Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate.

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production.

Effect of viscosity upon hydrodynamically controlled natural aggregates of animal cells grown in stirred vessels.

The effect of medium viscosity upon cell growth and aggregate characteristics of baby hamster kidney (BHK) cells cultivated in stirred tanks was evaluated. Two thickening agents were tested, 9300 MW dextran and a low-viscosity sodium (carboxymethyl)-cellulose; both were used in two different sets of experiments: (i) 250 cm3 Wheaton spinner flasks with a ball impeller operated at 45 rpm; (ii) 500 cm3 Corning spinner flasks with a paddle impeller, operated at constant power dissipation (88 cm2 s-3). Aggregate diameter and the fraction of cells in aggregates increased with the increase in viscosity. Power laws were applied to the experimental results. A dependence of aggregate size upon power dissipation of the order of -0.19 and kinematic viscosity of 0.34 and 0.49 for the constant agitation and constant power dissipation tests were obtained, respectively. A model based upon the entire universal-equilibrium range (i.e., the entire spectrum of isotropic eddies) was used to predict theoretical relationships between the variables studied. The model leads to a power dependence of -0.25 for the energy dissipated in the entire universal-equilibrium range and between 0.25 and 0.5 for the kinematic viscosity in the viscous dissipation subrange, depending on the energy correlation used; it also gives a good explanation for the dependence of aggregate size on the hydrodynamics of the vessel.

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 (mu). 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 mu 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 aggregrate, Qsproduct increased with increases in mu. In such systems, the cells have a rounded morphology. When cells were grown on microcarriers. Qsproduct decreased as mu increased. Cells growing attached to a surface are flat and elongated. The observed differences in the relationship of Qsproduct to mu are correlated with changes in cell morphology. The relationship between Qsproduct and mu is also affected by the choice of cell line.

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) physiochemical 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 1-1), plus the lowest values for bead size (2 mm) and inoculum density (10(7) m1-1). 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.

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 should be obtained using a fluidized 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 mu.

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.

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.

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.