Biotechnol. Rog. I O Q I , 7, 445-454
Manipulation of Heterogeneous Hybridoma Cultures for Overproduction of Monoclonal Antibodies Katherine L. McKinney,?Robert Dilwithf and Georges Belfort'tt Bioseparations Research Center, Howard P. Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180-3590, and Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201
In searching for ways to manipulate heterogeneous hybridoma cell cultures (ATCC HB124)t o obtain increased production of monoclonal antibodies (IgGa), we have selected for a higher secreting but slower growing subpopulation using the level of fluorescent surface-associated antibodies and a fluorescence-activated cell sorter. Cell surface fluorescence was found to be correlated with specific antibody secretion rate over the short term but not with intracellular antibody content. Also, the specific secretion rate of a heterogeneous population of hybridoma cells grown in batch culture has been shown to be inversely correlated with an increase in either the initial cell concentration or the medium antibody concentration. Several experiments suggest that an upper limit exists for medium antibody concentration, above which antibody is degraded at the same rate at which it is produced. Should other cell lines behave similarly, strategies for overproduction of monoclonal antibodies suggested herein could be profitably used in industry.
Introduction Hybridoma technology has made it possible to produce large amounts of monoclonal antibodies in vitro and thus has extended their use to many applications. As well as servingas a detection tool for molecular biologists, antibody specificityhas been exploited in such areas as affinity chromatography, antibody-mediated immunotherapy in the treatment of tumors and infectious diseases, and the use of antibodies as vaccines and even as enzymes that can catalyze chemical transformation of target molecules (Carlsson and Glad, 1989; Leist et al., 1990; Janda, 1990; Schultz, 1989). Due to complicated cell growth requirements as well as specialized downstream processing techniques,monoclonal antibody production is quite expensive, and methods for increasing production on a cellular level have been suggested. Researchershave observed a shift, for example, from cells that are high producers of immunoglobulin to those that are low producers, possibly due to chromosomal mutation or loss with culture age (Leibson et al., 1979; Andreeff et al., 1985;Altshuler et al., 1986b; Heath et al., 1990; Frame and Hu, 1990; Ozturk and Palsson, 1990b). Leibson et al. (1979) reported that cell surface IgA on a myeloma cell line decreased from initial levels over a 16month period to where a bimodal distribution in surface antibody developed in which 30% of the cells displayed an intermediate level of surface IgA. After 30 months, 90% had intermediate levels of surface IgA. Heath et al. (1990)reported a decline in intracellular antibody content as measured by flow cytometry over an 18-week period, while Ozturk and Palsson (1990b) reported a similar but reversible shift during a low serum adaptation study over a comparable time span. Frame and Hu (1990)suggested that a decrease in antibody production observed in continuous culture of hybridoma cells was due partly to the occurrence of a nonproducing subpopulation. Because
* Corresponding author. + Rensselaer Polytechnic Institute. t
New York State Department of Health. 8756-7938/91/3007-0445$02.50/0
of this shift to lower producing cultures, it has been suggested that cultures be periodically reseeded with cells that have been selected as high producers. Scientists have used cloning techniques such as limiting dilution as methods to isolate high-secreting clones; however, this is considered to be a tedious, labor-intensive process that is often unreliable (Underwood and Bean, 1988). Fluorescence-activatedcell sorting (FACS)has enabled larger quantities of cells with desired characteristics to be asepticallycollected in a rapid and efficient manner (Parks et al., 1979; Steinkamp, 1984). The criteria for selection of high-producing cells by flow cytometric methods have been based on the isolation of high-producing cells within gel microdroplets or of cells containing an increased amount of surface antibody. In the first procedure, single cells are encapsulated in an alginate or agarose matrix that contains immunobeads specific for the product and that is permeable to fluorescent antisera (Powell and Weaver, 1990). The product secreted by each cell is captured near the cell and can be quantified. The second technique involves staining the surface of the hybridoma cells directly with fluorescently labeled antisera and selecting for those cells displaying an increased amount of surface fluorescence (Leibson et al., 1979;Marder et al., 1990). This method would enable rapid and efficient sorting of large quantities of high-producingcells provided that surface fluorescence correlated with antibody secretion rate. Several recent papers have reported some contradictory results regarding the correlation between surface antibody content, intracellular antibody content, and antibody secretion rate (Meilhocet al., 1989;Senet al., 1990; Marder et al., 1990). Meilhoc et al. (1989)observed two populations with respect to surface antibody; however, specific antibody secretion rate did not correlate well with surface immunofluorescence or total cell-associated IgG. Sen et al. (1990)found a linear correlation between mean surface fluorescence and the specific antibody production rate, while Marder et al. (1990) used FACS to isolate clones
0 1991 American Chemical Society and American Instkute of Chemical Englneers
derived from highly fluorescent sorting regions and found enhanced immunoglobulin secretion from these clones. After summarizing the experimental details, we present and discuss results for four different experiments with the HB124 hybridoma culture. The objectives of the first experiment were to determine (i) if surface antibody was correlated with specific antibody secretion rate, (ii) whether high secretors could be segregated, and (iii) how long they could maintain their superior secreting capability. The purpose of the second experiment was to determine if intracellular antibody content correlated with surface-associated fluorescence, which represents antibody content on the surface of the cells. The goals of the third and fourth experiments were to determine whether antibody secretion rate was influenced by the hybridoma cell concentration and the external medium antibody concentration, respectively. The answers to each of these questions are summarized in the conclusions. Materials and Methods Cell Line and Growth Conditions. Murine hybridoma cells (ATCC HB124, or DB968, Rockville, MD) producing an IgG2, against bovine insulin were used in these studies. The cells were cultured at 37 "C in a humidified incubator with 5% COz saturation. Media consisted of RPMI-1640 (Gibco, Grand Island, NY) and 2.0 g/L sodium bicarbonate supplemented with 3.2 mL of 50X MEM amino acids, 1.6 mL of lOOX nonessential amino acids, and 1.6 mL of 1OOX MEM vitamins (Gibco, Grand Island, NY) per liter. Fetal bovine serum (Gibco, Grand Island, NY) was added a t approximately 10% (v/v), and gentamicin sulfate (Tri Bio Laboratories, State College, PA) was added at a concentration of 25 pg/mL. Cells were cultured in 25-cm2 and 75-cm2 T-flasks as well as 250-mL spinner flasks. Samples were withdrawn at appropriate intervals to determine cell count and viability measurements by using the trypan blue exclusion method and a hemacytometer. Supernatant antibody samples were frozen for later analysis by a modified ELISA (Altshuler et al., 1986a). Flow Cytometry. Analysis of surface antibody, intracellular antibody, and DNA distributions were performed on an EPICS C flow cytometer and an EPICS 752 flow cytometer (Coulter Electronics, Hialeah, FL). Excitation of cell samples at 488 nm from argon lasers was employed. The cytometers were calibrated with 10-pm fluorescent beads (Coulter Electronics, Hialeah, FL), with a coefficient of variance less than 2 % , prior to running samples each day. A dual staining procedure described by Altshuler et al. (1986b) allowed simultaneous analysis of intracellular antibody content and DNA distribution. The cells were fixed with ethanol and stained with a fluorescein isothiocyanate (FITC) conjugated goat anti-mouse (GAM) IgGl for the negative control sample and an FITC-conjugated GAM IgG2 (Meloy Laboratories, Springfield, VA) for the positive sample. The negative control sample generates a peak accounting for nonspecific binding of the antisera and cellular autofluorescence. All antibodies used in the flow cytometric analyses were polyclonal antibodies. Negative and positive samples were run separately, and 10 000 cells from each sample were analyzed. Propidium iodide (Sigma Chemical Co., St. Louis, MO) was used to stain the DNA at a concentration of 20 pg/mL. Antibody molecules on the surface of the cells were also stained with FITC GAM IgG, as the negative control and FITC GAM IgG2 as the positive stain. An FITCconjugated rabbit anti-mouse (RAM) IgGPb and FITC
Biotechnol. Prog., 1991, Vol. 7, No. 5
I ..-..= " ......... ......... ,,
f Log Green Fluorescence
Figure 1. Typical bitmaps constructed for sorting cells by surface antibody content. Forward angle light scatter, representing cell size, is plotted against log green fluorescence, which represents IgG,, content on the surface of the cells.
RAM IgG2, (ICN,Lisle, IL) were used in later experiments as the negative and positive stains, respectively. In the surface staining procedure (Heath, 1988), the cells remained viable and were kept sterile. The cells were initially washed twice with cold buffer containing 2 % fetal bovine serum and then incubated on ice with the appropriate antisera for approximately 30 min. The cells were washed again in the cold buffer and analyzed immediately on the flow cytometer. Antibody on the surface of the cells was also labeled by using fluorescent latex beads, 0.79-pm diameter (Baxter Healthcare, Mundelein, IL), that had been labeled with RAM IgGl and IgGz, (Zymed Laboratories, South San Francisco, CA). The cells were stained by layering the bead solution over cell suspensions contained in 24-well plates. The plates were centrifuged forming a monolayer of cells coated with beads (Parks et al., 1979; Heath, 1988). To ensure that cell sorting was carried out under sterile conditions, the flow cytometer sample lines were flushed with a 70% ethanol solution followed by sterile water prior to running samples. Labeled cells could then be sorted on the flow cytometer. Cell sorting was effected when droplets containing cells with the appropriate characteristics (Le., size, granularity, and green fluorescence) acquired a charge and were deflected into the appropriate containers. Bitmap gates, or windows, were created that enabled sorting a percentage of the cells with the highest and lowest surface antibody content. Bitmap windows were drawn by using forward-angle light scatter (FALS) representing cell size and 90° light scatter representing cell granularity for surface-stained cells in order to exclude doublets, cell debris, and nonviable cells from the analysis. Percent positive fluorescence was determined by considering the percentage of cells displaying fluorescence intensity levels above those displayed by the negative control peak. Sorting via surface antibody content was performed by drawing bitmap windows based on cell size and green fluorescence signals as shown in Figure 1. Care was taken so that only cells of the same size were sorted. FALS and red fluorescence measurements, representing DNA content, were considered when drawing bitmaps for internally stained cells. Comparison of surface antibody fluorescence on nonproducing myeloma cells (ATCC CRL 1581, Rockville, MD) and on the hybridomas revealed that background
Bbtechml. Rug..,1991, Vol. 7, No. 5
staining was approximately the same for the FITC GAM IgGl and IgG2 (Meloy Laboratories, Springfield, VA) and the FITC RAM Ig&b and IgGza (ICN, Lisle, IL), ensuring that IgG2a was providing the positive signal on the surface of the hybridomas. Cells were also stained with F(ab’)2 fragments and hence did not contain the Fc region of the antibody, to ensure that the presence of Fc receptors on the cell surface was not interfering with surface fluorescence measurements. Staining with FITC-conjugated goat anti-human (GAH) IgG F(ab’)2 and FITC GAM IgG (H L) F(ab’)2 (Boehringer Mannheim, Indianapolis, IN) as the negative and positive stains, respectively, showed very similar surface fluorescence measurements to those from the antisera containing intact antibodies. Antibody Purification. Immunoglobulin from the culture medium of the hybridomas was purified and used in later experiments. The supernatant was passed through a 0.2-pm filter to remove any debris remaining after centrifugation of the culture fluid. Ultrafiltration was then performed with a rotating filter apparatus (Benchmark, Membrex, Garfield, NJ) with a 30 000 molecular weight cutoff membrane. The supernatant was concentrated approximately 15-fold by this procedure. Purified antibody was then obtained by using affinity chromatography and the protein A Maps I1 buffer system (Bio-Rad Laboratories, Richmond, CA). Purified IgG2a at a concentration of approximately 0.7 mg/mL was obtained as determined by ELISA and analysis of total protein. A dye-binding assay was used to determine the total protein concentration (Bio-Rad Laboratories, Richmond, CA). It is unlikely that other medium components were concentrated along with the antibody due to the specificity of the purification method used.
Experimental Procedure Batch and Fed-BatchSuspension Cultures of Cells Sorted by Surface Antibody Content. The surface antibody of hybridoma cells stained with FITC GAM IgGl and IgG2 was analyzed, and cells were sorted on the flow cytometer. Separate flasks containing 4% of the cells with the highest and lowest surface antibody as determined by surface fluorescence measurements were obtained. An unsorted population of cells was collected as well by running cells through the flow cytometer in order to exclude nonviable cells from the population. Care was taken to obtain the same number of cells in each flask so that all cultures would be subjected to similar conditions. Cells were cultured in 250-mL T-flasks, and samples were taken each day over a 6-day period to determine cell count, viability, and medium antibody Concentration. Cultures were seeded at approximately 4 X lo4viable cells/mL and 95 5% viability. Surface antibody content was measured via the FITC-labeled antisera on day 4 of each batch culture. The surface antibody measurement obtained by staining cells with the FITC-labeled antibody did not give as strong a signal as was desired for separation of the high from low surface antibody containingcells. Consequently, fluorescent beads were used in place of the FITCconjugated antibody, and cells were sorted on the flow cytometer. Cultures were seeded at approximately 6 X lo4 viable cells/mL and 90% viability. Use of the fluorescent beads as the surface antibody marker expanded the fluorescence level of the positive sample beyond the control; however, considerable difference in antibody secretion rates for the two batch studies was not observed. A similar procedure was followed for the fed-batch suspension culture of sorted cells. The antisera used in this procedure was the FITC RAM IgGzb and IgGh. Four
percent of the cells with the highest and lowest surface antibody content were collected as well as an unsorted population. After 2 days, the sorted cells were counted and centrifuged in order to obtain essentially zero initial antibody concentration. The cells were seeded a t 1X lo4 viable cells/mL in 250-mL T-flasks with 15-mL working volume. Fresh medium was added periodically, and samples for cell count, viability, and medium antibody concentration were obtained over a 10-day period. Intracellular Antibody Content of Cells Sorted by Surface Antibody Content. Cells were sorted after surface staining with antisera (Meloy Laboratories, Springfield, VA) as described previously, and 10% of the cells with the highest and lowest surface antibody content were collected. An unsorted population was collected as well. A larger percentage of the population than previously used was collected in this experiment so that enough cells would be available for internal staining. Sorted cells were immediately stained internally for antibody content and DNA distribution and analyzed once again on the flow cytometer. I t should be noted that the contribution of the surface fluorescenceto the overall cellular fluorescence is so small that fluorescence signals would fall in the negative range when surface-stained cells are analyzed a t cytometer settings used for intracellular fluorescence analysis. Therefore, the surface staining procedure should not interfere with the intracellular fluorescence measurements. Variation of Cell Density in Batch Suspension Culture. Cells were seeded a t densities of 1 X 104, 5 X lo4, 1 X lo6, 5 X los, and 1 X los viable cells/mL in 250mL T-flasks with 70-mL workingvolume. The inoculation procedure involved centrifuging the cells and resuspending them in the appropriate medium, as the study was conducted in both conditioned and unconditioned media. The conditioned medium was prepared by seeding 250mL spinner flasks with 5 X 104viablecells/mL and growing them for 2 days. The medium was aseptically collected and centrifuged for use in the experiment. The cultures were incubated overnight, stained with FITC RAM Ig&b and IgGz,, and analyzed for surface antibody content on the flow cytometer. Samples were taken for cell count and analysis of medium antibody concentration. Variation of Antibody Concentration in Batch Suspension Culture. Cells were seeded at 2 X 10Sviable cells/mL in 25-cm2T-flasks with 10-mL working volume after centrifugation and resuspension in fresh medium. Purified antibody was added to the cultures in concentrations of 0,28,56, and 112 pg/mL for experiment 1and 0,21,42, and 70 pg/mL for experiment 2. The cultures were incubated overnight and analyzed for surface antibody content after staining with FITC RAM IgGzb and IgGza. Samples were taken to obtain cell counts and medium antibody concentrations.
Results Batch and Fed-BatchSuspension Cultures of Cells Sorted by Surface Antibody Content. In order to determine whether batch-cultured cells with high or low surface antibody levels retain their characteristic levels after cell sorting, we labeled, sorted and recultured the cells to obtain three different populations: those with high surface antibody content, unsorted, and those with low surface antibody content. The levels of surface antibody fluorescence for the three populations after the cells were cultured were then determined and compared. The results are shown in Figure 2 for two different labeling methods: one performed with FITC-labeled antibody as the positive
Biofechnol. Prog., 1991, Vol. 7, No. 5
I 23% P o s l r l v r
L))g Green Fluorescence
L o g Green Fluorescence
Figure 2. Surface antibody (IgG,,) histograms of sorted populations of HB124 hybridoma cells grown in batch culture that were analyzed on day 4 postseed after being stained with FITC-labeled antisera. (a), (b), and (c) are the high, unsorted, and low surface antibody populations, respectively, derived from cells stained with FITC-labeled antisera; (d), (e), and (f) are the high, unsorted, and low surface antibody populations, respectively, derived from cells stained with antibody-labeled fluorescent beads, 0.79 pm in diameter. Surfaces were stained with FITC GAM IgG, (negative distribution) and IgG2 (positive distribution). The difference is reported as percent positive.
antiserum and the other with antibody-labeled fluorescent beads. Cells originally with high surface antibody content (Figure 2a,d) remained high, at about the same or at a slightly higher percentage of positive surface antibody fluorescence as compared to the unsorted population (Figure 2b,e). Those cells originally with low surface antibody content (Figure 2c,f), however, showed a significant drop, by half, in positive percentage as compared to the unsorted population. At least over several generations the cells retained their characteristic levels of surface antibody This result is confirmed for both sets of cultures, each with different labeling methods. The batch results shown in Figure 2 are summarized in Table I together with specific IgG secretion rates and final medium antibody concentrations for the three populations described above. The cell viabilities for each culture were very similar (data not shown). The secretion rates are calculated according to d,IgG]/dt = k X ( t ) (1) where k is the specific antibody secretion rate and X is the viable cell concentration (Renard et al., 1988; Meilhoc et al., 19891. The initial specific IgG secretion rates were higher for the cells with the highest surface antibody content and dropped 4-7-fold during the batch run. The medium antibody concentration after 6 days of culture was 25 f 0.4 pg/mL for all the cultures with no dependence on the sorting by surface antibody or the label used. One possible explanation is that after 6 days, with very low cell viability and low culture pH, acid proteases become active
and degrade the IgG (Schlaeger et al., 1987; Karl et al., 1990). Another is that amino acid depletion in culture media may affect regulation of proteolysis, as amino acids have been found to inhibit protein degradation (MacLennan et al., 1988; Al-Rubeai and Emery, 1990). Finally, in most sorted cultures, the cells displaying low surface antibody fluorescence exhibited higher initial growth rates than both unsorted cultures and cultures displaying high surface antibody fluorescence (data not shown). Cell growth, medium antibody concentration, and antibody secretion rates in fed-batch mode for the three sorted populations described above are shown in Figure 3. Although all the cultures were seeded at approximately the same cell density, the population originally with low surface antibody content grew faster and reached higher cell densities than the other two cultures (Figure 3a). Also, because of the higher cell concentration, the antibody titer for this culture was higher than the other two (Figure 3b). The specific secretion rates after 1 day, however, are the highest for the cells from the culture with the highest level of surface antibody. The cumulative average specific antibody secretion rates are plotted in Figure 3c. This figure sh0v.s that over the course of the fed-batch run the cells displaying high surface antibody fluorescence secreted more antibody per cell on average. Clearly, one strategy for maximizing antibody production from a cell culture in fed-batch mode could be to separate and concentrate the cells with high surface antibody content. This could possibly be achieved by a serial sorting scheme in which cells with high surface antibody fluorescence are selected
BbrechnoL Rog., 1991, Vol. 7, No. 5
Table I. Antibody (IgGa) Production in Batch Culture by HB124 Hybridoma Cells Sorted via Fluorescent Surface Antibody Content surface fluorescence4 FITC-labeledsort bead-labeledsort high unsorted low high unsorted low 35 R positive fluorescence on day 4 postaeedb 34 17 23 18 9 96.5 95.4 79.8 41.6 30.8 21.5 initial specific IgG secretion rate, (pg/day) per 106 cellsC 17.6 final specific IgG secretion rate, (pg/day) per 106 cellsd 15.5 16.7 6.5 4.3 4.9 25.4 25.2 24.6 24.6 25.7 24.9 IgG concn on day 6 postseed, pg/mL a Removed 4 % of cells with highest and lowest surface antibodyas determinedby surface fluorescencemeasurements. Surfaceswere stained with FITC GAM IgGl (control) and IgGh or with antisera adsorbed onto 0.79-pm diameter fluorescent latex beads. b Approximately 10 OOO cells were analyzed for each sample. Secretion rates for days 0-1 for FITC-labeledsort and days e 2 for bead-labeled sort. d Secretion rates for days 1-3 for FITC-labeled sort and days 2-3 for bead-labeled sort.
. , . , . I 8
Figure 3. HB124 hybridoma growth and antibody (IgGa) production for sorted cells, (0) high, (v) unsorted, and ( 0 )low fluorescent surface antibody, grown in fed-batch culture: (a) viable cell concentration, (b) medium antibody concentration, and (c)cumulative average antibody secretion rate. The decrease in viable cell count and antibody concentration on some days representa media additions.
from the population by many rounds of sorting, allowing the cells to recover between each analysis. Such a procedure may enable a stable population of high secretors to be obtained. The feasibility of using such a technique to obtain higher titers of antibody, however, needs to be verified.
Intracellular Antibody Content of Cells Sorted by Surface AntibodyContent. Altahuleretal. (1986b)were one of the earliest to report that the internal antibody content of a hybridoma culture (ATCC HB124) displayed a bimodal distribution. Since then several others have confirmed this finding with the same (Heath et al., 1990) and other cell lines (Ozturk and Palsson, 1990b;Sen et al., 1990). In order to determine whether surface and internal antibody levels are correlated, HB124 cells were sorted by surface fluorescence to obtain cultures containing high, unsorted, and low levels of surface antibody and then stained for internal IgG content. The internal antibody distributions for these three populations were indistinguishable (data not shown). Variation of Cell Density in Batch Suspension Culture. Surface antibody histograms for cultures grown at different initial cell densities in unconditioned and conditioned medium show a decrease in the surface fluorescence with an increase in initial cell density (Figure 4). This is also accompanied by a decrease in the specific IgG secretion rate (Table 11). In conditioned medium, the specific secretion rate is much higher at low initial cell concentrations when compared to unconditioned medium. The specific antibody secretion rate increased 100-fold for cultures seeded in conditioned medium with 1 X 104 versus 1 x los cells/mL. The equivalent increase in unconditioned medium was just over 6-fold. Viabilities for all cultures were between 85 % and 95%. Variation of Antibody Concentration in Batch Suspension Culture. By adding purified IgGb from HB124 hybridoma supernatant into cell cultures, 0-112 bg/mL for experiment 1and 0-70 pg/mL for experiment 2, the effect of medium antibody concentration on the surface antibody fluorescence and specific antibody secretion rate for a fixed cell concentration was determined (Table 111). Increasing the medium antibody concentration resulted in a decrease in the surface antibody content. Specific IgG secretion rate also decreased and even became negative at high enough medium antibody concentrations. Since specific IgG production rate is defined as the difference between inherent secretion and degradation, a negative value implies that degradation is greater than inherent secretion. The two rates were approximately equal at a medium IgG concentration of around 32 pg/ mL. Discussion In an effort to understand the relationship between surface-associated, internal, and secreted antibody and the effect of cell density as well as medium antibody concentration, we have measured these parameters and derived specific IgG secretion rates for the HB124 hybridoma cell line. It is important to note that we are interested in understanding in what kind of environment
Blotechnol. Prog., lQQl, Vol. 7, No. 5
Table 111. Surface Antibody (IgGt.) Content and Antibody Production for HB124 Hybridoma Cultures Grown at Varying Initial Medium Antibody Concentrations.
medium antibody concentration, pg/mL experiment 1: experiment 2
Experiment 1 9% positive surface 44 43 fluorescenceb specific IgG secretion rate, 42.4 3.0 (pg/day)per 106 cells
Experiment 2 9% positive surface 40 26 10 12 fluorescence* specific IgG secretion rate, 38.1 2.6 -5.4 -63.2 (pg/day)per 1Oe cells a Initial cell density was 2 X 1@ cells/mL for all flasks. b Approximately 10 OOO cells were analyzed for each sample 1day after seeding flasks.
Log Oreen Fluorescence
Log Oreen Fluorescence
Figure 4. Variation of cell density in batch culture. Fluorescent surface antibody (18023 histograms for HB124 hybridoma cultures grown at different initial cell densities in (a) unconditioned and (b) conditioned media. Cells were analyzed 1 day after flasks were seeded. Placement of the cursor represents the point at which cells are considered to have positive fluorescence, as the negative peaks are not shown. Table 11. Fluorercent Surface Antibody (IgGt.) Content and Antibody Production for Batch HB124 Hybridoma Culturer Grown at Varying Initial Cell Densities seeding density (cells/mL) 1x 104
Unconditioned Media $1positive surface
fluorescence0 specific IgG secretion rate, (pg/day)per 106 cells medium antibody concn,
rg/mL Conditioned Media % positive surface 66 43 fluorescencea specific IgG secretion rate, 593.9 102.9 (pg/day)per 106 cells medium antibody yield, 9.8 9.0
pg/mLb a Approximately 10 OOO cells were analyzed for each sample 1day after seedingflasks. b Valuesarefinal IgG concentration minus initial conditioned medium IgG concentration.
the cells are likely to overproduce or underproduce antibody. Many of the results presented have shown that increased specific antibody secretion rate is associated with lower final yield in the culture medium. We believe these
results are important in designing culture methods and possibly purification schemes for hybridoma cell cultures. The importance of nutrient replenishment, waste removal, and product removal is demonstrated by the results presented. It should also be noted that we are mainly concerned with reproducible trends observed in these experiments. We have shown that cells which were separated into populations containing high, unsorted, and low levels of surface immunoglobulin are relatively stable over a t least several generations. Not only do the cells with high surface antibody fluorescence maintain high surface content in culture, but they also exhibit higher initial IgG secretion rates (Table I and data not shown). IgG yield, however, on the final day of the batch run was approximately the same, 25 Fg/mL, for the six cultures. As nutrients were depleted and waste products and antibody accumulated in the medium around day 3, all cultures became stressed and the cells could not perform optimally. Because the secretion rates were significantly different for the first 2 days only, a fed-batch run of sorted cells was performed in order to retain high cellular productivity for a longer period. The cultures could then be more reliably compared. Results in Figure 3 from the fed-batch study showed that the cells maintained secretion rates corresponding to their surface antibody fluorescenceand that the cells sorted from the low fluorescence region had higher growth rates than those from the unsorted and high surface fluorescence regions. It has been shown that cells often produce more product a t slowed growth rates, because cells are able to utilize more of their metabolic energy for antibody production than for cell growth. Suzuki and Ollis (1990) found that monoclonal antibody production could be increased by 50-120% in cultures treated with growth inhibitors that did not have an effect on antibody synthesis directly. In a long-term study performed by Ozturk and Palsson (1990b), losses in antibody productivity were observed as cells were adapted to low serum levels with improvement in growth rate. Heath et al. (1990) showed that IgG secretion rates increased as the percentage of serum in media was decreased for cells grown in batch culture. This was thought to be the result of a decreased rate of exponential growth. This study suggested that overgrowth of lower producing cells may cause loss in culture productivity over time, stressing the importance of reseeding cultures with cells selected to be high producers.
Biotechnol. Rw., 1991, Vol. 7, No. 5
Heath et al. (1990) as well as Ozturk and Palsson (1990b) also observed that internal antibody content of hybridoma cells decreased with culture age, along with a decline in medium antibody concentration. Therefore, it is possible that internal antibody content may be an indicator of productivity. Considering this postulate and assuming that surface and secreted antibody were correlated, cells sorted by surface antibody content were assayed for internal antibody content. This could be done immediately after sorting, because the fluorescently labeled surface antibody represents just a small fraction of the total cellassociated IgG. Because the populations all had similar internal IgG distributions, the limiting factor in productivity seems to involve the secretion mechanism and may not be strictly dependent on internal IgG content. Furthermore, DNA distributions for cells sorted by surface antibody content were found to be nearly identical. This does not ensure that IgG synthesis or secretion is not cell cycle dependent but rather that we are not selecting for cells that are in a particular phase of the cell cycle. Likewise, Altshuler et al. (1986b) did not find a change in the internal IgG distribution with cell cycle phase (Gl, S, G2, or M), suggesting that the internal IgG concentration was stable over the short term. Other investigators have proposed that antibody synthesis is dependent on cell cycle events and that maximum synthesis may occur in late G1 and early S phases of the cycle. Therefore, methods to accumulate hybridoma cells in G1 phase of the cell cycle have been recommended in order to optimize antibody production (Al-Rubeai and Emery, 1990; Ramirez and Mutharasan, 1990; Suzuki and Ollis, 1989). I t has been suggested that posttranslational processing steps may regulate protein secretion rate in eukaryotic cells (Gebhart and Ruddon, 1986). In order for hybridoma cells to secrete increased amounts of immunoglobulin under conditions of slowed growth during which protein translation rate is decreased, the rate of assembly or secretion steps must be increased (Bibila, 1991). Such essential steps may include processing in the endoplasmic reticulum (ER) or transport of the assembled antibody from the ER to Golgi apparatus or from the Golgi to the extracellular medium. The heavy-chain binding protein (BiP), also referred to as the glucose-regulated protein GRP78, and protein disulfide isomerase (PDI) are proteins that mediate antibody assembly in the ER (Munro and Pelham, 1986; Goochee and Passini, 1988; Flynn et al., 1989;Rothman, 1989; Bibila, 1991). BiP is constitutively expressed and is induced under conditions in which misfolded or mutant proteins accumulate in the ER. PDI functions to catalyze the formation of disulfide bonds in immunoglobulins. Interestingly, PDI levels have been shown to correlate with antibody secretion rates (Roth and Koshland, 1981). Therefore, productivity optimization, in which flow cytometry could play an important role, may conceivably be dependent on the assembly and secretory processes within the cell. Also, because the fluorescent antisera used in staining may bind unassembled antibody chains, internal IgG content may not be as good an indicator of productivity as surface fluorescence measurements. Density of cells in the culture flask has also been shown to influence cell surface fluorescence and antibody productivity (Figure 4 and Table 11). Very low cell densities showed the highest surface fluorescence, which decreased with increasing cell density (Figure 5a). As expected, Figure 5b shows increased antibody yield for cultures with higher initial cell densities. Specific secretion rates generally decreased with increasing cell density, with the
0 ' Ib4
. . ......
Figure 5. Variation of cell density in batch culture for unconditioned (a) and conditioned (e)media: (a) correlations between fluorescence on the HB124 hybridoma cell surface and seeding density for cells grown in unconditioned medium (r2 = 0.872), and for cells grown in conditioned medium (9 = 0.898), (b) effect of seeding density on antibody yield for unconditioned medium (r2 = 0.972), and (c) specific antibody secretion rate correlated with seeding density in unconditioned medium (9= 0.823) and in conditioned medium (data from Table 11). highest secretion rate seen for the culture seeded at 1 X lo4 cells/mL in conditioned medium (Figure 5c). The conditioned medium contains important growth factors and nutrients that otherwise have to be produced by the cells when they are suspended in fresh medium. In contrast, Ozturk and Palsson (1990a)found little difference in antibody production rate for cells grown in batch culture at varying initial cell densities (from 10s to lo5cells/mL). Figure 6a shows that cell surface fluorescence is inversely correlated with medium antibody concentration for the cells cultured in unconditioned medium. These results suggest that cells should optimally be kept at low cell density with continued removal of product in order to produce IgG at high rates. As mentioned previously, this may not correspond to the highest medium antibody yield obtainable; however, consideration of environments in which the cells are likely to overproduce will be important in designing new cell culture methods. Therefore, ex-
Biotechno/. Rog., 1991, Vol. 7, No. 5 80 70
I . I
1 . 1
. , v.v, . , . , . 20
Figure 6. Correlationsbetween cell surfacefluorescence,medium antibody concentration, and specific antibody secretion rate for cells grown in unconditioned (m) and conditioned (e) media: (a) cell surface fluorescence correlated with medium antibody concentration for cells grown in unconditioned medium (rZ = 0.859),(b)specificantibodysecretionrate correlatedwithmedium antibody concentrationfor cellsgrown in unconditioned medium (r2 = 0.825),and (c) specific antibodysecretion rate versus percent positive surface fluorescence (data from Table 11).
ploiting these high secretion rates in batch culture may not be advantageous due to the fact that antibody yield (micrograms per milliliter) per unit time at low cell density is likely to be less than at high cell density. There are obviously optimum conditions in which cells will secrete to their highest capacity, perhaps growing cells in perfusion culture with removal of product and recycle of conditioned medium. The necessity of product removal is also demonstrated by the correlation of specific antibody secretion rate with medium antibody concentration as observed in Figure 6b. Secretion rates diminish drastically as medium antibody concentrations reach their maximum observed levels. The medium antibody concentration of 27 gg/mL predicted for zero secretion rate correspondswell with the maximum
Figure 7. Variation of antibody concentration in batch sus-
pension culture. Correlationsbetween medium antibody (IgGh) concentration,percent positive surface fluorescence,and specific antibody secretion rate for batch HB124 hybridoma cultures, experiment 1 (m) and experiment 2 (e),with varying initial mediumantibody concentration: (a)positive surfacefluorescence correlated with medium antibody concentration [r2= 0.897 (1) andr2 = 0.801 (2)],(b)specificantibody secretion rate correlated with medium antibody concentration [r2 = 0.961 (1) and r2 = 0.949 (2)],and (c) specificantibody secretion rate correlatedwith positive surface fluorescence [rZ = 0.758 (1) and rz = 0.628 (211 (data from Table 111). levels normally observed in HB124 cell cultures. Figure 6c shows that specific antibody secretion rate increases with cell surface fluorescence, suggesting the plausibility of using flow cytometric techniques to monitor culture productivity. Finally, in order to use surface fluorescence as a measure of productivity, it is important to compare cells grown under similar conditions. For the study involving variation of antibody concentration in the medium, a dectease in positive surface fluorescence correlated with increasing concentration of IgG (Figure 7a). This suggests that a feedback mechanism may be important in antibody production and that
Biotectwwl. Rag,, 1991, Vol. 7, No. 5
a maximum concentration may exist above which medium antibody is degraded as fast as it is produced. In experiment 1, the specific antibody secretion rate was highest, 42.2 pg/(106 cells-day), for the culture containing 0pg/mL and decreased to near zero [3.0 pg/ (10scells-day)] in the culture containing 28 pg/mL antibody. In the remaining cultures the secretion rate became increasingly negative with increased concentration of antibody in the medium. Similar results were observed in experiment 2. Figure 7b shows a strong correlation between specific antibody secretion rate and initial medium antibody concentration. There seemed to be an upper limit for antibody concentration in the culture medium as apparent secretion rate dropped to zero around 27-36 pg/mL antibody. This apparent upper limit of monoclonal antibody concentration may also help to explain why the sorted cultures mentioned previously all yield around 25 pg/mL antibody on the final day of batch culture (Table I) and why the data in Figure 6b predict an upper limit of 27 pg/mL as specific antibody secretion rate decreases to zero. Therefore, the necessity of product removal from cell cultures to improve productivity is once again reinforced. I t is important to note that surface antibody fluorescence correlates with secretion rates (Figure 7c), but the lowest surface fluorescencemeasurements observed are unexpectedly high for cells that are apparently not secreting antibody. Antibody production depends on inherent secretion minus degradation. Where net negative production rates are observed, the cells may still be secreting some antibody, as displayed by surface fluorescence measurements, but the rate at which antibody is degraded may have been stimulated above the secretion rate. As mentioned previously, the chemical environment is likely to play a role in regulation of proteolysis (MacLennan et al., 1988; Al-Rubeai and Emergy, 1990). Proteases have also been shown to be active, especially at pH less than 4.5, during production of antibodies by some hybridoma cultures (Karl et al., 1990; Schlaeger et al., 1987). As mentioned previously, Leibson et al. (1979) saw significant decreases in surface antibody over a 30-month period. All experiments discussed here were performed with hybridomas that were revived from liquid nitrogen storage between 17 and 42 weeks prior to being used in experiments. Only a unimodal distribution in surface antibody content has been observed for HB124 hybridomas, although researchers have observed bimodal distributions for other cell lines (Leibson et al., 1979; Meilhoc et al., 1989;Marder et al., 1990;Ozturkand Palsson, 1990b; Sen et al., 1990). Greater differences in productivity may be observed as HB124 cells age, because their antibodyproducing capability may diminish. These and other results reported herein suggest the following strategies for maximizing antibody production from an HB124 cell culture. Cells should optimally be kept in a nutrient-rich environment, preferably with conditioned medium, at low cell growth with continuous removal of antibody. To reduce the potential overgrowth of the culture by fast-growingand lowdgG-producingcells, periodic flow cytometric analysis of the surface-associated antibodies should be undertaken. One way to do this and maintain high volumetric productivity is to use suspension cultures with high rates of perfusion of recycled conditioned medium from which the antibody has been extracted.
Conclusions In this study, not only do we show that surface antibody fluorescence is correlated with specific antibody secretion
rate for an HB124 hybridoma culture but also that we can select for these cells using a fluorescence-activated cell sorter. The sorted high producers retain their high specific antibody secretion rate and lower growth rate in fed-batch culture. Since we were unable to correlate surface antibody with intracellular Concentration,we concludethat secretion rather than intracellular production is the rate-limiting step for antibody production. Additionally, in an attempt to understand factors affecting antibody productivity, we have discovered that the surface fluorescence and specific IgG secretion rate are inversely correlated with initial cell seeding density. Also, higher seeds produce more absolute antibody in the medium, albeit a t a slower rate per cell than for lower seeds. This is one of the main reasons why entrapped cell cultures with their high cell concentration are of commercial interest. Artificially increasing the medium antibody concentration had a profound effect on specific antibody secretion rates. The secretion rate dropped from about 40 pg/(106 cells-day) with no added antibody to zero at about 32 pg/ mL antibody. This is also around the maximum predicted antibody concentration for the initial cell-seeding studies. The secretion rate eventually dropped to negative values between -63 and -101 pg/(106 cellsoday). These results must have a bearing on reactor design, especially entrapped bioreactors that use membranes with a molecular weight cutoff lower than the molecular weight of the IgG molecules, 155 000, so that the product is retained in the cell compartment. These new results suggest the following ways to manipulate heterogeneous hybridoma cultures (HB124) to produce higher titers of monoclonal antibodies (IgGe): (i) Use a fluorescence-activated cell sorter to select for high producers on the basis of their level of fluorescent surfaceassociated antibodies. Serial sorting may provide a stable culture of highproducers. (ii) Periodically test for changes in the culture secretion rate (or level of fluorescent surfaceassociated antibodies) to ensure that the faster growing but low-producing cells are not overgrowing the culture. (iii) Use a high-rate perfusion bioreactor to maintain a relatively low IgG medium concentration (as close to zero as possible) so as to obtain the highest specific antibody secretion rate and minimal antibody degradation rate. This can be done by removing the desired antibody from the medium and returning the conditioned medium to the perfusion bioreactor. Eventually, of course, waste products will have to be removed from the medium. A bleed and feed with unconditioned medium could also be used. In conclusion, the experiments reported here are relatively straightforward, yet they have strong potential bearing on commercial productivity of monoclonal antibodies. Care should be taken in generalizing these results to other cell lines. However, from previous research, HB124 is not considered to be an unusual hybridoma cell line (Altshuler et al., 1986b; Heath et al., 1990), and it is possible that other hybridoma cell lines will behave in a similar manner.
Acknowledgment We thank Gordon Altahuler and Carole Heath for laying the foundation for this work and Carole Heath and Denry Sat0 for valuable discussions. One of the authors (K.L.M.) is grateful to Howard P. Isermann for financial support.
Biotechnol. Prog., 1991, Vol. 7, No. 5
Literature Cited Al-Rubeai, M.; Emery, A. N. Mechanisms and Kinetics of Monoclonal Antibody Synthesis and Secretion in Synchronous and Asynchronous Hybridoma Cell Cultures. J.Biotechnol. 1990, 16,67-86. Altshuler, G. L.; Dziewulski, D. M.; Sowek, J.; Belfort, G. Continuous Hybridoma Growth and Monoclonal Antibody Production in Hollow Fiber Reactors-Separators. Biotechnol. Bioeng. 1986a, 28, 646-658. Altshuler, G. L.; Dilwith, R.; Sowek, J.; Belfort, G. Hybridoma Analysis at Cellular Level. Biotechnol. Bioeng. Symp. 198613, 17, 725-736. Andreeff, M.; Bartal, A.; Feit, C.; Hirshaut, Y. Clonal Stability and Heterogeneity of Hybridomas: Analysis by Multiparameter Flow Cytometry. Hybridoma 1985,4 (3), 277-287. Bibila, T. A. Structured Model for Monoclonal Antibody Synthesis in Exponentially Growing and Stationary Phase Hybridoma Cells. Biotechnol. Bioeng. 1991, 37, 210-226. Carlsson, R.; Glad, C. Monoclonal Antibodies into the '90's: The All-Purpose Tool. Bio/ Technology 1989, 7,567-573. Flynn, G. C.; Chappell, T. G.; Rothman, J. E. Peptide Binding and Release by Proteins Implicated as Catalysts of Protein Assembly. Science 1989, 245, 385-390. Frame, K. K.; Hu, W.-S. The Loss of Antibody Productivity in Continuous Culture of Hybridoma Cells. Biotechnol. Bioeng. 1990, 35,469-476. Gebhart, A. M.; Ruddon, R. W. What Regulates Secretion of Non-Stored Proteins by Eukaryotic Cells? BioEssays 1986, 4 (51, 213-218. Goochee, C. F.; Passini, C. A. Intracellular Proteins Produced by Mammalian Cells in Response to Environmental Stress. Biotechnol. h o g . 1988, 4 (51, 189-201. Heath, C. Engineering Aspects of Improved Antibody Production by Hybridomas. Ph.D. Thesis, Rensselaer Polytechnic Institute, Troy, NY, 1988. Heath, C.; Dilwith, R.; Belfort, G. Methods for Increasing Monoclonal Antibody Production in Suspension and Entrapped Cell Cultures: Biochemical and Flow Cytometric Analysis as a Function of Medium Serum Content. J. Biotechnol. 1990, 15,71-90. Janda, K. D. New Strategies for the Design of Catalytic Antibodies. Biotechnol. Prog. 1990, 6, 178-181. Karl, D. W.; Donovan, M.; Flickinger, M. C. A Novel Acid Proteinase Released by Hybridoma Cells. Cytotechnology 1990, 3, 157-169. Leibson, P. J.; Loken, M. R.; Panem, S.; Schreiber, H. Clonal Evolution of Myeloma Cells Leads to Quantitative Changes in Immunoglobulin Secretion and Surface Antigen Expression. Proc. Natl. Acad. Sci. U.S.A. 1979, 76 (6), 2937-2941. Leist, C. H.; Meyer, H.-P.; Fiechter, A. Potential and Problems of Animal Cells in Suspension Culture. J. Biotechnol. 1990, 15, 1-46. MacLennan, P. A.; Smith, K.; Weryk, B.; Watt, P. W.; Rennie, M. J. Inhibition of Protein Breakdown by Glutamine in Perfused Rat Skeletal Muscle. FEBS Lett. 1988, 237, (l),133136. Marder, P.; Maciak, R. S.; Fouts, R. L.; Baker, R. S.; Starling, J. J. Selective Cloning of Hybridoma Cells for Enhanced Im-
munoglobulin Production Using Flow Cytometric Cell Sorting and Automated Laser Nephelometry. Cytometry 1990, 11, 498-505. Meilhoc, E.; Wittrup, K. D.; Bailey, J. E. Application of Flow Cytometric Measurement of Surface IgG in Kinetic Analysis of Monoclonal Antibody Synthesis and Secretion by Murine Hybridoma Cells. J. Zmmunol. Methods 1989,121,167-174. Munro, S.; Pelham, H. R. B. An Hsp 70-like Protein in the E R Identity with the 78 kd Glucose-Regulated Protein and Immunoglobulin Heavy Chain Binding Protein. Cell 1986, 46, 29 1-300. Ozturk, S. S.; Palsson, B. 0. Effect of Initial Cell Density on Hybridoma Growth, Metabolism, and Monoclonal Antibody Production. J. Biotechnol. 1990a, 16, 259-278. Ozturk, S. S.;Palsson, B. 0.Loss of Antibody Productivity During Long-Term Cultivation of a Hybridoma Cell Line in Low Serum and Serum-Free Media. Hybridoma 1990b, 9 (2), 167-175. Parks, D. R.; Bryan, V. M.; Oi, V. T.; Herzenberg, L. A. AntigenSpecific Identification and Cloning of Hybridomas with a Fluorescence-Activated Cell Sorter. Proc. Natl. Acad. Sci. U.S.A. 1979, 76 (4), 1962-1966. Powell, K. T.; Weaver, J. C. Gel Microdroplets and Flow Cytometry: Rapid Determination of Antibody Secretion by Individual Cells Within a Cell Population. Biol Technology 1990,8, 333-335. Ramirez, 0. T.; Mutharasan, R. Cell Cycle- and Growth PhaseDependent Variations in Size Distribution, Antibody Productivity, and Oxygen Demand in Hybridoma Cultures. Biotechnol. Bioeng. 1990, 36, 839-848. Renard, J. M.; Spagnoli, R.; Mazier, C.; Salles, M. F.; Mandine, E. Evidence that Monoclonal Antibody Production Kinetics is Related to the Integral of the Viable Cells Curve in Batch Systems. Biotechnol. Lett. 1988, 10 (2), 91-96. Roth, R. A.; Koshland, M. E. Role of Disulfide Interchange Enzyme in Immunoglobulin Synthesis. Biochemistry 1981, 20,6594-6599. Rothman, J. E. Polypeptide Chain Binding Proteins: Catalysts of Protein Folding and Related Processes in Cells. Cell 1989, 59, 591-601. Schlaeger, E. J.; Eggimann, B.; Gast, A. Proteolytic Activity in the Culture Supernatants of Mouse Hybridoma Cells. Dev. Biol. Stand. 1987, 66, 403-408. Schultz, P. G. Catalytic Antibodies. Angew. Chem., Znt. Ed. Engl. 1989, 28, (lo), 1283-1295. Sen, S.; Hu, W.-S.; Srienc, F. Flow Cytometric Study of Hybridoma Cell Culture: Correlation Between Cell Surface Fluorescence and IgG Production Rate. Enzyme Microb. Technol. 1990, 12, 571-576. Steinkamp, J. A. Flow cytometry. Rev. Sci. Znstrum. 1984,55 (9), 1375-1400. Suzuki, E.; Ollis, D. F. Cell Cycle Model for Antibody Production Kinetics. BiotechnoL Bioeng. 1989, 34, 1398-1402. Suzuki, E.; Ollis, D. F. Enhanced Antibody Production at Slowed Growth Rates: Experimental Demonstration and a Simple Structured Model. BiotechnoL Prog. 1990, 6, 231-236. Underwood, P. A.; Bean, P. A. Hazards of the Limiting-Dilution Method of Cloning Hybridomas. J.Zmmunol. Methods 1988, 107, 119-128. Accepted June 25, 1991.