-intensive clonal propagation through the use of modified air-lift, bubble column, bioreactors (a balloon-type bubble bioreactor), together with temporary immersion systems for the propagation of shoots, bud-clusters and somatic embryos. Propagation of Anoectochilus, apple, Chrysanthemum, garlic, ginseng, grape, Lilium, Phalaenopsis and potato is described. In this chapter, features of bioreactors and bioreactor process design specifically for automated mass propagation of several plant crops are described, and recent research aimed at maximizing automation of the bioreactor production process is highlighted.     

Design of solid state fermentor for production of fungal metabolites on large scale

Summary A large scale solid state fermentor of 150 tray capacity is designed with appropriate control system. It gave better productivity even at higher scale of operation as compared to lab and large scale units of similar type and largely overcomes some operational problems.     

New bacteriophage vectors for the large scale production of single stranded insert DNA

SSEV 18 and SSEV 19, derivatives of the bacteriophages M13mp18/19, are new versatile cloning vectors allowing the large scale preparation of single stranded (ss) insert DNA. Replacing the original multiple cloning site by a synthetic 96 bp DNA fragment, a new polylinker region has been introduced containing complementary sequences designed to form a stem structure where single stranded insert fragments can be excised via a 'master restriction' site. The usefulness of such a vector has been demonstrated by the cloning of a 900 bp HindIII fragment derived from the Mycoplasma hyorhinis 23 S rRNA gene. After excision the single stranded insert was labeled isotopically and tested for sensitivity and specificity in detecting homologous sequences in pure DNA or cellular material immobilized on filters.     

History,taxonomy and culture of the pineapple

Pineapples, native to tropical America, have been most extensively produced in Hawaii which accounted for 85% of the 12 1/4 million cases of canned pineapple produced in 1947 by all countries of the world.     

Large scale cultivation of Bordetella pertussis in submerged culture for production of pertussis toxin

Summary Large quantities of purified Bordetella pertussis toxin were required to develop an investigational toxoid vaccine. To overcome the difficulties associated with the large scale growth of Bordetella pertussis in submerged culture, we employed an aeration strategy using compressed oxygen and an antifoam agent. The microorganism grown in this way in 1001 batches for 20–24 h yielded 4–6 mg·1^-1 pertussis toxin.     

Cellcube: A new system for large scale growth of adherent cells

A new disposable cell culture unit for adherent cell lines, the CellCube, was used to grow a variety of mammalian cell lines. A small unit with 2m² growth surface area generated up to 4·10⁹ cells. The disposable system consists of a series of polystyrene plates, mounted into a cubical container. A simple construction consisting of a spinner system for medium conditioning, rotameters for gas mixing and a peristaltic pump for medium circulation provided conditions for culture growth.Abbreviations COS M6 Green Monkey Kidney Cells - SKNMC Human Neuroblastoma Cell Line - BHK Baby Hamster Kidney Cells - CHO Chinese Hamster Ovary Cells - EDTA Ethtylenediaminetetraacetic Acid - PBS Phosphate Buffered Saline - FCS Fetal Calf Serum - MEM Minimum Essential Medium, Alpha Modification     

凤梨科植物的离体培养（综述）

本文主要从快速繁殖,育种探索和种质保存三个方面介绍凤梨离体培养研究的进展,并简要介绍其他凤梨科植物离体培养的研究概况,在此基础上对一些问题进行探讨并提出展望。     

Improved production and stability ofE. coli recombinants expressing transketolase for large scale biotransformation

Summary TheE.coli tkt gene has been subcloned into high copy number vectors. In fed batch fermentations up to 4gL^–1 of soluble intracellular transketolase was produced representing 43% of the total cell protein. Increased plasmid stability during fed-batch fermentations was obtained by using kanamycin resistant pBGS vectors rather than the ampicillin resistant pUC vectors. Plasmid stability was maintained throughout growth in a complex medium without any selective pressure by incorporating thecer region fromColE1 into the expression construct.