Batt Et Al Inclined Sedimentati

4sa Biotechnol. Prog. 1990, 6, 458-464

Inclined Sedimentation for Selective Retention of Viable Hybridomas in a Continuous Suspension Bioreactor

Brian C. Batt, Robert H. Davis, and Dhinakar S. Kornpala' Department of Chemical Engineering, University of Colorado, Boulder, Colorado 80309-0424

The continuous separation of nonviable hybridoma cells from viable hybridoma cells by using a narrow rectangular channel tha t is inclined from the vertical has been investigated experimentally. The effectiveness of the settler in selectively retaining viable hybridomas in the bioreactor while permitting the removal of nonviable hybridomas has been shown to depend on the flow rate through the settler. Intermediate flow rates through the settler have been found to provide the highest removal of nonviable hybridomas relative to viable hybridoma retention. At high dilution rates through the chemostat, over 95 % of the viable cells could be partitioned to the bottom of the settler while over 50 5% of the nonviable cells are removed through the top of the settler. This successful separation is due to the significantly larger size of the viable hybridomas than the nonviable ones. A continuous perfusion experiment was performed in which an external inclined settler was used to retain virtually all of the viable hybridomas in the culture, while selectively removing from the culture approximately 20% of the nonviable cells that entered the settler. A stable viable cell concentration of 1.0 X lo7 cells/ mL was achieved, as was an antibody productivity of over 50 pg/(mL-day). These represent 3- and 6-fold increases, respectively, over the values obtained from a chemostat culture without cell retention.

Introduction Early studies in batch hybridoma culturing showed that

monoclonal antibody production is proportional to the number of viable cells in the culture (1-6). Furthermore, it has recently been shown that nonviable or dead hybridomas do not lose significant amounts of antibody and, consequently, contribute negligibly to the productivity of a culture (7). Thus, high productivity of monoclonal antibody in suspension culture can be attained not by maximal cell growth rate but by maintaining high viable cell concentrations.

High viable cell concentrations in suspension bioreactors are currently achieved by using various cell retention or recycle devices. These enable bioreactors to be per- fused without cell washout a t dilution rates greater than the maximum specific growth rate of the cells. A typical cell retention device employs an internal spin filter, which is attached to the reactor's impeller shaft and permits the removal of cell-free culture medium (8-10). Cell recycle devices typically employ an external tangential flow filter in order to concentrate the cell suspension and recycle it back to the bioreactor (11,12). Another alternative passes the reactor effluent through a vertical sedimentation column that is sufficiently long to allow suspended cells to settle back into the bioreactor before they can be washed out (13-15).

Filter devices can be detrimental to long-term culture productivity because the hybridomas are subjected to excessive shear forces, resulting in higher cell death rates. Moreover, all of the methods mentioned above are characterized by the continuous accumulation of unpro- ductive dead cells in the bioreactor, which forces the intermittent removal of a culture fraction containing both viable and nonviable cells. This is especially imperative in the case of filtering devices when cell density becomes

* Author to whom correspondence should be addressed.

sufficient to cause difficulty in filtering spent medium (11, 12). The accumulation of nonviable cells and the removal of viable cells along with nonviable cells limit the culture productivity.

It is conceivable that the limitations in culture productivity may be overcome by continuously removing only nonviable hybridomas from the reactor while selectively retaining all viable cells in the reactor, provided that a method may be developed for accomplishing a selective separation of these subpopulations. It is known that faster- growing, viable mammalian cells are larger in average cell volume (12) and specific mass (16) than slower-growing and dead cells. Therefore, it is expected that the desired selective cell separation may be accomplished by exploiting the different sedimentation velocities of viable and nonviable hybridomas by using inclined sedimentation.

An inclined settler is a long and narrow tube or channel that is inclined from the vertical. Larger cells are removed from suspension by settling onto the upward-facing surfaces of the settler, where they form thin sediment layers that slide down to be collected a t the bottom of the vessel. Smaller cells, which do not settle as rapidly, are collected from the top. A kinematic theory for inclined sedimentation was developed more than a half-century ago (1 7, 28). This theory states that the volumetric production rate of clarified fluid from an inclined channel due to particle sedimentation is equal to the vertical settling velocity of the particles multiplied by the horizontal projected area of the channel surface available for sedimentation. For a rectangular channel, this implies that

S(u) = uw(L sin 0 + b cos 0) (1) where S(u) is the volumetric rate of production of fluid clarified of particles with settling velocity u, w is the width of the settler, L is the length of the settler, b is the spacing

8756-7938/90/3006-0458$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers

Eiofechnol. Prw... 1990, Vol. 6, No. 6

between the inclined walls of the settler, and 0 is the angle of inclination of the settler from the vertical.

For a channel of fixed dimensions and inclination angle, whether a cell of a given Stokes velocity settles out of suspension or is instead washed out of the channel is controlled by the volumetric flow rate through the channel, which determines the residence time of the suspended cells. Differences in Stokes velocity between cells of equal density are determined primarily by differences in cell diameter, as indicated by Stokes’ law:

(2) where d is the cell diameter, pc is its density, p is the fluid density, p is the fluid viscosity, and g is the gravitational acceleration constant. Thus, by adjusting the settler overflow rate so that the residence time of the suspension in the settler is in between the settling times of two subpopulations of different sizes, a selective cell separation may be accomplished. The benefit of inclined sedimentation is that cells need to settle only a distance on the order of the narrow spacing between the inclined walls of the settler in order to be removed from suspension, rather than a distance on the order of the settler height as in vertical sedimentation.

Inclined sedimentation has been applied successfully in a variety of bioreactor designs for selective cell retention: maintenance of a stable continuous culture containing a mixed bacteria/yeast population (19), selective recycle of flocculant yeast (20) and bacteria (21), and the separation of nondividing and dividing yeast (22). The advantages of applying an inclined settler as a cell retention device are the elimination of a high shear environment in the cell retention process, faster cell sedimentation rates than vertical settlers, and reduced accumulation of nonviable cells in the bioreactor. Furthermore, by continuously removing nonviable cells while selectively retaining viable cells in the culture, a perfusion bioreactor can in principle be operated as a steady-state process.

This paper describes a study of the feasibility of using inclined sedimentation to selectively separate nonviable from viable hybridomas in a suspension hioreactor. The performance of an inclined settler in promoting high viable cell concentration and antibody productivity, while preferentially removing nonviable cells from a perfusion hybridoma bioreactor, is also described.

Mater ia ls a n d Methods Cell Line and Medium. Cell line AB2-143.2 is a mouse

hybridoma derived from Sp2/0 myeloma that produces IgC2a antibodies against benzenearsonate (23). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% each lOOX MEM nonessential amino acids and 10 mM sodium pyruvate solution. The glucose concentration was 22 mM, and extra glutamine was added to give a concentration of 4.8 mM. No antibiotics were used in the medium. Cell size measurements (see below) indicated that the nonviable cells have an average diameter of approximately 8 pm, whereas that of the viable cells is approximately 12-14 pm, with the latter depending on the cell growth rate. The cell density was not determined exactly, but a crude neutral buoyancy measurement indicated that it is approximately 1.06 g/cm3. From eq 2, the average sedimentation velocity of the nonviable cells (d = S pm) is 1.1 cm/h, while that of the viable cells (d = 13 pm) is 2.9 cm/h.

Cell Cul ture Reactor a n d Inclined Sedimentation Channel. A 1.5-L reactor (Celligen, New Brunswick Scientific) equipped with a floating surface aerator (24,

u = d%, - p ) g / 1 8 ~

Figure 1. Schematic of the hioreactor confwation for studying the effect of the settler overflow rate on viable and nonviable hybridoma separation. During perfusion operation, the settler overflow stream is the reactor effluent instead of recycling it back to the reactor.

25) and marine impeller was used in all experiments. Agitation was controlled at 120 * 1 rpm, and the temperature was maintained at 37.0 f 0.1 OC. An interactive, four- gas control system designed for use with the Celligen reador was employed to control the dissolved oxygen (DO) concentration and pH. During chemostat operation, DO was maintained at 50% of air saturation by gas transfer from the headspace. During perfusion reactor operation, DO was maintained at 10% * 3% of air saturation by sparging oxygen periodically into the culture medium as a supplement to gas overlay in the headspace. The pH was maintained a t 7.20 * 0.01 by regulating the flow of COz into the reactor headspace and by adding 0.5 M sodium bicarbonate.

Two inclined sedimentation channels were used in this study. Each was made of glass and had the same rectangular dimensions of 5 cm in width and a 0.5-cm separation between the two inclined surfaces. One sedimentation channel had a length of 37 em, while the other was 23 cm long. From eq 1, the clarification rates for the average- sized nonviable and viable hybridoma cells are predicted to he S = 100 and 270 mL/h, respectively, for the longer settler and S = 64 and 170 mL/h, respectively, for the shorter settler. Each settler was tapered in width at the lower end to facilitate the return of all settling cells to the bioreactor. Each settler was also equipped with a water jacket through which 37 OC water was circulated during the experiment in order to maintain the cells in the settler a t the same temperature as in the reactor. To minimize the adherence of settling cells to the glass surfaces of the channel, each settler was siliconized with dimethyldi- chlorosilane prior to use.

The bioreactor configuration for this study is shown in Figure 1. The external inclined sedimentation channel WBS kept a t a constant angle of 30° from the vertical, in order to provide sufficient area for sedimentation while allowing the sediment to easily slide down the inclined wall. Flow through the inclined settler was generated by a peristaltic pump a t the settler outlet. Cells that settle completely from suspension are returned to the bioreactor by gravity flow of the sediment layer. Smaller cells that do not have

460

sufficient time to be removed from suspension are washed out in the settler overflow stream.

Two different studies were conducted, and the operation of the bioreactor was dependent on the particular study. The first experiment was to assess the separation of nonviable from viable hybridomas in the inclined settler as a function of the overflow rate from the settler. It was essential that the cells in the reactor be maintained at a constant growth ra te t o ensure a uniform cell size distribution for comparison purposes as the overflow rate was varied. Therefore, the reactor was operated as a conventional chemostat with the settler overflow returned to the reactor via a separate port to comprise an internal recycle loop for maintenance of a constant culture volume (Figure 1). The settler was allowed to operate a t each overflow rate studied for a minimum of 3 h before sampling to assure steady-state conditions. A range of overflow rates was chosen to span the clarification rates predicted by eq 1 for the nonviable and viable cells. Two different dilution rates were used: 0.39 day-' (for the 37-cm-long settler) and 0.89 day-' (for the 23-cm-long settler). The second experiment was to operate the system as a perfusion culture. Instead of recycling the settler overflow stream as shown in Figure 1, the overflow stream became the reactor effluent. The feed rate was adjusted to exceed the overflow rate slightly, with the effluent tube employed in chemostat operation used as a level control to maintain constant culture volume.

Sample Analyses. Cell concentrations were determined by counting in a hemacytometer, and viability fractions were determined by trypan blue staining (26). During the study of cell separation versus settler overflow rate, samples were taken from both the reactor and overflow stream at each overflow rate. A t least two samples were taken from the overflow stream a t each overflow rate so that the reported concentrations of both viable and nonviable cells are the average of two cell counts. During the perfusion culture, samples were taken initially twice a day, and then once a day after the dilution rate was set a t its final value, from both the reactor and settler overflow stream. After the cells were counted, the perfusion reactor sample was centrifuged and the supernatant was analyzed for antibody concentration by using a sandwich ELISA procedure in which alkaline phosphatase was used as the conjugated enzyme and the absorbance was read at 405 nm (27). The standard error for each reported antibody concentration was propagated from the point of half-maximal absorbance of the dilution curve of both the standard antibody and sample.

Determination of Cell Size Distributions. Cell size distributions and mean cell diameters were determined by using an Elzone 180XY particle size analyzer (Particle Data, Inc.). Reactor and settler overflow samples were diluted to a concentration of 3000-5000 cells/mL in phosphate-buffered saline (PBS) and then counted in the size analyzer with a 120-pm orifice.

Size and Viability Measurements with Flow Cy- tometry. The distributions of viable and nonviable hybridomas in reactor and overflow samples from the perfusion culture were determined by using an EPICS 541 flow cytometer with a Coherent argon ion laser. Samples were centrifuged and resuspended in cold PBS containing 0.5 pg of propidium iodide/mL and incubated for 30 min (28). Samples were then filtered through 62-pm nylon mesh just prior to analysis. Propidium iodide does not enter the viable cells but passes readily into the nonviable cells and stains their DNA. The dye in the nonviable cells is excited by the laser a t 488 nm and fluoresces

p 0.5 -

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Biotechnol. Prog., 1990, Vol. 6, No. 6

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d o D I 0.59 day-'

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Figure 2. Effect of settler overflow rate on the concentrations of viable and nonviable cells in the overflow stream relative to those in the reactor at a chemostat dilution rate of 0.39 day1. An inclined settler 37 cm in length at an angle of inclination of 30" was used in the study.

at 540 nm. Two discrete cell populations (viable and nonviable) were resolved in a two-dimensional forward- angle light scatter versus log integrated red fluorescence histogram for the reactor sample, and contour maps were drawn around the viable and nonviable populations in order to determine the separate distributions for the two cell populations in both the reactor and overflow samples.

Results and Discussion Effect of Overflow Rate on Cell Separation. The

effect of the overflow rate from the inclined sedimentation channel on the removal of nonviable hybridomas from, and the retention of viable hybridomas within, a suspension chemostat culture was assessed by determining the concentration of each in the overflow stream over a range of overflow rates. Because cell size is dependent on specific growth rate (12), the cell separation achievable by inclined sedimentation was determined at two dilution rates (in separate cultures). The two dilution rates chosen, 0.39 and 0.89 day-', provided specific growth rates of 0.65 and 0.97 day-', respectively, that represent low and high values relative to the maximum specific growth rate of 1.10 day-l determined for these cells in batch culture. At a low growth rate there is less difference in size between viable and nonviable cells, which implies that a cell separation is more difficult because there is a smaller difference in the Stokes settling velocity of each cell type. The converse is expected to be true a t a high growth rate, for which the size difference between viable and nonviable hybridomas is greater.

At a dilution rate of 0.39 day-', the total cell concentration in the reactor was 3.9 X 106 cells/mL, and the viable fraction was 0.60, when the inclined settling experiments were initiated. The concentrations of viable and nonviable hybridomas in the settler overflow stream from the longer settler, relative to those in the feed stream from the bioreactor, are shown in Figure 2 over a range of overflow rates from 16 to 603 mL/ h. All cells were retained in the settler for overflow rates below 55 mL/h, and only nonviable cells were detected in the overflow for overflow rates between 55 and 107 mL/h. From eqs 1 and 2, this range corresponds to diameters between 6 and 8 pm for the largest cells predicted to reach the overflow. As the overflow rate was increased further, the relative concentration of both cell types in the settler effluent increased, with that for the nonviable cells generally being higher


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Figure 3. .Mean cell diameter (circles) and viable fraction (triangles) in reactor and overflow stream samples over a range of settler overflow rates. The chemostat dilution rate was 0.39 day-1 and the inclined settler was 37 cm long at an angle of inclination of 30° throughout the study.

because they settle more slowly than the viable cells. These results are as expected because, as the overflow rate is increased, cells in suspension have a shorter residence time in the settler, and thus a smaller fraction has sufficient time to settle out of suspension. A t very high overflow rates, the residence time is small and the concentrations of both subpopulations in the settler overflow are seen in Figure 2 to approach their respective concentrations in the bioreactor. These results show tha t operation a t an intermediate overflow rate provides the best separation of nonviable cells from the culture.

The mean cell diameter was calculated from the cell size distribution generated by the particle size analyzer for samples from both the reactor and overflow stream at four overflow rates. These results along with the viable cell fraction in each overflow and reactor sample are plotted in Figure 3. The 99 76 confidence interval for each mean was less than f0.06 pm because the number of cells counted in each sample was greater than 10 000. The results show that chemostat operation maintained the cells in the reactor a t a relatively constant mean cell diameter of 11.2-11.3 pm that was not disrupted by the recycle loop through the inclined settler. However, the mean diameter and viable fraction of cells removed in the settler overflow increased with increasing overflow rate. Because the Stokes settling velocity of a cell is determined primarily by its diameter, only the smallest cells in the culture failed to settle completely during their residence time in the inclined settler a t low overflow rates. As seen in Figure 3 for an overflow rate of 107 mL/h, these small cells are primarily nonviable. The reduction in residence time caused by increasing the overflow rate allowed larger, viable cells to be washed out so that, a t the highest overflow rate studied, the mean cell diameter and the viable fraction in the overflow were nearly the same as those in the reactor.

In addition to the increase in cell size in the settler effluent with overflow rate, the viable fraction in the overflow stream increased as well. The viable fraction in the settler effluent remained less than that in the reactor a t all but the highest overflow rate studied. The viable fraction in the reactor decreased slightly as the overflow rate increased, presumably because larger viable cells were increasingly retained as sediment in the settler and returned more slowly to the reactor compared to nonviable cells, which were returned quickly to the reactor in the recycled overflow.

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- - o o o e 0.0 ,: ,?.0,4 . , . , . 1 , . , . , , . , , , , , , > . , I

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Figure 4. Effect of settler overflow rate on the concentrations of viable and nonviable cells in the overflow stream relative to those in the reactor at a chemostat dilution rate of 0.89 day1. An inclined settler 23 cm in length at an angle of inclination of 30' was used in the study.

In the second culture, the chemostat was operated at a dilution rate of 0.89 day-' and had a total cell concentration of 2.5 x 106 cells/mL with a viable fraction of 0.92. The relative concentrations of viable and nonviable hybridomas in the overflow stream as a function of the overflow rate are plotted in Figure 4. The shorter settler (23 cm long) was used in order to reduce the required throughput of culture medium and also because the viable cell sizes, and hence settling velocities, were larger for the higher dilution rate. Hybridomas began to wash out of the culture at an overflow rate of 31 mL/h. As the overflow rate was increased, the cell concentration in the settler effluent increased also, as was observed in the previous experiments. At overflow rates less than 123 mL/h, the nonviable cell concentration in the overflow stream was greater than that for viable cells even though the viable cell concentration in the reactor was nearly 12 times greater. The nonviable cells were being removed preferentially while viable cells were retained by the inclined settler a t these overflow rates. From Figure 4, it may be concluded that operation at intermediate overflow rates between about 70 and 120 mL/h gives very good separation in which the settler retains more than 95% of the viable cells while removing through its overflow more than 50% of the nonviable cells. From eqs 1 and 2, this range corresponds to diameters between 8 and 11 pm for the largest cells predicted to reach the overflow. This separation is better than that achieved in the previous experiment a t the lower dilution rate (see Figure 2 for comparison) because the viable cells were larger, on average, a t the higher dilution rate and were therefore more easily separated from the smaller nonviable cells by differential sedimentation.

The results a t the two different dilution rates demonstrate that selective removal of nonviable hybridomas from a mixed culture is achievable with inclined sedimentation. The cell separation is affected by varying the overflow rate through the inclined settler, which controls the residence time of suspended cells in the settling channel. The degree of nonviable cell removal is constrained by the difference in size distribution between viable and nonviable cells. Therefore, it is desirable to maintain the cells at a high specific growth rate, which maximizes the size of the viable cells.

Having demonstrated tha t nonviable cells can be preferentially removed even at a relatively low growth rate is significant for applications to perfusion culture. The

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Figure 5. Viable and total hybridoma concentrations in the reactor during the perfusion culture. Perfusion was initiated on day one at a dilution rate of 0.89 day-', which was increased incrementally to 1.65 day1 over the next 2'12 days. The dilution rate was increased to 1.70 day-' on day 5 and stayed there for the remainder of the study.

perfusion rate may be high relative to the washout dilution rate, but the growth rate could still be submaximal due to increased utilization of substrate caused by the greater viable cell concentration reached in a perfusion culture. Since monoclonal antibody production is non-growth- associated, however, it is desirable to maximize the viable cell concentration rather than the growth rate. The next phase of the study was to demonstrate the utility of inclined sedimentation in perfusion culture to promote a high viable cell concentration and monoclonal antibody productivity by selectively removing nonviable cells.

Perfusion Culture with Inclined Sedimentation. The perfusion culture was initiated approximately 3 days following the previous cell separation study to ensure a stable baseline in the cell concentration. During the interim, in which the bioreactor was operated as a chemo- s ta t with a dilution rate of 0.89 day-l, the total cell concentration reached 3.1 X lo6 cells/mL, the viability was 92%, and the antibody concentration was 10.4 pg/mL. A t the onset of perfusion, the dilution rate was maintained at 0.89 day-1 because this corresponded to an overflow rate of 37.8 mL/h, which in the previous study was found to retain practically all viable cells in the culture. The concentrations of total and viable hybridomas in the bioreactor are plotted over the duration of the perfusion culture in Figure 5, whereas Figure 6 shows the relative viable and nonviable cell concentrations in the settler effluent stream. At this low dilution rate, 100% of the viable cells, and approximately 95 7; of the nonviable cells, were returned to the bioreactor by the settler. Consequently, both the viable and nonviable cell concentrations increased in the bioreactor. Since no viable cells were observed in the effluent, the dilution rate was increased to 1.10 day-l (corresponding to an overflow rate of 46.7 mL/h) after almost 2 days in order to increase the number of nonviable cells removed. Over the next 2'/2 days, the dilution rate was increased incrementally to 1.65 day-'. The nonviable cell concentration in each overflow sample progres- sively increased with increasing dilution rate so that 20 % of the nonviable cells entering the settler were removed through the overflow a t the end of this period. In contrast, at most one viable cell was counted in any of these samples, indicating tha t the viable cells were being retained preferentially in the culture.

On the fifth day, the dilution rate was raised to 1.70 day-' (corresponding to a settler overflow rate of 72.2 mL/h and

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Biotechnol. Prog., 1990, Vol. 6, No. 6 483

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Figure 7. Forward-angle light scatter histograms of the viable and nonviable cell populations in the bioreactor (solid lines) and the nonviable cell population in the settler overflow stream (dashed line) on day 8 of the perfusion culture at a dilution rate of 1.70 dayl. There were so few viable cells in the overflow sample that the histogram corresponds to the bottom axis line. The peak in the reactor viable cell histogram is on the right in a higher- numbered channel than the peak in the reactor nonviable cell histogram at left. The peak in the overflow nonviable cell histogram is slightly to the left of that for the reactor nonviable cell population.

Cell Size a n d Viability i n Perfusion Cul ture by Flow Cytometry. The samples for analysis by flow cytometry were taken on the eighth day of the perfusion culture, after the dilution rate was set a t 1.70 day-l. Data were collected by gating around the separate viable and nonviable cell populations to permit later comparison of these distinct cell populations. Figure 7 shows the forward- angle light scatter (FALS) histograms of the viable and nonviable cell populations in the reactor sample (solid lines). The extent of forward-angle light scatter from a particle or cell is correlated to the square of its diameter such that the larger the particle, the higher the channel number in which it falls in the forward-angle light scatter measurement (29). Determining the size distribution in this way is not as quantitatively accurate as the Elzone 180 XY particle size analyzer, bu t it does provide qualitative cell size distributions of the two separate subpopulations in a mixture of viable and nonviable cells. As seen in Figure 7, the viable cells in the perfusion culture were typically larger than the nonviable cells, although there was some overlap in cell size between the two populations. Thus, complete removal of nonviable cells by the inclined sett ler is not attainable without a considerable loss of viable cells, which is consistent with the results in Figures 2 and 4. However, it is possible to obtain partial removal of the nonviable cells while retaining virtually all of the viable cells, provided that the overflow rate is chosen properly. This was the case for the conditions of Figure 7. There were very few viable cells in the overflow sample, and the viable cell histogram is nearly identical with the ordinate axis on this scale. This result concurs with the cell count data and provides further evidence that a negligible number of viable cells were lost in the settler effluent. The FALS histogram of the nonviable cell population in the overflow sample is shown as the dashed line in Figure 7. The overflow sample was also analyzed in the Elzone 180 XY particle size analyzer to determine the nonviable cell size distribution. The mean cell diameter was calculated to be 7.9 pm, which is in contrast to the mean diameter of 11 pm for the mixture of 50% viable and 50% nonviable cells in the bioreactor. A comparison of the FALS histograms of the nonviable cell populations in the reactor and settler overflow stream shows that the two populations are similar, as expected. The peak in the overflow distribution is shifted slightly to the left of the peak for the reactor distribution because viable cell retention in the culture necessitated the use of

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Figure 8. The antibody concentration (open triangles) and specific antibody productivity (solid triangles) in the reactor during the perfusion culture. The error bars show the standard error for each antibody concentration.

a perfusion rate that allowed retention of larger nonviable cells that were similar in size to the smaller viable cells.

Antibody Concentration in Perfusion Culture. The antibody concentration in the reactor during the perfusion culture is shown in Figure 8. The antibody concentration increased from 10.4 pg/mL in the chemostat to 30.4 pg/mL on the final day of the perfusion culture. The standard error associated with each concentration determination (shown as error bars) varied between 6.5 % and 20%. This result demonstrates that the reactor antibody concentration can be enhanced nearly 3-fold in this perfusion bioreactor over a chemostat because of the greater viable cell concentration achieved by selective retention.

The increase in antibody concentration during the perfusion culture reflects not only the increased viable cell concentration but also an increase in specific antibody productivity per viable cell as well. The variation in specific antibody productivity also is plotted in Figure 8. The specific antibody productivity nearly doubled over the duration of the perfusion culture. This observed increase in specific antibody productivity over time is consistent with an earlier experimental finding that the antibody productivity of this cell line decreases as the specific growth rate increases (30). The total antibody productivity per culture volume increased from 8.6 pg/(mL.day) in the chemostat to 52 pg/(mL.day) at the time that the perfusion culture was terminated.

Conclusions The results of this study clearly show that selective

retention of viable cells and removal of nonviable cells from a suspension hybridoma culture is practical with inclined sedimentation. For a given cell culture growing a t a particular rate, the degree of cell separation was found to depend on the overflow rate through the settler, which determines the residence time of suspended cells in the sedimentation channel. The cell separation was also found to be affected by the available area for sedimentation, since the overflow rate at which hybridomas were first washed out was lower with the 23-cm-long settler than with the 37-cm-long settler. The specific growth rate of the culture also determined the degree of cell separation. The enhanced viable cell retention a t higher specific growth rate was due to the associated increase in size of the viable cells relative to the generally smaller diameter of the nonviable cells.

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(12) Goebel, N. K.; Kuehn, R.; Flickinger, M. C. Methods for Determination of Growth-Rate-Dependent Changes in Hy- bridoma Volume, Shape, and Surface Structure During Continuous Recycle. Cytotechnology, in press.

(13) Sato, S.; Kawamura, K.; Hanai, N.; Fujiyoshi, Production of Interferon and Monoclonal Antibody Using a Novel Type of Perfusion Vessel. In Proceedings o f the International Symposium on Growth and Differentiation of Cells in Defined Environment; Murakami, H., Yamane, I., Barnes, D., Mather, J., Hayashi, I., Sato, G., Eds.; Springer-Verlag: New York, 1085;

(14) Kitano, K.; Shintani, Y.; Ichimori, Y.; Tsukamoto, K.; Sa- sai, S.; Kida, M. Production of Human Monoclonal Antibodies by Heterohybridomas. Appl. Microbiol. Biotechnol. 1986,24,

(15) Takazawa, Y.; Tokashiki, M.; Hamamoto, K.; Murakami, H. High Cell Density Perfusion Culture of Hybridoma Cells Recycling High Molecular Weight Components. Cytotech- nology 1988, 1, 171-178.

(16) Tovey, M.; Brouty-Boy6, D. Characteristics of the Chemo- stat Culture of Murine Leukemia L1210 Cells. Exp. Cell Res.

(17) Ponder, E. On Sedimentation and Reouleaux Formation. Q. J . Exp. Physiol. 1925, 15, 235-253.

(18) Nakamura, N.; Kuroda, K. La Cause de l’hcceleration de la Vitesse de Sedimentation des Suspensions sans les Recipients Inclines. Keijo J. Med. 1937, 8, 256-296.

(19) Davison, B. H.; San, K.-Y.; Stephanopoulos, G. Stable Competitive Coexistence in a Continuous Fementor with Size- Selective Properties. Biotechnol. Prog. 1985,l (4), 260-269.

(20) Davis, R. H.; Parnham, C. S. Competitive Yeast Fermen- tation with Selective Flocculation and Recycle. Biotechnol. Bioeng. 1989, 33, 767-776.

(21) Henry, K. L.; Davis, R. H. Continuous Recombinant Bacterial Fermentation Utilizing Selective Flocculation and Recycle. Biotechnol. Prog. 1990, 6, (l), 7-12.

(22) Lee, C. Y.; Davis, R. H.; Sclafani, R. A. Cell Separations of Nondividing and Dividing Yeast Using an Inclined Settler. In Proceedings of the 19th Annual Biochemical Engineering Symposium; Bajpai, R., Ed.; University of Missouri: Columbia,

(23) Hornbeck, P. V.; Lewis, G. K. Idiotype Connectance in the Immune System 11. A Heavy Chain Variable Region Idio- type that Dominates the Antibody Response to the p-Azoben- zenearsonate group is a Minor Idiotype in the Response to Tri- nitrophenyl Group. J. Exp. Med. 1985, 161, 53-71.

(24) de Bruyne, N. A. The Design of Bench-Scale Reactors. Anim. Cell Biotechnol. 1988, 3, 141-176.

(25) Hu, W. S.; Meier, J.; Wang, D. I. C. Use of Surface Aerator to Improve Oxygen Transfer in Cell Culture. Biotechnol. Bioeng. 1986,28, 122-125.

(26) Phillips, H. J. Dye Exclusion Tests for Cell Viability. In Tissue Culture Methods and Applications; Kruse, P. F., Patter- son, M. K., Eds.; Academic Press: New York, 1973; pp 406- 408.

(27) Catty, D.; Raykundalia, C. ELISA and Related Enzyme Im- munoassays. In Antibodies: A Practical Approach; Catty, D., Ed.; IRL Press: Oxford, England, 1988; Vol. 11, pp 97-154.

(28) Sasaki, D. T.; Dumas, S. E.; Engleman, E. G. Discrimination of Viable and Non-Viable Cells Using Propidium Iodide in Two Color Immunofluorescence. Cytometry 1987,8, 413-420.

(29) Shapiro, H. M. Practical Flow Cytometry, 2nd ed.; Alan R. Liss, Inc.: New York, 1988.

(30) Miller, W. M.; Blanch, H. W.; Wilke, C. R. A Kinetic Analysis of Hybridoma Growth and Metabolism in Batch and Contin- uous Suspension Culture: Effect of Nutrient Concentration, Dilution Rate, and pH. Biotechnol. Bioeng. 1988, 32, 947- 965.

pp 123-127.

282-286.

1976,101, 346-354.

MO, 1989; pp 123-132.

Perfusion culture results showed that the inclined settler performed very well as a viable cell retention device, as the maximum viable cell density reached in this study was comparable to those found by other investigators (1 1- 15). However, inclined sedimentation was shown to be advantageous over total cell retention devices in that approximately 20 % of the nonviable cell concentration entering the inclined settler was continuously removed from the culture, while less than 0.1% of the viable cell concentration was lost. Thus, continuous removal of nonviable cells from the culture by inclined sedimentation allows the possibility of o erating a perfusion reactor a t steady state. This woulcf eliminate interuption of the culture to purge the accumulated cells necessitated by clogging of filters used to retain cells (12) and would possibly prevent the decrease in antibody productivity observed in conventional perfusion cultures of long duration (14). Because selective removal of nonviable cells was shown to be possible a t low as well as high specific growth rates, erfusion cultures using inclined sedimen-

antibody productivity has been found to be greater (30). tation could Yl e operated a t low growth rates, where

Acknowledgment We acknowledge the support of Grants NBSRAHS-

OH130 from the Department of Commerce and BCS- 8857719 from the National Science Foundation.

Literature Cited (1) Fazekas de St. Groth, S. Automated Production of Mono-

clonal Antibodies in a Cytostat. J. Immunol. Methods 1983,

(2) Boraston, R.; Thompson, P.; Garland, S.; Birch, J. Growth and Oxygen Requirements of Antibody Producing Mouse Hy- bridoma Cells in Suspension Culture. Deu. Bid. Stand. 1984, 55,103-111.

(3) Reuveny, S.; Velez, D.; Riske, F.; Macmillan, J.; Miller, J. Production of Monoclonal Antibodies in Culture. Deu. Biol. Stand. 1985, 60, 185-197.

(4) Birch, J.; Thompson, P.; Lambert, K.; Boraston, R. The Large Scale Cultivation of Hybridoma Cells Producing Mono- clonal Antibodies. In Large-Scale Mammalian Cell Culture; Tolbert, W., Feder, J., Eds.; Academic Press: New York, 1985;

(5) Luan, Y.; Mutharasan, R.; Magee, W. Strategies to Extend Longevity of Hybridomas in Culture and Promote Yield of Monoclonal Antibodies. Biotechnol. Lett. 1987,9 (lo), 691- 696.

(6) Renard, J. M.; Spagnoli, R.; Mazier, C.; Salles, M. F.; Man- dine, 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.

(7) Reddy, S.; Miller, W. M. Effects of Environmental Stress on Hybridoma Antibody Production and Metabolism. AIChE Annual Meeting, November 1989.

(8) van Wezel, A,; van der Veldon-de Groot, C.; de Haan, H.; van den Heuvel, N.; Schasfoort, R. Large Scale Animal Cell Cultivation for Production of Cellular Biologicals. Deu. Biol. Stand. 1985,60,229-236.

(9) Reuveny, S.; Velez, D.; Macmillan, J.; Miller, L. Comparison of Cell Propagation Methods for their Effect on Monoclonal Antibody Yield in Fermentors. J. Immunol. Methods 1986, 86,61-69.

(10) Tolbert, W.; Lewis, C.; White, P.; Feder, J. Perfusion Culture Systems for Production of Mammalian Cell Biomolecules. In Large-Scale Mammalian Cell Culture; Tolbert, W., Feder, J., Eds.; Academic Press: New York, 1-98; pp 97-119.

(11) Brennan, A,; Shevitz, J.; Macmillan, J. A Perfusion System for Antibody Production by Shear-Sensitive Hybridoma Cells in a Stirred Reactor. Biotechnol. Tech. 1987, 1, (3), 169- 174.

57, 21-36.

pp 1-16.

Accepted October 2, 1990.

Batt Et Al Inclined Sedimentati

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