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Effects of Abrupt and Gradual Osmotic Stress on Antibody Production and Content in Hybridoma Cells That Differ in Production Kinetics Sridhar Reddy and William M. Miller* Department of Chemical Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3120
AB2-143.2 cells exhibit non-growth-associated antibody production (constant specific antibody production rate (qAb) throughout batch culture), while INDl cells exhibit growth-associated production (decrease in q A b during stationary phase). AB2-143.2 cells increase QAb following abrupt batch osmotic stress. Enhanced antibody production in INDl cells after batch osmotic shock is evidenced as maintenance of the higher exponential phase q A b into the stationary phase. AB2-143.2 cells also increase QAb in response to gradual osmotic stress in continuous culture. In contrast, INDl cells decrease QAb during gradual osmotic stress. Although the two cell lines differ in antibody production, they have very similar intracellular antibody content profiles. Both show (1)constant antibody content during the exponential phase in control culture with a decrease as cells enter stationary phase; (2) maintenance of exponential-phase antibody content into the stationary phase after batch osmotic shock; and (3)no change in antibody content in response to gradual osmotic stress.
Introduction Hyperosmotic stress resulting from sodium chloride addition has been shown to increase the specific antibody production rate @Ab) in hybridoma cultures (Nuss and Koch, 1976;0yaas et al., 1989;Ozturk and Palssonf1991a; Oh et al., 1993). From an industrial point of view, this is a very feasible method to increase production. It is very easy to implement an increase in osmolality. Control of a process at a specified osmolality would also be straightforward. Furthermore, almost no additional cost would be incurred because all that is required is sodium chloride. Hyperosmotic stress can be imposed as a sudden shock or be introduced gradually. Most studies on the effects of osmolality on antibody production have been limited to abrupt osmotic shock using sodium chloride or other compounds. The short-term response to hyperosmotic shock is the passive exit of water from the cell, which could be detrimental. Gradual hyperosmotic stress allows time for osmoregulation systems to help the cells adapt (Yanceyet al., 1982). Experimentswith yeast suggest that cells can tolerate much higher osmolalities if the increase is gradual (Gervais et al., 1992). Adaptation to hyperosmotic conditions may include the production of proteins resembling heat-shock proteins and the intracellular accumulation of amino acids (Silvotti et al., 1991). Most studies reporting enhanced QAb after osmoticshock have been conducted using hybridomasthat exhibit nongrowth-associated QAb. We have previously shown that enhancement of QAb by osmotic shock in a cell line that exhibits growth-associated production is evident as an extension of the exponential-phase QAb into the decline phase (Reddy et al., 1992). Here we compare the effects of abrupt osmotic shock in batch culture and gradual osmotic stress in continuous culture on cell growth, antibody production, and intracellular antibody content in two cell lines that differ in antibody production kinetics. Author to whom correspondence should be addressed. Telephone: (708)491-4828.FAX: (708)491-3728. 07587930/94/3010-0165$04.50/0
Materials a n d Methods Cell Lines and Media. All reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless noted otherwise. Tissue culture quality water (Milli-&Water System, Millipore, MA) was used in all cases. The hybridoma cell lines used in this work are designated AB2-143.2 and IND1. AB2-143.2 is a mouse-mouse hybridoma cell line formed by the fusion of a B cell and an Sp2/0 myeloma (Hornbeck and Lewis, 1985). It produces an IgGk monoclonal antibody against benzenearsonate. INDl is also amousemouse hybridoma cell line (provided by Xoma Corporation, Berkeley, CAI, resulting from the fusion of a BALB/c spleen cell and a P3x63-Ag8 myeloma. It produces an IgGb monoclonal antibody against melanoma tumor associated antigens. The medium used for both cell lines consisted of a 1:l mixture of DMEM (Sigma D777) and Ham's F12 (Sigma N6760) supplemented with 2.44 g/L (3.40 g/L for AB2-143.2) sodium bicarbonate, 5 pg/mL bovine insulin (Sigma 166341, 30 pg/mL (25 pg/mL for AB2-143.2) bovine transferrin (Sigma T1283), 20 pM ethanolamine, 20 nM sodium selenite, 1.15% (v/v) MEM nonessential amino acid (lOOX) solution, and 3.5 mM glutamine (6mM final concentration). INDl cultureswere additionally supplementedwith 0.1 % bovine serum albumin (BSA, Sigma A-60031, while AB2-143.2 cultures were supplementedwith 2 % FBS (Irvine Scientific,Santa Ana, CAI. No antibiotics were used in any of the cultures. Stock cultures were maintained in 250-mL glass Erlenmeyer culture flasks (Bellco, Vineland, NJ) in a humidified incubator at 37 "C and 5 % C02. Stock cultures were expanded in 100-mL spinner flasks (Bellco) for inoculation of the bioreactors. Bioreactor Operation. All bioreactor experiments were performed with a 3-L Applicon (Foster City, CA) bioreactor with surface aeration and a working volume of -1 L. The temperature was controlled at 37 f 0.1 "C by an electric heating jacket and a cold finger, the pH was maintained at 7.2 f 0.1 by manipulation of the C02 flow rate, and the dissolved oxygen (DO) was controlled at 40 f 2% of air saturation by manipulation of the oxygen flow rate to the reactor. The balance of the gas phase was
0 1994 American Chennical Society and American Instttute of Chemical Engineers
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nitrogen, which was maintained at 100 cmVmin. The agitator was equipped with a marine-type impeller and operated at 100 rpm, which was low enough to avoid vortexing and, hence, hydrodynamic damage to the cells (Kunas and Papoutsakis, 1990). A Minipuls 3 peristaltic pump (Gilson, Middleton, WI) equipped with two heads was used for chemostatoperation. Inlet and outlet tubing were connected to the same pump, but the inlet tubing inner diameter was smaller. The higher outlet flow rate maintained the desired level in the reactor. Inlet medium flow rate was measured using a 0.2-mL pipet connected to the feed line. The standard deviationof the mean of replicate flow rate measurements was typically less than 1%of the mean flow rate. Results are shown for individualcontrol and osmotically stressed experiments. For the INDl batch osmotic shock experiment, 6.15 g of sodium chloride was dissolved in 30 mL of medium and aseptically added to the reactor 88.25 h after inoculation with exponential-phase cells. The osmolality was initially 312 mOsm/kg and was increased to 448 mOsm/kg after osmotic shock. For the AB2-143.2 batch osmotic shock experiment, 4.00 g of sodium chloride was dissolved in 20 mL of medium containing 2% FBS. This was added aseptically to the reactor 86.25 h after inoculation. The osmolality increased from 352 to 488 mOsm/kg following osmotic shock. The batch osmotic shock results shown for each cell line are representative of replicate experiments (Reddy, 1993). For the continuous culture experiments, cells were initially grown to steady-state cell density in normalgrowth medium. Osmotic stress was implemented by replacing the feed bottle with one containing normal medium supplemented with 6 (IND1cultures) or 5 g/L (AB2-143.2 cultures) sodium chloride. The dilution rate was kept constant for the duration of each experiment. Results indicated in the text as "not shown" may be found in Reddy (1993). Assays. Total cell count was determined with a Coulter Multisizer (Coulter Electronics, Hialeah, FL) equipped with a 100-pmorifice. The standard deviation of the mean of replicate cell counts was typically less than 5% of the mean. Viability was determined by the trypan blue exclusion method using a hemacytometer. The standard deviation of the mean of replicate viabilitydeterminations was typically less than 2% of the mean. Secreted antibody was assayed by protein A affinity chromatographyusing a Waters HPLC system (Millipore, Bedford, MA) with an Affi-Prep column (no. 125-0460, Bio-Rad, Richmond, CA). Buffer A (0.1 M glycine and 0.2 M sodium chloride in water, pH 9) was used for binding, and buffer B (0.1M glycine and 0.2 M sodium chloride in water, pH 3) was used for elution. Only freshly made buffers were used, so that no biocides were added. The mobile-phase flow rate was kept constant at 1 mL/min. The volume of sample injected was always 1mL, while the volume of standard was varied to allow the injection of different amounts of standard antibody. For INDl experiments,purified INDl antibody (providedby Xoma) was used as the standard. For AB2-143.2 experiments, polyclonal mouse immunoglobulin G (Sigma 1-5381)was the standard. The column was initially equilibrated in 1009% buffer A. The followinggradient was automatically initiated when the sample was injected linear change from 100% A to 100% B over 5 min; 100% B for 5 min; linear change from 100% B to 100% A over 5 min. The next sample was injected 25 min after the previous sample was injected. The standard error for antibody concentration
Biotechnol. Rog., 1994, Vol. 10, No. 2
is estimated to be less than f15% on the basis of the variation of IgG standards. For the continuous culture experiments, QAb was calculated from
where D is the dilution rate (h-l), [Ab] is the antibody concentration (pg/mL), and X, is the viable cell concentration (cells/mL). To assess the amount of cell damage in a culture, the lactate dehydrogenase (LDH) concentration in the supernatant was measured with an enzymatic assay (Sigma 228-UV). The assayreagent was reconstituted as described by the manufacturer. INDl cell lysate was used as a standard. Two million total INDl cells of 98% viability were suspended in 1mL of PBS and sonicated. The debris was removed by centrifugation, and the lysate was stored at -20 "C in aliquots of 0.5 mL. LDH activity usually can be detected before the viability drops below 90 % (Legrand et al., 1992). Medium osmolality was measured with an Advanced DigiMaticModel 3D2 osmometer (AdvancedInstruments, Needham Heights, MA). Flow Cytometry. Centrifugationsteps were performed with an Eppendorf microcentrifuge (Model 5415, Brinkmann Instruments, Inc., Westbury, NY) for 5min at 700g. Viability staining with ethidium monoazide (EMA, Molecular Probes, Eugene, OR) was done only for the INDl batch experiments. Antibody content in dead cells has been presented elsewhere (Reddy et al., 1992)and will not be discussed here. Samples (either stained with EMA or directly from culture) containing 2 million total cells were washed once in PBB (0.1% BSA in PBS). The cell pellet was resuspended in the residual liquid by tapping the tube to disrupt the pellet. One milliliter of ice-cold (-20 "C) methanol was added dropwise to the cells while vortexing to prevent clumping. Samples were stored at -20 OC. Samples stored in methanol were washed once with 1 mL of cold (4 "C) PBS (PBB should not be used because residual methanol can cause BSA to precipitate out of solution). Next, the cells were resuspended in 300 pL of cold goat anti-mouse-IgG antibody-FITC conjugate (Sigma F-0257, diluted 150 in PBB) and incubated at 4 "C for 30 min. Samples were then washed twice with 1mL of cold PBB. For nonspecific staining, the same protocol was used except that a goat anti-rabbit-IgG antibodyFITC conjugate (Sigma F-0511, diluted 150 in PBB) was used as the label. The samples were resuspended in 1mL of cold PBB. Samples were kept cold and in the dark until analysis, which was always done on the same day as staining. Fluorescence measurements were performed using an EPICS Profile flow cytometer (Coulter). Laser excitation was at 488 nm (15 mW). Green and red fluorescencewere measured simultaneously with a 550-nm dichroic longpass filter. Green fluorescence (FITC) was detected through a 530-nm short-pass and 525-nmband-pass filter. A bitmap gate was set on the basis of the orthogonal and forward light-scattering characteristics of the cells. On a plot of log-side scatter versus forward scatter, cells show up in the center while debris and clumps appear at the extremes of the plot. Data acquisition was stopped when 10 000-20 O00 cells were accumulated. For quantitative analysis, the flow cytometer must be properly calibrated. For this purpose, calibration beads (Superbright microspheres, lot 5017, Coulter) were run and the photomultipliertube voltageswere varied to adjust the mean log green fluorescence to 145. This corrected
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culture time (hours) Figure 1. Unstressed batch reactor experimentsshowing total (A)and viable (A)cell concentrations: (A) AB2-143.2 cells; (B) INDl cella. for day-to-day variation in the instrument, but it is also necessary to account for variations in the staining procedure. To this end, a biological standard was prepared from INDl cells from a spinner-flask culture, as described previously (Reddyet al., 1992). The variation in the mode of the green fluorescence for standards run on different days was typically less than *15%, so no correctionswere made, especially since only relative changes between samples stained and run on the same day are discussed.
Results Unstressed Batch Culture. Typical batch growth curves for the two cell lines in normal medium are shown in Figure 1. The specificgrowth rates for AB2-143.2 (0.039 h-1) and INDl (0.038 h-l) cells are similar. The main difference between the two growth curves is a much longer stationary phase for INDl cells. Constant total cell numbersduring the declinephase for both cell lines suggest that the dead cells are not lysed. Figure 2 shows a plot of the extracellular antibody concentration versus the area (integral) under the viable cell curve for each cell line. The slope of such a plot is equal to q A b if the production rate is constant (Renard et al., 1988). AB2-143.2 cells (Figure 2A) displaynon-growbhassociated antibody production kinetics (constant qAb throughout batch culture). This pattern has been previously reported for AB2-143.2 cells (Miller et al., 1988) and many other cell lines (Dalili and Ollis, 1988,1989; de St. Groth, 1983; Hayter et al., 1992; Lee et al., 1989; McQueen and Bailey, 1990,Ozturk and Palsson, 1991a,b). Figure 2B shows that INDl cells also have a constant q A b during exponential growth. However, after reaching stationary phase, QAb decreased from 6.5 X lo-‘ to 0.5 X lo” pglcelllh, which is indicative of growth-associated antibody production. Note that the final *cumulative viable cell hour”value for the INDl culture is much higher
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Figure 2. Accumulated antibody concentration versus cumulative viable cell hours for the unstressed batch reactor experiments described in Figure 1: (A) AB2-143.2 cells; (B) IND1 cells. The area under the viable cell versus time curve was approximated by a s u m of rectangles with the height given by the cell concentration at the later time point (cumulativeviable cell hours). The slope of each plot is equal to the specificantibody production rate. The two lines for INDl cella representantibody production in the exponential and stationary/declinephases of the culture. Arrows indicate the approximate time that each culture entered stationary phase. than that for the AB2-143.2 culture due to the extended stationary phase of INDl cells (Figure 1). AB2-143.2 and INDl cells both exhibit bimodal intracellular antibody content distributions during exponential growth (seebelow). Figure 3 shows the change in the mode of the high-content peak during unstressed batch culture for each cell line. There was little change in AB2-143.2 cell antibody content throughout exponentialgrowth,with the high-content mode remaining at a value of about 180 (Figure 3A). As the culture began to enter stationary phase, there was a drop in the mode to about 110 (73 h). As the culture entered death phase (96 h), there was a continuing decline in the mode to a final value of about 15 (after 121 h). The change in intracellular antibody content over time for unstressed INDl batch culture has been previously illustrated by a series of flow-cytometry histograms (Reddy et al., 1992). During exponential growth, the high-contentmode remained constant at about 60 (Figure 3B). As the culture began to enter stationary phase (99 h), there was a drop in the mode to about 30, which was maintained throughout stationary phase. As the culture entered death phase (182 h), there was a decrease in the mode to a final value of about 15. These results are consistent with those of other investigators (Dalili and Ollis, 1990,Len0 et al., 19911,who demonstrated a relatively constant antibody content during exponential growth with a continuing decline as the culture entered stationary phase. For both cell lines there was much less change in the low-content mode, so that the bimodal distribution was lost by the end ofthe culture (not shown).
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Figure 5. Accumulated antibody concentration versus cumulative viable cell hours for the osmotic shock experimenta described in Figure 4 (A) AB2-143.2 cells; (B)INDl cells. The twolines for AB2-143.2cellsrepresentantibodyproductionbefore and after osmotic shock. Arrows indicate the time that each culture entered stationary phase after salt addition.
a 50 5% overlap between the exponential- and death-phase antibody distributions (Reddy et al., 1992; Rsddy, 1993). Hyperosmotically Stressed Batch Culture. Figure 4 showsthe effeds of a 136 mOsm/kg increase in osmolality on the batch growth of AB2-143.2 and INDl cells. In both cases, the osmotic shock was sufficient to inhibit cell growth, but mild enough to maintain a constant viable cell concentration for about 50 h after the shock. We have previouslyshownthat an osmolalityincreaseof 182 mOsm/ kg causes a rapid decrease in INDl cell viability (Reddy et al., 1992). Figure 5 illustrates the specificantibodyproduction rate. After osmotic shock, the AB2-143.2 cell QAb increased approximately50 7% above the preshock value (Figure 5A). In contrast, the INDl cell line exhibited no increase in qAb followinghyperosmotic shock (Figure 5B). Although there was no increase in 4Ab, maintenance of the exponentialphase qAb value into the stationary phase does represent an enhancement of antibody production relative to that in unstressed batch culture (Figure 2B). Similar maintenance of the exponential-phase q A b value hae been observed following more severe osmotic shock (Reddy et al., 1992). Figure 6 shows representative intracellular antibody distributions before and after osmotic shock. For both cell lines, there was no change in the antibody content throughout exponential growth (not shown), consistent with the unstressed batch reactor experiments. As for the unstressed cultures, the bimodal distribution was lost when the cells entered stationary phase. However, in contrast to unstressed cultures, this was due to the lowcontent population shifting to the level of the high-content population, so that there was no decrease in the intracellular antibody content. Similar results have been
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green fluorescence Figure 6. Intracellular antibody content distributionsfor the osmotic shock experiments described in Figure 4 (A) AB2-143.2 cell distribution before osmotic shock at 85 h; (B) AB2-143.2 distributionafter osmotic shock at 130 h; (C) INDl cell distribution before osmotic shock at 71 h; (d) INDl distributionafter osmotic shock at 121 h. All histograms represent the green fluorescence (antibody) distribution on a log scale. Cell frequency scales are arbitrary. Culture viabilitiesare shown in the lower left comer of each histogram. previously reported after a more severe osmotic shock of INDl cells (Reddy et al., 1992). Gradual Osmotic Stress in Continuous Culture. It is difficult to evaluate the effects of external stimuli on cells in batch culture because of the transient nature of batch culture. This is especially true for gradual changes in conditions. For example,gradual osmotic stress cannot be effectively evaluated in batch culture because so many things would change during the change in osmolality that it would be difficult to isolate the effects due to osmotic stress. Limited culture time also decreases the feasibility of evaluating gradual stress in batch culture. For these reasons, it is advantageous to investigate the effects of hyperosmotic stress in continuous culture. Osmotic stress can be administered after steady state has been attained, so that any changes in cell density and antibody production can be attributed to hyperosmotic effects. The major problem with continuous culture is that cells wash out of the reactor if growth is inhibited. By imposing osmotic stress in a gradual manner, there is more time for the cells to respond before the osmolality becomes so great that the cells either die or wash out of the reactor. Retention devices such as spin filters could be used to retain cells in the reactor, but these systems will not be discussed here. Figure 7A,B, respectively,shows the cell concentration and viability plots for a gradual osmotic stress continuous culture experiment with AB2-143.2 cells. The cell concentration and viability remained at control levels until the osmolality increased above 400 mOsm/kg. The viable cell concentration and viability began to decrease after 220 h and reached plateau values of 1.12 X lo6 cells/mL (25% decrease)and 71 7% viable cells at 250 h. These values were maintained until the osmolality reached 475 mOsm/ kg at 290 h, after which time the cell concentration and viability decreased again. Similar threshold values for the initial decrease in cell concentration were observed in two additional replicate experiments (not shown). In neither case was an intermediate plateau observed for cell
concentration or viability. However, in one case with an initial viability of 90%,there was no significant decrease in viability until the osmolality reached 480 mOsm/kg. As for batch osmotic shock, the AB2-143.2 cell QAb increased in response to gradual osmotic stress (Figure 7C). The specific antibody production rate began to increase when the cell concentration began to decrease at 220 h and was twice as great as the control qAb after 6 days. Increases in q A b of similar magnitude were observed in two additional replicate experiments, but in one case the increase was transient, such that QAb reached a maximum after 3 days and returned to control levels after 4 days (not shown). A bimodal intracellular antibody content distribution similar to that shown in Figure 6A was maintained throughout the experiment (not shown). Except for an increase in the high-content mode at the end of the experiment, the high- and low-content modes did not vary significantly. The effectsof gradual hyperosmoticstress on INDl cells are shown in Figure 8. A slight decrease in cell number was noted when the osmolality rose above 400 mOsm/kg (Figure 8A). A more extensive decrease was seen after 140 h when the osmolality was about 460 mOsm/kg. Interestingly, no decrease in viability was apparent until about 170 h (480 mOsm/kg, Figure 8B). This suggests initial growth inhibition and washout rather than cell death. Even at the end of the experiment, the viability had dropped to only 93 % , again suggesting primarily growth inhibition and not cell death. The high viabilities cannot be attributed to the lysis of dead cells because no LDH activity was detected at any sample point (not shown). Figure 8C showsthe QAb profile for the INDl continuous culture experiment. The decrease in qAb with increasing osmolality and growth inhibition is consistent with the growth-associated antibody production observed in unstressed batch culture (Figure 2B), but contrasts with maintenance of the midexponential q A b value following osmotic shock in batch culture (Figure5B). This contrasts with enhanced qAb by AB2-143.2 cells after both abrupt
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relative culture time (hours) Figure 7. AB2-143.2gradual hyperosmotic stress continuous cultureexperiment: (A) viable cell concentration( 0 )and reactor osmolality ( 0 )profile; (B)viability profile; (C)specific antibody production rate. Only samples for cell count were taken during batch growth, and the initialtransient period after medium flow was initiated. 0 hours correspondsto the time when periodic cell counta suggested that steady state had been achieved and extensive reactor sampling was begun. This was 10 days after inoculation. At 203.5 h (vertical line),the normal feed medium was switched to medium containing5 g/L sodium chloride. The dilution rate was kept constant at 0.023 h-l. (Figure5A)and gradual (Figure 7C)osmotic stress. Some variation in the percent of INDl cells with high intracellular antibody content was observed (not shown). However, there was essentially no change in the highcontent mode (average of 63)or low-content mode (about 10)over the course of the experiment. This is essentially the same pattern as that observed for AB2-143.2 cells. Similar results were also obtained for the INDl batch osmotic shock experiment, even though qAb behaved differently. In both INDl cases,the intracellular agtibody content remained high, although qAb decreased in continuous culture and remained constant in batch culture following osmotic stress.
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relative culture time (hours) Figure 8. INDlgradualhyperosmoticstress continuous culture experiment: (A) viable cell concentration ( 0 ) and reactor osmolality ( 0 )profile; (B)viability profie; (C)specificantibody production rate. Only samples for cell count were taken during batch growth, and the initialtransientperiod after medium flow was initiated. 0 hours corresponds to the time when extensive reactor sampling wae begun. This was 7 days after inoculation. At 94.5 h (vertical line),the normal feed medium was switched to medium containing6 g/L sodium chloride. The dilution rate was kept constant at 0.031 h-l.
Discussion The results presented here provide a comparison of two different hybridoma cell lines. AB2-143.2 cells exhibit non-growth-associated antibody production in batch culture (Figure 2A), while INDl cells possess growthassociated antibody production kinetics (Figure 2B). In the former case, qAb is constant throughout batch culture, independent of growth rate. In the latter case, QAb is constant during exponential growth, but decreases by a factor of 10 as cell growth ceases in stationary phase. Although the two cell lines differ in their antibody production kinetics, they both show enhanced qAb after osmotic shock in batch culture reltive to that in unstressed batch culture. With AB2-143.2c e h , qAb increases in the stationary phase following hyperosmotic shock, while it remains constant in unstressed culture (compare Figures 5A and 2A). Other investigators have demonstrated
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Table 1. Specific Antibody Production Rates and Percent High-Content Cells for Various Batch and Continuous AB2-143.2 Bioreactor Exwriments. 5% high IgG content culture mode description source qAb (NglceWh) 4.06 x 10-7 80 batch before osmotic shock Reddy, 1993 63 Figures 5A and 6A batch before osmotic shock 5.71 X control 70 4.07 X le7 Figure 2 4 Reddy, 1993 batch continuous before anoxia 26 2.02 x 10-7 Reddy, 1993 continuous after anoxia 10 4.6 X 1O-a Reddy, 1993 12 continuous after anoxia 3.4x 1O-a Reddy, 1993 continuous before low-pH shock 38 2.04 X le7 Reddy, 1993 Reddy, 1993 continuous after low-pH shock 33 2.69 X 10-7 Reddy, 1993 continuous before gradual osmotic stress 45 4.37 x le7 8.14 X 10-7 80 Figure 7C; Reddy, 1993 continuous before gradual osmotic stress a For batch experiments, qAb was obtained from the slope of a plot of antibody concentration versus cumulative viable cell hours. Preshock values were used for the batch osmotic shock experiments. The percent high-antibody-content reported for the batch experiments ia the average of samples immediately before osmotic shock. For the continuous culture experiments, only steady-state antibody data were used, so eq 1 waa simplified to QAb = D[Ab]/X,. In the gradual osmotic shock experiments, steady state was achieved before the osmotic strew was introduced. No steady state was achieved after introduction of osmotic stress. Therefore, the data for these experiments represent values of qAb and percent high antibody content before osmotic shock. The low-pH shock and anoxia continuous culture experiments were established in a manner similar to the gradual osmotic shock experimente, except that a spin filter was used to retain the cells after anoxic exposure. The cell concentration and antibody content returned to steady values following recovery from low-pH (6.9)shock (Reddy, 1993). In contrast, the cell concentration continued to increase after recovery from anoxia, even though the antibody concentration remained constant (Reddy, 1993).
increased QAb values for Cells inoculated directly into hyperosmotic medium (Nuss and Koch, 1976; 0yaas et al., 1989;Ozturk and Palsson, 1991a;Oh et al., 1993).With INDl cells, QAb remains at the exponential-phase value following hyperosmotic shock and does not decrease in stationary phase as it does in unstressed culture (compare Figures 5B and 2B). Different effects of osmotic stress on qAb in continuous culture were observed for the two cell lines. AB2-143.2 cells showed increased QAb under gradual osmotic stress (Figure 7C), consistent with the batch osmotic shock results. In contrast, INDl cell QAb was not increased by gradual hyperosmotic stress. Rather, the stressed INDl continuous culture displayed growth-associatedantibody production kinetics, similar to INDl unstressed batch culture. As cell growth decreased due to osmotic stress, QAb also decreased (Figure 8). This inconsistency with INDl osmotic shock batch culture is most likely due to the manner in which the stress was imposed (gradualversus abrupt). The difference between cell lines in response to gradualosmotic stress in continuousculture may be related to the fact that antibody production is growth-associated in INDl cells, but not in AB2-143.2 cells. It has been previously demonstrated that QAb increases nearly 3-fold for the AB2-143.2 cell line under abrupt low-pH stress in continuous culture (Miller et al., 1988). Despite their differences in antibody production kinetics, both cell lines behave similarly with respect to changes in the intracellular antibody content. For example, the intracellular antibody content is constant during exponential phase, but decreasesduring stationary phase in unstressed batch culture for both cell lines (Figure 3). When either cell line is subjectedto batch hyperosmotic shock, the intracellularantibody content of the low-content population shifts to that of the high-content population, which remains at the exponential-phase value, even into stationary phase (Figure 6). The higher qAb values in stationary phase followingbatch hyperosmotic shock correspond to the higher intracellular antibody content relative to unstressed cultures for both cell lines. Thus, for a given cell line, a drop in the intracellular antibody content during stationary phase of unstressed culture may indicate a decrease or no change in QAb, depending on whether the particular cell line exhibits growth-associatedor non-growth-associatedantibody production. However, in general it may not be concluded that a change in intracellular antibody content
reflects a change in QAb. Recall that the intracellular antibody content decreased in AB2-143.2 cellswhen there was no change in QAb, while the intracellular antibody content decreased in INDl cells when there was a decrease in QAb. Similarly, the intracellular antibody content remained constant after batch osmotic shock when there was an increase in QAb in AB2-143.2 cells, but no change in QAb in INDl cells. The intracellular antibody content also remained the same in both cell lines following gradual osmotic stress in continuous culture, even though QAb increasedfor AB2-143.2 cells but decreasedfor INDl cells. Although there may not be a generalcorrelationbetween intracellularantibody content and QAb, it has been reported that the percentage of cells with high antibody content in bimodal populations is proportional to qAb (Ozturk and Palsson, 1990; Lee and Palsson, 1990; Heath et al., 1990). The lower content population of the bimodal distribution is often describedas a non-antibody producing population. To investigate whether the fraction of high-antibodycontent cells is related to qAb for AB2-143.2 and INDl cells, data were compiled from various batch and continuous culture experiments not confounded by osmotic stress. Data for AB2-143.2 experiments are included in Table 1, while INDl results are summarized in Table 2. Figure 9A reveals a nearly linear relationship between qAb and the percent high-content AB2-143.2 cells. The culture conditions were different in each experiment, so some scatter in the qAb values is expected. These results are consistent with the increase in qAb that accompanied the shift of the low-antibody-content population to the high-antibody-content population in the batch osmotic shock experiment (Figure6A3). It has been demonstrated that qAb decreased over time as AB2-143.2 cells were repeatedly passaged (Miller,1987)or grown in continuous culture (Miller et al., 1988),but no intracellular antibody measurements were made. This indirectly implies that the percent high-content cells also decreased with time, since it was demonstrated above (Figure 9A) that the two parameters are related in AB2-143.2 cultures. Others have reported that qAb decreased linearly as the percent highcontent cells decreased for other hybridoma cell lines (Ozturk and Palsson, 1990;Lee and Palsson, 1990; Heath et al., 1990). Figure 9B shows that, in contrast to the AB2-143.2 results, there does not appear to be a correlation between percent high-content cells and qAb in INDl cultures, although qAb was greater for continuous culture experi-
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Table 2. Specific Antibody Production Rater and Percent High-Content Cells for Various Batch and Continuour INDl Bioreactor and SDinner-Flask Exwriments. source % high IgG content culture mode descriDtion QAb (ccglcelllh) batch control and osmotic spinners Reddy et al., 1992 6.5 X 1V' 19 Reddy et al., 1992 batch control and osmotic reactors 82 1.9 x 10-7 Figures 6B and 6C batch before osmotic shock 8.6 X lo-' 64 Reddy and Miller, 1992 continuous dilution rate exp 12.6 X 10-7 81 Reddy and Miller, 1992 continuous dilution rate exp 69 11.2 x 1 ~ 7 11.6 X lV7 58 Reddy and Miller, 1992 continuous dilution rate exp continuous dilution rate exp 13.9 X lo-' Reddy and Miller, 1992 65 continuous before gradual osmotic strew 12.0 x 10-7 80 Figure 8C; Reddy, 1993 a qAb valuea were calculated as discussed in the legend to Table 1. The preshock values were used for all batch experimenta. In the gradual osmotic shock experiment, steady state was achieved before the osmotic stress was introduced. No steady state was achieved after the introductionof osmotic stress. Therefore,the data for this experimentrepresent vduea of QAb and percent high antibody content before osmotic shock. All specific antibody production rates were calculated on the basis of the Sigma IgG standard (Reddy et al., 1992). Antibody data for the unstressed and osmotic shock reactor experiments, as well as the gradual osmotic strew Continuous culture experiment, discueeed in the text were based on the INDl antibody standard. Theae were converted to a Sigma standard basii by multiplying by the ratio of the dope of the Sigma standard curve to the slope of the INDl standard curve. The percent high antibody content reported for the batch experiments is the average of all available data before osmotic shock. The percent high-antibody-content valuea for the dilution rate continuous culture experiment were reported previously (Reddy and Miller, 1992). The percent high antibody content reported for the gradual osmotic strew continuous culture experiment is the average of steady-state data before the introduction of osmotic stress. ~
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that there was no correlation between the percent highcontent cells and qAb for a quadroma cell line, as well as for ita two parent hybridoma cell lines, even though the percent high-content cells decreased over extendedculture periods. Intuitively, one might expect that cells with higher antibody production rates would also have higher intracellular antibody levels. A near-linearcorrelation between percent high-content cells and qAb is generdy (but not always) reported in the literature. Our results indicate tht the relationship between percent high-content cells and QAb is cell-line-dependent. A h e a r correlation was demonstrated for the AB2-143.2 cell line (Figure 9A), but no such correlation was observed for the INDl cell line (Figure 9B). On the basis of the two cell lines studied here, this relationship may be related to whether the particular cell line exhibits growth-associated or nongrowth-associatedantibodyproduction in unstressed batch culture.
Acknowledgment A
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.
1
20 40 60 80 100 ?LO high antibody content
Figure 9. Relationship between qAb and percent high-antibodycontent celk (A) AB2-143.2batch (A)and continuous ( 0 )culture experiments described in Table 1; (B) INDl batch (A) and culture experiments described in Table 2. continuous (0)
menta than for batch experiments. Apparently, the antibody content is not related to QAb in INDl cells. This would explain why a shift of the low-antibody-content population to the high-antibody-content population did not coincide with an increase in q A b in the batch osmotic shock experiment (Figures5B and6C,D). It has previously been shown that the percent high-content cells decreases over time in extended INDl continuous culture (Reddy and Miller, 1992). However, there was essentially no change in QAb during this period. This was unexpected since the decrease in percent high-content cells over time seemed to suggest the presence of a low- or non-producer clone. The INDl results are consistent with the findings of Salazar-Kish and Heath (1993). They demonstrated
The authors thank Xoma Corporationfor providing the INDl cells. Flow cytometry analysis was performed at the Quantitative Cytology Program directed by Dr. Kenneth D. Bauer at Northwestern University Medical SchooL Financial support was provided by the National Science Foundation (Grants BCS-9058416 and BCS8903688), Eli Lilly and Company, Schering Plough Research, Lederle-Praxis Biologicals, and Abbott Laboratories.
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?
Accepted November 23,1993.
* Abstract published in Advance ACS Abstracts, February 16, 1994.
