some aspects of hybridoma cell cultivation

11
Appl Microbiol Biotechnol (1991) 35:165-175 0175759891001095 Applied ., Microbiology Biotechnology © Springer-Verlag 1991 Some aspects of hybridoma cell cultivation H. Graf I and K. Schiigerl 2 1 BASF, Ludwigshafen, Federal Republic of Germany 2 Institut for Technische Chemie, Universit~it Hannover, Callinstrasse 3, W-3000 Hannover, Federal Republic of Germany Received 30 November 1990/Accepted 17 December 1990 Summary. Two hybridoma cell lines were cultivated in an indirectly aerated 10-1 reactor in batch, fed-batch and continuous (perfusion) operations and in spinner flasks. The medium in the reactor was sampled either by an aseptic cross-flow filtration module integrated into a loop or by an in-situ tubular filter. The glucose concentration was monitored by an on-line flow injec- tion analyser and the ammonia concentration by an ion-selective electrode. Since the membrane transmis- sion of the high-molecular components decreased dur- ing cultivation, the product, a monoclonal antibody, was enriched in the reactor. During cultivation, the concentrations of cells, viable cells, glucose, lactase, acetate, citrate, ammonia, urea, amino acids, proteins, and monoclonal antibodies were determined off-line. The specific growth rate, specific production, and con- sumption rates of the medium components were in- fluenced considerably by the medium composition, es- pecially by the type and amount of serum used. Introduction Culture medium composition has considerable in- fluence on mammalian cell growth and product forma- tion (e.g., Glacken et al. 1986; Miller et al. 1989a, b; Wagner et al. 1987). On-line monitoring and control of medium components in microbial cultivation media are now commonplace on a laboratory scale (Schiigerl 1988). However, except for temperature, pO2, pH, and stirrer speed, no other process variables are controlled by in-situ or on-line measurements (e.g., Wallberg et al. 1987). There are only a few publications on the applica- tion of on-line monitoring for mammalian cell cultiva- tion (Schiigerl et al. 1990). The present paper reports on the development and application of substrate concen- tration control during mammalian cell cultivation. Offprint requests to: K. Schiigerl Materials and methods Cell lines. Two mouse-mouse cell lines (F34 and 3C2) were pre- pared by P. Nabet (personal communication) by the fusion of myeloma cells of cell line P3X63-Ag8 with activated B-lympho- cytes of a mouse. The mouse-mouse 3C2 cells produce mono- clonal antibody (MAB) immunoglobulin G-1 (IgG-1) against the hormone Human-Choriongonadotropin. The cell line F34 does not produce antibodies. The hybridoma cell stock cultures were stored at 37 ° C and 5% CO2 in air in Roux flasks (Falcon, Cockeyville, USA) in incuba- tors (Heraeus Type B 5060 EK CO2; Hanau) and were diluted twice a week with fresh medium to 105 cells ml h -1. These cells were used as seeds for the precultures in Bellco (Vineland, N J, USA) spinner flasks. Culture media. The basic medium for F34 and 3C2 was RPMI 1640 powder (Gibco, Grand Island, USA), supplemented with 0.328 g 1-1 glutamine (Serva, Heidelberg, FRG), 0.132 g 1-1 so- dium pyruvate (Riedel de Haen, Seelze), 10 lxg 1-1 2-mercaptoe- thanol (Serva), and 2.0 g 1-1 NaHCO3 (Riedel de Haen). For cul- tivation in the 10-1 reactor 0.060 g 1 -I gentamicin (Serva) in dis- tilled water (ASTM-Type 1, R= 18 M~ cm -1) was also added. Before inoculating the medium, it was sterile-filtered, and var- ious amounts of either foetal calf serum (FCS; Gibco) or horse serum (HS; Gibco) was added to the medium. The pH value was controlled by addition of gaseous CO2 or 0.1 N NaOH. Bioreactor. A 10-1 bioreactor (Biostat E, Braun, Melsungen, FRG) with a low stirrer speed (< 200 rpm), 15.2 m silicone tubing (3 mm diameter and 0.4 mm wall thickness) wound around a cylindrical basket of 16 cm diameter for indirect aeration and 10 m hydro- philized microporous polytetrafluorethylene (PTFE) tubing (Gore, Putzbrunn, FRG) (2 mm diameter 0.4 mm wall thickness) for medium exchange with perfusion, was used for cultivation (Fig. 1). The oxygen transfer rate and pH were controlled by the gas composition (N2:O2:CO2) in the silicone tubing. On-line analysis. Two aseptic sampling systems were used: (a) a cross-flow flat filtration module (Millipore, Freehold, N J, USA) (Fig. 2) with a pump (Watson-Marlow 101 UR, Eschborn, FRG) in the outer loop, and (b) a tubular filter developed in the Techni- cal Chemistry Institute (TCI) at the University of Hannover and sold by ABC (Puchheim, FRG) (Fig. 3). In the Millipore module, a hydrophilic flat membrane (GVWP 04700, Millipore) was used. In the tubular filter, polypropylene microfiltration tubing (Enka, Wuppertal, FRG) was used. The sampling modules are character- ized in Table 1.

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Page 1: Some aspects of hybridoma cell cultivation

Appl Microbiol Biotechnol (1991) 35:165-175

0175759891001095 Applied ., Microbiology Biotechnology © Springer-Verlag 1991

Some aspects of hybridoma cell cultivation H. Graf I and K. Schiigerl 2

1 BASF, Ludwigshafen, Federal Republic of Germany 2 Institut for Technische Chemie, Universit~it Hannover, Callinstrasse 3, W-3000 Hannover, Federal Republic of Germany

Received 30 November 1990/Accepted 17 December 1990

Summary. Two h y b r i d o m a cell lines were cult ivated in an indirect ly aerated 10-1 reactor in batch, fed-ba tch and con t inuous (perfusion) opera t ions and in spinner flasks. The m e d i u m in the reactor was sampled ei ther by an asept ic cross-f low filtration modu le integrated into a loop or by an in-situ tubular filter. The glucose concen t ra t ion was moni to red by an on-l ine f low injec- t ion analyser and the a m m o n i a concent ra t ion by an ion-selective electrode. Since the m e m b r a n e t ransmis- sion o f the h igh-molecu la r componen t s decreased dur- ing cult ivation, the product , a monoc lona l an t ibody, was enr iched in the reactor. Dur ing cult ivation, the concent ra t ions o f cells, viable cells, glucose, lactase, acetate, citrate, ammonia , urea, amino acids, proteins, and m o n o c l o n a l ant ibodies were de te rmined off-line. The specific g rowth rate, specific p roduc t ion , and con- sumpt ion rates o f the med ium componen t s were in- f luenced cons iderab ly by the m e d i u m compos i t ion , es- pecial ly by the type and amoun t o f serum used.

Introduction

Culture m e d i u m compos i t ion has considerable in- f luence on m a m m a l i a n cell growth and p roduc t fo rma- t ion (e.g., G lacken et al. 1986; Miller et al. 1989a, b ; Wagne r et al. 1987). On-l ine moni tor ing and control o f m e d i u m c o m p o n e n t s in microbial cult ivat ion media are now c o m m o n p l a c e on a labora tory scale (Schiigerl 1988). However , except for temperature , pO2, pH, and stirrer speed, no other process variables are cont ro l led by in-situ or on-l ine measurements (e.g., Wallberg et al. 1987). There are only a few publ ica t ions on the appl ica- t ion o f on-l ine moni to r ing for m a m m a l i a n cell cultiva- t ion (Schiigerl et al. 1990). The present pape r reports on the deve lopment and appl ica t ion o f substrate concen- t rat ion control dur ing m a m m a l i a n cell cultivation.

Offprint requests to: K. Schiigerl

Materials and methods

Cell lines. Two mouse-mouse cell lines (F34 and 3C2) were pre- pared by P. Nabet (personal communication) by the fusion of myeloma cells of cell line P3X63-Ag8 with activated B-lympho- cytes of a mouse. The mouse-mouse 3C2 cells produce mono- clonal antibody (MAB) immunoglobulin G-1 (IgG-1) against the hormone Human-Choriongonadotropin. The cell line F34 does not produce antibodies.

The hybridoma cell stock cultures were stored at 37 ° C and 5% CO2 in air in Roux flasks (Falcon, Cockeyville, USA) in incuba- tors (Heraeus Type B 5060 EK CO2; Hanau) and were diluted twice a week with fresh medium to 105 cells ml h -1. These cells were used as seeds for the precultures in Bellco (Vineland, N J, USA) spinner flasks.

Culture media. The basic medium for F34 and 3C2 was RPMI 1640 powder (Gibco, Grand Island, USA), supplemented with 0.328 g 1-1 glutamine (Serva, Heidelberg, FRG), 0.132 g 1-1 so- dium pyruvate (Riedel de Haen, Seelze), 10 lxg 1-1 2-mercaptoe- thanol (Serva), and 2.0 g 1-1 NaHCO3 (Riedel de Haen). For cul- tivation in the 10-1 reactor 0.060 g 1 -I gentamicin (Serva) in dis- tilled water (ASTM-Type 1, R= 18 M~ cm -1) was also added.

Before inoculating the medium, it was sterile-filtered, and var- ious amounts of either foetal calf serum (FCS; Gibco) or horse serum (HS; Gibco) was added to the medium. The pH value was controlled by addition of gaseous CO2 or 0.1 N NaOH.

Bioreactor. A 10-1 bioreactor (Biostat E, Braun, Melsungen, FRG) with a low stirrer speed (< 200 rpm), 15.2 m silicone tubing (3 mm diameter and 0.4 mm wall thickness) wound around a cylindrical basket of 16 cm diameter for indirect aeration and 10 m hydro- philized microporous polytetrafluorethylene (PTFE) tubing (Gore, Putzbrunn, FRG) (2 mm diameter 0.4 mm wall thickness) for medium exchange with perfusion, was used for cultivation (Fig. 1). The oxygen transfer rate and pH were controlled by the gas composition (N2:O2:CO2) in the silicone tubing.

On-line analysis. Two aseptic sampling systems were used: (a) a cross-flow flat filtration module (Millipore, Freehold, N J, USA) (Fig. 2) with a pump (Watson-Marlow 101 UR, Eschborn, FRG) in the outer loop, and (b) a tubular filter developed in the Techni- cal Chemistry Institute (TCI) at the University of Hannover and sold by ABC (Puchheim, FRG) (Fig. 3). In the Millipore module, a hydrophilic flat membrane (GVWP 04700, Millipore) was used. In the tubular filter, polypropylene microfiltration tubing (Enka, Wuppertal, FRG) was used. The sampling modules are character- ized in Table 1.

Page 2: Some aspects of hybridoma cell cultivation

166

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Fig. 2. Cross-flow flat membrane filtration module for on-line aseptic sampling

The on-line flow injection analyser (FIA) system for glucose analysis consisted of a 16-channel peristaltic pump (Skalar, Erke- lenz, FRG) tygon-tubing, motor valve (Latek-TMV, Eppenheim, FRG), injector valve (Lee Hydraulic Miniature Components, Frankfurt, FRG) and a YSI-glucose analyser (Model 23A, Yellow Spring Instruments, Ohio, USA) modified for on-line operation (TCI, Hannover). The operation of the FIA system was controlled by a microprocessor (Motorola, Type 68 000) and a suitable soft- ware package (FERAS; Wieneke 1989).

Connection tube

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Page 3: Some aspects of hybridoma cell cultivation

167

Table 1. Properties of the sampling modules

Parameters Flat Tubular Millipore Enka module module

Free filtration area (cm 2) 7.2 30 Dead volume on the per- meate side (ml) 1.0 8 Response time (min) 1.0 9 Membrane material Polyvinylidene Polypropylene

fluoride Mean membrane thickness (mm) 0.125 1.5 Mean membrane pore diameter (txm) 0.22 0.2

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Results

Determination of specific consumption rates of medium components by cell line F34 NP

Reducing sugar was determined in a continuous air-segmented analyser (Skalar) by means of p-hydroxybenzoic acid hydrazide (p-HBAH) (Schmidt et al. 1985) and a photometer at 410 nm (Type V5, Julabo Labortechnik, Seelbach, FRG) (Fig. 4). The de- termination of the ammonia concentration was carried out by an ion-selective electrode (Type 9512, Orion Research, Cambridge, Mass., USA) and the pO2 by an 02 electrode (Bauermeister 1978).

The transfer, control, and evaluation of on-line analysis data were performed by a computer system (VME 68 000/RTOS oper- ating system, and PDP ll/23+/RSX-11M + operating system, Fig. 5) as well as by FERAS and CASFA (Dors 1989) software packages.

Off-line analysis. The cell concentration was determined by a Thoma chamber, the vitality with trypan blue, the concentration of glucose by off-line FlA. The following off-line analyses were carried out: the Giuco-DH method (Mercotest 14335, Merck, Darmstadt, FRG), lactate (Boehringer, Mannheim, FRG; BM 149993), urea (urease), citrate (BM 139076), acetate (BM 14826), ammonia (BM 542 946). The analysis of protein was performed by the Bicinchonin acid-method of Pierce (Smith et al. 1985), the am- ino acids by the HPLC-o-phthaldialdehyde method (Kretzmer 1986) and the MAB with sandwich ELISA (Nabet, personal com- munication).

The cells were cultivated in RPMI 1640 medium, sup- plemented with 2.5 g 1-1 glucose, NaHCO3, Na-pyru- vate, 2-mercaptoethanol, glutamine, gentamicin and 4% HS at 37°C and pH 7.2 in batch operation with an ini- tial cell concentration of 6.104 cells ml- 1. After an 18 h lag phase, 100 h exponential growth (specific growth rate,/z = 0.0278 h - 1), and a 62 h stationary phase, a via- ble cell concentration of 1.02.10 -6 cells m1-1 was at- tained. In Fig. 6, the viable cell concentration and oxy- gen uptake rate (OUR) are shown as a function of the cultivation time. They attained a maximum at 125 h and/or 150 h and had similar courses. The specific sub- strate consumption rates during exponential growth are shown in Table 2.

The essential amino acids (arginine, leucine, valine, threonine, isoleucine, methionine and lysine) were con- sumed at fairly high rates. It was not possible to deter- mine histidine because its small peak appeared together with the high peak of glutamine in the HPLC spec- trum.

During fed-batch cultivation with 3 g 1-1 glucose, 4% HS and an initial cell concentration of 5.104 viable cells m1-1 after a 20 h lag, 90-100 h exponential growth (/J = 0.026-0.0285 h- l ) and the stationary phase, 1.3-106 viable cells m1-1 were attained. In Fig. 7, the

Page 4: Some aspects of hybridoma cell cultivation

168

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Fig. 8. Logarithm of viable cell concentration and oxygen con- sumption rate as a function of the cultivation time. Batch cultiva- tion of F34 cell line

Table 2. Specific substrate consumption rates (qs) in nM (10 6 cells h)-1 of F34 cells during the exponential growth phase

Substrate qs Substrate qs

Oxygen 125.30 Arginine 11.20 Glucose 70.90 Alanine - 12.50 Lactate 489.80 Tyrosine 0.66 Aspartate 1 . 4 8 Methionine 1.40 Glutamate - 0.88 Valine 2.28 Asparagine 1 . 8 3 Tryptophan 0.28 Serine 1.62 Phenylalanine 0.52 Glutamine 59.60 Isoleucine 1.76 Glycine - 1.79 Leucine 3.t3 Threonine 2.11 Lysine 1.01

The + symbol indicates consumption and the - symbol indicates production

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Fig. 7. Variation in the logarithm of the viable cell concentration and the glucose concentration measured enzymatically off-line and with the p-hydroxybenzoic acid hydrazide (p-HBAH) method on-line as functions of the cultivation time. The F34 cell line was used with RPMI 1640 medium

logarithm of the viable cell concentration, and the glu- cose concentration measured on-line by p-HBAH and off-line by enzyme FIA are shown as a function of the cultivation time. After about 125 h, the stationary phase was attained. Glucose consumption was fairly high dur- ing the stationary phase. After 200 h, the glucose was consumed. In Fig. 8, again the logarithm of the viable cell concentration and OUR, as well as the specific oxy- gen utilization rate qo2 are shown. During the exponen- tial phase, the qo2 was nearly constant [4.5 ~tg 0 2 (10 6 cells h ) - 1].

A comparison of the results of batch and fed-batch cultivations shows no dramatic differences. Only the lag phase was longer in the fed-batch culture. However, this deviation could be caused by differences in the inoculum quality.

After 6 min direct aeration of the cell-containing cul- ture medium, the viable cell concentration dropped to 50% of its original value. No dead cells, but only cell fragments were present. With increasing column height /diameter ratio, the viable cell concentration in- creased (J~mmrich 1988).

Cultivation o f mouse-mouse hybridomas 3C2

Batch cultivations were performed with RPMI 1640 medium containing 10 and 5% FCS as well as 4% HS. The initial cell concentrations were 5.104 cells m1-1 and the dissolved oxygen saturation was 50%. During the exponential phase, the viable cell fraction was 100%. With 10% FCS after 95 h, the cell viability began to diminish, by 107 h it was only 66% and by 150 h had decreased to below 10%. The rapid reduction in the vi- able cell fraction after depletion of the glucose is typ- ical for culture media of low amino acid content. The glutamine content was already exhausted at 90 h, i.e., 10 h before the glucose was spent.

Page 5: Some aspects of hybridoma cell cultivation

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Fig. 9. Viable and total cell concentrations as well as immunoglo- bulin G (IgG) concentration as a function of the batch cultivation time of 3C2 cells

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Fig. 11. Oxygen consumption rate calculated from the dissolved oxygen concentrations measured at the top and in the bottom (B) of the bioreactor as a function of the batch cultivation time of 3C2 cells

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Fig. 12. Glucose consumption rate (GUR) and lactate production rate (LPR) as functions of the batch cultivation time of 3C2 cells

In Fig. 9, the cell and viable cell concentrations as well as the IgG concentration are shown as a function of the cultivation time. After 75 h, the difference be- tween overall and viable cell concentration became ap- parent, and above 100 h, the deviation increased quick- ly. The MAB concentration attained its maximum at 125 h. A comparison of Fig. 9 and Fig. 10, in which glu- cose and lactate concentration courses are shown, indi- cates the close relationship between the glucose con- centration and the reduction in the viable cell fraction. A comparison of Fig. 9 and Fig. 11 again demonstrates the close relationship between the viable cell concentra- tion and OUR. On the other hand, Fig. 12 indicates that most of the glucose was converted into lactate.

Table 3. Influence of the serum type and concentration on the growth and production of mouse-mouse-hybridoma 3C2 in RPMI 1640 medium

Parameters Biostat E Spinner

10%FCS 5%FCS 4%HS 10%FCS

Lag phase (h) 13 13 15 5 /~ (h -1) 0.04 0.033 0.035 0.039 Max. MAB conc (mg 1-1) 56 15 19 30 Max. viable cell conc (10 6 cells m1-1) 1.01 0.95 0.58 0.8

MAB, monoclonal antibody; FCS, foetal calf serum; HS, horse serum

Page 6: Some aspects of hybridoma cell cultivation

170

Comparison of batch cultures of 3C2 with FCS and HS

Cell line 3C2 has been adapted during several years to 10% FCS. Therefore, it was expected that the medium with 10% FCS would give a better performance than that with HS. In Table 3, batch cultures in a spinner flask with 10% FCS, and in Biostat E with 10%, 5% FCS and 4% HS are compared.

Cultures in a spinner flask with 10% FCS had the shortest lag phase and yielded the highest MAB pro- ductivity. The culture performance in Biostat E with 10% FCS was better than with 5% FCS and 4% HS, but unexpectedly, 4% HS gave better results than 5% FCS.

Cyclic batch cultivation of 3C2

The supplemented RPMI 1640 medium was used with 4% HS. The initial cell concentration of 5. | 0 4 cells m1-1 increased with # = 0.036 h-1 to 6. l0 s viable cells ml-1 after 100 h. The medium exchange was performed as soon as the glucose concentration dropped below 0.1 g 1-1 (Fig. 13). The volume was reduced from 10 1 to 5 1 and made up with fresh medium to 10 1. In general, after each cycle, a viable cell concentration of 1.10 -6 and MAB concentration of 20 mg 1-1 were attained (Fig. 14). The lactate concentration variation was a mir- ror image of the glucose concentration course (Fig. 15). With a sampling flow rate of 0.4 ml min-1 and a vol- ume of the tubular sample filter of 8 ml, the response time of the sampling system was 20 min. The off-line and on-line measured glucose concentrations agreed

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Fig. 13. Variation in glucose concen- tration during cyclic batch cultiva- tion of the 3C2 cell line. Glucose was determined by three different methods: flow injection analysis (Yellow Springs Instruments, YSI), reducing sugar analysis (on-line) and enzymatic analysis

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Page 7: Some aspects of hybridoma cell cultivation

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satisfactorily (Fig. 13). For the balancing of the medium components the volume loss by sampling was taken into account.

Continuous (perfusion) cultivation of 3C2

The supplemented RPMI medium was used with 4% HS. The initial cell concentration of 3.104 cells m1-1 increased after a 20-h lag phase and 50 h exponential growth with #=0.039 h -1 to 1.1-10 6 cells m1-1. After 250 h continuous culture with a medium throughput of about 2.6 1 per day, the cultivation was changed to cy- clic batch culture.

During the stationary phase at a constant glucose concentration level of 0.18 g 1 -~, the specific oxygen consumption rate attained values between 3 and 6 t~g

171

(10 6 viable cells h) -1. The MAB concentration in- creased to 60 mg l-1 and attained a maximum level of 90 mg 1-1, an average MAB production rate of 456 ng (10 6 cells h) -1, which was only 10% lower than that in the exponential growth phase. Glucose was partly con- verted into lactate, and during the oxidation of glutam- ine, ammonia was formed (Fig. 16). The lactate concen- tration attained a maximum of 12.1 mM 1-1 at t - - 134 h, and during the stationary phase it decreased to 7 mM 1-1, probably due to a change in metabolism after the reduction in the glucose concentration.

The reduction in the lactate concentration (Fig. 16) and glutamine concentration was accompanied by a rise in the alanine and glycine concentrations (Fig. 17). The amino acids present in the culture medium can be divided into four groups: 1. Amino acids formed: alanine and glycine (Fig. 17). 2. Amino acids consumed at a high rate: leucine, iso- leucine, valine, lysine, threonine and methionine, in this order at a decreasing rate (Figs. 18, 19, 20). 3. Amino acids consumed at a low rate: aspartate, phe- nylalanine and tyrosine (Figs. 18, 20). 4. Amino acids with nearly constant concentrations: serine, aspartate and glutamate.

In Table 4 is shown the concentrations and specific consumption rates of the amino acids and metabolites by 3C2 cells during the exponential growth and the sta- tionary phase. This run was repeated with a higher ini- tial cell concentration (1.10 6 viable cells ml-1). At a perfusion rate of 4 1 per day at a steady state, a viable cell concentration of 1.87-10 6 cells m1-1 at a constant ammonia concentration of 30-40 mg 1-1 (Fig. 21) was attained.

The specific growth rate ~ = 0.0153 h - 1) amounted to only about 40% of that measured during the expon- ential growth phase in the cyclic batch culture and con- tinuous culture with a low initial cell concentration (Ta- ble 5). At the end of the cultivation, the MAB concen- tration increased steeply to 113 mg 1-1 (Fig. 21). This

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Page 8: Some aspects of hybridoma cell cultivation

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~" ~,~,,. ~, / - ~ X~X~'~.X i .i XN.~'/

~ ,.j,.~Z, . _ - . - . . . .

I I , 1 ~ 1 SO I00 ISO 200 2S0

cultivation time [h]

' ~ o i i

300d ~

300=; o

30~

o

Fig. 17. Glutamine, alanine, and glycine con- centrations as functions of the cultivation time during continuous perfusion cultivation of the 3C2 cell line

Fig. 18. Threonine, tyrosine and phenyl- alanine concentrations as functions of the cultivation time during continuous perfusion cultivation of the 3C2 cell line

Fig. 19. Methionine, valine, and lysine con- centrations as functions of the cultivation time during continuous perfusion cultivation of the 3C2 cell line

Page 9: Some aspects of hybridoma cell cultivation

~ ~ ' ~ ,. ~ - ~ ,_ ,,\-,~\ .~

/ - - .... ,

'., • ~ ~ !7 = ~ 5 . . ~ ~ ~ X'Xii'l~'ll'l X'X~

~ i ~# ~ I I I I ~ SO I00 I~0 200 2S0

cultivation time [hi

~ o

30~ ~

173

Fig. 20. Isoleucine, leucine and asparagine concentrations as functions of the cultivation time during continuous perfusion cultivation of the 3C2 cell line

Table 4. Initial concentrations (in mM l-i), concentrations (in % of the initial concentration) and specific consumption rates [in nM (10 6 cells h) -l] of amino acids and metabolites of the 3C2 cells and MAB concentration (mg 1-1) and production rates [ng (10 6 cell h) -1] during exponential growth (1), end of exponential growth (2) shortly before the feeding of fresh medium (3) and shortly after the feeding of fresh medium (4) during the stationary phase

Amino Culture Specific Concentrations acids medium consumption

[mM 1-1] rate (1) (2) (3) (4) [nM (10 6 [O/o ] [O/o ] [o,~] cells h)- 1]

Asp 0.157 - 10.1 189.0 83.4 122.0 Giu 0.153 - 20.1 285.0 1 8 5 . 0 238.0 Asn 0.349 11.8 52.0 50.0 41.0 Ser 0.289 - 10.6 151.0 69.0 119.0 Gin 3.663 199.1 22.8 0.3 14.0 Gly 0.164 - 41.2 454.0 372.0 391.0 Thr 0.151 8.1 24.0 13.0 18.0 Ala 0.026 - 55.8 3143.0 3170.0 3302.0 Thr 0.100 4.5 37.0 24.0 29.0 Met 0.091 4.9 24.5 6.9 7.4 Val 0.151 9.6 10.4 4.8 4.8 Phe 0.081 4.9 13.6 8.7 13.6 Ile 0.339 20.3 15.9 6.3 12.6 Leu 0.338 21.2 22.3 3.1 9.5 Lys 0.119 8.8 37.9 17.7 26.5

Other components

Glucose 10.68 188.0 75.0 6.4 8.0 Lactate 0.21 - 563.0 3883.0 4971.0 4206.0 Ammonia 1.18 - 99.8 217.0 294.0 284.0 Acetate 0.13 - 9.3 147.0 28.5 62.5

[rag 1-1] [ng (106 [°R] [°/0] [°/0] cell h) - a]

MAB (IgG) 2.60 -510.5 376.0 1507.0 1969.0

IgG, immunoglobulin G

was caused by retent ion o f M AB by the per fus ion mi- crofi l t rat ion membrane , since after long opera t ion it acts like an ultrafi l tration membrane , as can be seen in Fig. 22. This change is irreversible unde r cult ivat ion condit ions.

Except for the con t inuous culture at high initial cell concent ra t ion , the specific growth rate o f the cells was fairly constant ~ = 3 . 5 to 4 .10 -2 h - l ) . The low/~ value o f this run could be caused by the high th roughpu t rate (growth factors p r o d u c e d by the cells kept at a low lev- el) or by the different qualities o f the HS or inoculum.

The M AB concent ra t ions at ta ined in cycl ic-batch opera t ion were 40% higher than those in the ba tch op- eration. In the per fus ion operat ion, the M AB was en- r iched and therefore cannot be c o m p a r e d with the M AB concent ra t ions at tained in o ther runs.

The aim o f these investigations was the improve- ment o f the process analysis o f h y b r i d o m a cell cultiva- t ion and M AB product ion . On-l ine and off-line ana- lyses a l lowed the moni tor ing o f several m e d i u m com- ponen ts and the calculat ion o f their c o n s u m p t i o n / p r o - duc t ion rates in different process phases.

A compar i son o f cultivations with different sera and in different opera t ion modes indicated that the investi- ga ted h y b r i d o m a cells with an R P M I 1640 basic me- d ium supp lemented with 10% FCS showed the highest per formance . Batch cultures in spinner flasks per- fo rmed more efficiently than in the 10-1 stirred tank in- vestigated. In cycl ic-batch operat ions , h igher p roduc t concent ra t ions were at ta ined than in ba tch cultures. Dur ing cont inuous per fus ion M AB was enr iched in the medium, because the macrof i l t ra t ion per fus ion mem- brane changed its character irreversibly after longer op- erat ions and acted like an ultrafi l trat ion membrane .

Acknowledgements. Part of these investigations were carried out within the project 0132 D of Biotechnology Action Program of the European Community in cooperation with J. Lehmann, Bielefeld, J. M. Engasser, P. Nabet (Nancy), and J. Hache (Plaisir). The au-

Page 10: Some aspects of hybridoma cell cultivation

174

o ~

/ . / - I <~ . . . . . . . : '>"~:-"~ , / ' _ - x . . . . ><.x ~.#

' ~xk~ ~ ~ " ~ x ,

] e - %.,...,, I • N ~ ', ! ~ x"x. / /

~ x.. / z e l x, ~ _ _ ~ / ~ ~P k / / ~ / ~i / / ~ l

l ~ . ~ ~ . . . . ~"~.. .~ .~

~ r _ ~ _ _ ~ _ i I I ~ I 1 x ~ d ' i I

~0 50 tO0 150 200

cul t ivat ion t ime [h]

=~ =-tn x

o ° _~,.

-7 ~ ~ m ~ ~

r~ ~

tD _ ~ ~1

f~ E E

Fig. 21. Logarithms of viable cell con- centration, glucose, ammonia and IgG concentrations as functions of time during continuous perfusion cultiva- tion of the 3C2 cell line with 4% horse serum

Table 5. Comparison of the cultivation of the 3C2 cell line with RPMI 1640 medium and 4% HS under different conditions

Operation Initial Lag Specific MAB viable phase growth concentration cell conc [hi rate [mg 1 - 1] [ml] [h]

Batch 5 . 1 0 4 15 3.53' 10 -2 19 Cyclic batch 5. I0 4 13 4.01.10-2 26 Continuous (2.6 1 per day) 3 . 1 0 4 20 3.90' 10 -2 51 Continuous (4:01 per day) 1.105 15 1.53.10 -2 113 a

a Final concentration after 200 h perfusion operation with part enrichment of MAB

100.

90.

80.

70.

60.

50.

40.

30.

20.

10.

0

Mr

glucose culture BSA mouse IgG dexlran, blue supernatant

180 - 67 000 145 000 2 000 000

Fig. 22. Transmission of different components across the PTFE perfusion membrane after long-term operation: BSA, bovine se- rum albumin; Mr, molecular weight

thors gratefully acknowledge the support of the Behringwerke, Marburg, and H. Graf expresses his thanks to the Bundesministe- rium for Forschung und Technologie, Bonn, for the DECHEMA scholarship.

References

Bauermeister GD (1978) German patent no. P 28 01223.9-52 Dors M (1989) Entwicklung und Einsatz eines interaktiven Sy-

stems zur Offline Verarbeitung von Bioprozessen. Disserta- tion, University of Hannover

Glacken MW, Fleischacker RJ, Sinskey AJ (1986) Reduction of waste product excretion via nutrient control: possible strate- gies for maximizing product and cell yields on serum in cul- tures of mammalian cells. Biotechnol Bioeng 28:1376-1389

Jammrich U (1988) Mechanische Beanspruchung von Suspen- sionszellen und ihre Charakterisierung. MS thesis, University of Hannover

Kretzmer G (1986) Aminosaureanalytik bei Fermentationen mit der HPLC. MS thesis, University of Hannover

Miller WM, Wilke CR, Blanch HW (1989a) Transient responses of hybridoma cells to nutrient additions in continuous culture. I. Glucose pulse and step changes. Biotechnol Bioeng 33:477- 486

Miller WM, Wilke CR, Blanch HW (1989b) The transient re- sponse of hybridoma cells to nutrient additions in continuous culture. II. Glutamine pulse and step changes. Biotechnol Bioeng 33:487-499

Schmidt WJ, Kuhlmann W, Schiigerl K (1985) Automated deter- mination of glucose in fermentation broths with p-HBAH. Appl Microbiol Biotechnol 21:78-84

Sch~gerl K (1988) Measurement and bioreactor control. In: Du- rand G, Bobichon L, Florent J (eds) 8th International Biotech- nology Symposium, Paris 1988. Socirt6 Francaise de Micro- biologie, pp 547-562

SchOgerl K, Graf H, Kretzmer G, Freitag R, Scheper T, Wentz D (1990) Monitoring nutrient component, metabolite, product concentrations, and the behavior of animal cells in bioreac- tots. In: Christiansen HC, Munck L, Villadsen J (eds) 5th Eu- ropean Congress on Biotechnology, Copenhagen, July 8-13, 1990. Munksgaard, Vol I, pp 486-489

Page 11: Some aspects of hybridoma cell cultivation

175

Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NN, Olson B J, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85

Wagner R, Krafft H, Ryll T, Lehmann J (1987) Variation of amino acid concentrations in the medium of human interleukin 2 producing cell lines. In: Neissel OM, Meer RR van der, Luyben KChAM (eds) Proceedings of the 4th European Con- gress on Biotechnology, June 1987, vol 4. Elsevier Science Publishers, Amsterdam, pp 576-579

Wallberg C, Rosenquist J, Billig D (1987) Process control for the optimization of animal cell cultures. In: Neissel OM, Meer RR van der, Luyben KChAM (eds) Proceedings of the 4th Euro- pean Congress on Biotechnology, June 1987, vol 4. Elsevier Science Publishers, Amsterdam, pp 417-420

Wieneke KH (1989) FERAS - Ein flexibles Front-End-System zur Automatisierung chemischer Reaktoren. Dissertation, Univer- sity of Hannover