Protein-free human-human hybridoma cultures in an intercalated-spiral alternate-dead-ended hollow fiber bioreactor

Batch cell cultures of a human-human hybridoma line in a convective flow dominant intercalated-spiral altetnate-dead-ended hollow fiber are compared with those using conventional axial-flow hollow fiber bioreactors and a stirred-tank bioreactor. Relatively short-term fed-batch and perfusion cell cultures were also employed for the intercalated-spiral bioreactor. When operating conditions of a batch intercalated-spiral bioreactor were properly chosen, the cell growth and substrate consumption paralleled that of a batch stirred-tank culture. The results verified the premise of the intercalated-spiral hollow fiber bioreactor that nutrient transport limitations can be eliminated when the convective flux through the extracapillary space is sufficiently high.(c) John Wiley & Sons, Inc.     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

Comparison of cell growth in T-flasks, in micro hollow fiber bioreactors, and in an industrial scale hollow fiber bioreactor system

In this article, cell growth in a novel micro hollow fiberbioreactor was compared to that in a T-flask and theAcuSyst-Maximizer®, a large scale industrial hollowfiber bioreactor system. In T-flasks, there was relativelylittle difference in the growth rates of one murine hybridomacultured in three different media and for three other murinehybridomas cultured in one medium. However, substantialdifferences were seen in the growth rates of cells in themicro bioreactor under these same conditions. These differencecorrelated well with the corresponding rates of initial cellexpansion in the Maximizer. Quantitative prediction of thesteady-state antibody production rate in the Maximizer was moreproblematic. However, conditions which lead to faster initialcell growth and higher viable cell densities in the microbioreactor correlated with better performance of a cell line inthe Maximizer. These results demonstrate that the microbioreactor is more useful than a T-flask for determining optimalconditions for cell growth in a large scale hollow fiberbioreactor system.     

Hybridoma cells in a protein-free medium within a composite gel perfusion bioreactor

A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×10⁷ cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 g per 10⁶ viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.     

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     

Hybridoma growth in a new generation hollow fibre bioreactor: antibody productivity and consistency

This paper analyses the performance of MAbMax^TM/Tricentric^TM, a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process parameters were optimised using a mouse hybridoma, IgG1_K secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long period of time and a continuously high production of antibodies was detected over several months. Foetal bovine serum concentration was reduced to 1\% and the effects of weaning of cells from serum were monitored in terms of cell metabolism and antibody productivity. Antibody harvests collected at regular intervals throughout the run (2 to 12 weeks) were purified using affinity chromatography on a recombinant protein A/G matrix and then analysed in terms of antigen binding properties, isoelectric forms and oligosaccharide structures, in order 1) to control antibody quality consistency as a function of time and serum concentration and 2) to compare antibody characteristics as a function of culture conditions, in vitro bioreactor cultivation versus in vivo mouse ascite cultivation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antibody production in packed bed reactors using serum-free and protein-free medium

The present work demonstrates the utility of packed bed reactors for the production of monoclonal antibody. We present data from a continuous process run for the production of over 100 grams of antibody, using serum-free medium. An additional pilot run also demonstrates the potential for continued antibody production under protein-free conditions, using a standard basal medium.     

Calcium alginate immobilized hybridomas grown using a fluidized-bed perfusion system with a protein-free medium

Hybridoma SPO1 cells were immobilized in calcium alginate beads and were further grown in a fluidized-bed perfusion system with a protein-free medium. The presence of serum in the steps of entrapment was shown to be helpful for the preservation of cell viability. Each step during immobilization was investigated with respect to the extent of cell damage caused. The immobilization process using small beads caused a lower cell viability initially but allowed a higher rate of cell growth subsequently, compared to those in large beads. In a perfusion system for the continuous production of monoclonal antibodies (MAb), the viable cell density reached 2×10⁷ cells per ml of beads with a viability of 40%. Compared with the cells in suspension culture, the immobilized SPO1 cells showed higher viable cell based specific rates of substrate uptake (glucose and glutamine) and of MAb production. A significant drop in the formation of lactate after the cell growth entered a steady state suggested a higher activity of the Tricarboxylic Acid Cycle in the cells when the cell density became high.     

Influence of bcl-2 on antibody productivity in high cell density perfusion cultures of hybridoma

Apoptosis is an active, genetically determined death mechanism which can be induced by a wide range of physiological factors and by mild stress. It is the predominant form of cell death during the production of antibodies from murine hybridoma cell lines. A number of studies have now demonstrated that the suppression of this death pathway, by means of over-expression of survival genes such as bcl-2, results in improved cellular robustness and antibody productivity during batch culture. In the present study, the influence of bcl-2 expression on hybridoma productivity in two high density perfusion bioreactor systems was investigated. In the first system, a fixed-bed reactor, the DNA content in the spent medium was 25% higher in the control (TB/C3-pEF) culture than that found in the bcl-2 transfected (TB/C3-bcl2) cultures at all perfusion rates. This is indicative of a higher level of cell death in the control cell line. The average antibody concentration for the TB/C3-pEF cell line was 14.9 mg L-1 at perfusion rates of 2.6 and 5.2 d-1. However, for the TB/C3-bcl2 cell line it was 33 mg L-1 at dilution rates of 2 and 4 d-1. A substantial increase in antibody concentration was also found in the Integra Tecnomouse hollow fibre reactor. The antibody titre in the TB/C3-bcl2 cassette was nearly 100% higher than that in the TB/C3-pEF cassette during the cultivation period which lasted 6 weeks. Clearly, these results demonstrate the positive impact of bcl-2 over-expression on production of antibody in hybridoma perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor

Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acysyst-P^® (Endotronic) with a total fiber surface area of 7.2 m² (6×1.2 m²) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics-and serum-free IMDM medium, supplemented with 50g/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20–40 g protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.     

Supermacroporous polymer‐based cryogel bioreactor for monoclonal antibody production in continuous culture using hybridoma cells

Cryogel matrices composed of different polymeric blends were synthesized, yielding a unique combination of hydrophilicity and hydrophobicity with the presence or absence of charged surface. Four such cryogel matrices composed of polyacrylamide–chitosan (PAAC), poly(N‐isopropylacrylamide)–chitosan, polyacrylonitrile (PAN), and poly(N‐isopropylacrylamide) were tested for growth of different hybridoma cell lines and production of antibody in static culture. All the matrices were capable for the adherence of hybridoma cell lines 6A4D7, B7B10, and H9E10 to the polymeric surfaces as well as for the efficient monoclonal antibody (mAb) production. PAAC proved to be relatively better in terms of both mAb production and cell growth. Further, PAAC cryogel was designed into three different formats, monolith, disks, and beads, and used as packing material for packed‐bed bioreactor. Long‐term cultivation of 6A4D7 cell line on PAAC cryogel scaffold in all the three formats could be successfully done for a period of 6 weeks under static conditions. Continuous packed‐bed bioreactor was setup using 6A4D7 hybridoma cell line in the three reactor formats. The reactors ran continuously for a period of 60 days during which mAb production and metabolism of cells in the bioreactors were monitored periodically. The monolith bioreactor performed most efficiently over a period of 60 days and produced a total of 57.5 mg of antibody in the first 30 days (in 500 mL) with a highest concentration of 115 μg mL^?1, which is fourfold higher than t‐flask culture. The results demonstrate that appropriate chemistry and geometry of the bioreactor matrix for cell growth and immobilization can enhance the reactor productivity. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Use of hollow fiber systems for rapid and direct scale up of antibody production from hybridoma cell lines cultured in CL-1000 flasks using BD Cell MAb medium

The combination of BD Cell MAb medium with the CL-1000 flask is increasingly being used to generate a few hundred milligram of antibody for early stage research projects. Cells are inoculated at 2 million per ml, and the antibody is harvested after 15 days or when the antibody concentration reaches above 10 mg ml⁻¹, whichever comes first. Currently, there is no means to scale up beyond this production level using this technology. In this study, we evaluated hollow fiber technology as the scale up alternative. The hollow fiber system was run in batch mode to mimic the method used for the CL-1000 with BD MAb medium. The FL-NS murine hybridoma cell line was simultaneously inoculated at 2 million cells per ml in a CL-1000 and the Maximizer hollow fiber bioreactor system, a 21-fold theoretical scale up over the CL-1000. The Maximizer produced 23-fold more antibody, very close to the expected theoretical amount. However, production was complete after 9 days in the Maximizer, while the CL-1000 required the full 15 days for production. In summary, these results demonstrate successful scale up of antibody production from the CL-1000 to a hollow fiber system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Perfluorocarbon facilitated O2 transport in a hepatic hollow fiber bioreactor

A mathematical model describing O₂ transport in a hepatic hollow fiber (HF) bioreactor supplemented with perfluorocarbons (PFCs) in the circulating cell culture media was developed to explore the potential of PFCs in properly oxygenating a bioartificial liver assist device (BLAD). The 2‐dimensional model is based on the geometry of a commercial HF bioreactor operated under steady‐state conditions. The O₂ transport model considers fluid motion of a homogeneous mixture of cell culture media and PFCs, and mass transport of dissolved O₂ in a single HF. Each HF consists of three distinct regions: (1) the lumen (conducts the homogeneous mixture of cell culture media and PFCs), (2) the membrane (physically separates the lumen from the extracapillary space (ECS), and (3) the ECS (hepatic cells reside in this compartment). In a single HF, dissolved O₂ is predominantly transported in the lumen via convection in the axial direction and via diffusion in the radial direction through the membrane and ECS. The resulting transport equations are solved using the finite element method. The calculated O₂ transfer flux showed that supplementation of the cell culture media with PFCs can significantly enhance O₂ transport to the ECS of the HF when compared with a control with no PFC supplementation. Moreover, the O₂ distribution and subsequent analysis of ECS zonation demonstrate that limited in vivo‐like O₂ gradients can be recapitulated with proper selection of the operational settings of the HF bioreactor. Taken together, this model can also be used to optimize the operating conditions for future BLAD development that aim to fully recapitulate the liver's varied functions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A novel alginate hollow fiber bioreactor process for cellular therapy applications

Gel‐matrix culture environments provide tissue engineering scaffolds and cues that guide cell differentiation. For many cellular therapy applications such as for the production of islet‐like clusters to treat Type 1 diabetes, the need for large‐scale production can be anticipated. The throughput of the commonly used nozzle‐based devices for cell encapsulation is limited by the rate of droplet formation to ～0.5 L/h. This work describes a novel process for larger‐scale batch immobilization of mammalian cells in alginate‐filled hollow fiber bioreactors (AHFBRs). A methodology was developed whereby (1) alginate obstruction of the intra‐capillary space medium flow was negligible, (2) extra‐capillary alginate gelling was complete and (3) 83 ± 4% of the cells seeded and immobilized were recovered from the bioreactor. Chinese hamster ovary (CHO) cells were used as a model aggregate‐forming cell line that grew from mostly single cells to pancreatic islet‐sized spheroids in 8 days of AHFBR culture. CHO cell growth and metabolic rates in the AHFBR were comparable to small‐scale alginate slab controls. Then, the process was applied successfully to the culture of primary neonatal pancreatic porcine cells, without significant differences in cell viability compared with slab controls. As expected, alginate‐immobilized culture in the AHFBR increased the insulin content of these cells compared with suspension culture. The AHFBR process could be refined by adding matrix components or adapted to other reversible gels and cell types, providing a practical means for gel‐matrix assisted cultures for cellular therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A three-phase pattern in growth, monoclonal antibody production, and metabolite exchange in a hybridoma bioreactor culture

The use of partial cubic spline data interpolation for the calculation of volumetric metabolite exchange rates suggested the existence of three distinct metabolic phases during bioreactor culture of a hybridoma cell line. During phase 1, a rapid amino acid uptake rate and ammonia release rate were observed. The growth rate was low and glutamine synthetase activity fell. In phase 2, maximum growth rate and minimum glutamine assimilation and ammonium production rates were observed. Attempts to corroborate the apparent ammonia assimilation in this phase using (15)NH(4)Cl resulted in low incorporation rates into alanine and glutamine. Maximum glutamine synthetase activity took place during this period. Maximum antibody production rate was observed during phase 3 during which peaks in glutamine assimilation, ammonia release, and glutamine synthetase activity were observed. The apparent existence of the three phases prompted us to carry out Northern blot analysis of glutamine synthetase RNA at appropriate times during the process. This revealed a pattern of appearance and dis-appearance of mRNA consistent with the three phases indicated by the fermentation parameters. (c) 1993 John Wiley & Sons, Inc.     

Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators

Growth of a hybridoma culture, along with production of monoclonal antibody, was demonstrated over extended periods in polysulfone hollow fiber membrane modules. The molecular weight cutoffs of the membranes were 70,000, 50,000, and 100,000 daltons. The hybridoma cell line, designated 65/26, produced IgG (2b/kappa) directed at mouse thymus cell surface antigen, TL.1. Cell growth occurred in the shell space of the reactor, using supplemented RPMI 1640 (20% fetal bovine serum) supplied from a separate reservoir vessel through the hollow fiber lumen. The reservoir contained 125 mL media, which was changed every 4 days. Concentrations of immunoglobulin were determined by an enzyme immunoassay (using protein A and alkaline phosphatase-labeled antibody conjugate). For the 10K, 50K, and 100K hollow fiber membrane modules, the maximum IgG concentrations detected in the 2.5-mL shell space were 47.5-80, 510, and 740 mug/mL, respectively. In the 125-mL reservoir for the 100K hollow fiber membrane module, the IgG concentration was measured at 260 mug/mL These values compare with an IgG concentration of 1 mug/mL when grown in a standard tissue culture flask and 3.2-7.6 mug/mL when grown in 100 ml media in a spinner flask. In addition, 10K and 50K hollow fiber membrane modules were run in a mode that decreased the fetal bovine serum supplement with time. Differences between these systems suggest that it is possible to obtain high IgG accumulation rates, both during and after the exponential growth phase of the hybridoma population.     

Protein adsorption in polysulfone hollow fiber bioreactors used for serum-free mammalian cell culture

The recovery of serum-free medium proteins from poly-sulfone hollow fiber bioreactors (HFBRs) was investigated. More than 99% of the initial transferrin was adsorbed to the hydrophobic hollow fibers within 2 h of HFBR operation. A methodology to minimize transferrin adsorption by pre-adsorption of bovine serum albumin (BSA) was developed. BSA adsorption on suspended cut fibers was virtually complete within 1 h. BSA-coated fibers adsorbed only 5% of the transferrin within 10 days, whereas uncoated cut fibers adsorbed more than 99% of the transferrin within 1 h. An improved HFBR startup procedure, using a BSA-coating step before inoculation, resulted in substantially higher transferrin recovery. Additional factors influenced extracapillary space (ECS) transferrin concentrations. Pronounced downstream polarization of transferrin was observed in the ECS. In addition, the 30-kDa nominal molecular weight cutoff ultrafiltration membranes rapidly leaked transferrin from the ECS to the lumen. (c) 1993 John Wiley & Sons, Inc.     

Detoxification of organophosphate nerve agents by immobilized dual functional biocatalysts in a cellulose hollow fiber bioreactor

A whole-cell technology for detoxification of organophosphates based on genetically engineered Escherichia coli cell expressing both cellulose-binding domain (CBD) and organophosphorus hydrolase (OPH) onto cell surface was reported recently (Wang et al., 2002). This study reports the application of these biocatalysts when immobilized in a cellulose hollow fiber bioreactor (HFB) for the biodetoxification of a model organophosphate, paraoxon, in a continuous flow mode. In 24 h, 0.79 mg wet cell/cm2 fiber surface were immobilized onto cellulose fibers specifically and strongly through the cellulose binding domain, forming a monolayer demonstrated by Scanning Electronic Micrograph, and essentially no cell was washed away by washing buffer. The immobilized biocatalyst had a high performance of detoxifying paraoxon solution of 5,220 mumol/h x L reactor or 990 mumol/h x m2 reactor. The immobilized biocatalysts maintained a stable degradation capacity for 15 uses over a period of 48 days with only 10% decline in degradation efficiency under operating and storage conditions. In addition, the bioreactor was easily regenerated by washing with 1% sodium dodecyl sulfate (SDS), with 86.7% immobilization capacity and 93.9% degradation efficiency recovery. This is the first report using the HFB in a non-traditional way, immobilizing whole-cell biocatalysts by specific adhesion thus rendering the catalysis operation the advantages of low pressure drop, low shear force, and low energy requirement. The successful application of this genetically engineered dual functional E. coli strain in a model bioreactor shows its promise in large-scale detoxification of organophosphate nerve agents in bulk liquid phase.     

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.     

Examination of primary metabolic pathways in a murine hybridoma with carbon-13 nuclear magnetic resonance spectroscopy

Primary metabolism of a murine hybridoma was probed with (13)C nuclear magnetic resonance (NMR) spectroscopy. Cells cultured in a hollow fiber bioreactor were serially infused with [1-(13)C] glucose, [2-(13)C] glucose, and [3-(13)C] glutamine. In vivo spectroscopy of the culture was used in conjunction with off-line spectroscopy of the medium to determine the intracellular concentration of several metabolic intermediates and to determine fluxes for primary metabolic pathways. Intracellular concentrations of pyruvate and alanine were very high relative to levels observed in normal quiescent mammalian cells. Estimates made from labeling patterns in lactate indicate that 76% of pyruvate is derived directly from glycolysis; some is also derived from the malate shunt, the pyruvate/melate shuttle associated with lipid synthesis and the pentose phosphate pathway. The rate of formation of pyruvate from the pentose phosphate pathway was estimated to be 4% of that from glycolysis; This value is a lower limit and the actual value may be higher. Incorporation of pyruvate into the tricarboxylic acid (TCA) cycle appears to occur through only pyruvate dehydrogenase; no pyruvate carboxylase activity was detected. The malate shunt rate was approximately equal to the rate of glutamine uptake. The rate of incorporation of glucosederived acetyl-CoA into lipids was 4% of the glucose uptake rate. The TCA cycle rate between isocitrate and alpha-ketoglutarate was 110% of the glutamine uptake rate. (c) 1994 John Wiley & Sons, Inc.