J.B. Griffiths

Use of lactate dehydrogenase release to assess changes in culture viability

This study reports the use of lactate dehydrogenase release to monitor changes in culture viability in flask culture and fixed bed, porosphere bioreactor systems. Lactate dehydrogenase release shows good agreement with increase in non-viable cell numbers and decline in glucose utilisation in flask cultures. Studies with the immobilised system show that lactate dehydrogenase release can detect loss of viability which is not always indicated by a decrease in glucose utilisation. The data show that culture viability in a repeated-feed-and-harvest system is influenced markedly by both a) the medium change regime itself and b) the use of an immobilised bioreactor compared to a flask system for the same medium change regime.

Animal cell culture processes - batch or continuous?

Batch culture is the traditional and, for most purposes, the preferred production technology for animal cells. The reasons for this are: (a) it developed from vaccine manufacture where lytic systems made batch culture the only option, human diploid cells were the only acceptable cell line for biologicals and they had a very limited life-span, and the technology available in the 1960s was suited only to batch culture; (b) when scale-up was needed, particularly from multiple roller bottles, the simplest way forward was to adopt the bacterial-type stirred tank reactor

Studies on monoclonal antibody production by a hybridoma cell line (C1E3) immobilised in a fixed bed, porosphere culture system

The aim of this study was to investigate the potential of fixed beds of macroporous glass spheres as a production process for animal cell products. The growth, metabolism and monoclonal antibody expression of a mouse-mouse hybridoma cell line was investigated in order to both test the potential of and to optimise the system. After the initial growth phase, the culture went into a steady-state phase brought on by glutamine limitation. An event occurred after 120-160 h of steady-state operation which destabilised the culture, causing a decline in productivity, after which the culture recovered. This event was analysed in detail to determine its cause, and whether a major switch in metabolic function had occurred. The parameter which correlated most closely to antibody production rate was oxygen, but as this was kept constant in the void medium of the bed it has to be concluded that oxygen diffusion into the spheres was the regulatory factor. A comparison of the fixed bed and a flask culture identified interesting differences in glucose metabolism between the two systems. The data gave strong indications as to how the productivity of the fixed bed system can be further improved. This includes optimisation of the glutamine concentration and modifying the porous structure of the spheres to improve diffusion characteristics.

An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells.

Abstract Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. “mono-”, “bi-” and “tri-cistronic” vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in anti-tumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation – time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals.

Expression of recombinant antibody and secreted alkaline phosphatase in mammalian cells. Influence of cell line and culture system upon production kinetics

The growth and productivity of an Sp2/0 cell line, F3b10, expressing a recombinant antibody (rAb) and BHK21 cells expressing either the same rAb from the same plasmids (BHK.IgG) or secreted alkaline phosphatase (SEAP) (BHK.SEAP) were investigated. The F3b10 line was grown as a single cell suspension. The BHK lines were grown either as suspended natural aggregates or on Cytodex 3 microcarriers. The data for F3b10 showed that the cell-specific rAb production rate (QsrAb) increased in parallel with increases in the specific growth rate (μ). A similar result was obtained for suspended aggregate cultures of both recombinant BHK cell lines. In contrast, for microcarrier cultures of both BHK cell lines, Qsproduct increased as μ decreased. This report shows that the relationship between cell growth and Qsproduct for the cell lines and products studied is dependent upon the culture process. In systems where recombinant cells are growing as a single cell suspension or within a natural suspension aggregate, Qsproduct increased with increases in μ. In such systems, the cells have a rounded morphology. When cells were grown on microcarriers, Qsproduct decreased as μ increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qsproduct to μ are correlated with changes in cell morphology. The relationship between Qsproduct and μ is also affected by the choice of cell line.

Maximisation of perfusion systems and process comparison with batch-type cultures. Maximisation of perfusion cultures.

A comparison of cell yields and monoclonal antibody productivity from the same hybridoma has been made in a wide range of cell bioreactors including both batch and continuous perfusion cultures. The most productive systems were based on porous microcarriers in fixed and fluidised beds which can be operated with a high degree of efficiency and reliability from the physico-chemical engineering point of view. Further improvements should be possible by improving the physiological environment in dense cell cultures, as indicated by the preliminary studies that are described. These include experimental data showing the relationship between monoclonal antibody production rates with glucose, glutamine, ammonia, and oxygen levels in microporous beads.The results strongly indicate that perfusion processes that are scaleable in both volume and cell density can significantly reduce production costs. Manufacturers of biologicals from animal cells now have a choice between the proven batch-type processes and reliable perfusion systems based on microporous beads.

Influence of ammonium ion and glucose on mAb production in suspension and fixed bed hybridoma cultures

Previous work indicated that mAb production by a mouse-mouse hybridoma, grown in a fixed bed of macroporous glass beads with a variable feed glutamine concentration, was correlated only to QvO2. The work reported in this study further investigated the metabolic parameters modulating mAb production using metabolic data from a continuous stirred tank reactor (CSTR) to interpret the behaviour of cells grown in a fixed bed bioreactor (FBR). For a FBR culture grown with a feed glutamine concentration of 3 mmol l-1, QvmAb was correlated to QvO2 and QvGln. However, if the feed glutamine concentration was switched between 1 and 3 mmol l-1, the above relationship did not hold, probably because QvO2 was at or near its maximum value. For both the FBR and CSTR, switches in the feed glutamine concentration suggested that maximum QvmAb values were associated with glucose concentrations above 12.8 mmol l-1 and an ammonium concentration of 2.0-2.5 mmol l-1.

Investigation of parameters affecting a fixed bed bioreactor process for recombinant cell lines

A BHK 21 cell line expressing a recombinant antibody was grown in a fixed bed reactor (FBR) system using a porous support made of Siran glass beads. The contribution of five process variables (bead and inoculum sizes; circulation and dilution rates; glutamine concentration of the feed) to the productivity of the process (defined as production rate, effluent product concentration or yield of product on medium supplied) was investigated using a partial factorial experimental design. Individually, none of the variables tested had a significant affect upon productivity. The combination of smaller bead and inoculum sizes, higher circulation and dilution rates, plus higher feed glutamine concentration gave a markedly higher productivity than any other combination of variable levels tested. This combination of variable levels suggested that better results shold be obtained using a fluidised bed reactor system. However, comparison of the productivities of the two systems showed that the FBR gave the better results. This result can be explained in terms of the relationship of QsrAb to μ.

Cultural and physiological factors affecting expression of recombinant proteins

The variability in expression of recombinant proteins has been analyzed with regard to (a) comparison of clones from the same transfection experiment; (b) comparison of the same genetic construct in different cell lines; (c) the effect of the culture system used (free suspension, aggregate suspension, and microcarrier); and (d) physicochemical parameters in long-term (100d) culture in a macroporous fixed bed bioreactor (FBR).Differences in product expression between clones were accompanied by differences in growth rates, metabolic kinetics, and ability to grow in suspension as opposed to attached culture. The single most important factor affecting product expression when comparing constructs (for SEAP and IgG), cell lines (BHK 21 and myeloma), and culture systems was whether cells were grown in an attached or suspension mode. Thus key factors could be related to cell morphology (suspension versus monolayer), the presence of microenvironments and physiological stress to control growth rate.The relationship of key process parameters to volumetric and specific rAb productivity of the FBR was investigated in a partial factorial experiment with a rBHK cell line. The highest productivity levels are associated with a combination of the highest values tested for re-cycle (195 ml min−1) and dilution rates (1 d−1) and glutamine concentration (2.5 mmol l−1), plus the lowest values for bead size (2 mm) and inoculum density (107 ml−1). Together with data from fluidised bed cultures, these results suggest that higher productivity is not primarily the result of greater cell numbers within the system but more the physicochemical definition of the system.

GENETIC AND BIOCHEMICAL ANALYSIS OF A MURINE HYBRIDOMA IN LONG-TERM CONTINUOUS CULTURE

Abstract Multilocus DNA-fingerprinting was used to monitor the genomic stability of a murine hybridoma grown in a CSTR for 83 days. Minor changes in the fingerprint were observed by day 20 with additional changes seen by day 83. The values of QsmAb at the beginning and end of the culture were not significantly different, neither was there a change in product heterogeneity as shown by IEF affinity immunoblot analysis. There was no change in either the isotype or specificity of the mAb during the culture. Therefore the genetic drift was not associated with marked changes in the product. The minor genetic drift observed by DNA-fingerprinting in long-term continuous cultures is therefore probably not significant.