Cytotechnology 8: 189-194, 1992. 9 1992KluwerAcademic Publishers. Printed in the Netherlands.
Transferrin recycling perfusion culture of hybridoma cells Yoshiharu Takazawa and Michiyuki Tokashiki Biotechnology Research Laboratories, Teijin Limited, Hino 191, Japan Received 13 December 1991; accepted in revised form 23 May 1992
Key words: transferrin, recycling, perfusion culture, serum-free, hybridoma, monoclonal antibody
Abstract A perfusion culture of hybridoma cells in serum-free medium recycling transferrin was carried out, which greatly reduced the level of transferrin that was needed. The culture was maintained even without supplying transferrin for nine days. IgG concentration reached 1.1 mg m1-1 in a month of recycling and its ratio to the total protein was 45.8%. The affinity of the antibody did not decrease and no degradation was observed after long recycling period. The cell density under recycling condition was 2-3 times higher than that without recycling. It was indicated that there was autocrine growth promoting activity in the culture supematant.
Introduction Various mammalian cell culture methods for large scale protein production have been developed. They can be classified into three types: batch, fed-batch and perfusion. The main reasons for loss of cell viability and protein productivity are said to be nutrient depletion and waste product formation (Lauffenburger and Cozens, 1989). In batch culture, the former is thought to be dominant. Fed-batch culture can eliminate nutrient depletion but not waste product accumulation. We think perfusion culture is best in that it supplies nutrients continuously, can keep waste product accumulation low by the raising perfusion rate, and can keep cell density higher than in other systems. We have developed perfusion culture systems using three different cell-medium separation methods: filtration (Tokashiki etal., 1987); gravity settling (Tokashiki et al., 1988); and centri-
fugal separation (Hamamoto et al., 1989), with good results. But a perfusion system also has disadvantages in that raising perfusion rates wastes medium which contains expensive components and lowers product concentration. So recycling of medium components are attracting increased attention (Lehmann et al., 1989). Although some perfusion culture systems like hollow fiber (Altshuler et al., 1985) or rnicrocapsule (Duff, 1985; Gilligan et al., 1988; King et al., 1987) have been reported to yield high product concentration, they are not suitable for large scale production. Suspension culture is desirable for this purpose. Previously we developed a suspension perfusion culture system in which high molecular weight components are recycled (Takazawa et al., 1988), and attained the high IgG concentration of 2 mg m1-1. This system can also reduce the consumption of high molecular weight components such as transferrin or other peptide growth factors. Transferrin is one of the most expensive
190 components in serum-free media. Some substitutes for transferrin have been reported (Basset et al., 1986; Rasmussen and Toftlund, 1986; Yabe, 1987), but they cannot necessarily be applied to all cell lines, whereas recycling high molecular weight components can. More interestingly, the increase in cell density was observed under recycling condition in the previous experiments. In this report, we describe more details of the culture properties of transferrin recycling culture of mouse-human hybridoma cells. We also investigated the growth promoting activity in the culture supematant.
was connected. The details of the culture conditions were the same as those described previously (Takazawa et al., 1988).
Assays for IgG The concentration of IgG and the affinity to the antigen were determined by ELISA, as described (Takazawa et al., 1988). The total protein concentration was assayed with Protein Assay Kit (BioRad). SDS-PAGE and Immunoblotting were done also as described (Takazawa et al., 1988).
Assay for transferrin Materials and methods
Cells Mouse-human hybridoma X87 producing antiherpes virus human IgG, which were established at Biomedical Research Institute, Teijin Ltd. was used. They were maintained in ITES-eRDF (Murakami et al., 1982; Murakami et al., 1984) (insulin 9 lag ml -I, transferrin 10 lag ml-1, ethanolamine 10 laM, sodium selenite 20 nM).
The concentration of transferrin was determined by a double antibody sandwich ELISA, using goat anti-human transferrin antiserum (Cappel) as the first antibody and goat anti-human transferrinperoxidase conjugate (E-Y Laboratories' as the second antibody.
Results
Transferrin requirement of X87 cells m static culture
Growth assays Exponentially growing cells from static culture in ITES-eRDF were inoculated into 2 ml of each medium described in results at the initial density of 5 • 104 cells ml -! in 35 mm~ culture plates. Cell number was determined with a cell counter.
Pelfusion culture Perfusion culture was carried out using ITESeRDF in a gravity settling culture vessel for continuous medium change (Tokashiki et al., 1988). The concentration of dissolved oxygen was maintained at 3 ppm by aeration with pure oxygen. Stirring speed was 35 rpm and temperature was 37~ For recycling high molecular weight components, ultrafiltration unit which had three 100 mm square Pellicon membrane packet (molecular weight limit 10,000, Millipore Co.)
The growth of X87 cells in ITES-eRDF at various transferrin concentration was examined in static culture to determine the suitable range. Cells were inoculated at 5 x 104 cells ml -l and counted after 7 days incubation. The result is shown in Fig. I. Cell density was almost the same between 1 to 20 lag ml -I of transferrin and lower below and above this range. The decrease of cell density at the concentration of 100 and 500 lag m1-1 was not the consequence of overgrowth because the growth rates at these concentrations were lower than those at 1 to 20 gg m1-1. Therefore 1 to 20 I.tg m1-1 was the optimum concentration in static culture.
Transferrin recycling culture Cells were inoculated into 280 ml of ITES-eRDF including 10 lag m1-1 of transferrin at the viable
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........
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Fig. 1. Transferrin requirement of X87 cells in static culture.
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Cells were inoculated at 5 x 104 cells m1-1 and counted after 7 days incubation in 35 mm~ plates. Determinations were made from duplicate cultures.
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cell density of 4.3 x 105 cells m1-1. The real culture volume in which cells existed was approximately 150 ml. Figure 2 shows the time course of cell density, transferrin concentration and IgG production. After 1 day from the inoculation, perfusion was started at the rate of 260 ml day -I and raised at the 4th day to 500 ml day -I. When cells entered the stationary phase, recycling was started. The medium was continuously discharged from the culture vessel at the rate of 500 ml day -l to the ultrafiltration unit. The concentrated medium was returned to the vessel at 100 ml day -l and the fresh medium was supplied at 400 ml day -l. The concentration of transferrin in the fresh medium was reduced as indicated in Fig. 2. Viable cell density was 9.3 x 106 cells ml -l when recycling was started and ~adually increased to 2.6 x 107 cells ml -l by daY 30. Although transferrin supply was stopped at day 30, cell density was maintained at around 2 • 107 cells ml -I for 9 days. When the concentration of transferrin in the culture medium became lower than 1.0 ~tg ml -I, cell density decreased rapidly. It recovered to 3.3 • 107 cells ml -I by starting transferrin supply again. Recycling was stopped at day 78, to examine its effect, and viable cell density dropped to approximately 1 x 107 cells ml -l from 2-3 • 107 cells ml -l at the end of recycling.
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80
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Fig. 2. Time course of the transferrin recycling culture of X87 cells. The culture volume was 280 ml0 of which 150 ml contained cells. Perfusion rate was 260 ml day -I for day I - day 4 and 500 ml day -] for day 4 - day 7. After the start of recycling, medium discharging rate was 500 ml day -I, and fresh medium was supplied at 400 ml day -I with concentrated medium through ultrafiltration unit at 100 ml day -1.
IgG concentration increased from 19 ~tg ml -I to 1.1 mg ml -l by day 32. It decreased at day 32 and day 37 due to the leak from tubing caused by mechanical trouble, but recovered. IgG productivity ranged from 2 to 10 ~tg 106 cells -l day -I. It tended to be relatively low when cell density was high.
Analysis of lgG The IgG produced in recycling culture was analyzed to examine degradation, purity and affinity to the antigen. The culture supernatant at day 7, 17, 26 and 37 and IgG purified from human plasma were applied to SDS-PAGE, followed by immunoblotting with anti-human IgG antisera
192
0.7 0.6 0.5 .n
r
o
0.4 0.3
0.2
0.1 ,
0 10 .3
(Fig. 3). In the S D S - P A G E , two clear bands of heavy and light chain were observed in the lanes of culture supematant and no major band other than IgG was found, even after 1 month from the start of recycling. Immunoblotting showed more clearly that there was no significant degradation under recycling condition, while a major band between heavy and light chain was observed in the lane of IgG from plasma. The ratio of IgG to the total protein before and after 30 days recycling is shown in Table 1. The ratio after 1 month recycling was 45.8%, which was much higher than that before recycling (8.1%). Affinity to the antigen was examined by E L I S A using antigen coated plates. The culture supernatant at the 9th day (just after the start of recycling) and the 48th day (after 41 days recycling) was assayed. IgG purified from the supernatant o f the conventional X87 perfusion culture
........
10 .2
t
........
1
10 0
1 0 "1
IgG (pg/ml)
Fig. 3. Analysis of the culture supematant of the recycling culture by SDS-PAGEand immunoblotting. Lane 1:1 mg ml-l of human IgG purified from human plasma, Lane 2, 3, 4 and 5: culture supernatant of the 7th, 17th, 26th and 37th day, respectively. The same volumes of each samples were applied to 10% polyacrylamide gel under reducing condition. Goat anti-human IgG antiserum was used for immunoblotting.
, , ,,,+,1
Fig. 4. Affinity of the IgG produced in recycling culture to the antigen. Human IgG produced by X87 cells in perfusion culture without recycling (A) and the culture supernatant of the recycling culture at 9th (171)and 48th (O) day was examined by ELISA with a plate coated with the antigen.
using settling culture vessel was used as a control. As shown in Fig. 4, there was no significant difference in the affinity a m o n g these three sampies.
Effect o f culture supernatant on the growth o f X 8 7 cells To confirm that the increase in cell density in recycling culture was due to the accumulation of some factor in the culture medium, the autocrine effect of culture supernatant of the recycling culture was examined in static culture. The culture supernatant at the 33rd day was concentrated 10fold with A m i c o n D I A F L O ultrafiltration m e m brane Y M 10 (molecular weight limit 10,000), and 20 I.tl of it containing 94 p.g m1-1 o f transferrin was added to the 2 ml of static culture containing 1 • 105 cells just after inoculation. Table 2 shows
Table 1. Purity of IgG before and during recycling Culture period (days)
IgG concentration (lag ml-I)
Total protein (lag m1-1)
IgG ratio (%)
7 37
19 1100
235 2400
8.1 45.8
The 7th day was just after the start of recycling and the 37th day was the day of maximum IgG concentration.
193 Table 2. Effect of culture supernatant on the growth of X87 cells in static culture
Additives
Cell density (cells m1-1)
Growth ratio (%)
None Supernatant
5.87 x 105 8.46 x 105
100.0 144.1
Twenty microlitersof concentratedsupernatant was added to 2 ml of ITES-eRDFcontaining 1 x 105 cells and incubated for 4 days in 35 mm~ plates. Determinations were made from duplicate cultures. the comparison o f cell density after 4 days incubation with and without the concentrated supernatant. The cell density o f the culture with it was 44% higher than that of the culture without it.
Discussion The critical concentration of transferrin for cell growth in recycling culture was about 1 ktg ml -I, while the cells grew enough at the concentration o f 0.1 I.tg m1-1 in static culture. This difference is probably due to environmental factors like shear stress. It will be necessary to keep transferrin concentration above 1 lag m1-1 in recycling culture. It is known that cells take in transferrin by endocytosis and it is released into lysosome and recycled by exocytosis (Klausner et al., 1983). But it was consumed at a certain rate in the recycling culture. The consumption rate calculated from the stable period was about 1.5/.tg -1 m1-1 day -l. When m e d i u m supply rate is 400 ml day -1, 1 ktg ml -l in the fresh medium is needed to maintain an even concentration. In other words, when the concentration of transferrin is 10 ktg m1-1 at the start of recycling, 1 l.tg m1-1 in the fresh medium will be enough to maintain 10 ktg ml -l in the culture. On the other hand, perfusion culture without recycling needs at least 2 ~tg m1-1 o f transferrin in the fresh m e d i u m to keep its concentration above I ~tg m1-1. Usually we carry out perfusion cultures with fresh medium containing 2 - 1 0 ktg ml -I o f transferrin (unpublished data). Therefore recycling will lead to a great reduction in transferrin requirement. It was apparent that recycling high molecular weight components increased cell density, from the fact that it rapidly decreased when recycling
was stopped. The increase of cell density was not due to the accumulation of transferrin because cell density reached 3.3 x 107 cells ml -I even when transferrin concentration was below 10 l.tg ml -I (the 52nd day). The accumulation o f high molecular weight autocrine factor may have affected the cell density. The reason for the delay in the increase of cell density after the start o f recycling was probably that it took a certain period for the factor to accumulate enough to increase cell density. The existence of the factor was supported by the growth promoting effect o f the concentrated supematant in static culture. The concentrated supernatant contained 94 p.g ml -l transferrin and only a trace amount of insulin. So it is obvious that the effect was not due to these growth factors. Further investigation will make it clearer. IgG concentration reached 1.1 mg ml -l in about a month. Such high concentration is the same level as those in hollow fiber (Alshuler e t al., 1985) or microcapsule (Duff, 1985). The purity o f IgG in the culture supernatant was reasonably high and there was no degradation and loss of afffmity to the antigen even after the long culture period. So it was proved that recycling did not cause any deterioration o f the antibody. IgG productivity tended to be low when cell density was high (about 2 - 3 x 107 cells ml-1). W e think this was probably due to nutrient shortage, although there might be the possibility o f the negative effect of recycling on antibody productivity. We intend to carry out recycling cultures in which nutrients and metabolites are monitored and controlled. Such improved culture will maintain high productivity. In conclusion, we carried out a transferrin recycling culture which compensate for the disad-
194 vantage of perfusion culture like wastage of medium or low product concentration. This culture also had an advantage of higher cell density than conventional the
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References Altshuler GL0 Dziewulski DM, Sowek JA and Belfort G (1985) Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators. Biotechnol. Bioeng. 28: 646-658. Basset P, Quesneau Y and Zwiller J (1986) Iron-induced L1210 cell growth: evidence of a transferrin-independent iron transport. Cancer Res. 46:1644 1647. Duff RG (1985) Microencapsulation technology: a novel method for monoclonal antibody production. Trends Biotechnol. 3: 167-170. Gilligan KJ, Littlefield S and Jarvis A Jr (1988) Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology. In Vitro 24: 35---41. Glacken MW (1988) Catabolic control of mammalian cell culture. Bio/Technology 6: 1041-1050. Hamamoto K, Ishimaru K and Tokashiki M (1989) Perfusion culture of hybridoma cells using a centrifuge to separate cells from culture mixture. J. Ferment. Bioeng. 67: 190-194. King GA, Daugulis AJ, Faulkner P and Goosen MFA (1987) Alginate-polylysine microcapsule of controlled membrane molecular weight cutoff for mammalian cell culture engineering. Biotechnol. Progr. 3: 231-240. Klausner RD, Renswoude JV, Ashwell G, Kempf C, Schechter AN, Dean A and Bridges KR (1983) Receptor-mediated endocytosis of transferrin in K562 cells. J. Biol. Chem. 258: 4715--4724.
Lauffenburger D and Cozens C (1989) Regulation of mammalian cell growth by autocrine growth factors: analysis of consequences for inoculum cell density effects. Biotechnol. Bioeng. 33: 1365-1378. Lehmann J, Kempken R, Lutkemeyer D and Buntemeyer H (1989) Economic aspects of medium recycling. Proceedings of the Second Annual Meeting of the Japanese Association for Animal Cell Technology; pp. 55-59. Murakami H, Masui H, Sato GH, Sueoka N, Chow TP and Kano-Sueoka T (1982) Growth of hybridoma cells in serumfree medium. Proc. Natl. Acad. Sci., USA 79:1158-1162. Murakami H, Shimomura T, Nakamura T, Ohashi H, Shirmhara K and Omura H (1984) Development of a basal medium for serum-free cultivation of hybridoma cells in high density. Nippon Nougeikagaku Kaishi 58: 575-583. Rasmussen L and Toftlund H (1986) Phosphate compounds as iron chelators in animal cell cultures. In Vitro 22: 177-179. Takazawa Y, Tokashiki M, Hamamoto K and Murakami H (1988) High cell density perfusion culture of hybridoma cells recycling high molecular weight components. Cytotechnology 1: 171-178. Tokashiki M, Takazawa Y and Hamamoto K (1987) High density culture of hybridoma cells using a perfusion culture vessel with filter. Hakko Kogaku Kaishi 65: 535-536. Tokashiki M, Hamamoto K, Takazawa Y and Ichikawa Y (1988) High-density culture of mouse-human hybridoma cells using a new perfusion culture vessel. Kagaku Kogaku Ronbunshu 14: 337-341. Yabe N (1987) Role of iron chelators in growth-promoting effect on mouse hybridoma cells in a chemically defined medium. Soshikibaiyou 13: 13-16.
Address for correspondence: Yoshiharu Takazawa, Biotechnology Research Laboratories, Teijin Limited, 4-3-2 Asahigaoka, Hino, Tokyo 191, Japan.
Transferrin recycling perfusion culture of hybridoma cells Yoshiharu Takazawa and Michiyuki Tokashiki Biotechnology Research Laboratories, Teijin Limited, Hino 191, Japan Received 13 December 1991; accepted in revised form 23 May 1992
Key words: transferrin, recycling, perfusion culture, serum-free, hybridoma, monoclonal antibody
Abstract A perfusion culture of hybridoma cells in serum-free medium recycling transferrin was carried out, which greatly reduced the level of transferrin that was needed. The culture was maintained even without supplying transferrin for nine days. IgG concentration reached 1.1 mg m1-1 in a month of recycling and its ratio to the total protein was 45.8%. The affinity of the antibody did not decrease and no degradation was observed after long recycling period. The cell density under recycling condition was 2-3 times higher than that without recycling. It was indicated that there was autocrine growth promoting activity in the culture supematant.
Introduction Various mammalian cell culture methods for large scale protein production have been developed. They can be classified into three types: batch, fed-batch and perfusion. The main reasons for loss of cell viability and protein productivity are said to be nutrient depletion and waste product formation (Lauffenburger and Cozens, 1989). In batch culture, the former is thought to be dominant. Fed-batch culture can eliminate nutrient depletion but not waste product accumulation. We think perfusion culture is best in that it supplies nutrients continuously, can keep waste product accumulation low by the raising perfusion rate, and can keep cell density higher than in other systems. We have developed perfusion culture systems using three different cell-medium separation methods: filtration (Tokashiki etal., 1987); gravity settling (Tokashiki et al., 1988); and centri-
fugal separation (Hamamoto et al., 1989), with good results. But a perfusion system also has disadvantages in that raising perfusion rates wastes medium which contains expensive components and lowers product concentration. So recycling of medium components are attracting increased attention (Lehmann et al., 1989). Although some perfusion culture systems like hollow fiber (Altshuler et al., 1985) or rnicrocapsule (Duff, 1985; Gilligan et al., 1988; King et al., 1987) have been reported to yield high product concentration, they are not suitable for large scale production. Suspension culture is desirable for this purpose. Previously we developed a suspension perfusion culture system in which high molecular weight components are recycled (Takazawa et al., 1988), and attained the high IgG concentration of 2 mg m1-1. This system can also reduce the consumption of high molecular weight components such as transferrin or other peptide growth factors. Transferrin is one of the most expensive
190 components in serum-free media. Some substitutes for transferrin have been reported (Basset et al., 1986; Rasmussen and Toftlund, 1986; Yabe, 1987), but they cannot necessarily be applied to all cell lines, whereas recycling high molecular weight components can. More interestingly, the increase in cell density was observed under recycling condition in the previous experiments. In this report, we describe more details of the culture properties of transferrin recycling culture of mouse-human hybridoma cells. We also investigated the growth promoting activity in the culture supematant.
was connected. The details of the culture conditions were the same as those described previously (Takazawa et al., 1988).
Assays for IgG The concentration of IgG and the affinity to the antigen were determined by ELISA, as described (Takazawa et al., 1988). The total protein concentration was assayed with Protein Assay Kit (BioRad). SDS-PAGE and Immunoblotting were done also as described (Takazawa et al., 1988).
Assay for transferrin Materials and methods
Cells Mouse-human hybridoma X87 producing antiherpes virus human IgG, which were established at Biomedical Research Institute, Teijin Ltd. was used. They were maintained in ITES-eRDF (Murakami et al., 1982; Murakami et al., 1984) (insulin 9 lag ml -I, transferrin 10 lag ml-1, ethanolamine 10 laM, sodium selenite 20 nM).
The concentration of transferrin was determined by a double antibody sandwich ELISA, using goat anti-human transferrin antiserum (Cappel) as the first antibody and goat anti-human transferrinperoxidase conjugate (E-Y Laboratories' as the second antibody.
Results
Transferrin requirement of X87 cells m static culture
Growth assays Exponentially growing cells from static culture in ITES-eRDF were inoculated into 2 ml of each medium described in results at the initial density of 5 • 104 cells ml -! in 35 mm~ culture plates. Cell number was determined with a cell counter.
Pelfusion culture Perfusion culture was carried out using ITESeRDF in a gravity settling culture vessel for continuous medium change (Tokashiki et al., 1988). The concentration of dissolved oxygen was maintained at 3 ppm by aeration with pure oxygen. Stirring speed was 35 rpm and temperature was 37~ For recycling high molecular weight components, ultrafiltration unit which had three 100 mm square Pellicon membrane packet (molecular weight limit 10,000, Millipore Co.)
The growth of X87 cells in ITES-eRDF at various transferrin concentration was examined in static culture to determine the suitable range. Cells were inoculated at 5 x 104 cells ml -l and counted after 7 days incubation. The result is shown in Fig. I. Cell density was almost the same between 1 to 20 lag ml -I of transferrin and lower below and above this range. The decrease of cell density at the concentration of 100 and 500 lag m1-1 was not the consequence of overgrowth because the growth rates at these concentrations were lower than those at 1 to 20 gg m1-1. Therefore 1 to 20 I.tg m1-1 was the optimum concentration in static culture.
Transferrin recycling culture Cells were inoculated into 280 ml of ITES-eRDF including 10 lag m1-1 of transferrin at the viable
191 1 0r
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1 0s 10 .3
........ ' 10 .2
........ i 1 0 "1
........
J
10 0
Transferrin
........
J
........
101
~
10 2
........
10 3
(#g/ml)
Fig. 1. Transferrin requirement of X87 cells in static culture.
lo'
Cells were inoculated at 5 x 104 cells m1-1 and counted after 7 days incubation in 35 mm~ plates. Determinations were made from duplicate cultures.
,...,.
., . . . . . .
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,
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' . . .
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cell density of 4.3 x 105 cells m1-1. The real culture volume in which cells existed was approximately 150 ml. Figure 2 shows the time course of cell density, transferrin concentration and IgG production. After 1 day from the inoculation, perfusion was started at the rate of 260 ml day -I and raised at the 4th day to 500 ml day -I. When cells entered the stationary phase, recycling was started. The medium was continuously discharged from the culture vessel at the rate of 500 ml day -l to the ultrafiltration unit. The concentrated medium was returned to the vessel at 100 ml day -l and the fresh medium was supplied at 400 ml day -l. The concentration of transferrin in the fresh medium was reduced as indicated in Fig. 2. Viable cell density was 9.3 x 106 cells ml -l when recycling was started and ~adually increased to 2.6 x 107 cells ml -l by daY 30. Although transferrin supply was stopped at day 30, cell density was maintained at around 2 • 107 cells ml -I for 9 days. When the concentration of transferrin in the culture medium became lower than 1.0 ~tg ml -I, cell density decreased rapidly. It recovered to 3.3 • 107 cells ml -I by starting transferrin supply again. Recycling was stopped at day 78, to examine its effect, and viable cell density dropped to approximately 1 x 107 cells ml -l from 2-3 • 107 cells ml -l at the end of recycling.
-
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g~
?~ o4 o2
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o
o
to
20
30
40
50
80
70
80
90
Ioo
Cullute time (days)
Fig. 2. Time course of the transferrin recycling culture of X87 cells. The culture volume was 280 ml0 of which 150 ml contained cells. Perfusion rate was 260 ml day -I for day I - day 4 and 500 ml day -] for day 4 - day 7. After the start of recycling, medium discharging rate was 500 ml day -I, and fresh medium was supplied at 400 ml day -I with concentrated medium through ultrafiltration unit at 100 ml day -1.
IgG concentration increased from 19 ~tg ml -I to 1.1 mg ml -l by day 32. It decreased at day 32 and day 37 due to the leak from tubing caused by mechanical trouble, but recovered. IgG productivity ranged from 2 to 10 ~tg 106 cells -l day -I. It tended to be relatively low when cell density was high.
Analysis of lgG The IgG produced in recycling culture was analyzed to examine degradation, purity and affinity to the antigen. The culture supernatant at day 7, 17, 26 and 37 and IgG purified from human plasma were applied to SDS-PAGE, followed by immunoblotting with anti-human IgG antisera
192
0.7 0.6 0.5 .n
r
o
0.4 0.3
0.2
0.1 ,
0 10 .3
(Fig. 3). In the S D S - P A G E , two clear bands of heavy and light chain were observed in the lanes of culture supematant and no major band other than IgG was found, even after 1 month from the start of recycling. Immunoblotting showed more clearly that there was no significant degradation under recycling condition, while a major band between heavy and light chain was observed in the lane of IgG from plasma. The ratio of IgG to the total protein before and after 30 days recycling is shown in Table 1. The ratio after 1 month recycling was 45.8%, which was much higher than that before recycling (8.1%). Affinity to the antigen was examined by E L I S A using antigen coated plates. The culture supernatant at the 9th day (just after the start of recycling) and the 48th day (after 41 days recycling) was assayed. IgG purified from the supernatant o f the conventional X87 perfusion culture
........
10 .2
t
........
1
10 0
1 0 "1
IgG (pg/ml)
Fig. 3. Analysis of the culture supematant of the recycling culture by SDS-PAGEand immunoblotting. Lane 1:1 mg ml-l of human IgG purified from human plasma, Lane 2, 3, 4 and 5: culture supernatant of the 7th, 17th, 26th and 37th day, respectively. The same volumes of each samples were applied to 10% polyacrylamide gel under reducing condition. Goat anti-human IgG antiserum was used for immunoblotting.
, , ,,,+,1
Fig. 4. Affinity of the IgG produced in recycling culture to the antigen. Human IgG produced by X87 cells in perfusion culture without recycling (A) and the culture supernatant of the recycling culture at 9th (171)and 48th (O) day was examined by ELISA with a plate coated with the antigen.
using settling culture vessel was used as a control. As shown in Fig. 4, there was no significant difference in the affinity a m o n g these three sampies.
Effect o f culture supernatant on the growth o f X 8 7 cells To confirm that the increase in cell density in recycling culture was due to the accumulation of some factor in the culture medium, the autocrine effect of culture supernatant of the recycling culture was examined in static culture. The culture supernatant at the 33rd day was concentrated 10fold with A m i c o n D I A F L O ultrafiltration m e m brane Y M 10 (molecular weight limit 10,000), and 20 I.tl of it containing 94 p.g m1-1 o f transferrin was added to the 2 ml of static culture containing 1 • 105 cells just after inoculation. Table 2 shows
Table 1. Purity of IgG before and during recycling Culture period (days)
IgG concentration (lag ml-I)
Total protein (lag m1-1)
IgG ratio (%)
7 37
19 1100
235 2400
8.1 45.8
The 7th day was just after the start of recycling and the 37th day was the day of maximum IgG concentration.
193 Table 2. Effect of culture supernatant on the growth of X87 cells in static culture
Additives
Cell density (cells m1-1)
Growth ratio (%)
None Supernatant
5.87 x 105 8.46 x 105
100.0 144.1
Twenty microlitersof concentratedsupernatant was added to 2 ml of ITES-eRDFcontaining 1 x 105 cells and incubated for 4 days in 35 mm~ plates. Determinations were made from duplicate cultures. the comparison o f cell density after 4 days incubation with and without the concentrated supernatant. The cell density o f the culture with it was 44% higher than that of the culture without it.
Discussion The critical concentration of transferrin for cell growth in recycling culture was about 1 ktg ml -I, while the cells grew enough at the concentration o f 0.1 I.tg m1-1 in static culture. This difference is probably due to environmental factors like shear stress. It will be necessary to keep transferrin concentration above 1 lag m1-1 in recycling culture. It is known that cells take in transferrin by endocytosis and it is released into lysosome and recycled by exocytosis (Klausner et al., 1983). But it was consumed at a certain rate in the recycling culture. The consumption rate calculated from the stable period was about 1.5/.tg -1 m1-1 day -l. When m e d i u m supply rate is 400 ml day -1, 1 ktg ml -l in the fresh medium is needed to maintain an even concentration. In other words, when the concentration of transferrin is 10 ktg m1-1 at the start of recycling, 1 l.tg m1-1 in the fresh medium will be enough to maintain 10 ktg ml -l in the culture. On the other hand, perfusion culture without recycling needs at least 2 ~tg m1-1 o f transferrin in the fresh m e d i u m to keep its concentration above I ~tg m1-1. Usually we carry out perfusion cultures with fresh medium containing 2 - 1 0 ktg ml -I o f transferrin (unpublished data). Therefore recycling will lead to a great reduction in transferrin requirement. It was apparent that recycling high molecular weight components increased cell density, from the fact that it rapidly decreased when recycling
was stopped. The increase of cell density was not due to the accumulation of transferrin because cell density reached 3.3 x 107 cells ml -I even when transferrin concentration was below 10 l.tg ml -I (the 52nd day). The accumulation o f high molecular weight autocrine factor may have affected the cell density. The reason for the delay in the increase of cell density after the start o f recycling was probably that it took a certain period for the factor to accumulate enough to increase cell density. The existence of the factor was supported by the growth promoting effect o f the concentrated supematant in static culture. The concentrated supernatant contained 94 p.g ml -l transferrin and only a trace amount of insulin. So it is obvious that the effect was not due to these growth factors. Further investigation will make it clearer. IgG concentration reached 1.1 mg ml -l in about a month. Such high concentration is the same level as those in hollow fiber (Alshuler e t al., 1985) or microcapsule (Duff, 1985). The purity o f IgG in the culture supernatant was reasonably high and there was no degradation and loss of afffmity to the antigen even after the long culture period. So it was proved that recycling did not cause any deterioration o f the antibody. IgG productivity tended to be low when cell density was high (about 2 - 3 x 107 cells ml-1). W e think this was probably due to nutrient shortage, although there might be the possibility o f the negative effect of recycling on antibody productivity. We intend to carry out recycling cultures in which nutrients and metabolites are monitored and controlled. Such improved culture will maintain high productivity. In conclusion, we carried out a transferrin recycling culture which compensate for the disad-
194 vantage of perfusion culture like wastage of medium or low product concentration. This culture also had an advantage of higher cell density than conventional the
most
p e r f u s i o n c u l t u r e . It w i l l b e o n e o f
ideal
culture
systems
for
producing
biologicals.
References Altshuler GL0 Dziewulski DM, Sowek JA and Belfort G (1985) Continuous hybridoma growth and monoclonal antibody production in hollow fiber reactors-separators. Biotechnol. Bioeng. 28: 646-658. Basset P, Quesneau Y and Zwiller J (1986) Iron-induced L1210 cell growth: evidence of a transferrin-independent iron transport. Cancer Res. 46:1644 1647. Duff RG (1985) Microencapsulation technology: a novel method for monoclonal antibody production. Trends Biotechnol. 3: 167-170. Gilligan KJ, Littlefield S and Jarvis A Jr (1988) Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology. In Vitro 24: 35---41. Glacken MW (1988) Catabolic control of mammalian cell culture. Bio/Technology 6: 1041-1050. Hamamoto K, Ishimaru K and Tokashiki M (1989) Perfusion culture of hybridoma cells using a centrifuge to separate cells from culture mixture. J. Ferment. Bioeng. 67: 190-194. King GA, Daugulis AJ, Faulkner P and Goosen MFA (1987) Alginate-polylysine microcapsule of controlled membrane molecular weight cutoff for mammalian cell culture engineering. Biotechnol. Progr. 3: 231-240. Klausner RD, Renswoude JV, Ashwell G, Kempf C, Schechter AN, Dean A and Bridges KR (1983) Receptor-mediated endocytosis of transferrin in K562 cells. J. Biol. Chem. 258: 4715--4724.
Lauffenburger D and Cozens C (1989) Regulation of mammalian cell growth by autocrine growth factors: analysis of consequences for inoculum cell density effects. Biotechnol. Bioeng. 33: 1365-1378. Lehmann J, Kempken R, Lutkemeyer D and Buntemeyer H (1989) Economic aspects of medium recycling. Proceedings of the Second Annual Meeting of the Japanese Association for Animal Cell Technology; pp. 55-59. Murakami H, Masui H, Sato GH, Sueoka N, Chow TP and Kano-Sueoka T (1982) Growth of hybridoma cells in serumfree medium. Proc. Natl. Acad. Sci., USA 79:1158-1162. Murakami H, Shimomura T, Nakamura T, Ohashi H, Shirmhara K and Omura H (1984) Development of a basal medium for serum-free cultivation of hybridoma cells in high density. Nippon Nougeikagaku Kaishi 58: 575-583. Rasmussen L and Toftlund H (1986) Phosphate compounds as iron chelators in animal cell cultures. In Vitro 22: 177-179. Takazawa Y, Tokashiki M, Hamamoto K and Murakami H (1988) High cell density perfusion culture of hybridoma cells recycling high molecular weight components. Cytotechnology 1: 171-178. Tokashiki M, Takazawa Y and Hamamoto K (1987) High density culture of hybridoma cells using a perfusion culture vessel with filter. Hakko Kogaku Kaishi 65: 535-536. Tokashiki M, Hamamoto K, Takazawa Y and Ichikawa Y (1988) High-density culture of mouse-human hybridoma cells using a new perfusion culture vessel. Kagaku Kogaku Ronbunshu 14: 337-341. Yabe N (1987) Role of iron chelators in growth-promoting effect on mouse hybridoma cells in a chemically defined medium. Soshikibaiyou 13: 13-16.
Address for correspondence: Yoshiharu Takazawa, Biotechnology Research Laboratories, Teijin Limited, 4-3-2 Asahigaoka, Hino, Tokyo 191, Japan.
