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Kinetic study on HBs-MAb production in continuous cultivation
journal of biotechnology ELSEVIER
Journal of Biotechnology34 (1094~ 227-235
Kinetic study on HBs-MAb production in continuous cultivation Minoru Harigae, Masatoshi Matsumura *, Hiroshi Kataoka btstitute of Applied Btochemtstry, U'ntt.erstt).'of T~ukuba Tennodat 1-1-1. Tsukuba. Ibarakt 305. Japan
l Rece~ved 19 Februa~' 1993:revision accepted 31 Ma.~ 1903)
Abstract
Continuous cultivation of mouse-mouse hybridoma, T0405 cells producing an lgG monoclonal antibod.~ (blAb) against hepatitis B surface antigen, was carried out in a membrane reactor with partial cell bleeding. By changing cell bleeding rate, viable cell concentrations were maintained at a high level in the range of 1.0 x 10" to 4.0 x 10~' ml -t, which is difficult to attain in a conventional continuous culture. The characteristics of MAb production, and metabolism of glucose and glutamine at high cell densit~ were in~.estigated under steady state. This cell line's MAb production was found to be growth associated. The calculation of ammonia yield on glutamine showed that ammonia was mainly produced when glutaminc was used for cell maintenance, while lactate .sield on glucose used for cell growth ssas higher than that for cell maintenance. Key words: HBs monoclonal antibody; Hybridoma: Antibody production rate: Continuous culture
1. Introduction
Antibody secretion by hybridoma is considered to continue as long as the cell is viable (Luan et al., 1987b). In accordance with this basic idea, much effort has been made to maintain viable cells at high concentration. In recent years, the development of serum-free medium and perfusion culture systems have brought about great improvements to animal cell growth and MAb production in vitro. There are many things that we have yet to understand, for example, the control mechanism of MAb production (Glacken and Synskey, 1985), the relationship between MAb production and cell growth (Linardos et al., 1991),
* Corresponding author.
and the influence of metabolic wastes on cell growth (Miller et al., 1988b). There ss,ere many previous studies on the kinetics of MAb production. Almost all of these studies, however, are based on results from unsteady state conditions like batch or perfusion culture, and there are many contradictions in the relationship b e ~ ' e e n cell growth and MAb production. Merten (1988) investigated the cell growth and MAb production kinetics in batch culture, and reported three different production patterns: pattern 1 showing the highest specific production rate at the beginning of the batch culture and decreasing continuously: pattern 2 showing the recover}, of production rate at the onset of the stationary and death phase; and pattern 3 showing a constant production rate independent from cell growth rate. Using a mem-
I)168-1656/04/$07.00 ,,~'. 190,1 Elsevier Science B.V. ,All rights reser,,ed SSDI 0168- 1656( 94)00017-7
"28
M Hart~ae ,'t al
Jmtrnal oI' B t o t e c h n o h ~ ' 34 l lqO4~ 22~-235
brane aided perfusion culture, Hagedorn and Kargi (1990) found that antibody production was higher during periods of slo~ growth and adverse culture conditions. In spite of using a similar cultkation system to that of Hagedorn, Seaman and Hu (1990) obtained a completel$ different result; specific MAb production rate ~,as independent of specific gro~,~th rate during slo~ growth, but the pattern of MAb production changed to growth-associated t],.'pe under specific gro~,,th rates higher than 0.025 h Secretion rate of MAb into the medium is influenced b~ permeation resistance of MAb in cell membrane (Reddy et aI., 1992). Under unsteady state condition, the accumulation rate of NL~b in the medium does not coincide with MAb production rate inside the cells. The exception is onl.v x~hen the permeation resistance is negligibk small, in spite of the lack of general methods to estimate the intensit.v of permeation resistance, not so much attention has been paid to this point in the previous works, which led to misinterpretation of the experimental results. Even in the experiments using continuous culture in ~hich the influence of m e m b r a n e permeation resistance is removed, we can see nlany contradicting results ILoss et a[., 1087, Miller et al., 1988a: Linardos et al., 1991; Robinson and Memmert, 1991). In a
--
]
2. Materials and methods
Cell line atul culture m e d m m The cell line used in this work was a mousemouse hybridoma T0405 derived from mouse h$bridoma NS-1 prepared by the institute of lmmunoloD..' (Tochigi. Japan). The cell secretes lgG monoclonal antibody which reacts with hepatitis B surface antigen (HBs-MAbL Serum-free E R D F medium (Kyokuto Pharmaceuticals, Japan) was used as the basal medium throughout this studs.. The growth factors ITES (Kyokuto Pharmaceuticals, J a p a n ) i n c l u d e 200 IU I -~ insulin, 10 mg I human transferrin, 10 p. mol I - I ethanolamine
tel It
]
~[edium /
long-term cultivation like continuous culture, it is also necessary to pay attention to the stabili~' of MAb production activity of hybridoma cells. In this work, continuous cultivation of HBsMAb producing mouse-mouse h~bridoma ~sas carried out in a membrane reactor with partial cell bleeding s~stem, and the kinetics of MAb production under high cell densit3. was studied based on the results obtained in steady-state continuou~ culture. The kinetic study on the metabolism of glucose and glutamine ~sere also performed.
- j,
L~---.~ d
~"
Effluent
-
[.~,
~
.,o~lr.,.C, .I,_
Te~ [,:,n ]ube ~l~_~brar,,
7 , ~ :.7- i~,.....
?~_:-~,~_ar,_ '
Fig. 1 Experimental ',el-up for continuous culture ,.,.ith partial cell bleeding.
AL Hangae et aL ,/Journal of Biotechnolo,~..' 34 ¢1994) 227-235
229
and 20 nmol l - t Na-selenite. Bovine serum albumin (BSA, 1 g I - l , Sigma) and sodium bicarbonate (1.13 g I - I ) were also supplemented to the serum-free medium.
separation by HPLC. Glutamine was analyzed b~ an Amino Acid Analyzer. HBs-MAb activity was assayed by PHA (passive haemagg[utination assay) and ELISA methods.
Cell c u h u r e reactor
D e t e r m i n a t i o n o f kinetic t'ahws in c o n t b m o u s culture coupled with U F m e m b r a n e separation
Continuous culture with partial cell bleeding was carried out by using a 500-ml stirred glass reactor (250 ml working volume) equipped with control devices for pH, DO, temperature and liquid level as shown in Fig. 1. A UF membrane with a molecular cut-off of 10000 Daltons (Diaflo, YMI0. Amicon) was fLxed at the bottom of the reactor. Agitation was provided by a magnetic impeller operating at 40 to 80 rpm. Air mLxed with 5%. C O , gas was supplied into the vessel surface, and also through a 1-m porous Teflon tube (TP-32, 70%. porosity, Sumitomo, Japan) for bubble-free aeration. D O was controlled at 6%. of air saturation by aerating pure o.~.'gen into the Teflon tube. The pH ~ a s maintained at 7.1 + 0.1 with 0.5 tool I-~ N a O H solution. The inoculum cell concentration was around 2 × 105 ml-~. The culture was initially operated in batch mode. When the viable cell concentration reached around 10 ° m l - ~, the supply of fresh medium into the reactor was started at a constant feed rate of 250 m l d - ~(dilution rate, D = 1 d - t ). Then continuous culture commenced by drawing out the reaction mLxture through an effluent tube, and cell-free and MAb-free spent medium through the UF membrane at a dilution rate of f D and (1 - f ) D , respectively. By varying the ceil dilution rate ]'19. it was possible to obtain various steady-state data of cell growth and MAb production. To maintain the total dilution rate D at l d -~. cell-free MAb-free spent medium was withdrawn through the UF membrane by means of a peristaltic p u m p controlled by a level sensor.
The specific growth rate g and specific death rate K o were calculated from the following material balances for total cell concentration X r and viable cell concentration X v. d XT/dt
= # )(~ - J D X T
dXv/dt
= # Xv - KoXv
( 1) - ]DXv
(2)
where cell b.sis was ignored. At the steady state. U =fD( XT/X v )
(3)
KD = U - / D
(4)
Similarly, the specific consumption rate ~'s and production rates rtr,lAt, and r/p were obtained from the following material balance equations: dS/dt
= D( S. - S) - ~,sXv
(5)
d P M A b / d t = rI M.~b X~ - f D P M A b
(6 )
dP/dt
(7)
= rlpX~ - D P
where S , and S are the substrate concentrations at the inlet and outlet of the reactor, P and Pr,~.~,t, are the concentrations of low molecular metabolic wastes and MAb, respectively. At the steady state. t's = D ( S,. - S ) / X ~
(8)
rl MAb = .tDP~I ~b/'X~
(9
rlp = D P / X ~
(10)
3. Results and discussion Cell m a i n t e n a n c e
S a m p l e analysis
Cell count and viability were determined b.~ T~.'pan blue exclusion using a hemae)¢ometer. Glucose and ammonia concentrations in the culture medium ~.ere determined by using test kits (Glucose-CII Test Kit, Ammonia Test Kit, Wako Pure Chemicals, Japan). Lactate was measured by the optical density method at 210 nm after
To make sure that the cells were free from contamination with mycoplasma, we cultivated the T0405 cells in a medium containing MC-210 (Dainihon Seiyaku Co. lot) for 6 d, before we started culturing in the membrane reactor. MC210 is a reagent which kills or inhibits the growth of mycoplasma. MC-210 did not have any adverse effect on the cell's MAb production abilit3.,.
23(J
M.
Hartgae et al. Journal of Biotechnolo.D' 34 IO04~ 227-235
To determine the effect of long-term culture on the cell's MAb producing ability, we continuousb subcultured the cells even, 3 d for 60 d. The specific PHA value is stable until around passage number 10 and then the cell's MAb-producing ability drops abruptly. Schmid et al. (1990) observed a loss of MAb-producing ability when the number of subcultures increased. Miller et al. (1988a) also observed a decreased in MAb productivity when increasing the passage number after thawing the cells. These results indicate that it is safe to run cultivation experiments in the bioreactor within 1 month.
Perhtsion and continuous culture When the cell bleeding is completely stopped ( f D = 0), the cultivation in the membrane reactor becomes a perfusion culture. A perfusion culture was carried out as a control experiment. Fig. 2 shows a typical time course of perfusion culture of hybridoma T0405 cells in the membrane reactor. Note that the viable cell concentration was maintained constant, while the MAb concentration increased. Sometimes the misconception that the MAb production was non-growth associated x,,as inferred from results similar to this (Hagedorn and Kargi, 1990). Fig. 3 shows a typical time course of continuous culture of hybridoma T0405 cells at a cell dilution rate fD = 0.0220 h -t. The culture ~ a s operated in batch mode until about 72 h. When
{.,,FI [ _ =_
[ FlU,JLIS
Cu I ~ute
L,,
, fC,=O I121'IIh -i-i-1
._-.
i--d-~-
i"
_ Z
L" L,,,
_ ,
~ul,
[ l ,T,~_ ' ~t ' Fig. 3. A D p i c a l
time course ol c o n t i n u o u s
culture of T( 4 ( S
cells in m e m b r a n e reactor at 0.0220 h i cell dilution rate. Symbols: ( 4 4 total cell: ( ." I ~iable cell.
the cell concentration reached the 10" ml ' level. the supply of fresh medium into the reactor and cell bleeding were started. The drop in the cell concentration from 216 to 240 h was caused b~ the malfunction of the feed pump, but when the error was corrected the cell concentration returned to the previous steady level. So we considered the data from 288 to 408 h as the steady-state data, The total and viable cell, and other medium components were held constant, indicating a real steady-state condition (Fig. 4). We regarded the
P e r fu~l,:,r, q[ultut,:
[,=Ida, -
-
'
, "
I¸
C
==_--
lime ,,u
tnu
t',
'
h'
u,,
Fig. 2. A t3,pical time course o f perfusion culture of T0405 cells m m e m b r a n e reactor. Symbols: (c,I total cell: ( L ) ~mble cell: (,>1 HBs-MAb: (Ell glucose; (o) glutamine.
Fig. 4. Steady state c o n c e n t r a n o n s of c o n t i n u o u s culture at 0.0220 h - ' d i l u u o n rate. Symbols: (:,) total cell: q ~ l viable cell: (<,,) HBs-MAb: (t~) glucose: ( • ) lactate: (e) glutamine; ( • ) ammonia.
M. Harigae et al. / Journal q[ Biotechnolog)' 34 (1994) 227-235
231
1 O0
[A ]
//--.-
:Z, ~
E 6,] >.
2
40 "~
L~
2~ _
,,[
i
i
i
t
i
,) ~,
g]
v ..... ,, . t
,, ii
[,l~uti(:,n
Fig. 6. Effect
of
dilution
. ,,
r~ze
rate
on
,, ,.,] f[.'
J
"
'h-''
HBs-MAb
concentration.
& (12
i]Jl g 0~
_
. ', ",1
~, ', 2
[tlut:,:,n
,., O~
r~te
i: ,)~
,, ,,~
fD ,'i7 - ~ '
Fig. 5. Effect of dilution rate on cell growth• (A) (O) viable cell concentration and ( zx ) viability; (B) ( zx ) specific growth rate and (,:.2,) specific death rate.
average of each of these points as the steady-state value.
Viable cell concentration, riabifit),, and specific growth a n d d e a t h rates
Viable cell concentration, viability, and specific growth ( # ) and death rates ( K D) were plotted against the cell dilution rate f D (Fig. 5). As shown in Fig. 5A, the maximum viable cell number of 4 × 10 6 ml- ~ was observed at the lowest fD of 0.00316 h-~, and the viability increased as the cell dilution rate fD increased. The specific growth rate increased with increasing fD. while the death rate increased with decreasing fD and it attained the same value as the specific growth rate at the lowest fD (Fig. 5B). The same trend was found in previous work (Robinson and Memmert, 1991).
steady-state values, the specific MAb production rates, r/MAb were calculated using Eq. 9. The results were plotted against specific growth rates as shown in Fig. 7. The specific HBs-MAb production rate increased with increasing specific growth rate. This indicates that this cell line's MAb production is growth associated. The t~o stray points (solid symbols) in Fig, 7, which were obtained in a long-term continuous culture of more than 1000 h, suggest a drop of the specific MAb production rate. These results indicate that the steady-state data obtained from extremely extended continuous culture is not suitable for kinetic studies and that attention should be paid to the stability of MAb production. Similar discussion was found in previous works (Hiller et al., 1991: Frame and Hu, 1990). Frame and Hu (1990) observed a continuous decrease in antibod.~ productivity over the course of a long-term continuous culture. Low et al. (1987) suggested that the
= E
Monoclonal antibod.v When the steady-state values of the MAb concentration at various cell dilution rates fD were plotted against fD, the expected decreasing pattern is obtained as shown in Fig. 6. Using these
;[.; ,i
11
Specific
u u_
. ,)]
. ,~
growth r a t e
,, ,i ~
,h ' ,
Fig. 7. Effect of specific grov~th rate on specific MAb production rate.
232
M H a r t g a e et al.
J o u r n a l ~t" B i o l e c h n o l o ~
Io~ specific MAb production rate at low specific growth rate was due to the degradation or inactivation of MAb. In this work, this was checked in a separate experiment, ~herein the MAb activity ~as maintained exen after 2 ~eeks of storage in a 37"-'C incubator. The kinetics of murine MAb production b.~ hybridoma have often been described as nongrowth associated (Miller et al.. 1988a: Linardos et al., 1991). Using continuous culture, Linardos et al. (1991)sho~,ed that the specific MAb production rate r/MAt, ~.as inversely dependent on the specific growth rate p., and r/mAh v, as correlated with specific death rate K D. They claimed that MAb production mainly occurs in the GI phase. The time the cell spends in GI phase is increased bx adverse environmental conditions that affect cell proliferation. The percentage of cells in the G I phase increased with a slow down in growth rate, which results in enhancement of ~LAb production. Using a cell line producing IgM-MAb against human blood group B antigen, Low et al. (1987) showed different MAb production kinetics; r/MA~, increased with increasing /1 up to a dilution rate of 0.02 h n but at higher specific growth rate. r/r,t~,t, leveled off and became independent of /1. Robinson and Memmert (1901) showed a growth-associated kinetics of a chimeric MAb. In their experiment, transformed m~eloma cells were cultivated on a serum-free medium containing methotrexate to maintain constant selection pressure. Therefore, the activity of MAb production of the cells must be stable during long-term continuous cultivation. For growth-associated MAb production, the com,entional long-term perfusion culture is not a suitable process, because cell growth is highly repressed under high cell densit3. In this case, a repeated-perfusion culture, as sho~n in Fig. 8, must be the best v, ay to satist3. the tx~o requirements of high productivity and concentrated MAb. As mentioned above, there are many contradicting results on MAb production kinetics exen when the.~ are obtained in continuous cultures. To a great extent, this difference may be attributed to the different cell lines used. However, knowledge of the relationship between flMA~, and p. is of critical importance in
34 I lqq41 .~. .' ."
~ 2+-
Ill o •
•
lip
Oo
o t
o o
0'41
•
~
•
e'm °
I Iq
•
111 -,ll
]H'~
I -,IP t IU'_
~11(I
~nll
"fllPl
,',it
~l ,
Fig. ~. Production of HBs-MAb in repeated-perlusion culture. Symbols: (e) ~iable cell concentratuon. ( " | PHA ~alue.
choosing reactor designs or operating strategies. Therefore, much more effort should be made for claril3.'ing the mechanism of antibody production and antibody transport.
Glucose and lactate, glutamine atzd ammonia Glucose and glutamine are the major energy and carbon sources for animal cell culture. When these substrates are metabolized, lactate and ammonia are produced. Glucose metabolism can be divided into two major pathways: lactate synthesis and ATP generation or biosynthesis through the TCA cycle. Glucose metabolism through the TCA c.vcle is ~,ell known to be more efficient for ATP generation compared to lactate s3,nthesis. Glutamine metabolism can provide 30 to 65% of the energ~ for mammalian cell growth (Reizer et al., 1979). The pathway of glutamine metabolism leads to biosynthesis and production of amino acids. The use of glutamine as an e n e r ~ ' source in animal cells proceeds via a pathway producing a-ketoglutarate, which is incorporated into the T C A cycle. There is a net production of 2 moles of ammonia from 1 mole of glutamine. Ammonia has serious inhibitor5.' effects on the growth of T0405 cells at a concentration of 5 mmol 1-J (Na~'e et al., 1991). In hybridoma cultures, lactate production and glucose consumption are tightly interrelated. Hybridomas are capable of carrying out glycol vsis even under aerobic conditions (kanks and LI, 1988). About 80c~ of glucose proceeded through the glycob, tic pathway to lactate, and only 4 to
M. Harigae et al. /Journal o/Biotechnolog3' 34 11994) 227-235
5%. entered the T C A cycle when the glucose concentration was greater than 1 mmol I - ' (Reizer et al., 1979). As shown in Fig. 9, the residual glucose concentration in this experiment was reD' high, about 5 to 11 m m o l l - ~ , therefore a high rate of glycolysis can be expected. The specific glucose consumption rate uGk and lactate production rate r/L~~ as a function of the specific growth rate are shown in Fig. 10A and B, respectively. The specific glucose consumption and lactate production rate seem to increase more rapidly at the higher specific growth rate. This unusually high glucose consumption rate at high specific growth rate was also found in the experiment by Robinson and Memmert (1991), in which MAb production kinetics was growth associated. In this work. we tried to evaluate the yield values of lactate on glucose for cell maintenance and cell growth. ~'G,c and r/r~ may be correlated with # as in the following equations except at extremely high glucose consumption regions (Pirt. 1985): t,Gic = aol~/.t + mt31c
( 11)
~Lac = aLac/d" + nlLac
(12)
where a is the slope of the line and m is the specific value for cell maintenance. A regression analysis performed on the specific glucose consumption and lactate production rate values for the lowest sLx specific growth rates gives a.c,~ = 0.17 mmol per cell, triG,~ = 0.51 mmol per cell per h. CtLac 0.22 mmol per cell, IHLac 0.25 mmol =
=
:/
..-.,
.-j "7-
Dilutb~,n rate fD , h - ' Fig. 9. Effect of dilution rate on steady-state glucose and lactate concentration. S.,,mbols: (c,) glucose: ( ." ) lactate.
233
I% m L
=
= ,
L
j'
~-
=
-
"T'
i ..... ' 04 [,ezif1,: grT,~'~n ~2[e L . . . . :,i:
,,~
Fig. 10. Effect of specific growth rate on ~'c,,~ the specific glucose consumption rate (A) and on r/Lo~ the specific lactate production rate (B).
per cell per h. The lactate yield values on glucose were calculated as follows: YL~c C,Ic = CrLac/aGIc
(13)
YmLac GI¢ = m L a c / m G i c
(14)
where YL~c.G~o is the lactate yield values on glucose during cell growth, and Y,nL~ Gl~ iS the lactate yield values on glucose for maintenance. YL~'G]c was 1.34 mol per tool and Ymt,~o G]¢ was 0.48 mol per mol. YmL~ GIc was much lower than Ytac G~" These values indicate that lactate produced from glucose which was used for cell maintenance is much smaller than that for cell growth. If we use lactate production as a gauge to evaluate glucose utilization, it can therefore be said that, in maintenance metabolism, glucose utilization was very efficient. The same obse~'ations in a hybridoma culture were reported by Luan et al. (1987a). They pointed out that during the initial batch growth, a large fraction of glucose was metabolized to lactate, while in stationaD.' phase, no net lactate was formed.
234
M H a r i e a e et al. . J o u r n a l o] Biotechnolog}' 3 4 q 1904J 2 2 7 - 2 3 5
were calculated using Eqs. 8 and 10. The results were plotted against the specific growth rate and are shown in Fig. 12A and B. Similar to the analysis of glucose and lactate, regression was performed which gives the following, t~Gl n 0.29 mmol per cell, mGi n = 7.67 X 10--' mmol per cell per h, C~NH~ = 0.13 mmoI per cell and raN, ' = 0.16 mmol per cell per h. The ammonia yield on glutamine for growth and maintenance are 0.46 and 2.02 mol per mol, respectively. The m a y imum theoretical value of the ammonia yield on glutamine is 2 tool per mol. This means that almost all the glutamine consumed for maintenance of the cell were deaminated for biosynthesis or ATP generation through the TCA cycle. From these results of yield values of lactate and ammonia, we can mention that in a perfusion culture with low growth rate and high cell densit5,, the accumulation of ammonia is enhanced and a selective ammonia removal system is deemed necessa~'. =
m
= m
r
Ii
ii 1
p 11 ~
,, r,.
'
ir
i~
i
I
Dilutior. rate fD ,h ~, Fig. I1. Effect of dilution rate on steady-state glutamine and ammonia concentration. Symbols: (7,) glutamine: ( ." ) ammonia.
Fig. 11 shows the plot of the steady-state values of glutamine and ammonia concentration under various dilution rates. Unlike glucose and lactate, glutamine concentration increased with increasing dilution rate. Using glutamine and ammonia steady-state value, the specific glutamine consumption rate and ammonia production rate
The authors would like to thank Mr. Usuda, Mr. Takahashi and Ms. lwanari of the Institute of Immunology for preparing the hybridoma cells and providing excellent technical advice about the antibody analysis. This work was supported in part by a research grant from the University of Tsukuba.
7
-.,
Acknowledgments
=_-
_-,
i
.~ ,~,
References
i
i
"l '"1
I
IJ (,.
Frame, K.K. and Hu, W.S. (1990) The loss of antibody productivits.' in continuous culture of hybridoma cells. Biotechnol. Bioeng. 35. 469-476. Glacken, M.W. and Synskey, A.J. 11985) Modeling and optimization of fed-batch cultures of hybridoma cells. AIChE Annual Meeting Chicago, IL. Hagedorn, J. and Kargi. F. (1990) Coiled tube membrane bioreactor for cultivation of hybridoma cells producing monoclonal antibodies. Enz%,me Microb. Technol. 12, 824-
,
IJ '.'?
~,-_,~z~fzz gr,z, wt~-~
I) ~.,~
raze
829.
,I-,-
Fig. 12. Effect of specific growth rate on l,'Gi n the specific glutamine consumption rate (A) and on r/NH, the specific ammonia production rate (B).
Hiller, G.W., Aeschlimann. A.D.. Clark, D.S. and Blanch. H.W. (19911 A kinetic analysis of hybridoma growth and metabolism in continuous suspension culture on serum free medium. Biotechnol. Bioeng. 38, 733-741.
M. Harigae et aL /Journal o]" Biotechnology 34 t lgq4J 227-235 Lanks, K.W. and LL, P.W. (1988) End products of glucose and glutamine metabolism by cultured cell lines. J. Cell. Phys. 135, 151-155. Linardos. T.I., Kalogerakis, N., Behie, L.A. and Lamontagne, L.R. (1991) The effect of specific growth rate and death rate on monoclonal antibody production in hybridoma chemostat cultures Can. J. Chem. Eng. 69, 429-438. Lov,, K.S., Harbour. C. and Barford. J.P. (1987) A study of hybridoma cell grov~lh and antibody production kinetics in continuous culture. Biotechnol. Techniq. 1,239-244. Luan. Y.q-., Mutharasan, R. and Magee, W.E. (1987a) Factors governing lactic acid formation in long term culti~,ation of h~,bridoma cells. Biotechnol. Lett. 9. 751-756. Luan. Y.T., Mutharasan. R. and Magee, W.E. (1987b) Strategies to extend Iongetivity of hybridomas in culture and promote }ield of monoclonal antibodies. Biotechnol. Lett. 9, 691-696. Merten. O.W. (1988) Batch production and growth kinetics of hybridomas. Cytotechnolog~ 1. 113-121. Miller, W.M.. Blanch, H.W. and Wilke, C.R. (1988a) A kinetic analysis of h.~bridoma growth and metabolism in batch and continuous suspensnon culture: effect of nutrient concentration, dilution rate. and pH. Biotechnol. Bioeng. 32, 947-965. Miller. W.M.. Wilke, C.R. and Blanch. H.W. (1988b) Tran-
235
sient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture. Bioprocess Eng. 3. 113-122 Nayve Jr.. F.R.P., Motoki, M., Matsumura, M. and Kataoka. H. (1991) Selective removal of ammonia from animal cell culture broth. Cytotechnolo~' 6, 121-130. Pirt. S.J. (1985) Principles of microbe and cell cultivation. Blaclc~ell Scientific Publications. Cambridge, UK. Reddy, S., Bauer. K.D. and Miller, W.M. (1992) Determination of antibody content in live versus dead hybridoma cells: analysis of antibody, production in osmotically stressed culture. Biotechnol. Bioeng. 40, 947-964. Reizer, L.J., Wice, BM. and Kennell, D. (1979) Evidence that glutamine, not sugar, is the major energy source for cultured Hela cells. J. Biol. Chem. 254, 2669-2676. Robinson, D.K. and Memmert, K.W. (1991) Kinetics of recombinant nmmunoglobulin production by mammalian cells in continuous culture. Biotechnol. Bioeng. 38, 072-976. Schmid, G.. Blanch. H.W. and Wilke, C.R. (1990) Hybridoma growth, metabolism, and product formation in HEPESbuffered medium-l, effect o1 passage number. Biotechnol. Lett. 12. 627-632. Seamans, I.G. and Hu. W.S. (1990) Kinetics of growth and antibody production by a hybridoma cell line in a perfusion culture. J. Ferment. Bioeng. 70, 241-245.
Journal of Biotechnology34 (1094~ 227-235
Kinetic study on HBs-MAb production in continuous cultivation Minoru Harigae, Masatoshi Matsumura *, Hiroshi Kataoka btstitute of Applied Btochemtstry, U'ntt.erstt).'of T~ukuba Tennodat 1-1-1. Tsukuba. Ibarakt 305. Japan
l Rece~ved 19 Februa~' 1993:revision accepted 31 Ma.~ 1903)
Abstract
Continuous cultivation of mouse-mouse hybridoma, T0405 cells producing an lgG monoclonal antibod.~ (blAb) against hepatitis B surface antigen, was carried out in a membrane reactor with partial cell bleeding. By changing cell bleeding rate, viable cell concentrations were maintained at a high level in the range of 1.0 x 10" to 4.0 x 10~' ml -t, which is difficult to attain in a conventional continuous culture. The characteristics of MAb production, and metabolism of glucose and glutamine at high cell densit~ were in~.estigated under steady state. This cell line's MAb production was found to be growth associated. The calculation of ammonia yield on glutamine showed that ammonia was mainly produced when glutaminc was used for cell maintenance, while lactate .sield on glucose used for cell growth ssas higher than that for cell maintenance. Key words: HBs monoclonal antibody; Hybridoma: Antibody production rate: Continuous culture
1. Introduction
Antibody secretion by hybridoma is considered to continue as long as the cell is viable (Luan et al., 1987b). In accordance with this basic idea, much effort has been made to maintain viable cells at high concentration. In recent years, the development of serum-free medium and perfusion culture systems have brought about great improvements to animal cell growth and MAb production in vitro. There are many things that we have yet to understand, for example, the control mechanism of MAb production (Glacken and Synskey, 1985), the relationship between MAb production and cell growth (Linardos et al., 1991),
* Corresponding author.
and the influence of metabolic wastes on cell growth (Miller et al., 1988b). There ss,ere many previous studies on the kinetics of MAb production. Almost all of these studies, however, are based on results from unsteady state conditions like batch or perfusion culture, and there are many contradictions in the relationship b e ~ ' e e n cell growth and MAb production. Merten (1988) investigated the cell growth and MAb production kinetics in batch culture, and reported three different production patterns: pattern 1 showing the highest specific production rate at the beginning of the batch culture and decreasing continuously: pattern 2 showing the recover}, of production rate at the onset of the stationary and death phase; and pattern 3 showing a constant production rate independent from cell growth rate. Using a mem-
I)168-1656/04/$07.00 ,,~'. 190,1 Elsevier Science B.V. ,All rights reser,,ed SSDI 0168- 1656( 94)00017-7
"28
M Hart~ae ,'t al
Jmtrnal oI' B t o t e c h n o h ~ ' 34 l lqO4~ 22~-235
brane aided perfusion culture, Hagedorn and Kargi (1990) found that antibody production was higher during periods of slo~ growth and adverse culture conditions. In spite of using a similar cultkation system to that of Hagedorn, Seaman and Hu (1990) obtained a completel$ different result; specific MAb production rate ~,as independent of specific gro~,~th rate during slo~ growth, but the pattern of MAb production changed to growth-associated t],.'pe under specific gro~,,th rates higher than 0.025 h Secretion rate of MAb into the medium is influenced b~ permeation resistance of MAb in cell membrane (Reddy et aI., 1992). Under unsteady state condition, the accumulation rate of NL~b in the medium does not coincide with MAb production rate inside the cells. The exception is onl.v x~hen the permeation resistance is negligibk small, in spite of the lack of general methods to estimate the intensit.v of permeation resistance, not so much attention has been paid to this point in the previous works, which led to misinterpretation of the experimental results. Even in the experiments using continuous culture in ~hich the influence of m e m b r a n e permeation resistance is removed, we can see nlany contradicting results ILoss et a[., 1087, Miller et al., 1988a: Linardos et al., 1991; Robinson and Memmert, 1991). In a
--
]
2. Materials and methods
Cell line atul culture m e d m m The cell line used in this work was a mousemouse hybridoma T0405 derived from mouse h$bridoma NS-1 prepared by the institute of lmmunoloD..' (Tochigi. Japan). The cell secretes lgG monoclonal antibody which reacts with hepatitis B surface antigen (HBs-MAbL Serum-free E R D F medium (Kyokuto Pharmaceuticals, Japan) was used as the basal medium throughout this studs.. The growth factors ITES (Kyokuto Pharmaceuticals, J a p a n ) i n c l u d e 200 IU I -~ insulin, 10 mg I human transferrin, 10 p. mol I - I ethanolamine
tel It
]
~[edium /
long-term cultivation like continuous culture, it is also necessary to pay attention to the stabili~' of MAb production activity of hybridoma cells. In this work, continuous cultivation of HBsMAb producing mouse-mouse h~bridoma ~sas carried out in a membrane reactor with partial cell bleeding s~stem, and the kinetics of MAb production under high cell densit3. was studied based on the results obtained in steady-state continuou~ culture. The kinetic study on the metabolism of glucose and glutamine ~sere also performed.
- j,
L~---.~ d
~"
Effluent
-
[.~,
~
.,o~lr.,.C, .I,_
Te~ [,:,n ]ube ~l~_~brar,,
7 , ~ :.7- i~,.....
?~_:-~,~_ar,_ '
Fig. 1 Experimental ',el-up for continuous culture ,.,.ith partial cell bleeding.
AL Hangae et aL ,/Journal of Biotechnolo,~..' 34 ¢1994) 227-235
229
and 20 nmol l - t Na-selenite. Bovine serum albumin (BSA, 1 g I - l , Sigma) and sodium bicarbonate (1.13 g I - I ) were also supplemented to the serum-free medium.
separation by HPLC. Glutamine was analyzed b~ an Amino Acid Analyzer. HBs-MAb activity was assayed by PHA (passive haemagg[utination assay) and ELISA methods.
Cell c u h u r e reactor
D e t e r m i n a t i o n o f kinetic t'ahws in c o n t b m o u s culture coupled with U F m e m b r a n e separation
Continuous culture with partial cell bleeding was carried out by using a 500-ml stirred glass reactor (250 ml working volume) equipped with control devices for pH, DO, temperature and liquid level as shown in Fig. 1. A UF membrane with a molecular cut-off of 10000 Daltons (Diaflo, YMI0. Amicon) was fLxed at the bottom of the reactor. Agitation was provided by a magnetic impeller operating at 40 to 80 rpm. Air mLxed with 5%. C O , gas was supplied into the vessel surface, and also through a 1-m porous Teflon tube (TP-32, 70%. porosity, Sumitomo, Japan) for bubble-free aeration. D O was controlled at 6%. of air saturation by aerating pure o.~.'gen into the Teflon tube. The pH ~ a s maintained at 7.1 + 0.1 with 0.5 tool I-~ N a O H solution. The inoculum cell concentration was around 2 × 105 ml-~. The culture was initially operated in batch mode. When the viable cell concentration reached around 10 ° m l - ~, the supply of fresh medium into the reactor was started at a constant feed rate of 250 m l d - ~(dilution rate, D = 1 d - t ). Then continuous culture commenced by drawing out the reaction mLxture through an effluent tube, and cell-free and MAb-free spent medium through the UF membrane at a dilution rate of f D and (1 - f ) D , respectively. By varying the ceil dilution rate ]'19. it was possible to obtain various steady-state data of cell growth and MAb production. To maintain the total dilution rate D at l d -~. cell-free MAb-free spent medium was withdrawn through the UF membrane by means of a peristaltic p u m p controlled by a level sensor.
The specific growth rate g and specific death rate K o were calculated from the following material balances for total cell concentration X r and viable cell concentration X v. d XT/dt
= # )(~ - J D X T
dXv/dt
= # Xv - KoXv
( 1) - ]DXv
(2)
where cell b.sis was ignored. At the steady state. U =fD( XT/X v )
(3)
KD = U - / D
(4)
Similarly, the specific consumption rate ~'s and production rates rtr,lAt, and r/p were obtained from the following material balance equations: dS/dt
= D( S. - S) - ~,sXv
(5)
d P M A b / d t = rI M.~b X~ - f D P M A b
(6 )
dP/dt
(7)
= rlpX~ - D P
where S , and S are the substrate concentrations at the inlet and outlet of the reactor, P and Pr,~.~,t, are the concentrations of low molecular metabolic wastes and MAb, respectively. At the steady state. t's = D ( S,. - S ) / X ~
(8)
rl MAb = .tDP~I ~b/'X~
(9
rlp = D P / X ~
(10)
3. Results and discussion Cell m a i n t e n a n c e
S a m p l e analysis
Cell count and viability were determined b.~ T~.'pan blue exclusion using a hemae)¢ometer. Glucose and ammonia concentrations in the culture medium ~.ere determined by using test kits (Glucose-CII Test Kit, Ammonia Test Kit, Wako Pure Chemicals, Japan). Lactate was measured by the optical density method at 210 nm after
To make sure that the cells were free from contamination with mycoplasma, we cultivated the T0405 cells in a medium containing MC-210 (Dainihon Seiyaku Co. lot) for 6 d, before we started culturing in the membrane reactor. MC210 is a reagent which kills or inhibits the growth of mycoplasma. MC-210 did not have any adverse effect on the cell's MAb production abilit3.,.
23(J
M.
Hartgae et al. Journal of Biotechnolo.D' 34 IO04~ 227-235
To determine the effect of long-term culture on the cell's MAb producing ability, we continuousb subcultured the cells even, 3 d for 60 d. The specific PHA value is stable until around passage number 10 and then the cell's MAb-producing ability drops abruptly. Schmid et al. (1990) observed a loss of MAb-producing ability when the number of subcultures increased. Miller et al. (1988a) also observed a decreased in MAb productivity when increasing the passage number after thawing the cells. These results indicate that it is safe to run cultivation experiments in the bioreactor within 1 month.
Perhtsion and continuous culture When the cell bleeding is completely stopped ( f D = 0), the cultivation in the membrane reactor becomes a perfusion culture. A perfusion culture was carried out as a control experiment. Fig. 2 shows a typical time course of perfusion culture of hybridoma T0405 cells in the membrane reactor. Note that the viable cell concentration was maintained constant, while the MAb concentration increased. Sometimes the misconception that the MAb production was non-growth associated x,,as inferred from results similar to this (Hagedorn and Kargi, 1990). Fig. 3 shows a typical time course of continuous culture of hybridoma T0405 cells at a cell dilution rate fD = 0.0220 h -t. The culture ~ a s operated in batch mode until about 72 h. When
{.,,FI [ _ =_
[ FlU,JLIS
Cu I ~ute
L,,
, fC,=O I121'IIh -i-i-1
._-.
i--d-~-
i"
_ Z
L" L,,,
_ ,
~ul,
[ l ,T,~_ ' ~t ' Fig. 3. A D p i c a l
time course ol c o n t i n u o u s
culture of T( 4 ( S
cells in m e m b r a n e reactor at 0.0220 h i cell dilution rate. Symbols: ( 4 4 total cell: ( ." I ~iable cell.
the cell concentration reached the 10" ml ' level. the supply of fresh medium into the reactor and cell bleeding were started. The drop in the cell concentration from 216 to 240 h was caused b~ the malfunction of the feed pump, but when the error was corrected the cell concentration returned to the previous steady level. So we considered the data from 288 to 408 h as the steady-state data, The total and viable cell, and other medium components were held constant, indicating a real steady-state condition (Fig. 4). We regarded the
P e r fu~l,:,r, q[ultut,:
[,=Ida, -
-
'
, "
I¸
C
==_--
lime ,,u
tnu
t',
'
h'
u,,
Fig. 2. A t3,pical time course o f perfusion culture of T0405 cells m m e m b r a n e reactor. Symbols: (c,I total cell: ( L ) ~mble cell: (,>1 HBs-MAb: (Ell glucose; (o) glutamine.
Fig. 4. Steady state c o n c e n t r a n o n s of c o n t i n u o u s culture at 0.0220 h - ' d i l u u o n rate. Symbols: (:,) total cell: q ~ l viable cell: (<,,) HBs-MAb: (t~) glucose: ( • ) lactate: (e) glutamine; ( • ) ammonia.
M. Harigae et al. / Journal q[ Biotechnolog)' 34 (1994) 227-235
231
1 O0
[A ]
//--.-
:Z, ~
E 6,] >.
2
40 "~
L~
2~ _
,,[
i
i
i
t
i
,) ~,
g]
v ..... ,, . t
,, ii
[,l~uti(:,n
Fig. 6. Effect
of
dilution
. ,,
r~ze
rate
on
,, ,.,] f[.'
J
"
'h-''
HBs-MAb
concentration.
& (12
i]Jl g 0~
_
. ', ",1
~, ', 2
[tlut:,:,n
,., O~
r~te
i: ,)~
,, ,,~
fD ,'i7 - ~ '
Fig. 5. Effect of dilution rate on cell growth• (A) (O) viable cell concentration and ( zx ) viability; (B) ( zx ) specific growth rate and (,:.2,) specific death rate.
average of each of these points as the steady-state value.
Viable cell concentration, riabifit),, and specific growth a n d d e a t h rates
Viable cell concentration, viability, and specific growth ( # ) and death rates ( K D) were plotted against the cell dilution rate f D (Fig. 5). As shown in Fig. 5A, the maximum viable cell number of 4 × 10 6 ml- ~ was observed at the lowest fD of 0.00316 h-~, and the viability increased as the cell dilution rate fD increased. The specific growth rate increased with increasing fD. while the death rate increased with decreasing fD and it attained the same value as the specific growth rate at the lowest fD (Fig. 5B). The same trend was found in previous work (Robinson and Memmert, 1991).
steady-state values, the specific MAb production rates, r/MAb were calculated using Eq. 9. The results were plotted against specific growth rates as shown in Fig. 7. The specific HBs-MAb production rate increased with increasing specific growth rate. This indicates that this cell line's MAb production is growth associated. The t~o stray points (solid symbols) in Fig, 7, which were obtained in a long-term continuous culture of more than 1000 h, suggest a drop of the specific MAb production rate. These results indicate that the steady-state data obtained from extremely extended continuous culture is not suitable for kinetic studies and that attention should be paid to the stability of MAb production. Similar discussion was found in previous works (Hiller et al., 1991: Frame and Hu, 1990). Frame and Hu (1990) observed a continuous decrease in antibod.~ productivity over the course of a long-term continuous culture. Low et al. (1987) suggested that the
= E
Monoclonal antibod.v When the steady-state values of the MAb concentration at various cell dilution rates fD were plotted against fD, the expected decreasing pattern is obtained as shown in Fig. 6. Using these
;[.; ,i
11
Specific
u u_
. ,)]
. ,~
growth r a t e
,, ,i ~
,h ' ,
Fig. 7. Effect of specific grov~th rate on specific MAb production rate.
232
M H a r t g a e et al.
J o u r n a l ~t" B i o l e c h n o l o ~
Io~ specific MAb production rate at low specific growth rate was due to the degradation or inactivation of MAb. In this work, this was checked in a separate experiment, ~herein the MAb activity ~as maintained exen after 2 ~eeks of storage in a 37"-'C incubator. The kinetics of murine MAb production b.~ hybridoma have often been described as nongrowth associated (Miller et al.. 1988a: Linardos et al., 1991). Using continuous culture, Linardos et al. (1991)sho~,ed that the specific MAb production rate r/MAt, ~.as inversely dependent on the specific growth rate p., and r/mAh v, as correlated with specific death rate K D. They claimed that MAb production mainly occurs in the GI phase. The time the cell spends in GI phase is increased bx adverse environmental conditions that affect cell proliferation. The percentage of cells in the G I phase increased with a slow down in growth rate, which results in enhancement of ~LAb production. Using a cell line producing IgM-MAb against human blood group B antigen, Low et al. (1987) showed different MAb production kinetics; r/MA~, increased with increasing /1 up to a dilution rate of 0.02 h n but at higher specific growth rate. r/r,t~,t, leveled off and became independent of /1. Robinson and Memmert (1901) showed a growth-associated kinetics of a chimeric MAb. In their experiment, transformed m~eloma cells were cultivated on a serum-free medium containing methotrexate to maintain constant selection pressure. Therefore, the activity of MAb production of the cells must be stable during long-term continuous cultivation. For growth-associated MAb production, the com,entional long-term perfusion culture is not a suitable process, because cell growth is highly repressed under high cell densit3. In this case, a repeated-perfusion culture, as sho~n in Fig. 8, must be the best v, ay to satist3. the tx~o requirements of high productivity and concentrated MAb. As mentioned above, there are many contradicting results on MAb production kinetics exen when the.~ are obtained in continuous cultures. To a great extent, this difference may be attributed to the different cell lines used. However, knowledge of the relationship between flMA~, and p. is of critical importance in
34 I lqq41 .~. .' ."
~ 2+-
Ill o •
•
lip
Oo
o t
o o
0'41
•
~
•
e'm °
I Iq
•
111 -,ll
]H'~
I -,IP t IU'_
~11(I
~nll
"fllPl
,',it
~l ,
Fig. ~. Production of HBs-MAb in repeated-perlusion culture. Symbols: (e) ~iable cell concentratuon. ( " | PHA ~alue.
choosing reactor designs or operating strategies. Therefore, much more effort should be made for claril3.'ing the mechanism of antibody production and antibody transport.
Glucose and lactate, glutamine atzd ammonia Glucose and glutamine are the major energy and carbon sources for animal cell culture. When these substrates are metabolized, lactate and ammonia are produced. Glucose metabolism can be divided into two major pathways: lactate synthesis and ATP generation or biosynthesis through the TCA cycle. Glucose metabolism through the TCA c.vcle is ~,ell known to be more efficient for ATP generation compared to lactate s3,nthesis. Glutamine metabolism can provide 30 to 65% of the energ~ for mammalian cell growth (Reizer et al., 1979). The pathway of glutamine metabolism leads to biosynthesis and production of amino acids. The use of glutamine as an e n e r ~ ' source in animal cells proceeds via a pathway producing a-ketoglutarate, which is incorporated into the T C A cycle. There is a net production of 2 moles of ammonia from 1 mole of glutamine. Ammonia has serious inhibitor5.' effects on the growth of T0405 cells at a concentration of 5 mmol 1-J (Na~'e et al., 1991). In hybridoma cultures, lactate production and glucose consumption are tightly interrelated. Hybridomas are capable of carrying out glycol vsis even under aerobic conditions (kanks and LI, 1988). About 80c~ of glucose proceeded through the glycob, tic pathway to lactate, and only 4 to
M. Harigae et al. /Journal o/Biotechnolog3' 34 11994) 227-235
5%. entered the T C A cycle when the glucose concentration was greater than 1 mmol I - ' (Reizer et al., 1979). As shown in Fig. 9, the residual glucose concentration in this experiment was reD' high, about 5 to 11 m m o l l - ~ , therefore a high rate of glycolysis can be expected. The specific glucose consumption rate uGk and lactate production rate r/L~~ as a function of the specific growth rate are shown in Fig. 10A and B, respectively. The specific glucose consumption and lactate production rate seem to increase more rapidly at the higher specific growth rate. This unusually high glucose consumption rate at high specific growth rate was also found in the experiment by Robinson and Memmert (1991), in which MAb production kinetics was growth associated. In this work. we tried to evaluate the yield values of lactate on glucose for cell maintenance and cell growth. ~'G,c and r/r~ may be correlated with # as in the following equations except at extremely high glucose consumption regions (Pirt. 1985): t,Gic = aol~/.t + mt31c
( 11)
~Lac = aLac/d" + nlLac
(12)
where a is the slope of the line and m is the specific value for cell maintenance. A regression analysis performed on the specific glucose consumption and lactate production rate values for the lowest sLx specific growth rates gives a.c,~ = 0.17 mmol per cell, triG,~ = 0.51 mmol per cell per h. CtLac 0.22 mmol per cell, IHLac 0.25 mmol =
=
:/
..-.,
.-j "7-
Dilutb~,n rate fD , h - ' Fig. 9. Effect of dilution rate on steady-state glucose and lactate concentration. S.,,mbols: (c,) glucose: ( ." ) lactate.
233
I% m L
=
= ,
L
j'
~-
=
-
"T'
i ..... ' 04 [,ezif1,: grT,~'~n ~2[e L . . . . :,i:
,,~
Fig. 10. Effect of specific growth rate on ~'c,,~ the specific glucose consumption rate (A) and on r/Lo~ the specific lactate production rate (B).
per cell per h. The lactate yield values on glucose were calculated as follows: YL~c C,Ic = CrLac/aGIc
(13)
YmLac GI¢ = m L a c / m G i c
(14)
where YL~c.G~o is the lactate yield values on glucose during cell growth, and Y,nL~ Gl~ iS the lactate yield values on glucose for maintenance. YL~'G]c was 1.34 mol per tool and Ymt,~o G]¢ was 0.48 mol per mol. YmL~ GIc was much lower than Ytac G~" These values indicate that lactate produced from glucose which was used for cell maintenance is much smaller than that for cell growth. If we use lactate production as a gauge to evaluate glucose utilization, it can therefore be said that, in maintenance metabolism, glucose utilization was very efficient. The same obse~'ations in a hybridoma culture were reported by Luan et al. (1987a). They pointed out that during the initial batch growth, a large fraction of glucose was metabolized to lactate, while in stationaD.' phase, no net lactate was formed.
234
M H a r i e a e et al. . J o u r n a l o] Biotechnolog}' 3 4 q 1904J 2 2 7 - 2 3 5
were calculated using Eqs. 8 and 10. The results were plotted against the specific growth rate and are shown in Fig. 12A and B. Similar to the analysis of glucose and lactate, regression was performed which gives the following, t~Gl n 0.29 mmol per cell, mGi n = 7.67 X 10--' mmol per cell per h, C~NH~ = 0.13 mmoI per cell and raN, ' = 0.16 mmol per cell per h. The ammonia yield on glutamine for growth and maintenance are 0.46 and 2.02 mol per mol, respectively. The m a y imum theoretical value of the ammonia yield on glutamine is 2 tool per mol. This means that almost all the glutamine consumed for maintenance of the cell were deaminated for biosynthesis or ATP generation through the TCA cycle. From these results of yield values of lactate and ammonia, we can mention that in a perfusion culture with low growth rate and high cell densit5,, the accumulation of ammonia is enhanced and a selective ammonia removal system is deemed necessa~'. =
m
= m
r
Ii
ii 1
p 11 ~
,, r,.
'
ir
i~
i
I
Dilutior. rate fD ,h ~, Fig. I1. Effect of dilution rate on steady-state glutamine and ammonia concentration. Symbols: (7,) glutamine: ( ." ) ammonia.
Fig. 11 shows the plot of the steady-state values of glutamine and ammonia concentration under various dilution rates. Unlike glucose and lactate, glutamine concentration increased with increasing dilution rate. Using glutamine and ammonia steady-state value, the specific glutamine consumption rate and ammonia production rate
The authors would like to thank Mr. Usuda, Mr. Takahashi and Ms. lwanari of the Institute of Immunology for preparing the hybridoma cells and providing excellent technical advice about the antibody analysis. This work was supported in part by a research grant from the University of Tsukuba.
7
-.,
Acknowledgments
=_-
_-,
i
.~ ,~,
References
i
i
"l '"1
I
IJ (,.
Frame, K.K. and Hu, W.S. (1990) The loss of antibody productivits.' in continuous culture of hybridoma cells. Biotechnol. Bioeng. 35. 469-476. Glacken, M.W. and Synskey, A.J. 11985) Modeling and optimization of fed-batch cultures of hybridoma cells. AIChE Annual Meeting Chicago, IL. Hagedorn, J. and Kargi. F. (1990) Coiled tube membrane bioreactor for cultivation of hybridoma cells producing monoclonal antibodies. Enz%,me Microb. Technol. 12, 824-
,
IJ '.'?
~,-_,~z~fzz gr,z, wt~-~
I) ~.,~
raze
829.
,I-,-
Fig. 12. Effect of specific growth rate on l,'Gi n the specific glutamine consumption rate (A) and on r/NH, the specific ammonia production rate (B).
Hiller, G.W., Aeschlimann. A.D.. Clark, D.S. and Blanch. H.W. (19911 A kinetic analysis of hybridoma growth and metabolism in continuous suspension culture on serum free medium. Biotechnol. Bioeng. 38, 733-741.
M. Harigae et aL /Journal o]" Biotechnology 34 t lgq4J 227-235 Lanks, K.W. and LL, P.W. (1988) End products of glucose and glutamine metabolism by cultured cell lines. J. Cell. Phys. 135, 151-155. Linardos. T.I., Kalogerakis, N., Behie, L.A. and Lamontagne, L.R. (1991) The effect of specific growth rate and death rate on monoclonal antibody production in hybridoma chemostat cultures Can. J. Chem. Eng. 69, 429-438. Lov,, K.S., Harbour. C. and Barford. J.P. (1987) A study of hybridoma cell grov~lh and antibody production kinetics in continuous culture. Biotechnol. Techniq. 1,239-244. Luan. Y.q-., Mutharasan, R. and Magee, W.E. (1987a) Factors governing lactic acid formation in long term culti~,ation of h~,bridoma cells. Biotechnol. Lett. 9. 751-756. Luan. Y.T., Mutharasan. R. and Magee, W.E. (1987b) Strategies to extend Iongetivity of hybridomas in culture and promote }ield of monoclonal antibodies. Biotechnol. Lett. 9, 691-696. Merten. O.W. (1988) Batch production and growth kinetics of hybridomas. Cytotechnolog~ 1. 113-121. Miller, W.M.. Blanch, H.W. and Wilke, C.R. (1988a) A kinetic analysis of h.~bridoma growth and metabolism in batch and continuous suspensnon culture: effect of nutrient concentration, dilution rate. and pH. Biotechnol. Bioeng. 32, 947-965. Miller. W.M.. Wilke, C.R. and Blanch. H.W. (1988b) Tran-
235
sient responses of hybridoma cells to lactate and ammonia pulse and step changes in continuous culture. Bioprocess Eng. 3. 113-122 Nayve Jr.. F.R.P., Motoki, M., Matsumura, M. and Kataoka. H. (1991) Selective removal of ammonia from animal cell culture broth. Cytotechnolo~' 6, 121-130. Pirt. S.J. (1985) Principles of microbe and cell cultivation. Blaclc~ell Scientific Publications. Cambridge, UK. Reddy, S., Bauer. K.D. and Miller, W.M. (1992) Determination of antibody content in live versus dead hybridoma cells: analysis of antibody, production in osmotically stressed culture. Biotechnol. Bioeng. 40, 947-964. Reizer, L.J., Wice, BM. and Kennell, D. (1979) Evidence that glutamine, not sugar, is the major energy source for cultured Hela cells. J. Biol. Chem. 254, 2669-2676. Robinson, D.K. and Memmert, K.W. (1991) Kinetics of recombinant nmmunoglobulin production by mammalian cells in continuous culture. Biotechnol. Bioeng. 38, 072-976. Schmid, G.. Blanch. H.W. and Wilke, C.R. (1990) Hybridoma growth, metabolism, and product formation in HEPESbuffered medium-l, effect o1 passage number. Biotechnol. Lett. 12. 627-632. Seamans, I.G. and Hu. W.S. (1990) Kinetics of growth and antibody production by a hybridoma cell line in a perfusion culture. J. Ferment. Bioeng. 70, 241-245.