Cytotechnology 6: 197-208, 1991. 9 1991 KluwerAcademic Publishers. Printed in the Netherlands.

Comparison of culture methods for human-human hybridomas secreting anti-HBsAg human monoclonal antibodies Yasushi Shintani, Yoh-Ichiro Kohno, Hidekazu Sawada and Kazuaki Kitano Microbiology Research Laboratories, Research & Development Division, Takeda Chemical Industries Ltd., 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532, Japan Received 26 September 1990; acceptedin revised form 14 May 1991

Key words: growth-associated production, hepatitis B virus, human-human hybridoma, immobilized culture, monoclonal antibody, perfusion culture Abstract Human-human hybridomas which secrete a human monoclonal antibody (h-MoAb) against hepatitis B virus surface antigen showed growth associated production kinetics. The rate of h-MoAb production rapidly decreased after cell growth was arrested in a perfusion culture, even if the perfusion rate was increased. A continuous suspended-perfusion culture, in which both culture broth and culture supernatant are continuously harvested and the same volume of fresh medium is continuously fed into the reactor, was developed to maintain continuous growing conditions during cultivation. In this culture system, the production of h-MoAb continued for more than 50 days with an average productivity of 5.0 mg/l of working volume/day. A semicontinuous immobilized-perfusion culture in which parts of the cells are repeatedly removed from the immobilized reactor was another useful technique for the long term cultivation of these h-h hybridomas. As an average h-MoAb production rate, 62 rag/1 of immobilized-bed volume/day was achieved for 65 days of cultivation using a ceramic matrix reactor, and 327 rag/l/day was achieved over 47 days of cultivation using a hollow fiber reactor equipped with Cultureflo M TM. Thus, the antibody productivity per reactor volume per day by the semicontinuous immobilizedperfusion culture was much higher than that of the continuous perfusion culture in an agitation reactor.

Introduction Human monoclonal antibodies (h-MoAbs) are expected to be useful in the therapy of various infectious diseases and cancers and also as a tool for in vivo imaging. For industrial production of monoclonal antibodies (MoAbs), the culture systems which stably provide high productivity for a long period and are capable of scaling up without difficulty should be developed. Perfusion culture

in which the spent medium is continuously replaced with fresh medium is one effective technique for cultivating hybridoma cells for a long period and of obtaining a high cell density and high productivity of the MoAb. Kitano et al. (1986, 1988) have successfully cultivated a mouse.human-human (m.h-h) heterohybridoma which produces an anti-tetanus toxoid (T.T.) human MoAb using this type of culture system. Takazawa et al. (1988) have also reported an effective perfusion

198 culture apparatus by which MoAb was continuously produced. In this paper, we first clarify the kinetics of the production of h-MoAb against hepatitis B virus surface antigen (HBsAg) by human-human (h-h) hybridomas and then propose two kinds of long-term culture methods. One is a suspension culture combining perfusion and continuous culture conditions, and the other is an immobilized culture combining perfusion culture and repeated-batch (semicontinuous) culture conditions.

Materials and methods Cell lines

A h-h hybridoma TFC-7 (Kitano et al., 1990) derived from HBW-4.16 (Ichimori et al., 1987; Harada et al., 1989) was used in all culture systems unless otherwise stated. Three other h-h hybridomas, W471-7.24, HBW-3.7 and HBW6.20 (Harada et al., 1989), were also used. All these hybridomas produce h-MoAbs against HBsAg.

Media

The serum-free medium PEG-86-1 (Shintani et al., 1988) was used unless otherwise stated. This medium consists of a mixture (1:1:2 by vol.) of Iscove's modified Dulbecco's medium (IMDM), Ham's F12 medium and L15 medium to which 2 mg/1 crystalline bovine insulin, 2 mg/l human transferrin, 2 x 10-6M ethanolamine, 2.5 x 10-SM sodium selenite (ITES), 4.5 g/1 HEPES, 1 g/l sodium bicarbonate and 1 g/1 polyethylene glycol 20,000 are added. The following media were also used: IsF medium, a mixture (1"1 by volume) o f IMDM and H a m ' s F12 medium plus 10% FCS; PEG-86-2 medium, PEG-86-1 medium supplemented with 0.3 g/l GFS (a 55% to 70% ammonium sulfate fraction of serum from adult cattle) as a serum substitute (Kitano et al., 1986); ASF103 medium (Murata et al., 1988) and ASF104 medium established by Ajinomoto Co. Inc. For

PEG-86-1 medium, PEG-86-2 medium and IsF medium, 100 mg/1 streptomycin and 1 x 105 units/1 penicillin G potassium were added into the medium. All these media were prepared with distilled water, ~idjusted pH to 7.2 and filtered through 0.2 ~tm filters.

Cultivation

Cells were inoculated at a concentration of 1 - 2 x 105/ml. Cells in plastic flasks were incubated at 37~ in a 5% CO 2 incubator. Cultivation in a 125 ml Techne spinner flask was carried out at 37~ at an agitation speed of 30 rpm. Perfused-suspension culture was performed using a 2 1 roundbottom jar fermentor equipped with a cell sedimentation column with a double jacket (Kitano et al., 1986). In the 2 1 jar fermentor cultures, pH and dissolved oxygen (DO) were controlled at pH 7.2 and over 1 ppm, respectively. For the immobilized-perfusion cultures, two different kinds of reactors were used. One was a ceramic matrix reactor, the Opticell T M culture system 5200R (Charles River Biochemical Services Inc., MA, U.S.A.) equipped with Opticore S-51 (the volume of extramatrix space is 200 ml). The other was a hollow fiber reactor system B625 (Tabal Espec Co., Inc., Osaka) equipped with a Vitafiber II cartridge (nominal molecular-weight cutoff, 50,000; volume of extracapillary space, 30 ml; Amicon Division, W.R. Grace & Co., MA) or Cultureflo M (pore size, 0.4 t.tm; 10 ml volume of extracapillary space; Asahi Medical Co., Ltd., Tokyo).

Assay methods

Total cells were counted with a Coulter counter (Coulter Electrics Inc.) or a haemocytometer. Viable ceils were counted with a haemocytometer after staining the cells with 0.5% trypan blue. Human antibodies (IgG and IgM) were determined by ELISA as described by Ichimori et al. (1985). Amino acids were analyzed using a high performance amino acid analyzer (Beckman, sys-

199 tem 6300). Glucose was measured using a Glucostat, lactate by an enzymatic method using lactate dehydrogenase and glutamate-pyruvate transaminase, and ammonia by an enzymatic method using glutamate dehydrogenase (Boehfinger Mannheim). The oxygen consumption rate (OCR) was used to monitor the cell growth in the ceramic matrix and the hollow fiber reactor. Taken with the flow rate of the medium, the difference between the In and Out oxygen concentration in the medium passing through the immobilized bed (Opticore or hollow fiber cartridge) is used to calculate the OCR (Lydersen et al., 1985).

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Growth associated production of human M o A b We have established a perfusion culture system by which m.h-h heterohybridomas are effectively cultivated for a long period and constantly produce h-MoAb (Kitano et al., 1986). However, when a h-h hybridoma was cultivated in PEG-861 medium using this system, the profile of antibody production was quite different from that of a heterohybridoma. Figure 1 shows the time course of h-MoAb production by h-h hybridoma TFC-7 in this culture. Active synthesis of h-MoAb occurred at the beginning of the culture. However, antibody production dropped rapidly day by day after 9 days of cultivation and was almost zero after 15 days, even though viable cell count was maintained at an almost constant level.

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IMDM and L15 medium were purchased from Gibco Laboratories, Ham's F12 medium from Nissui Seiyaku, Co., ASF 103 and 104 medium from Ajinomoto Co.,Inc., insulin and transferfin from Sigma Co., ethanolamine and polyethylene glycol from Wako Pure Chemical Ind., Ltd. and sodium selenite from Katayama Chemical Co. GFS was prepared at our Hikari Plant.

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Fig. 1. Time course of a perfusion culture of h-h hybridoma in a 2 1 jar fermentor. Cells were inoculated into 800 ml of PEG-861 m e d i u m at a concentration of 1 x 105/ml and cultivated at 37~ under the agitation of 30 rpm. The m e d i u m was perfused at the rate of 0.7 day -1 beginning the third day of cultivation. Dissolved oxygen and pH were controlled.

As the antibody production by h-h hybridomas is reported to be more stable than that of m.h-h hybridomas (Ichimori et al., 1987), we examined the productivity of each cell isolated from the broth cultured for 18 days in the perfusion culture system similar to that shown in Fig. 1. In this culture broth, 55% of total cells was viable though additional antibody production was not detectable. The cells were cloned in IsF medium by the limiting dilution method, and the MoAb productivity of these clones was determined after 4 days culture in IsF medium using 24-well plates. As shown in Fig. 2, the majority of these clones produced the antibody similarly to the cells cloned from the broth of a 4-day culture in a flask. When the other h-h hybridomas secreting antiHBsAg h-MoAbs, W471-7.24, HBW-3.7 and HBW-6.20 which were established using the same parental cell line, TAW-925 as that of TFC-7, were cultivated in the perfusion culture system,

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Chemostat culture is generally a useful technique to maintain continuous growing conditions during cultivation. W e first tried to cultivate TFC-7 by chemostat culture using a 2 1 round-bottom jar fermentor. Based on the data from batch cultures, it was presumed that the m a x i m u m specific growth rate of the h y b r i d o m a was about 0.6 day -1. Therefore, a dilution rate (culture-broth volume removed/working volume/day) of less than 0.5 day -1 was adopted. Figure 3 shows the time courses of h - M o A b production and cell growth in the chemostat cultures in which the dilution rate was controlled at 0 . 5 0 - 0 . 3 8 d a y - l ( A ) and 0 . 3 8 0.25 day -1 (B). With the higher dilution rate(A), the M o A b production continued for a longer period, but the productivity was low because of the low cell density. With the lower dilution rate(B), a higher cell density was maintained, but the production rate rapidly decreased.

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Fig. 2. Distribution of h-MoAb productivityof each cell which was cloned from the cells cultured in the perfusion system and in a 150 cm2 flask. A: cloned from cells perfusion-culturedfor 18 days; B: cloned from cells cultivated in a flask for 4 days. the production pattern was the same (data not shown). There were also no changes when other media such as IsF, PEG-86-2 and ASF103/104 were used in the perfusion culture (data not shown). T o maintain growing conditions during cultivation, a semi-continuous culture without perfusion was performed in a 125 ml Techne spinner flask. The antibody was produced repeatedly in accordance with repeated cell growth (data not shown). Thus it was confirmed that the synthesis o f h - M o A b in h-h hybridomas is associated with cell growth.

Short-term perfusion culture in a 2 1jar fermentor was successfully performed by increasing the perfusion rate [feed volume of fresh medium (=harvest volume o f culture supernatant)/working volume/day/ to 1.5 day -1 and maintaining a dissolved oxygen level of over 1 p p m as shown in Fig. 4. In the culture, the cells continued to grow for 17 days reaching a cell density of about 1 • 107 cells/ml and produced in total 125 mg/l o f h-MoAb. For cultivation longer than 17 days, it was necessary to r e m o v e a large part of the culture broth and to cultivate again after adding fresh medium.

Continuous perfusion culture T o maintain productivity effectively for a long period, we developed a continuous-perfusion culture system combining chemostat and perfusion cultures as schematically shown in Fig. 5. Figure 6 shows the time course o f the M o A b production in this culture. Both culture supernatant and culture broth were continuously harvested at the rate

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shown in the figure and fresh medium was continuously supplied at the same rate as the total of perfusion and dilution. The h-MoAb was continuously produced at a relatively high productivity during the cultivation for 50 days. The average rate of antibody production was calculated to be 5.0 mg/1 of working volume/day. Although this culture was stopped at the 50th day, it would be possible to continue the culture for a longer period.

Efficient immobilized culture systems f o r hlh hybridomas An immobilized culture generally has the advantage over a suspension culture of obtaining a

much higher cell density per unit volume of reactor. Lydersen et al. (1985) reported that a ceramic matrix which provides a high degree of surface area per unit volume is useful for the immobilization of mammalian cells. Hollow fiber modules have also been utilized to immobilize various mammalian cells ever since they were originally used by Knazek et al. (1972).

OpticelF M culture system We first performed an immobilized-perfusion culture using an Opticell TM culture system. In this system, cells are immobilized on a ceramic matrix (Opticore TM, 200 ml) whereby the medium flows directly over the cells. As the cell density in an immobilized reactor cannot be directly deter-

202 this culture system. The production of h - M o A b actively occurred during the growing phase when the O C R was increasing, but the production rate significantly decreased after the O C R reached a plateau, as expected f r o m the data on the suspension culture. T o maintain growing conditions during cultivation, a portion of the cells was r e m o v e d f r o m the ceramic matrix after the O C R reached a plateau by circulating a t r y p s i n - E D T A solution containing 1.2 m g / m l of trypsin and 0.5 m g / m l of E D T A in Ca, Mg-free PBS through the ceramic matrix for about 1 h. As s h o w n in F i g . 8, the antibody productivity increased in accordance with the repeated increase in the O C R for m o r e t h a n two months. T h e m a x i m u m cell density was estimated to be approximately 2 x 107 cells/ml o f the reactor based on the O C R and the specific respiration rate of the h y b r i d o m a cells. The average rate o f h - M o A b production was calculated to be 62 mg/1 o f the ceramic matrix/day.

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in a 2 liar fermentor. PEG-86-1 medium was used, and working volume was 11. Perfusion was initiated beginning the forth day and its rate was increased according to the increase in cell number as shown in the figure. Dissolved oxygen and pH were controlled.

A hollow-fiber cartridge is another useful module for retaining h y b r i d o m a cells. T h e m e d i u m is circulated through the intracapillary space (ICS) and at the s a m e time f r o m the extra-capillary space (ECS) to the ICS of the hollow fibers. W e first used a Vitafiber II (Model VF2-033) cartridge of which the antibody produced was accumulated

mined, O C R is used to m o n i t o r cultures. Figure 7 shows the time course o f h - M o A b production in

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in the ECS because of the 50,000 nominal molecular-weight cutoff membrane. A portion of the cells in the ECS was removed by vibrating the cartridge and greatly increasing the flow of the medium from the ICS to the ECS, when the OCR reached a plateau. Figure 9 shows the time course of the antibody production and the OCR in the culture using this system. The h-MoAb was produced in accordance with the gradual increase in the OCR, and a high production rate of 167 mg/1 of the reactor/day was achieved in the culture for 24 days. However, after the second removal of cells, the OCR did not recover to the original level and gradually decreased.

We next used a Cultureflo M T M cartridge (pore size, 0.4 ktm). The antibody passes through the fiber membranes in this cartridge and is recovered in the harvest medium. As shown in Fig. 10, the antibody was successfully produced for a period of 47 days in accordance with the repeated increase in the OCR, though the production rate decreased gradually. The maximum viable cell density was assumed to be roughly 1.8 x 108 cells/ml of the reactor based on the OCR. The average antibody production rate was calculated to be 327 rag/1 of the reactor/day which is 5 times as much as that in the ceramic matrix reactor.

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TM culture system. 2.5 x 108 cells were inoculated into an Opticore, and 3 1 of PEG-86-1 medium in a reservoir was circulated through the Opticore. Fresh medium was fed into the reservoir and spent medium was harvested at the speed of 100, 200 or 250 ml/h as shown as F/H in the figure, pO 2 inlet was controlled at 150 mmHg (designated 1), 160 mmHg (2) or 170 mmHg (3) as shown in the figure, and pO 2 outlet was controlled at 135 mmHg throughout the cultivation. A portion of the cells in the Opticore chamber was removed at the indicated times (,1,) by circulating trypsin-EDTA solution.

Discussion Analysis of production kinetics gives insight into the construction of an effective cultivation process. Merten (1988) classified antibody production patterns of mouse-mouse hybridomas in relation to cell growth phase. Up to now, although there have been many examples of hybridomas which exhibit non growth-associated production kinetics (Reuveny et al., 1986,Takazawa et al.,

1988, 1989, Suzuki and Ollis, 1989), only a few hybridomas have been reported to show growthassociated production (Kwong et al., 1989, Ray et al., 1989) and the kinetics have not been analyzed well. Here, we could clearly show that our h-h hybridomas exhibi~ the growth-associated production kinetics through perfusion cultures. IgG synthesis in these h-h hybridomas may be restricted to limited periods of cell cycle, as Buell and Fahey (1969) reported that production of IgG

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and IgM was greatest during the late G1 and S phases of cell cycle in a synchronized culture of human lymphoid cell line WI-L2 from which TAW-925, the parental cell line for our h-h hybridomas, was derived. For our h-h hybridomas, the usual perfusion culture was not useful as a long-term cultivation

method, because the h-MoAb production rate significantly decreased after cell growth was arrested. Some methods of suspension culture were tested in an effort to establish efficient culture systems. Table 1 summarizes the results of these tests. A short-term perfusion culture had the advantage of high cell density resulting in high production, though the duration of the culture was maximally 17 days. To continuously produce the antibody over a long period, we tried chemostat cultures, but this was not useful f o r cells which have a low growth;rate. For this reason, we here developed the continuous-perfusion culture system, in which perfusion and chemostat culture characteristics were combined. Though long term cultivation over 47 days was achieved, the average productivity per unit volume of reactor per day was higher than that of batch culture, but lower than that of short term perfusion culture. Immobilized-peffusion culture is one of the most effective method for cultivating hybridomas (Hopkinson, 1985; Altshuler et al., 1986; Merten, 1987). However, when a h-h hybridoma was cultivated by the standard method, antibody production did not continue for a long period, i.e. similar to the perfusion culture using an agitation

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207 Table 1. Human MoAb productivity in various reactors

Culture method

Culture volume (ml)

Suspension culture Batch culture 800 Perfusion culture (short term) 1000 Chemostat culture 800 Continuous perfusion culture 1000 Semi-continuous immobilized perfusion culture Ceramic Matrixa 200 Vitafiber IIb 30 Cultureflo Me 10

Culture period (day)

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h-MoAb productivity per reactor (mg/1)

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per reactor per day (rag/l/day) 4.0 7.4 2.0 5.0 62 167 327

per spent medium (mg/1) 32 13 7.0 4.2 2.9 2.7 3.6

aOpticell culture system (CRBS); bHollow fiber (Amicon); CHollowfiber (Asahi Medicals).

vessel. B y r e m o v i n g a portion o f the cells w h e n the O C R reached a plateau, w e succeeded in long t e r m cultivation. This cultivation m e t h o d was n a m e d semicontinuous i m m o b i l i z e d perfusion culture. T a b l e 1 also s u m m a r i z e s the results using a ceramic matrix or a hollow fiber cartridge. As the v o l u m e o f each c h a m b e r is different, it is difficult to directly c o m p a r e the results. H o w ever, Cultureflo M was superior to Vitafiber II, possibly due to a deficit in o x y g e n or nutrients in the E C S due to clogging o f the hollow fiber's m e m b r a n e s . With the ceramic matrix reactor, o x y g e n supply was the factor limiting the cell density. Therefore, productivity m u c h higher than 62 mg/1/day will be achieved by increasing the o x y g e n supply to the ceramic matrix, in any case, the antibody productivity per reactor volu m e per d a y b y i m m o b i l i z e d culture was m o r e than ten times than that o f the suspension cultures. Thus, the semi-continuous i m m o b i l i z e d perfusion culture was f o u n d to be the m o s t efficient culture m e t h o d for h - M o A b production b y h-h h y b r i d o m a s , based on the productivity per reactor p e r day. T h e productivity per unit v o l u m e o f spent m e d i u m b y the i m m o b i l i z e d perfusion culture was, h o w e v e r , m u c h l o w e r than that for batch suspension culture and for short t e r m perfused suspension culture. F o r e c o n o m i c a l cultiva-

tion, the a m o u n t o f m e d i u m c o n s u m e d for the perfusion should be reduced b y further investigation. Furthermore, there s e e m to be s o m e p r o b l e m s in scaling up the semicontinuous i m m o b i l i z e d perfusion culture. One such p r o b l e m is h o w to detach a portion of cells f r o m a large scale i m m o b i l i z e d reactor, as it m a y be not feasible to t r y p s i n i z e repeatedly the cells on a large scale. For the large scale production o f h - M o A b , short t e r m suspended-perfusion culture seems to be practical f r o m the v i e w point o f productivity per reactor per day, productivity per spent m e d i u m and simplicity o f the scale up. Studies on these points are n o w in progress.

Acknowledgements W e wish to thank Drs, Y.Sugino, M . N i s h i k a w a and H . O k a z a k i o f our Research & D e v e l o p m e n t Division for their interest and e n c o u r a g e m e n t throughout this work. W e also wish to thank Messrs. I-I.Suzuki and S.Sumi for their skillful technical assistance. This w o r k was supported b y funds f r o m N E D O ( N e w E n e r g y and Industrial Technology Development Organization)Japan.

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Address for offprints: H. Sawada, Microbiology Research Laboratories, Research & Development Division, Takeda Chemical Industries Ltd., 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532, Japan