Growth kinetics of hybridoma cells in high density culture

JOURNALOFFERMENTATIONANDBIOENGINEERING Vol. 73, No. 2, 159-165. 1992

Growth Kinetics of Hybridoma Cells in High Density Culture YOSHIHITO SHIRAI, KENJI HASHIMOTO,* anD HIROYUKI TAKAMATSU

Department of Chemical Engineering, Faculty of Engineering, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received 31 January 1991/Accepted 5 November 1991 The growth kinetics of a mouse-mouse hybridoma cell, 4C10B6, in a high density culture were compared with those in a low density culture. Lower glucose and glutamine consumption rates were observed in the high density culture, resulting in lower lactate and ammonium production rates. On the other hand, the specific oxygen consumption rate did not decrease in the high density culture. Higher growth yields against glucose and glutamine were obtained in the high density culture than in the low density one.

Hybridoma cells can produce monoclonal antibodies, one of the most promising new physiologically active components. Monoclonal antibodies are useful not only for diagnostic applications but also for therapeutic treatment of tumors and immunological diseases (1, 2). They have already been used in laboratories as ligands in affinity chromatography (3). However, the low proliferation rate of hybridoma cells makes effective production of monoclonal antibodies difficult in industry. High density culture of hybridoma cells is probably the best method to improve monoclonal antibody production on an industrial scale (4). Besides a low proliferation rate, hybridoma cells have another major drawback; their growth is inhibited by a slight amount of waste products such as ammonium or lactate (4, 5). In high density cultivation, the cells and medium used are usually separated in fermentors where only a part of the medium is removed, and fresh medium is supplied simultaneously to keep the concentration of waste products in the medium low enough so as not to inhibit growth. This kind of operation is called a perfusion culture. In perfusion culture, a key factor is to separate the cells from the medium. Membranes (6, 7), sedimentation tubes (8,9) or a centrifuge (10) have all been used for this purpose. Recently, several researchers have investigated the growth kinetics of hybridoma cells in high density culture (11, 26-28). This paper also deals with the growth kinetics of hybridoma cells in high density culture, and especially focuses on a comparison of the growth kinetics between high and low density culture to clarify the specificity of high density culture.

serum components. Triple distilled water treated with potassium permanganate was used for preparing the medium. The detailed composition of the medium used is given in a previous paper (13), but BSA was not added to the medium used here. Media (Kyokuto Seiyaku Co., Tokyo) from which glucose and glutamine were removed were used for cultivation of the 4C10B6 cells in which the glucose and glutamine concentrations were regulated. Cultivation of the cells The cells were cultivated statically in a flask as well as in a stirred fermentor, shown in Fig. 1, under suspension conditions. A perfusion system was used in the high density culture. The hybridoma cells were separated by means of a membrane sheet (Cellulose Nitrate Membrane Filter, Toyo Co., Tokyo) set at the bottom of the fermentor. The pore size of the membrane was 0.45/~m, and the volume of cell suspension in the fermentor was kept constant at 15 ml by adjusting the supply and withdrawal of medium using two pumps. The perfusion rate was between 1 d-~ and 8 d-~. The oxygen concentration in the medium was maintained above half the air saturation level at atmospheric pressure. The cell suspension was stirred at a rate of 50 rpm and kept in a thermostatic chamber at 37°C. Cells proliferating in the exponential growth phase were adopted as seed cells. The inoculum was cultivated for one day at a concentration from ca. 1 x 105 cells/ml to 3 x 105 cells/ml to allow uniform conditioning before all the culture experiments. It was then centrifuged and resuspended in the experimental medium. The initial pH levels in the medium were adjusted by 0.1 M HC1 solution before cultivation. Assay The concentrations of glucose, glutamine, lac-

air,5%C0 2 gasvent )ump

MATERIALS AND METHODS Materials A mouse myeloma P3/X63-AgS-Ul was fused with mouse spleen cells to produce mouse-mouse hybridoma cells, 4C10B6, which were supplied by Teijin Company Limited. A non-serum medium developed by Murakami et al. (12) was used for cultivation of the cells: d mixture of RPMI 1640 (Gibco Co., USA), F12 (Nissui Co., Tokyo) and DME (Nissui Co., Tokyo) media with a weight ratio of 2: 1 : 1 , in which only insulin, transferrin, monoethanolamine and sodium selenite were included as non-

mediu

~edium O.Z.5pm filter

* Correspohding author.

FIG. I.

159

Fermentor used for batch and perfusion culture systems.

160

S H I R A I ET AL.

J. FERMENT. BIOENO.,

tate and ammonium were measured using an enzyme reaction. Oxygen concentration was measured by a D.O. electrode. The oxygen consumption rate of the hybridoma was measured in a small cell described in a previous paper (14), in which a D.O. electrode was inserted and the hybridoma cell suspension was mixed. The measurement of the oxygen consumption rate of the cells cultured in high density was performed in the medium used in the high density culture with a part of the cells removed from the cell suspension. This was done to avoid too rapid a change in oxygen concentration during the measurement owing to the oxygen uptake of many cells.

lO6 E

8

ulos

GROWTH KINETICS The growth kinetics of the hybridoma were investigated based on the data obtained in the exponential growth phase in a batch culture. Some models have been proposed to describe the growth characteristics of animal cells (15-19). The following equations were proposed for expressing the culture results of two different cell lines (5):

(1)

d X / d t = p ( S , , PI, P2)X

i = 1, 2

dS~/dt = (-- 1/ Y~) (dX/dt) dP~/dt= Y~dX/dt

(2)

i = 1, 2

(3)

where

Y,a = AXIAS~

i = 1, 2

(4)

Yi= ~P~/AX

i = 1, 2

(5)

where p is the specific growth rate and expressed in complex terms of substrate and product concentrations (20)./1 was assumed to be constant in this work because the growth kinetics were examined based only on the data obtained from the exponential growth phase. Combining Eqs. 1 and 2 or 3 yields

-(dSi/dt)/X=ui=p/Y,j (dPi/dt)/X=JTi=l~Yi

i = I, 2

(6)

i = l, 2

dX/dt = pX

(1)

- - d S i / d t = u . , X - ( F / g ) (S~o--Si) i = 1, 2

i = 1, 2

i

0

i

40 60 80 Cultivation lime l: h ]

100

FIG. 2. Growth of 4C10B6 hybridoma ceils in static and suspension batch culture. Symbols: e , suspension culture; O, static culture.

kinetics of the 4C10B6 cells cultivated in low density (5 × 104-1 x 106 cells/ml) was examined both in static culture in a T-flask (Nunc, Denmark) and in suspension culture in the fermentor shown in Fig. 1. Figure 2 shows the growth curves in the static culture ((3) and the suspension culture ( • ) . The cells were confirmed to proliferate exponentially. The specific growth rates of the cells cultivated statically and in suspension were determined from Fig. 2 to be 0.043 h -1 and 0.041 h -1, respectively. Therefore, mixing has only a slight effect on the growth rate of the cells cultivated 12 ~i0

,-112 ~

slope=-1.7x108

s,ope°77 ,0

~I0i

/t

"4'8

% x 6

d

u 2

32

slope=_7~x10 7 . J ' " ~

ceUs/rr~nol u -

oc

,._12t(c).....

The growth

/

¢

i

4 8 12 16 20 Lactate conc.En'wnolll]

(31ucose conc. [mmo[ll]

(9)

RESULTS

t

0

2 .(d)' ' '~ slope=7.4x108 /

.~.'~° ce,s~ ,-~

(8)

These differential equations can be simultaneously solved analytically when/l is constant. The u~and ~i were obtained from the concentration changes in substrates or waste products during the period in which the specific growth rate of the hybridoma cells is constant. In general, the death rate of the cells should affect the growth kinetics. However, any death term is neglected in the above equations because the data were collected only when the cell viability was above 90%.

Growth kinetics in low density culture

2

(7)

where ~i and ~ri are the specific glucose or glutamine consumption rate and the specific lactate or ammonium production rate of the hybridoma cell, respectively. Mass balances in the perfusion culture can be written as follows:

dP~/dt=rr.,X-FP~/V

I

I

0

-61xI0 cellslmmo,

8

co JO

"~6

o4

0

0~,

0.8

1.2

1.6

20

G l u t a m i n e conc.[mmo(ll]

214

0

1 2 3 4 NH 4" conc. [mrnolll]

FIG. 3. Relationships between substrate or waste product concentrations and cell concentration in 4C10B6 cell batch culture. (a) Glucose cone. vs. cell cone.; (b) lactate cone. vs. cell cone.; (c) glutamine cone. vs. cell cone.; (d) a m m o n i u m cone. vs. cell cone.

VOL. 73, 1992

HIGH DENSITY CULTURE OF HYBRIDOMA CELLS

TABLE 1. Specific substrata consumption rates and growth yields

Early stage of culture Late stage of culture

v, x l 0 ,0 yx, x 10-~ v2x 10,, y,.2x 10-s (mmol/cell h) (cells/mmol) (mmol/cell h) (cells/mmol) 5.6 7.4 18 2.3 2.4

17

6.8

6. l

v,: Specific glucose consumption rate, v2: specific glutamine consumption rate, Y~I: growth yield for glucose, Y~2:growth yield for glutamine. Specific waste production rates and waste yields

Early stage of culture Late stage of culture

Itlx 10'0 YIxl0 s /t2x 10H Yzxi09 (mmol/cell h) (mmol/cell) (mmol/cell h) (mmol/cell) 12 2.8 20 4.8 5.4

1.3

5.6

1.4

~q: Specific lactate production rate, ~t~:specific ammonium production rate, Y,: lactate yield, ]"2: ammonium yield. in suspension. The relationships between the cpncentrations o f substrates (glucose and glutamine) and waste products (lactate and a m m o n i u m ) and cell concentration are shown in Fig. 3. Each relationship can be represented by a linear equation which has a change in slope at some points, regardless o f the culture methods. F r o m the slopes in Fig. 3, the growth yields for glucose and glutamine and the lactate and a m m o n i u m yields are calculated directly by Eqs. 4 and

161

5. These values are changed significantly between the,early stage and the late stage o f the culture. The specific substrate consumption and waste production rates obtained by Eqs. 6 and 7 are summarized in Table 1 with the growth yields for glucose and glutamine and the waste yields. A n averaged value of/z (0.042 h - ~) was adopted for the calculation o f the specific rates shown in Table 1. Both the growth yields for glucose and glutamine increase, whereas the waste yields and each specific consumption rate decrease at the late stage o f the culture, in spite o f the identical growth rate in the course o f the culture. Growth kinetics in high density culture Figures 4 and 5 show the growth curves o f the cells and changes in substrates (glucose and glutamine) and waste products (lactate and a m m o n i u m ) during two series o f high density perfusion culture, respectively. The numbers at the b o t t o m o f the growth curves in Figs. 4 and 5 indicate the perfusion rate (ratio o f the medium feeding rate to the culture volume, unit: d - l ) . Exponential growth was observed up to 7.5 x 106 cells/ml even when the perfusion culture was performed with low perfusion rates (about 2 d-~). The growth kinetics were estimated based on the data obtained in the exponential growth phase. The specific substrata consumption rates, the specific waste production rates and the growth yields for glucose an.d glutamine are summarized in Tables 2 and 3 for the low perfusion rate and high perfusion rate culture, respectively. These tables show that both the consumption and production rates decrease with the cultivation time, and the growth yields increase, regardless of the perfusion rate. The high perfusion rate system maintains high glucose

E

~107

~107 c .g

/

r" ®

E 8

o U

~1. 20.

1.8

I

16

i

23 (d-~)

I i

I

I

i

i

i

I i

i

i

i

i

0 20 4O 6O 80 I00

2O 40 6O 8O I00 120 Time rh'l

Time r h ]

8_

[.J

g Io(

i

I

I

,

I

I

I

I

I

¢-

.g

_]5

I

l

l

l

l

l

l

l

i lO(

48

8

i ~q 20

i .<

4O 6O 8O II30o Time [h3 FIG. 4. 4C10B6 growth, substrate consumption and waste production in the perfusion system with lower perfusion rates. Symbols: O, glucose; zx, ammonium; n, lactate; v, glutamine.

2~

O< 4O 60 8O 1(30 Time I'h3 FIG. 5. 4CIOB6 growth, substrata consumption and waste production in the perfusion system with higher perfusion rates. Symbols: O, glucose; A, ammomum; n, lactate; v, glutamine. (1

20

162

SHIRAI ET AL.

J. FERMENT.BIomqG.,

TABLE 2. Growth kinetics in high density culture (Run 1) Time (h) F/V(I/d) Yxl (cells/mmol) Y,,2 (ceUs/mmol) vj (mmol/cell h) v2 (mmol/cell h) Vo2 (mmol/cell h) 7t, (retool/cell h) nz (retool/ceil h)

0-24

24-48

Batch culture

2.1 1.9 x IOs 4.7 x los 1.7 x lO-,o 0.7 x lO-,o -2.7 x tO-I° 0.7 x 10-,o

2.0 4.7 x los l.l x los 0.7 x lO-'° 0.3 x lO-jo 3.0x 10-I° 1.0 x lO-I° 0.3 x 10-'o

0 7.4 x IO7 2.3 x los 5.6 x lO-,o 1.8 x lO-,o 2.1 x 10-'o 1.2 x tO-9 2.0x 10-.o

/z=0.033 h-' in the exponential growth phase. Vo2: Specific oxygen consumption rate. and glutamine concentrations while preventing waste product accumulation. This is favorable for hybridoma cell growth, as shown by the higher growth rate in the culture with a high perfusion rate. However, the higher growth yields for glucose and glutamine were obtained in the culture with a low perfusion rate. Tables 1 and 2 indicate that the values of the growth yields estimated from high density culture even at the early stage are higher than that obtained from low density culture. In high density culture, the specific growth rate did not decrease in spite of decreases in the 'specific consumption and production rates, resulting in an increase in the growth yields. The specific consumption rate of oxygen of the hyb r i d o m a cells cultivated in high density culture was 3 x 10-~°mmol/cell h, about 1.5 times higher than measured in low density culture (14). Only the specific oxygen consumption rate did not decrease in high density culture, in contrast to the other specific consumption rates shown in Table 2. To further examine these changes in the growth yields and the specific consumption or production rates, the following were investigated: the effect of pH in the fermentation medium, the effect of variation in the medium components, and the effect of medium conditioned by the 4C10B6 cells. Effects of pH on the growth kinetics Recently, McQueen and Bailey found that the growth yield for glucose of a hybridoma cultivated at pH 6.8 increased by three to ten times or more compared to the culture at pH value 7.6, but that the growth yield for glutamine is similar at all pH values examined (21). These trends were also reported by Miller et al. (17). The pH level comes down in the course of a batch fermentation of hybridoma ceils because of the acid produced. Batch experiments with various initial pH values were carried out to check the effects of p H on the growth kinetics of 4C10B6 hybridoma ceils. Table 4 lists the changes in glucose concentration as well as the growth yield estimated. Table 4 indicates that a major change in the growth yield with the various pH values was not ob-

TABLE 3. Growth kinetics in high density culture (Run 2) Time (h) F / V (l/d)

Yxl Y~2 vl v2 rt, n2

(cells/mmol) (cells/mmol) (mmol/cell h) (mmol/cell h) (mmol/cell h) (mmol/cell h)

0-24 8.0 5.4x I07 2.0x l0s 7.6x 10-'° 2.1 × 10-1° 1.7x 10-9 3.7 x 10-t°

24-48 6.8 1.4x los 3.4x los 2.9x 10-'° 1.2x 10-l° 6.5 x 10-10 1.9x l0 -j°

48-72 6. ! 2.2x los 5.9x l0s 1.9× 10-1° 0.7 x 10-10 3.4x 10-'° 0.8 x 10-10

/.t=0.041 h-' in the exponential growth phase. served in the culture between pH values. The growth yield increases with the cultivation time regardless of the pH values. Effects of variation in medium components on the growth kinetics The variation in medium components should affect the growth kinetics. For example, a high lactate concentration inhibits the flux of glucose to the metabolic pathway. The effects of glucose, glutamine, lactate and a m m o n i u m concentrations on the 4C10B6 hybridoma cell growth were investigated by adjusting the initial concentration of each component in the medium. The growth yields for glucose and glutamine and the specific growth rate obtained from the early stage of batch culture are summarized in Table 5. Table 5 is divided into several groups: glucose concentration in the medium was changed in group I, glutamine concentration was changed in group II, a m m o n i u m and lactate were initially added to the medium in groups III and IV, respectively. Both a m m o n i u m and lactate were simultaneously added in group V, and each initial concentration was varied in group VI. The high growth yields observed in the late stage of the batch culture shown in Fig. 1 or in high density culture listed in Tables 2 and 3 are not found in Table 5 except for two values shown in group VI. The very high growth yields that were obtained from the late stage of the high density culture with a low perfusion rate were never reached in the batch experiments summarized in Table 5. In the concentration rages examined, the specific growth rate decreases with reductions in the concentrations of glucose and glutamine, whereas it does not decrease so much when a m m o n i u m and lactate are added. Hybridoma growth in a conditioned medium A conditioned medium was prepared by cultivating the 4C10B6 cells for 1 d at an initial cell concentration of 1 x 105 cells/ml and then separating the medium and the cells. Glutamine was added to the conditioned medium to supply the glutamine consumed by the cells in one day. By this treatment, the culture condition was changed from that in an ordinary batch culture after 1 d. Figure 6 shows the relationship between the glucose and cell concentrations. A linear relationship can be found in Fig. 6, with no change in the slope as was seen in Fig. 3. It

TABLE 4. Effects of pH on growth kinetics Run no. Time (h)

pH (--)

0 24 48

7.4 7.0 6.6

pH-I ASt (mM)

Yx, x 10-7 (cells/mmol)

pH

pH-2 asl

-3.7 22

7.0 6.6 6.4

-2.8 2.3

-4.5 2.6

Initial cell concentration: 1 x 105cells/ml. ' AS, =Amount of glucose consumed.

Yxl x 10-v

pH

pH-3 ASI

-6.1 23

6.6 6.6 6.6

-2.4 2.1

gx × 10-v -4.6 19

HIGH DENSITY CULTURE OF HYBRIDOMA CELLS

VoL 73, 1992

163

TABLE 5. Effects of variation of medium components on growth yields Initial concentrations

YxJ x 10-s (cells/mmol)

Y,~x 10-s (cells/mmol)

// (h-t)

0.54 0.96 0.57 0.43

3.5 4.0 2.5 1.5

0.042 0.041 0.039 0.030

0.3 0.3

0.35 0.33

2.8 2.7

0.035 0.030

0 0 0

1.6 2.3 2.8

0.80 0.68 0.68

1.4 1.2 1.5

0.040 0.039 0.039

2.3 2.3 2.3

1.6 3.2 6.4

0.3 0.3 0.3

0.69 0.67 0.95

2.3 2.5 3.1

0.040 0.039 0.039

9.7 9.7

2.3 2.3

4.5 8.7

1.0 2.0

0.68 0.68

2.0 2.2

0.041 0.041

8.0 5.1 6.3 2.6

1.9 i.2 1.3 0.7

4.8 10.4 7.0 9.6

0.8 1.8 1.8 2.8

0.48 0.96 1.30 0.88

2.2 6.3 3.4 3.0

0.042 0.041 0.042 0.021

9.7

2.3 0 standard medium

0.3

0.74" 1.70b

2.3 6.1

0.043 0.043

GIc.

Gin.

Lac.

(raM) 7.9 3.2 1.6 0.6

(mM) 1.9 1.9 1.9 1.9

(mM) 0 0 0 0

II

7.9 7.9

1.0 0.5

0 0

III

9.7 9.7 9.7

2.3 2.3 2.3

IV

9.7 9.7 9.7

V VI

I

NIL +

(raM) 0.3 0.3 0.3 0.3

Glc, Glucose; Gin, glutamine; Lac, lactate, NH4 +, ammonium.

a Early stage of batch culture. b Late stage of culture. was found from calculation using Fig. 6 that the growth yield for glucose is estimated to be 1.5 x 108 cells/mmol for culture in the conditioned medium, even at the early stage o f culture. DISCUSSION The specific glucose and glutamine consumption rates decrease at the identical specific growth rate in the course o f high density culture, resulting in an increase in the growth yields for glucose and glutamine. The decrease in substrate consumption rates results in a decrease in the specific lactate and a m m o n i u m production rates. As shown in Table 1, a similar p h e n o m e n o n was observed in low density culture: the growth yields for glucose and glutamine are changed in the late period o f the low density-culture I

i

i

I

I

×6 ta 4

slope=-1

0

I

0

2 4 6 G:lucose conc. [mmolll]

"

8

FIG. 6. Relationship between glucose concentration and concentration of 4C1~)B6 ceils cultivated in a conditioned medium.

when compared to the early period. Enhancement o f the growth yield has already been reported by McQueen and Bailey (21). They found that it occurred at under low p H or in the presence o f a m m o n i um. The p H level in a cell decreases under such conditions (22). The enhancement o f the growth yields was attributed to a decline in the activity o f phosphofructokinase in the glycolytic pathway in a cell owing to a decrease in p H and to a change in the energy source from glucose to glutamine, resulting in an increase in the growth yield for glucose. A similar trend is, however, not found in Table 4. Such high growth yields as shown in Table 2 were not obtained by changing the initial p H level in the medium. Therefore, the explanation o f McQueen and Bailey (21, 22) would not be applicable to our case. The growth yield for glucose in the early stage o f cell culture also increased more than that in a conventional batch culture when the h y b r i d o m a cells were cultivated in the conditioned medium. Since a change in the initial glucose concentration in the medium affected the growth yield only slightly, as shown in Table 5, the enhancement o f the growth yield can not be ascribed to the change in the initial glucose concentration. Autocrine components contribute to animal cell growth (23-25); for example, growth o f fibroblast cells depends on the concentration o f the platelet-derived growth factor produced by the cells themselves (25). The presence o f autocrine components may contribute to the enhancement o f the growth yield o f the 4C10B6 cells, thus explaining the conditioned medium p h e n o m enon. Accumulation o f the autocrine components in the late period o f culture, or in the conditioned medium would contribute to the enhancement o f the growth yield. In high density culture the value o f the growth yield estimated for the perfusion system with lower perfusion rates was

164

SHIRAI ET AL.

J. FERMENT.BIOEt~O.,

higher than that with higher perfusion rates. The concentration of the autocrine components must be higher in the system with the lower perfusion rates; the resulting growth yield would thus increase. This is a possible hypothesis to explain the increase in the growth yields in high density culture and the late stage of a batch culture. This hypothesis has not yet been confirmed clearly by an experiment, but results are expected shortly. Another possible explanation for the change in the growth yields is that the concentration changes of the medium components would have something to do with the increase in the growth yields. High growth yields are rare in Table 5, which summarizes the changes in growth yields obtained by changing the concentrations of only a few important medium components. For instance, glutamine was the only amino acid examined. Perhaps some other component plays a significant role. Similar growth yields for glucose and glutamine to those obtained at the late stage of batch culture were found in Table 5-VI, indicating that a special combination of the components composing the medium might exigt to increase the growth yields. This should be also confirmed experimentally. The purpose of this research is to clarify the difference in the growth kinetics between high density culture and low density,culture of the 4C10B6 hybridoma cells. The substrate consumption and waste production rates decreased whereas the growth yields for glucose and glutamine increased in high density culture, indicating that high density culture is more advantageous for the cultivation of hybridoma cells than conventional batch culture because lower amounts of nutrients are consumed and lower amounts of toxic wastes are produced. High density cultures are found to b,e more efficient for hybridoma cells with respect to energy. NOMENCLATURE F : feeding rate of fresh medium, m3/s Pt : concentration of lactate, m o l / m 3 P2 : concentration of a m m o n i u m , m o l / m 3 St : concentration of glucose, m o l / m 3 $2 : concentration of glutamine, m o l / m 3 V : volume of cell suspension, m 3 Yxl : growth yield for glucose, cells/mol Y~2 : growth yield for glutamine, cells/mol Yt : lactate yield, mol/cell Y2 : a m m o n i u m yield, mol/cell p : specific growth rate, l / s ul : specific glucose consumption rate, mol/cell s u2 : specific glutamine consumption rate, mol/cell s v02 : specific oxygen consumption rate, mol/cell s ~rt : specific lactate production rate, mol/cell s ~r2 : specific a m m o n i u m production rate, mol/cell s (subscript) 0 : fresh medium ACKNOWLEDGMENT The authors wish to thank Teijin Limited, Tokyo, Japan for providing the 4C10B6 hybridoma cells. REFERENCES I. Olsson, L. and Mathe, G.: Emerging immunological approaches to treatment of neoplastic diseases, p. 334-337. In Mathe, G., Bonadonna, G., and Salmon, S. (ed.), Recent results in cancer re-

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