Iournal oflmmunalogk'al Methods, 146 (1992) I 11-120
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© 1992 ElsevierScience Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06161
Propagation of a mouse myeloma cell line J558L producing human CD4 immunoglobulin G1 E r n s t - J i i r g e n S c h l a e g e r and BEnEdictc S c h u m p p Pharma Research, New Tedmologies, F. Hoffinatm-LaRtn'he Ltd., 4002 Basle; Switzerland
(Received5 April 1991, revised received22 July 1991,accepted 5 September 1991)
Transfected mouse myeloma cells are of increasing interest for the production of a wide variety of solubilised recombinant fusion proteins. A stably transfected J558L mouse mycloma subclone (J558LCD4) secreting human CD4-immunoglobulin type G1 receptor ( C D 4 - H y l ) was employed as a model system for cell suspension ct Iture and expression of chimaeric molecules. Cells were grown up to 3 - 5 × 10 ~ celis/ml in serum-free and protein-reduced DH! medium consisting of a mixture of DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL and Piuronic F68. Primatonc RL was the essential growth-promoting factor in protein-free medium. The soluble C D 4 - H y l receptor, the production of which was not growth-associated, accumulated in the medium to concentrations of 4 0 / . t g / m l with a specific formation rate of 0.18/.Lg/10 ~ cells/h in conventional cultures. The cell density was further increased by growing the cells in dialysis tubing or by using a peffusion system with cell retention. Because of the continuous exchange of nutrients and metabolic end-products average concentrations of 35 × 10 ~ cells/ml were achieved. CD4-HTI accumulated in the dialysis tubing up to 1.3 m g / m l . After an initial rapid growth period, a ten-fold reduction in specific nutrient consumption rates and metabolic end-product formation was observed. Chimaeric proteins purified by protein G chromatography from conventional and perfusion cultures were indistinguishable when compared by SDS-PAGE, limited proteolysis and isoeleetric focusing analysis (isoelectric point: 8.5-8.6). Key words: Myelomacell, mouse; Proliferation; Soluble chimeric CD4-Hyl receptor
Correspondence to: E.-J. Schlaeger,Pharma Research, New Technologies, F. Hoffmann-LaRoehe Ltd., PRTM 66/108, CH-4002 Basle, Switzerland (Tel.: 061-68854 65; Fax: 061-691 93 91). Abbreviations: CD4-Hyl, human CD4-immanoglobulin type (31 chimaerie fusion protein; FCS, fetal calf serum; gpt, guanidyl phosphoribosyl transferase; HIV, human immunodeficiency virus; IEF, isoelectric focusing; lg, immunoglobulin; J558L-CD4, transfected J558L mouse myelomaclone producing CD4-Hyl.
Introduction The use of mouse myeloma cells for expression of recombinant fusion proteins is based on four main rationales (Traunecker et al., 1991). First, myeloma cells are dedicated secretory cells, the malignant counterparts of immunoglobulin (Ig) secreting plasma cells. Second, myeloma cells have the machinery to correctly glycosylate heter-
112 ologous proteins. Third, the genetic elements required for high Ig expression in myeloma cells are well characterized. Fourth, myeloma cells grow well in suspension, are robust, readily manipulated and'easy to transfect. Chimaeric molecules composed of ectodomains of ligand binding proteins and of constant regions of human lg chains are of great interest for producing soluble forms of proteins and receptors of cells for structure/function studies, for generating monoclonal or polyclonal antibodies and for serving eventually as therapeutic agents. For example, the CD4 receptor-lgGl (CD4-Hyl) fusion protein was constructed to confer on the soluble CD4 receptor some properties of lgG1, notably a long plasma half-life, Fc receptor binding, complement activation, placental transfer, etc. (Traunecker et ai., 1989; Byrn et ai., 1990). These properties were in addition to the gpl20binding and HIV-biocking properties of recombinant soluble CD4 (Traunecker et al., 1988; Robey and Axel, 1990). The two external CD4 domains which bind the gpl20 protein were linked to the hinge, Cy2 and Cy3 constant regions of the IgG1. After transfection into J558L mouse myeloma cells the chimaeric CD4-Hyl protein was secreted as homodimer into the cell culture supernatant. Other chimaeric molecules have also been expressed in mouse myeloma cells, including the homodimeric CD4-Hy3 and the pentameric CD4-Hp. proteins (Traunecker et al., 1991). Our interest has focused on the proliferation and production characteristics of the J558L-CD4 mouse myeloma cell line from the laboratory to the 20 litre bioreactor scale. Cell growth in serum-free and protein-reduced media, cellular metabolism including amino acid utilization, stability of the CD4-Hyl production and growth to high cell densities were investigated. In order to test the homogeneity of CD4-Hyl expression, proteins from a low cell density culture were compared to proteins produced in high cell density systems by SDS-PAGE, limited tryptic proteolysis and isoelectric focusing analysis after purification by protein G affinity' chromatography.
Materials and methods
Cells The transfected mouse myeloma cell line J558L-CD4 expressing the human chimaeric molecule CD4-Hyl was obtained from A. Traunecker and K. Karjalainen (Basel Institute for Immunology, Basel, Switzerland). The cell line J558L, which constitutively secretes Ig,Al light chain, was selected for expression of a chimaeric gene consisting of part of the gene encoding the extracellular gpl20 binding domains I and 2 of the surface protein CD4 inserted upstream of the hinge and both the Cy2 and Cy3 constant region coding sequences of IgG1. Transfection was performed by protoplast fusion and subsequent selection with mycophenolic acid and xanthine (Traunecker et al., 1991). The cells were routinely maintained in T flasks incubated at 37°C under 5% CO2-balance air and 96% water saturation. Growth experiments and pre-culture for bioreactor inoculation were performed in roller bottles incubated under the same conditions as the flasks. Media and chemicals DHI medium (Gibco BRL, Basel, Switzerland) which is a mixture of DMEM HamFl2, and IMDM media in the respective proportions: 1 : l : 2 (v/v/v) containing 5 mM L-glutamine and 25 mM glucose was supplemented with insulin (5 /.~g/ml), selenite (20 nM), ethanolamine (20/.t M), human transferrin (6 ~.g/ml, Boehringer Mannheim, Rotkreuz, Switzerland), Primatone RL ultrafiltered (10 K) (2.5 mg/ml, Humko Sheffield Products Norwich, NY, U.S.A.), Piuronic F68 (0.1 mg/mi, Serva, Heidelberg, Germany) and fl-mercaptoethanoi (2.5 mM). Mycophenolic acid and xanthine were added at 4 ~ g / m l and 250 p.g/ml, respectively. The medium was sterilized by Durapore membrane filtration (0.2 p.m) (Miilipore, KIoten, Switzerland). Fetal calf serum (Amimed, Muttenz, Switzerland) was added to the medium in various concentrations up to 2%. The feeding solution consisted of glucose (5.5 raM), glutamine (1 mM), Primatone RL
113 (0.25 mg/ml) and vitamins (Gibco). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.) unless otherwise specified.
Growth determination and sample analysis Growth counts were determined by the use of a hemocytometer and cell viability was assessed by exclusion of Trypan Blue (Patterson, 1979). The measurements of glucose, amino acids, lactate and ammonia were performed on cell-free culture supernatants as previously described (Schumpp and Schlaeger, 1990). Soluble CD4H y l molecules were quantified using a modified ELISA sandwich type assay in which a sheep anti-human CD4 was used as coating antibody and a peroxidase conjugated sheep anti-human CD4 as detecting agent (B. Wipf, unpublished).
Dialysis tubing growth system Pieces of dialysis tubing (Spectra/Por6, 2 × 103 molecular weight cut-off, 10 mm diameter, Medical Industries, Los Angeles, CA, U.S.A.) were prepared as described before (Schumpp and Schlaeger, 1989). For growth experiments logarithmically growing cells were suspended in culture medium and 1 ml aliquots were dispensed into each portion of dialysis tubing. Usually 20 pieces of dialysis tubing were incubated in roller bottles in 300 ml growth medium, which was changed at regular intervals.
Product analysis Polyacrylamide gel electrophoresis of the CD4Hyl protein. SDS-PAGE was performed according to Laemmli (1970) under non-reducing conditions on 10% gels, using the Mini Gel System of Bio-Rad Laboratories (Giattburg, Switzerland). The gels were stained with Coomassie Blue. Low molecular weight standards were purchased from Bio-Rad. Purification of the CD4-Hyl protein. CD4H y l molecules were isolated via the Fc part of their Ig tail by protein G affinity chromatography at 4°C (Pharmaeia/LKB Biotechnology, Uppsala, Sweden). The column was first equilibrated with PBS (phosphate-buffered saline), pH 7.5. Cells were removed by centrifugation and the 0.45/zm
filtered cell culture supernatant was passed through the column using a flow rate of I ml/min. Three washing steps were carried out, the first with PBS and 0.1% Triton X-100 and the two last with PBS alone. Elution was achieved with 0.9% NaCI, pH 2.8, controlled by acetic acid addition at a flow rate of (1.3 ml/min ~3 column volumes). The column was regenerated by intensive washing steps with PBS before reutilization. The fractions were immediately neutralized with NaOH. Using this method, recoveries ranged from 75 to 85% and the proteins added to the initial medium were found in the flow through (not shown). Tryptic digest analysis. 10-20 /~g purified CD4-Hyl protein (10/~g/mi) were incubated at 37°C in 0.3 M phosphate buffer pH 8.0 and 2 /zg/ml trypsin added to start the reaction. The reaction was stopped by mixing aliquots with ! vol. of SDS sample buffer and heating at 100°C for 5 min. The digests were analysed by non-reducing SDS-PAGE. Isoelectric focusing (IEF). Isoelectric focusing analysis was performed with precast gels (PAGPlate 3.5-9.5, Pharmacia/LKB) as described by the manufacturer. The concentration of the samples tested was approximately ! mg/ml. An IEF marker preparation (4.5/10.5) was purchased from BDH Limited (Poole, England).
Culture in bioreactors 20 litre batch cultures were performed in airlift bioreactors (Chemap, Zurich, Switzerland). The dissolved oxygen level was kept at 30% air saturation. The pH value dropped from 7.2 to 6.9 during the fermentation process. Feeding solution was added daily in order to prolong the viability of the cells in the non-dividingstationary phase. High cell density cultures were run as described before (Schumpp and Schlaeger, 1990). Briefly, a 2 litre airlift type Bellco bioreactor was connected to a Minikros hollow fibre cartridge (Microgon, Brussels, Belgium) which was used as a cross-flow filtration module (0.2 # m pore diameter) for the removal of cell-free waste medium. Cells were recycled using a peristaltic pump in the reactor which was perfused at the same rate as cell-free supernatant was removed (1-2 vols./ day).
114
Results and discussion Characte~:ation o r re°! grow'th in D H I m e d i u m The growtF' p~c,~>::rfie,~of the J558L-CD4 cells were tested t::~;ciY: selection ple~za;'e with mycophenolic acid and xanthine in complete DHI medium which consisted in a mixture of DMEM, Ham F12 and i M D M media supplemented with several additives such as transferrin, insulin and Primatone RL, as described in the materials and methods section. Addition of 2% fetal calf serum (FCS) supported cellular growth of this subclone tO a maximum cell density of 4 x 106 cells/ml whereas the product titre of the chimaeric protein accumulated to 43 m g / I . Both cell density and C D 4 - H y l content were usually slightly decreased in the absence of serum (Fig. 1). In the presence of serum the doubling time of the cell population from 106 to 2 x 106 cells/ml was 17 h which corresponds to a growth rate of 0.043 h - ~. The specific formation rate of the soluble receptor under these conditions was 0.182 /J.g/106 cells/h. It should be mentioned that additional feeding of glucose and a mixture ot' amino acids at the end of the exponential phase did not increase the cell numbers significantly but prolonged the cell viability in the stationary phase, leading to a slightly higher product titre. Shifting the osmotic pressure of the culture medium from 320 to 400 mosmol/kg did not lead to any improvement in the productivity of the batch culture as observed from several monoclonal antibody producing mouse hybridomas (unpublished lOO O%FCS
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Fig. I. Proliferation and production kinetics of C D 4 - H y l producing cells in DH1 medium containing 0% (left) or 2% (right) fetal calf serum. Cells were grown in 200 ml DHI medium in roller bottles. Cell proliferation ( o - - - - - o ) , CD4H'),I production (if] - - D ) .
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Fig. 2. Response of recombined J558L cells to the addition of specific supplements in serum-free basal medium. Cells were
cultured in 200 ml basal DHI medium in roller bottles. Influence of transferrin (T), insulin (1) and Primatone (P) is shown in .4. B shows the effect of increasing Primatone concentrations in the medium without any supplement (C). Cells were first harvested by centrifugation and thoroughly washed in 37°C warm basal DH1 medium before inoculation in fresh media. results; Oyaas et al., 1989). Gel electrophoretic analysis of cell-free culture supernatant revealed that in the absence of serum two dominant protein bands were present, the C D 4 - H y l fusion protein with a molecular weight of about 98 kDa and the constitutively expressed A light chain with a molecular weight of 26 kDa (not shown). In the following series of experiments in serum-free basal medium the growth promoting effect of several additives such as transferrin, insulin and Primatone RL was studied in more detail (Fig. 2A). The results clearly showed that Primatone RL was the most important supplement, since it had a significant growth promoting effect even when added alone. Addition of Primatone R L together with the growth hormone insulin increased the cell number slightly. Neither transferrin nor insulin alone supported cell growth, suggesting that the iron-binding property of transferrin was not essential for this subclone. However, the growth rate was significantly increased when insulin and transferrin were added together with Primatone RL. Therefore, J558LCD4 cells were grown in the presence of Primatone RL, transferrin and insulin in all experiments unless otherwise specified. Primatone RL is a tryptic meat extract containing some essential nutrients of low molecular weight. In general Primatone RL when used in cell culture as an
amino acid source or as a serum alternative significantly prolongs the viability of the cells at the end of the growth phase and reduces the rate of decline (not shown). In Fig. 2B the optimal concentration of Primatone RL was determined in serum-free medium without any other sapplement. As can be seen 0.3% Primatone RL in the culture medium appeared to be optimal for growth. A similar optimal concentration has been determined for a mouse hybridoma cell line (Velez et al., 1986). Concentrations higher than 0.4% decreased the growth rate indicating that some compounds were growth inhibitory when added at critical concentrations. Metabolic actit'ities Measurements of glucose, glutamine, lactate and ammonia showed expected decreases in glucose and glutamine concentrations with concomitant increases in concentrations of lactate and ammonia (not illustrated). The availability of amino acids in culture medium, especially at the end of the exponential growth phase, is of great importance in obtaining high yields of cells and CD4-H71 proteins. Therefore, the amino acid utilization of the mouse myeloma J558 L cell line was measured by a kinetic study in medium supplemented with 2% serum. Fig. 3 compares the change of each amino acid tested at the end of the growth culture
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Fig. 3. Changes in concentrations of amino acid, in a J558LCD4 conventional culture. Final consumption (negative values) or production (positive values) as percentages of the initial concentrations in the DHI medium supplemented with 2% fetal calf serum after 5 days of culture.
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Passage Passage Fig. 4. Production stability of CD4-.H Y I producing J558L-CD4 cells. Cells were grown in duplicate in 6-well plates in 4 ml DHI medium supplemented with 0.5% fetal calf ~rum and in the presence or absence of 4 pg/ml mycophenolic acid (MPA) and ~ 0 pg/ml xanthine (X). shown in Fig. l B . T h e c o n s u m p t i o n o r f o r m a t i o n
of amino acids is shown as the percentages of the initial concentrations in DHI medium. The results of this study demonstrated that only glutamine was totally consumed and had to be fed during the late phase of the growth culture. In addition to the major group of amino acids which were poorly consumed (5-30%), another group of eight amino acids more than doubled in concentration during growth. An example of this group was alanine, which increased constantly during the growth period up to almost fourfold its initial concentration. There is strong evidence that the formation of alanine serves as a detoxification mechanism for ammonia, which is liberated by the process of glutaminolysis (Lanks and Li, 1988). The high concentration of alanine at the end of growth has been reported for most other cell lines (Butler et al., 1983; Wagner et al., 1988; Miller et ai., 1989a, b; Schumpp and Schlaeger, 1990; Bi)ntemeyer et al., 1991). In general the utilization of amino acids in a given medium depends on the cell line and differences between subclones from the same origin may be possible. The production stability of the C D 4 - H y l producing subclone was analysed (1) with selection pressure and (2) in the absence of gpt selection. The specific formation rate of the recombinant protein was measured over a one month period corresponding approximately to seven passages (Fig. 4). The production rates remained stable in both cases, suggesting that no significant loss of productivity should be expected in a scale up
process without selection pressure. The slight decrease in the specific formation rate of C D 4 - H y l compared to the values found in roller bottle cultures may have been due to the fact that the cells were grown in 6-well plates in which growth and production conditions are not optimal.
Growth in dialysis tubing In order to analyse the formation of soluble C D 4 - H y l receptor at high cell density, cells were grown in pieces of dialysis tubing which were incubated in a roller bottle in excess of culture medium (Adamson et al., 1983; Schumpp and Schlaeger, 1989). The molecular weight cut-off of the dialysis membrane was 2 × 103, indicating that only low molecular weight substances could cross the membrane. Using this simple culture system cells grew without problems of nutrient deficiency or interference from accumulated toxic metabolic end-products. The growth kinetics and the corresponding CD4-H~/1 titre are shown in Fig. 5. The cells grew continuously to cell numbers of 20-30 x 106 cells/ml with a doubling time of 40.5 h which corresponds to a growth rate of 0.017 h - I . The reason for the increased doubling time is not clear. Other cell lines did not show different doubling times when grown in dialysis tubing compared to conventional cultures
(Schumpp and Schlaeger, 1989). The high cell density phase could be maintained with a viabiliity of 50% over at least 200 h demonstrating that the exchange of low molecular substances partially prevents entry into the decline phase. Factors limiting further cell growth in this membrane system may have included the insufficient availability of oxygen, clogging of the dialysis membrane and formation of loose cell aggregates. It appears that at a critical cell concentration the mieroenvironment in the tubing was irreversibly changed so that further growth was almost impossible. However, the survival of the non-dividing culture was established with a rather low death rate. Results obtained with other cell lines indicate that the critical point at which the cells shift into the so-called dense stationary phase is different for each individual cell line. High cell concentrations in the dialysis tubing (more than 60 X 106 cells/ml) were correlated with a high viability in the stationary phase (70-80%), whereas cell lines which only grew to 30 × 106 celis/mi always had a greater death rate (Schumpp and Schlaeger, 1989). Using the J558LCD4 cells in a high cell density perfusion system (see below), cell numbers similar to the dialysis tubing experiment were observed, supporting our suggestion that the limitation in the high cell
Production of CD4-71 in dialysis tubing (1% FCS) 100 A
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:i Time (h) Fig. 5. ,4: proliferation of CD4-HT! producing J558L-CD4 cells in dialysis tubing. 15 pieces of dialysis tubing containing [ ml cells in DH] medium supplemented with 0.5% fetal calf serum were incubated in 300 ml of this same medium changed every other day.
Each point shows the average of duplicates. Viable cell number (e e), CD4-Hyl production (D D). B: SDS-PAGE analysis of CD4-H'),I containing supernatants of J558L-CD4 cells grown in dialysis tubing after 1/2 dilution in a 10% gel under non-reduced conditions. Lane h molecularweight markers; lanes 2-7: culture supernatants, lane 8: purified standard CD4-HTI. The molecularweightsof the standard proteins were: 97.4, 66.2, 45, 31 and 21.5 kDa, from the top to the bottom, respectively.
density culture is a specific property of the cell line used. Thus the dialysis tubing system offers a simple means for obtaining preliminary data on the behaviour of individual cell lines in a high density process with the advantage of parallel sample analysis. The kinetics of CD4-H~,I production (Fig. 5A) showed that synthesis and cellular release of *.he recombinant protein occurred during the exponential growth phase (0.177/.tg/106 c e l l s / h ) as well as in the non-dividing phase (0.142 /~g/106 c e l l s / h ) with roughly similar rates. This finding implies that the rate of C D 4 - H y l formation is not linked to the growth phase of the cells although a slightly higher rate was observed during the active dividing phase. However, the decrease of productivity may also have been caused by factors accumulated in the dialysis tubing. The electrophoretic analysis showed the accumulation of the C D 4 - H y l fusion proteins as well as the IgG1 light chain in the dialysis tubing during growth. It should be mentioned that the marker protein BSA exhibits a different gel eletrophoretic mobility u n d e r non-reduced or reduced conditions and the molecular weight standards always contained 100 m M DTT. (Fig. 5B). Bovine serum albumin, below the CD4-H3,1, was the third major band.
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Time (h) Fig. 7. Perfusion culture with cell retention in a 2 litre hioreactor. The perfusion culture was started with a flow rate of 1.5 litre/day which was increased each day up to 4 litres/ day. The medium with 7 mM glutamine and 30 mM glucose was supplemented with 0.5% fetal calf serum without gpt selection. The hollow fibre was exchanged on the day indicated by the arrow. A: viable cell number (e e), CD4H'yl production per day (D ra). B: specific rates of glucose (o o) and glutamine (o O) consumption and lactate ( • • ) and ammonia (D []) production.
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Fig. 6. Batch culture in a 20 litre airlift reactor. Cells were grown in DHI medium with 2% fetal calf serum without gpt selection. Feeding occurred daily from the day indicated by the arrow. Viable cell number (e e), CD4-H.),I production ([] [] ).
Scale-up studies in airlift bioreactors were performed in the batch cultivation mode without gpt selection. A typical batch culture in a 20 litre bioreactor is shown in Fig. 6. A cell density of 5.5 x 106 c e l l s / m l was achieved in D H I medium containing 2% FCS. Generally, the growth properties as well as the yield of CIM-H~,I receptor in the culture medium (0.775 g for a 22 litre medium volume) under controlled conditions were similar to the results obtained in the roller bottle cultures (Fig. 1), indicating that conditions can be optimized in small culture vessels in the laboratory. Subclones producing other chimaeric CD4-Ig proteins with other Ig isotype chains such as "),3 or p., secreted as a homodimer or a pentamer,
respectively, have been constructed (Traunecker et al., 1991). Growth studies showed that cell yields were similar, but product yields varied widely with the construct and the subclone selected: 20 /.tg/ml for a CD4-/z subcione to 80 / z g / m l for a CD4-y3 subclone (Schlaeger and Schumpp, 1991). The mouse myeloma cells were also grown in a 2 litre bioreactor using a continuous culture mode with cell retention. A tangential cross-flow filtration module was connected to the bioreactor as an external loop (Velez et al., 1989; Murphy, 1990; Schumpp and Schlaeger, 1990). The culture medium containing the cells was permanently circulated through the hollow fibre unit using a peristaltic pump. The membrane pore diameter of 0.2 p.m permitted the removal of cell-frce
Tryptic digestion of CD4-y1
conditioned medium containing also the ehimaeric CD4-HT1 product. The bioreactor volume was held constant by adding fresh medium at the same rate as the permeate was drawn off. Under these conditions there was an exponential growth phase up to a cell concentration of 20 × 106 cells/ml with a growth rate of 0.024 h-~ (Fig. 7A). The perfusion rate was constantly increased from 1.5 litre/day to 4.0 litre/day leading to a maximum cell concentration of 35 x 10 ~ cells/ml which could be maintained for several days. The production of C D 4 - H y l per day increased in parallel with the cell number to a maximum level and then decreased, while product accumulated inside the bioreactor as observed by electrophoretic analysis (not shown). Threefold values were found in the permeate after connecting
proteins
IEF analysis of CD4-71 proteins M
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Fig. 8. Analysis of CD4-H~,I proteins. CD4-Hyl moleculespurified from a low cell density (Icd) culture (4x 10t~ cells/ml) and from a high cell density(hcd) culture (35 x 106 cells/ml) by affinitychromatographywere compared by limited tryptic digestion(,4) and by isoelectriefocusing(B).
119 a new hollow fibre module (Fig. 7A). We believe that retained components, such as debris of dead cells, accumulated on the membrane surface generating a layer which entrapped a large amount of protein. We therefore suggest the connection of several modules in parallel to increase the filtration capacity and culture time of the system. Specific glutamine and glucose consumption rates and ammonia and lactate production rates were always maximal in the early logarithmic growth phase and decreased approximately tenfold during cultivation (Fig. 7B). This has been described in other investigations (Miller et al., 1989a,b; Schumpp and Schlaeger, 1990; Wagner et ai., 1990). It appears that specific consumption rates increase when high glucose or glutamine concentrations are available leading to high production rates of the metabolites.
Comparison of CD4-H),I proteins from low t,ersus high cell density cultures CD4-H~,I containing culture supernatants from a low cell density culture with a final cell concentration of 4 x 106 cells/ml and from a high cell density culture with a tenfold higher cell concentration were purified by protein G affinity chromatography as described in the materials and methods section. Proteolytic fingerprints were made to characterize the CD4-H~,I molecules. The tryptic patterns were identical in both cases indicating a similar sensitivity to trypsin digestion and therefore similar native structures (Fig. 8A). The SDS-PAGE analyses were also similar (not shown). Isoelectric focusing (IEF) permits analytical studies of protein heterogeneity based on different isoelectric points. I E F gels showed one very dominant band and a few minor bands (Fig. 8B). In both eases the proteins migrated at the same isoelectric point of 8.5-8.6. This very basic value was expected from the amino acid sequence of the CD4 receptor. The J558L-CD4 cells were grown in serum-reduced DHI medium for several months in the absence of selection pressure. The CD4-H~,I production, which was not growth-associated, remained stable during this time. If serum-free or protein-reduced conditions are required, it ap-
pears to be relatively simple to wean these cells from serum-containing medium by adding transferrin, insulin and Primatone RL. These are important considerations if chimaeric proteins are developed for therapeutical use. Recombined CD4-H~/I molecules purified from low or high cell density cultures were similar with respect to their molecular weight, tryptic digest pattern and isoelectric point, indicating a high homogeneity in their native structures. From the cell culture point of view the mouse myeloma cell line J558L appears to be a good candidate as an expression system for the production of soluble chimaeric forms of membrane proteins.
Acknowledgements We are grateful to A. Traunecker and K. Karjalainen at the Basel Institute for Immunology for providing the J558L-CD4 cell line and for their interest in this investigation. We also thank S. Mathews and H. Loctscher for their critical reading of the manuscript,
References Adamson, S.R., Fitzpatrick, S.L., Ikhie, LA., Gaucher, G.M. and Lesser, B.H. (1983) In vitro production of high titre monoclonal antibody by bybridomacells in dialysisculture. Biotechnol. Len. 5, 573-578. Biintemeyer, H., Liitkemeyer, D. and Lehmann, J. (1991) Optimization of serum-free fermentation processes for antibody production. Cytotechnol., in press. Butler, M., Imamura, T., Thomas, J. and Thilly,W.G. (1983). High yields from microcarrier cultures by medium peffusion. J. Cell Sci. 61,351-363. Byrn, R.A., Mordenti, J., Lucas, C., Smith, D., Marsters, S.A., Johnson, J.S., Cossum, P., Chamow, S.M, Wurm, F.M., Gregory, T., Groopman, J.E. and Capon, D.J. (1990) Biological properties of a CD4 immunoadhesin. Nature 344, 667-670. Laemmli, U.K. (1970) Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 227, 680-685. Lanks, K.W. and Li, P.-W. (1988) End products of glucose and glutamine metabolism by cult,ared cell lines. J. Cell. Physiol. 135, 151-155. Miller, W.M., Wilke, C.R. and Blanch, H.W. (1989a) Transient responses of hybridoma cells to nutrient additions in continuous culture. I. Glucose pulse and step changes. Biotechnol. Biocng. 33, 477-486.
120 Miller, W.M., Wilke, C.R. and Blanch, H.W. (1989b) Transient responses of hybridoma cells to nutrient additions in continuous culture. II. Glutamine pulse and step changes. Biotechnol. Bioeng. 33, 486-499. Murphy, T.J. (1989) Continuous perfusiun of stirred or airlift bioreactors using hollow fibers for cell separation and oxygenation. Presented at the Cell Culture Engineering II Meeting. December 3-8,1989, Santa Barbara, CA, U.S.A., abstract no. 62. Patterson, M.K. (1979). Measurement of growth and viability of cells in culture. Methods EnzymoL 58, 141-152. Robey, E. and Axel, R. (1990) CD4: collaborator in immune recognition and H1V infection. Cell 60, 697-700. Schlaeger, E.-J. and Schumpp, B. (1991) Propagation of human CD4-immunoglobulin producing cell lines. In: R.E. Spier, J.B. Griffiths and B. Meignier (Eds.), Production of Biologicals from Animal Cells in Culture. ButterworthsHeinemann, Sevenooks, Kent, pp. 18-25. Schumpp, B. and Schlaeger, E.-J. (1989) Physiological studies of high cell density culture of different cell lines. In: R.E. Spiel-. J.B. Griffiths, J. Stephenne and P.J. Crooy (Eds.), Advances in Animal Cell Biology and Technology for Bioprocesses. Butterworths, Sevenoaks, Kent, pp. 224-229. Schumpp, B. and Schlaeger, E.-J. (1990). Optimization of culture conditions for high cell density proliferation of HL-60 human promyelocytic leukemia cells. J. Cell Sci. 97, 639-647.
Traunecker, A., Liike, W. and Karjalainen, K. (1988). Soluble CD4 molecules neutralize human immunodeficiency virus type 1. Nature 331, 84-86. Traunecker, A., Schneider, J.. Kiefer, H. and Karjalainen, K. (1989) Highly efficient neutralization of HIV with recombinant CD4-immunoglobulin molecules. Nature 339, 6870. Traunecker, A., Oliveri, F. and Karjalainen, K. (1991) Myeloma based expression system for production of large mammalian proteins. TIBTECH 9, 109-113. Velez, D., Miller, L. and Macmillan, J.D. (1989) Use of tangential flow filtration in perfusion propagation of hybridoma cells for production of monoclonal antibodies. Biotechnol. Bioeng. 33, 938-940. Velez, D., Reuveny, S., Miller, L. and Macmillan, J.D. (1986) Kinetics of munoclonal antibody production in low serum growth medium. J. lmmunol. Methods 86, 45-52. Wagner, R., Ryll, T., Krafft, H. and Lehmann, J. (1988) Variation of amino acid concentrations in the medium of HU/3-1FN and HU IL-2 producing cell lines. Cytotechnology I, 145-150. Wagner, A., Marc, A., Engasser, J.M., Villermaux, S. and Einsele, A. (1990) Continuous production of prourokinase in feed harvest and perfusion cultures. Biotechnol. Bioeng. 36, 623-629.
I| 1
© 1992 ElsevierScience Publishers B.V. All rights reserved 0022-1759/92/$05.00
JIM 06161
Propagation of a mouse myeloma cell line J558L producing human CD4 immunoglobulin G1 E r n s t - J i i r g e n S c h l a e g e r and BEnEdictc S c h u m p p Pharma Research, New Tedmologies, F. Hoffinatm-LaRtn'he Ltd., 4002 Basle; Switzerland
(Received5 April 1991, revised received22 July 1991,accepted 5 September 1991)
Transfected mouse myeloma cells are of increasing interest for the production of a wide variety of solubilised recombinant fusion proteins. A stably transfected J558L mouse mycloma subclone (J558LCD4) secreting human CD4-immunoglobulin type G1 receptor ( C D 4 - H y l ) was employed as a model system for cell suspension ct Iture and expression of chimaeric molecules. Cells were grown up to 3 - 5 × 10 ~ celis/ml in serum-free and protein-reduced DH! medium consisting of a mixture of DMEM, HamF12 and IMDM media supplemented with transferrin, insulin, Primatone RL and Piuronic F68. Primatonc RL was the essential growth-promoting factor in protein-free medium. The soluble C D 4 - H y l receptor, the production of which was not growth-associated, accumulated in the medium to concentrations of 4 0 / . t g / m l with a specific formation rate of 0.18/.Lg/10 ~ cells/h in conventional cultures. The cell density was further increased by growing the cells in dialysis tubing or by using a peffusion system with cell retention. Because of the continuous exchange of nutrients and metabolic end-products average concentrations of 35 × 10 ~ cells/ml were achieved. CD4-HTI accumulated in the dialysis tubing up to 1.3 m g / m l . After an initial rapid growth period, a ten-fold reduction in specific nutrient consumption rates and metabolic end-product formation was observed. Chimaeric proteins purified by protein G chromatography from conventional and perfusion cultures were indistinguishable when compared by SDS-PAGE, limited proteolysis and isoeleetric focusing analysis (isoelectric point: 8.5-8.6). Key words: Myelomacell, mouse; Proliferation; Soluble chimeric CD4-Hyl receptor
Correspondence to: E.-J. Schlaeger,Pharma Research, New Technologies, F. Hoffmann-LaRoehe Ltd., PRTM 66/108, CH-4002 Basle, Switzerland (Tel.: 061-68854 65; Fax: 061-691 93 91). Abbreviations: CD4-Hyl, human CD4-immanoglobulin type (31 chimaerie fusion protein; FCS, fetal calf serum; gpt, guanidyl phosphoribosyl transferase; HIV, human immunodeficiency virus; IEF, isoelectric focusing; lg, immunoglobulin; J558L-CD4, transfected J558L mouse myelomaclone producing CD4-Hyl.
Introduction The use of mouse myeloma cells for expression of recombinant fusion proteins is based on four main rationales (Traunecker et al., 1991). First, myeloma cells are dedicated secretory cells, the malignant counterparts of immunoglobulin (Ig) secreting plasma cells. Second, myeloma cells have the machinery to correctly glycosylate heter-
112 ologous proteins. Third, the genetic elements required for high Ig expression in myeloma cells are well characterized. Fourth, myeloma cells grow well in suspension, are robust, readily manipulated and'easy to transfect. Chimaeric molecules composed of ectodomains of ligand binding proteins and of constant regions of human lg chains are of great interest for producing soluble forms of proteins and receptors of cells for structure/function studies, for generating monoclonal or polyclonal antibodies and for serving eventually as therapeutic agents. For example, the CD4 receptor-lgGl (CD4-Hyl) fusion protein was constructed to confer on the soluble CD4 receptor some properties of lgG1, notably a long plasma half-life, Fc receptor binding, complement activation, placental transfer, etc. (Traunecker et ai., 1989; Byrn et ai., 1990). These properties were in addition to the gpl20binding and HIV-biocking properties of recombinant soluble CD4 (Traunecker et al., 1988; Robey and Axel, 1990). The two external CD4 domains which bind the gpl20 protein were linked to the hinge, Cy2 and Cy3 constant regions of the IgG1. After transfection into J558L mouse myeloma cells the chimaeric CD4-Hyl protein was secreted as homodimer into the cell culture supernatant. Other chimaeric molecules have also been expressed in mouse myeloma cells, including the homodimeric CD4-Hy3 and the pentameric CD4-Hp. proteins (Traunecker et al., 1991). Our interest has focused on the proliferation and production characteristics of the J558L-CD4 mouse myeloma cell line from the laboratory to the 20 litre bioreactor scale. Cell growth in serum-free and protein-reduced media, cellular metabolism including amino acid utilization, stability of the CD4-Hyl production and growth to high cell densities were investigated. In order to test the homogeneity of CD4-Hyl expression, proteins from a low cell density culture were compared to proteins produced in high cell density systems by SDS-PAGE, limited tryptic proteolysis and isoelectric focusing analysis after purification by protein G affinity' chromatography.
Materials and methods
Cells The transfected mouse myeloma cell line J558L-CD4 expressing the human chimaeric molecule CD4-Hyl was obtained from A. Traunecker and K. Karjalainen (Basel Institute for Immunology, Basel, Switzerland). The cell line J558L, which constitutively secretes Ig,Al light chain, was selected for expression of a chimaeric gene consisting of part of the gene encoding the extracellular gpl20 binding domains I and 2 of the surface protein CD4 inserted upstream of the hinge and both the Cy2 and Cy3 constant region coding sequences of IgG1. Transfection was performed by protoplast fusion and subsequent selection with mycophenolic acid and xanthine (Traunecker et al., 1991). The cells were routinely maintained in T flasks incubated at 37°C under 5% CO2-balance air and 96% water saturation. Growth experiments and pre-culture for bioreactor inoculation were performed in roller bottles incubated under the same conditions as the flasks. Media and chemicals DHI medium (Gibco BRL, Basel, Switzerland) which is a mixture of DMEM HamFl2, and IMDM media in the respective proportions: 1 : l : 2 (v/v/v) containing 5 mM L-glutamine and 25 mM glucose was supplemented with insulin (5 /.~g/ml), selenite (20 nM), ethanolamine (20/.t M), human transferrin (6 ~.g/ml, Boehringer Mannheim, Rotkreuz, Switzerland), Primatone RL ultrafiltered (10 K) (2.5 mg/ml, Humko Sheffield Products Norwich, NY, U.S.A.), Piuronic F68 (0.1 mg/mi, Serva, Heidelberg, Germany) and fl-mercaptoethanoi (2.5 mM). Mycophenolic acid and xanthine were added at 4 ~ g / m l and 250 p.g/ml, respectively. The medium was sterilized by Durapore membrane filtration (0.2 p.m) (Miilipore, KIoten, Switzerland). Fetal calf serum (Amimed, Muttenz, Switzerland) was added to the medium in various concentrations up to 2%. The feeding solution consisted of glucose (5.5 raM), glutamine (1 mM), Primatone RL
113 (0.25 mg/ml) and vitamins (Gibco). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.) unless otherwise specified.
Growth determination and sample analysis Growth counts were determined by the use of a hemocytometer and cell viability was assessed by exclusion of Trypan Blue (Patterson, 1979). The measurements of glucose, amino acids, lactate and ammonia were performed on cell-free culture supernatants as previously described (Schumpp and Schlaeger, 1990). Soluble CD4H y l molecules were quantified using a modified ELISA sandwich type assay in which a sheep anti-human CD4 was used as coating antibody and a peroxidase conjugated sheep anti-human CD4 as detecting agent (B. Wipf, unpublished).
Dialysis tubing growth system Pieces of dialysis tubing (Spectra/Por6, 2 × 103 molecular weight cut-off, 10 mm diameter, Medical Industries, Los Angeles, CA, U.S.A.) were prepared as described before (Schumpp and Schlaeger, 1989). For growth experiments logarithmically growing cells were suspended in culture medium and 1 ml aliquots were dispensed into each portion of dialysis tubing. Usually 20 pieces of dialysis tubing were incubated in roller bottles in 300 ml growth medium, which was changed at regular intervals.
Product analysis Polyacrylamide gel electrophoresis of the CD4Hyl protein. SDS-PAGE was performed according to Laemmli (1970) under non-reducing conditions on 10% gels, using the Mini Gel System of Bio-Rad Laboratories (Giattburg, Switzerland). The gels were stained with Coomassie Blue. Low molecular weight standards were purchased from Bio-Rad. Purification of the CD4-Hyl protein. CD4H y l molecules were isolated via the Fc part of their Ig tail by protein G affinity chromatography at 4°C (Pharmaeia/LKB Biotechnology, Uppsala, Sweden). The column was first equilibrated with PBS (phosphate-buffered saline), pH 7.5. Cells were removed by centrifugation and the 0.45/zm
filtered cell culture supernatant was passed through the column using a flow rate of I ml/min. Three washing steps were carried out, the first with PBS and 0.1% Triton X-100 and the two last with PBS alone. Elution was achieved with 0.9% NaCI, pH 2.8, controlled by acetic acid addition at a flow rate of (1.3 ml/min ~3 column volumes). The column was regenerated by intensive washing steps with PBS before reutilization. The fractions were immediately neutralized with NaOH. Using this method, recoveries ranged from 75 to 85% and the proteins added to the initial medium were found in the flow through (not shown). Tryptic digest analysis. 10-20 /~g purified CD4-Hyl protein (10/~g/mi) were incubated at 37°C in 0.3 M phosphate buffer pH 8.0 and 2 /zg/ml trypsin added to start the reaction. The reaction was stopped by mixing aliquots with ! vol. of SDS sample buffer and heating at 100°C for 5 min. The digests were analysed by non-reducing SDS-PAGE. Isoelectric focusing (IEF). Isoelectric focusing analysis was performed with precast gels (PAGPlate 3.5-9.5, Pharmacia/LKB) as described by the manufacturer. The concentration of the samples tested was approximately ! mg/ml. An IEF marker preparation (4.5/10.5) was purchased from BDH Limited (Poole, England).
Culture in bioreactors 20 litre batch cultures were performed in airlift bioreactors (Chemap, Zurich, Switzerland). The dissolved oxygen level was kept at 30% air saturation. The pH value dropped from 7.2 to 6.9 during the fermentation process. Feeding solution was added daily in order to prolong the viability of the cells in the non-dividingstationary phase. High cell density cultures were run as described before (Schumpp and Schlaeger, 1990). Briefly, a 2 litre airlift type Bellco bioreactor was connected to a Minikros hollow fibre cartridge (Microgon, Brussels, Belgium) which was used as a cross-flow filtration module (0.2 # m pore diameter) for the removal of cell-free waste medium. Cells were recycled using a peristaltic pump in the reactor which was perfused at the same rate as cell-free supernatant was removed (1-2 vols./ day).
114
Results and discussion Characte~:ation o r re°! grow'th in D H I m e d i u m The growtF' p~c,~>::rfie,~of the J558L-CD4 cells were tested t::~;ciY: selection ple~za;'e with mycophenolic acid and xanthine in complete DHI medium which consisted in a mixture of DMEM, Ham F12 and i M D M media supplemented with several additives such as transferrin, insulin and Primatone RL, as described in the materials and methods section. Addition of 2% fetal calf serum (FCS) supported cellular growth of this subclone tO a maximum cell density of 4 x 106 cells/ml whereas the product titre of the chimaeric protein accumulated to 43 m g / I . Both cell density and C D 4 - H y l content were usually slightly decreased in the absence of serum (Fig. 1). In the presence of serum the doubling time of the cell population from 106 to 2 x 106 cells/ml was 17 h which corresponds to a growth rate of 0.043 h - ~. The specific formation rate of the soluble receptor under these conditions was 0.182 /J.g/106 cells/h. It should be mentioned that additional feeding of glucose and a mixture ot' amino acids at the end of the exponential phase did not increase the cell numbers significantly but prolonged the cell viability in the stationary phase, leading to a slightly higher product titre. Shifting the osmotic pressure of the culture medium from 320 to 400 mosmol/kg did not lead to any improvement in the productivity of the batch culture as observed from several monoclonal antibody producing mouse hybridomas (unpublished lOO O%FCS
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Fig. I. Proliferation and production kinetics of C D 4 - H y l producing cells in DH1 medium containing 0% (left) or 2% (right) fetal calf serum. Cells were grown in 200 ml DHI medium in roller bottles. Cell proliferation ( o - - - - - o ) , CD4H'),I production (if] - - D ) .
I+ o:+ 0.2 % 0.1% 0%
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Fig. 2. Response of recombined J558L cells to the addition of specific supplements in serum-free basal medium. Cells were
cultured in 200 ml basal DHI medium in roller bottles. Influence of transferrin (T), insulin (1) and Primatone (P) is shown in .4. B shows the effect of increasing Primatone concentrations in the medium without any supplement (C). Cells were first harvested by centrifugation and thoroughly washed in 37°C warm basal DH1 medium before inoculation in fresh media. results; Oyaas et al., 1989). Gel electrophoretic analysis of cell-free culture supernatant revealed that in the absence of serum two dominant protein bands were present, the C D 4 - H y l fusion protein with a molecular weight of about 98 kDa and the constitutively expressed A light chain with a molecular weight of 26 kDa (not shown). In the following series of experiments in serum-free basal medium the growth promoting effect of several additives such as transferrin, insulin and Primatone RL was studied in more detail (Fig. 2A). The results clearly showed that Primatone RL was the most important supplement, since it had a significant growth promoting effect even when added alone. Addition of Primatone R L together with the growth hormone insulin increased the cell number slightly. Neither transferrin nor insulin alone supported cell growth, suggesting that the iron-binding property of transferrin was not essential for this subclone. However, the growth rate was significantly increased when insulin and transferrin were added together with Primatone RL. Therefore, J558LCD4 cells were grown in the presence of Primatone RL, transferrin and insulin in all experiments unless otherwise specified. Primatone RL is a tryptic meat extract containing some essential nutrients of low molecular weight. In general Primatone RL when used in cell culture as an
amino acid source or as a serum alternative significantly prolongs the viability of the cells at the end of the growth phase and reduces the rate of decline (not shown). In Fig. 2B the optimal concentration of Primatone RL was determined in serum-free medium without any other sapplement. As can be seen 0.3% Primatone RL in the culture medium appeared to be optimal for growth. A similar optimal concentration has been determined for a mouse hybridoma cell line (Velez et al., 1986). Concentrations higher than 0.4% decreased the growth rate indicating that some compounds were growth inhibitory when added at critical concentrations. Metabolic actit'ities Measurements of glucose, glutamine, lactate and ammonia showed expected decreases in glucose and glutamine concentrations with concomitant increases in concentrations of lactate and ammonia (not illustrated). The availability of amino acids in culture medium, especially at the end of the exponential growth phase, is of great importance in obtaining high yields of cells and CD4-H71 proteins. Therefore, the amino acid utilization of the mouse myeloma J558 L cell line was measured by a kinetic study in medium supplemented with 2% serum. Fig. 3 compares the change of each amino acid tested at the end of the growth culture
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Fig. 3. Changes in concentrations of amino acid, in a J558LCD4 conventional culture. Final consumption (negative values) or production (positive values) as percentages of the initial concentrations in the DHI medium supplemented with 2% fetal calf serum after 5 days of culture.
Medium+MPA/X
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2
3
4
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6
7
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Passage Passage Fig. 4. Production stability of CD4-.H Y I producing J558L-CD4 cells. Cells were grown in duplicate in 6-well plates in 4 ml DHI medium supplemented with 0.5% fetal calf ~rum and in the presence or absence of 4 pg/ml mycophenolic acid (MPA) and ~ 0 pg/ml xanthine (X). shown in Fig. l B . T h e c o n s u m p t i o n o r f o r m a t i o n
of amino acids is shown as the percentages of the initial concentrations in DHI medium. The results of this study demonstrated that only glutamine was totally consumed and had to be fed during the late phase of the growth culture. In addition to the major group of amino acids which were poorly consumed (5-30%), another group of eight amino acids more than doubled in concentration during growth. An example of this group was alanine, which increased constantly during the growth period up to almost fourfold its initial concentration. There is strong evidence that the formation of alanine serves as a detoxification mechanism for ammonia, which is liberated by the process of glutaminolysis (Lanks and Li, 1988). The high concentration of alanine at the end of growth has been reported for most other cell lines (Butler et al., 1983; Wagner et al., 1988; Miller et ai., 1989a, b; Schumpp and Schlaeger, 1990; Bi)ntemeyer et al., 1991). In general the utilization of amino acids in a given medium depends on the cell line and differences between subclones from the same origin may be possible. The production stability of the C D 4 - H y l producing subclone was analysed (1) with selection pressure and (2) in the absence of gpt selection. The specific formation rate of the recombinant protein was measured over a one month period corresponding approximately to seven passages (Fig. 4). The production rates remained stable in both cases, suggesting that no significant loss of productivity should be expected in a scale up
process without selection pressure. The slight decrease in the specific formation rate of C D 4 - H y l compared to the values found in roller bottle cultures may have been due to the fact that the cells were grown in 6-well plates in which growth and production conditions are not optimal.
Growth in dialysis tubing In order to analyse the formation of soluble C D 4 - H y l receptor at high cell density, cells were grown in pieces of dialysis tubing which were incubated in a roller bottle in excess of culture medium (Adamson et al., 1983; Schumpp and Schlaeger, 1989). The molecular weight cut-off of the dialysis membrane was 2 × 103, indicating that only low molecular weight substances could cross the membrane. Using this simple culture system cells grew without problems of nutrient deficiency or interference from accumulated toxic metabolic end-products. The growth kinetics and the corresponding CD4-H~/1 titre are shown in Fig. 5. The cells grew continuously to cell numbers of 20-30 x 106 cells/ml with a doubling time of 40.5 h which corresponds to a growth rate of 0.017 h - I . The reason for the increased doubling time is not clear. Other cell lines did not show different doubling times when grown in dialysis tubing compared to conventional cultures
(Schumpp and Schlaeger, 1989). The high cell density phase could be maintained with a viabiliity of 50% over at least 200 h demonstrating that the exchange of low molecular substances partially prevents entry into the decline phase. Factors limiting further cell growth in this membrane system may have included the insufficient availability of oxygen, clogging of the dialysis membrane and formation of loose cell aggregates. It appears that at a critical cell concentration the mieroenvironment in the tubing was irreversibly changed so that further growth was almost impossible. However, the survival of the non-dividing culture was established with a rather low death rate. Results obtained with other cell lines indicate that the critical point at which the cells shift into the so-called dense stationary phase is different for each individual cell line. High cell concentrations in the dialysis tubing (more than 60 X 106 cells/ml) were correlated with a high viability in the stationary phase (70-80%), whereas cell lines which only grew to 30 × 106 celis/mi always had a greater death rate (Schumpp and Schlaeger, 1989). Using the J558LCD4 cells in a high cell density perfusion system (see below), cell numbers similar to the dialysis tubing experiment were observed, supporting our suggestion that the limitation in the high cell
Production of CD4-71 in dialysis tubing (1% FCS) 100 A
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:i Time (h) Fig. 5. ,4: proliferation of CD4-HT! producing J558L-CD4 cells in dialysis tubing. 15 pieces of dialysis tubing containing [ ml cells in DH] medium supplemented with 0.5% fetal calf serum were incubated in 300 ml of this same medium changed every other day.
Each point shows the average of duplicates. Viable cell number (e e), CD4-Hyl production (D D). B: SDS-PAGE analysis of CD4-H'),I containing supernatants of J558L-CD4 cells grown in dialysis tubing after 1/2 dilution in a 10% gel under non-reduced conditions. Lane h molecularweight markers; lanes 2-7: culture supernatants, lane 8: purified standard CD4-HTI. The molecularweightsof the standard proteins were: 97.4, 66.2, 45, 31 and 21.5 kDa, from the top to the bottom, respectively.
density culture is a specific property of the cell line used. Thus the dialysis tubing system offers a simple means for obtaining preliminary data on the behaviour of individual cell lines in a high density process with the advantage of parallel sample analysis. The kinetics of CD4-H~,I production (Fig. 5A) showed that synthesis and cellular release of *.he recombinant protein occurred during the exponential growth phase (0.177/.tg/106 c e l l s / h ) as well as in the non-dividing phase (0.142 /~g/106 c e l l s / h ) with roughly similar rates. This finding implies that the rate of C D 4 - H y l formation is not linked to the growth phase of the cells although a slightly higher rate was observed during the active dividing phase. However, the decrease of productivity may also have been caused by factors accumulated in the dialysis tubing. The electrophoretic analysis showed the accumulation of the C D 4 - H y l fusion proteins as well as the IgG1 light chain in the dialysis tubing during growth. It should be mentioned that the marker protein BSA exhibits a different gel eletrophoretic mobility u n d e r non-reduced or reduced conditions and the molecular weight standards always contained 100 m M DTT. (Fig. 5B). Bovine serum albumin, below the CD4-H3,1, was the third major band.
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Fig. 6. Batch culture in a 20 litre airlift reactor. Cells were grown in DHI medium with 2% fetal calf serum without gpt selection. Feeding occurred daily from the day indicated by the arrow. Viable cell number (e e), CD4-H.),I production ([] [] ).
Scale-up studies in airlift bioreactors were performed in the batch cultivation mode without gpt selection. A typical batch culture in a 20 litre bioreactor is shown in Fig. 6. A cell density of 5.5 x 106 c e l l s / m l was achieved in D H I medium containing 2% FCS. Generally, the growth properties as well as the yield of CIM-H~,I receptor in the culture medium (0.775 g for a 22 litre medium volume) under controlled conditions were similar to the results obtained in the roller bottle cultures (Fig. 1), indicating that conditions can be optimized in small culture vessels in the laboratory. Subclones producing other chimaeric CD4-Ig proteins with other Ig isotype chains such as "),3 or p., secreted as a homodimer or a pentamer,
respectively, have been constructed (Traunecker et al., 1991). Growth studies showed that cell yields were similar, but product yields varied widely with the construct and the subclone selected: 20 /.tg/ml for a CD4-/z subcione to 80 / z g / m l for a CD4-y3 subclone (Schlaeger and Schumpp, 1991). The mouse myeloma cells were also grown in a 2 litre bioreactor using a continuous culture mode with cell retention. A tangential cross-flow filtration module was connected to the bioreactor as an external loop (Velez et al., 1989; Murphy, 1990; Schumpp and Schlaeger, 1990). The culture medium containing the cells was permanently circulated through the hollow fibre unit using a peristaltic pump. The membrane pore diameter of 0.2 p.m permitted the removal of cell-frce
Tryptic digestion of CD4-y1
conditioned medium containing also the ehimaeric CD4-HT1 product. The bioreactor volume was held constant by adding fresh medium at the same rate as the permeate was drawn off. Under these conditions there was an exponential growth phase up to a cell concentration of 20 × 106 cells/ml with a growth rate of 0.024 h-~ (Fig. 7A). The perfusion rate was constantly increased from 1.5 litre/day to 4.0 litre/day leading to a maximum cell concentration of 35 x 10 ~ cells/ml which could be maintained for several days. The production of C D 4 - H y l per day increased in parallel with the cell number to a maximum level and then decreased, while product accumulated inside the bioreactor as observed by electrophoretic analysis (not shown). Threefold values were found in the permeate after connecting
proteins
IEF analysis of CD4-71 proteins M
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hcd
Fig. 8. Analysis of CD4-H~,I proteins. CD4-Hyl moleculespurified from a low cell density (Icd) culture (4x 10t~ cells/ml) and from a high cell density(hcd) culture (35 x 106 cells/ml) by affinitychromatographywere compared by limited tryptic digestion(,4) and by isoelectriefocusing(B).
119 a new hollow fibre module (Fig. 7A). We believe that retained components, such as debris of dead cells, accumulated on the membrane surface generating a layer which entrapped a large amount of protein. We therefore suggest the connection of several modules in parallel to increase the filtration capacity and culture time of the system. Specific glutamine and glucose consumption rates and ammonia and lactate production rates were always maximal in the early logarithmic growth phase and decreased approximately tenfold during cultivation (Fig. 7B). This has been described in other investigations (Miller et al., 1989a,b; Schumpp and Schlaeger, 1990; Wagner et ai., 1990). It appears that specific consumption rates increase when high glucose or glutamine concentrations are available leading to high production rates of the metabolites.
Comparison of CD4-H),I proteins from low t,ersus high cell density cultures CD4-H~,I containing culture supernatants from a low cell density culture with a final cell concentration of 4 x 106 cells/ml and from a high cell density culture with a tenfold higher cell concentration were purified by protein G affinity chromatography as described in the materials and methods section. Proteolytic fingerprints were made to characterize the CD4-H~,I molecules. The tryptic patterns were identical in both cases indicating a similar sensitivity to trypsin digestion and therefore similar native structures (Fig. 8A). The SDS-PAGE analyses were also similar (not shown). Isoelectric focusing (IEF) permits analytical studies of protein heterogeneity based on different isoelectric points. I E F gels showed one very dominant band and a few minor bands (Fig. 8B). In both eases the proteins migrated at the same isoelectric point of 8.5-8.6. This very basic value was expected from the amino acid sequence of the CD4 receptor. The J558L-CD4 cells were grown in serum-reduced DHI medium for several months in the absence of selection pressure. The CD4-H~,I production, which was not growth-associated, remained stable during this time. If serum-free or protein-reduced conditions are required, it ap-
pears to be relatively simple to wean these cells from serum-containing medium by adding transferrin, insulin and Primatone RL. These are important considerations if chimaeric proteins are developed for therapeutical use. Recombined CD4-H~/I molecules purified from low or high cell density cultures were similar with respect to their molecular weight, tryptic digest pattern and isoelectric point, indicating a high homogeneity in their native structures. From the cell culture point of view the mouse myeloma cell line J558L appears to be a good candidate as an expression system for the production of soluble chimaeric forms of membrane proteins.
Acknowledgements We are grateful to A. Traunecker and K. Karjalainen at the Basel Institute for Immunology for providing the J558L-CD4 cell line and for their interest in this investigation. We also thank S. Mathews and H. Loctscher for their critical reading of the manuscript,
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