Cytotechnology 5: 201-209, 1991. 9 1991 Kluwer Academic Publishers. Printed in the Netherlands.

Passive release of monoclonal antibodies from hybridoma cells Sudesh B. Mohan and Andrew Lyddiatt School of Chemical Engineering, University of Birmingham, P.O. Box 363, Birmingham B15 277, UK Received 23 July 1990; acceptedin revisedform 16 October 1990

Key words: continuous culture, monoclonal antibodies, passive MCAB release

Abstract

Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. This release is promoted at room temperature. Passively released MCAB is indistinguishable from that released by actively growing cells, as judged by SDS-polyacrylamide gel electrophoresis. The significance of these observations in relation to the continuous culture of hybridoma cells is discussed. Maximum MCAB content of TB/C3 hybridoma cells is about 55pg per cell, any additional MCAB produced is secreted. Abbreviations: MCAB - monoclonal antibodies; PBS - phosphate buffered saline; RT - room temperature; SDS - sodium dodecyl sulphate

Introduction

Monoclonal antibodies (MCAB) are important biological products which have gained widespread usage in many technical and medical fields (Carlsson and Glad, 1989). Currently most of the demand is met by culturing hybridoma ceils in batch fermentation. Various authors have reported that in batch cultures antibody production continues during decline phase of growth and is often maximal during this phase (Birch et al., 1985; Velez et al., 1986). This increase has been attributed to secretion from living cells as opposed to passive release from dying ceils (Birch et al., 1987; Renard et al., 1988). Recently, an apparatus was described for the continuous-flow culture of hybridoma cells with integrated product recovery for laboratory scale

production of MCAB (Rudge et al., 1987; Fig. 1). Essentially, hybridoma ceils were grown at 37~ in a constant volume of medium and the spent medium was collected in a small reservoir (50 mL volume) at 4~ This reservoir served two functions. Firstly, it allowed cells and any cell debris to settle prior to application of the spent medium to an immunoaffinity column. Secondly, it was hoped that additional secretion of MCAB by the settling cells, as anticipated from the batch culture studies, would increase the recovered yield of MCAB. The above preliminary study indeed reported the expected increase in MCAB levels (Rudge et al., 1987). However, subsequent studies described in this paper suggest that such increase is not consistently obtained and provide evidence that the possibility of passive release of MCAB by hybridoma cells cannot be ruled out.

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Materials and methods

Continuous culture of TB/C3 hybridoma cells for the production of monoclonal antibodies Murine hybridoma cells (TB\C3) which produce monoclonal antibodies (MCAB) specific to C 2 domain of human IgG (obtained from Dr. R Jefferies, Department of Immunology, University of Birmingham) were grown in batch and continuous-flow culture as described by Rudge et al. (1987) except that the 5% foetal calf serum was replaced by 1% foetal calf and 4% IgG-depleted serum (Gibco and Applied Protein Products, respectively). For continuous-flow culture, the culture vessel was connected to the on-line recovery process described by the above authors (Fig. 1). Cells were grown at 37~ in a flat-bottomed flask, in a constant volume of medium (0.71) maintained by a side arm which was used to pump out the spent medium. Ports for medium addition, air venting, headspace gassing, sparging, sample re-

moval and pH and dissolved oxygen probes were connected to the culture vessel. Temperature was controlled by immersing the flask in a water bath. The flask was inoculated with cells to a density of 1 x 10-5/mL and stirred by a magnetically driven stirrer. Medium was gassed by headspace gassing (100 mL air/min) initially and then by sparging at a low rate (10 mL air/min) to maintain dissolved oxygen level of 50%. W h e n the c e l l density reached 4-5 x 10-5/mL, fresh medium (stored at 4~ was pumped in at the same rate as the spent medium was pumped out (18 mL/hr; dilution rate of 0.025). The latter was collected in a cell settler (working volume 50 mL, maintained by a side arm) at 4~ where the cells were allowed to settle by gravity. Clarified medium was pumped on to an affinity column which was developed when saturated with MCAB. Samples, withdrawn daily from the culture vessel and from the clarified post cell settler medium (a and b, respectively, Fig. 1), were used to monitor cell counts and MCAB levels.

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Cell counts Viable and non-viable cell numbers were obtained using trypan blue exclusion method (Patterson, 1979). The number of particles, attributable to cell disruption were also recorded.

Antibody assay Enzyme linked immunosorbent assay (ELISA) described by Voller et al. (1976) was used to estimate MCAB concentrations. Microtitre plates were coated with 5 p.g/mL human IgG and blocked with 2% casein. Incubation with sheep anti-mouse IgG-horse radish conjugate was followed by substrate, o-phenylene diamine (0.08% w/v in 0.1 M citrate phosphate buffer, pH 4.3, containing 0.08% H202). After incubation for 10-60 min, the reaction was stopped with 12% H2SO 4 and the absorbance at 405nm was measured with a Titertek Multiscan photometer (Flow Laboratories). MCAB concentrations were calculated with reference to a standard curve obtained simultaneously using murine IgG (SeroTec).

Preparation of samples from the cell settler and batch culture Spent medium was carefully decanted from the cell settler and the cells washed once by suspension in phosphate buffered saline (PBS, 0.02 M potassium phosphate, pH 7.4, containing 0.15 M NaC1 and 15 mM sodium azide) followed by centrifugation (1000 g, 5 min). The supematant was discarded and cells resuspended in fresh PBS. The suspension was divided into four aliquots: 1. Cells from one aliquot were collected by centrifugation and lysed in the original volume of water. One-half of this sample was centrifuged and the supematant used as the 0 time control. The other half of the sample was stored at 4~ until required. 2. A second aliquot was used as a 0 time PBS control. Cells and particles present in this sample

were counted, prior to centrifugation to obtain a clarified supernatant. 3 & 4. The remaining two samples were stored at 4~ and room temperature, respectively. When required cells were resuspended and aliquots withdrawn for measuring the various parameters. Cells from the batch culture were collected by centrifugation (1000 g, 10 min), washed with PBS and the various samples prepared as described above. All parameters were measured in triplicate and expressed as the total present in the volume used initially to resuspend the washed ceils.

Purification of MCAB by affinity chromatography MCAB was purified by affinity chromatography on Sepharose CL-4B coupled to human-IgG. The adsorbent was prepared as described by March et al. (1974). Samples (batch culture supematant and cell settler lysate) were applied to the adsorbent (2 ml) which had previously been equilibrated in PBS. The column was washed until no protein was detected in the eluate, followed by 10 to 15 volumes each of PBS containing 1 M NaC1 (tO remove any non-specifically adsorbed protein) and PBS. Adsorbed protein was eluted with 2 ml potassium thiocyanate (3M, dissolved in PBS) and desalted immediately on a Sephadex G-25 column (10 ml) connected downstream of the affinity column.

SDS-polyacrylamide gel electrophoresis 10-15% gradient gels were run on the PhastSystem (Pharmacia-LKB) and stained by silver according to the manufacturer's protocols.

Results

Monoclonal antibody levels detected in the continuous culture of hybridoma cells Various parameters monitored during two inde-

204 maintained for 10 days (Fig. 2a i). During this time 20-30 gg/mL of MCAB was detected in samples taken from both the culture vessel and

pendent experiments are compared in Fig. 2. In the first experiment (Fig. 2a), high cell densities (6 x 10-5/mL) and viability (above 90%) were

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Passive release of monoclonal antibodies from hybridoma cells.

Evidence is presented for the passive release of monoclonal antibodies (MCAB) from hybridoma cells grown in either batch or continuous-flow culture. T...
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