Cytotechnology 7: 33-38, 1991. 9 1991 KluwerAcademic Publishers. Printed in the Netherlands.
Hybridoma growth and monoclonal antibody production in iron-rich protein,free medium: Effect of nutrient concentration Franti~ek Fran~k and Jana Dotm'kov~i Department o f Fundamental Cytotechnology, Institute of Molecular Genetics, Czechoslovak Academy of Sciences, CS-14220 Praha 4, Czechoslovakia Received 15 July 1991; acceptedin revised form 9 August 1991
Key words: mouse-mouse hybridoma, protein-free supplement, basal medium, apoptosis, bioreactor culture Abstract
The iron-rich (500 g M ferric citrate) protein-free supplement was added to six different basal media. Cell growth and monoclonal antibody production of a mouse-mouse hybridoma were investigated in 1.3 1 batch cultures performed in a laboratory bioreactor with automatic control of pH and dissolved oxygen concentration. RPMI 1640 served as the control medium: Fortification of the basal medium by balanced mixtures of amino acids and vitamins showed higher positive effect than daily supplementation by glucose and glutamine. Strongly fortified medium, based on RPMI 1640, was found superior to other basal media. The viability index increased by a factor of 3.04 and the total antibody production by a factor of 2.82, relative to the control.
Introduction
The development of various protein-free formulations omitting both serum and purified proteins in the culture medium represents a fundamental innovation in hybridoma culture mad in monoc!onal antibody production. Protein-free media opens the way to qualitatively higher exploration of hybridoma cell physiology and biochemistry, because the culture fluid is devoid of background external macromolecules. The employment of protein-free media in monoclonal antibody production contributes to the economy of the production process and to the biosafety of the product. The iron-rich (500 I.tM ferric citrate) proteinfree medium supplement developed in this laboratory ( K o v ~ and Fran~k, 1987) enables storage
of cells adapted to protein-free growth in frozen state. Starting from cryopreserved vials, the hybridoma clone is propagated for large-scale bioreactor culture in absence of external proteins. Adapted cells may be easily re-cloned without the necessity of adding serum or proteins. Whereas the RPMI 1640 basal medium was found sufficient for stationary cultures ( K o v ~ and Fran~k, 1987), the achievement of high cell densities in spinner flasks or in a stirred bioreactor requires basal medium of higher concentration of nutrients and vitamins. RPMI 1640 was enriched with glucose, glutamine, essential amino acids and vitamins, or combined with DMEM (1:1) (Fran~k et al., 1990; Fran~k and Dolm'kov~i, 1991). The aim of this work was to investigate the effect of increased concentrations of nutrients
34 on growth and antibody secretion in a stirred suspension culture carried out in the protein-free iron-rich medium.
Materials and methods Cell line
A mouse-mouse hybridoma TSH-5.07 producing an IgG1 kappa antibody to human thyrotropin was used in this work. This hybridoma has been generated in this laboratory with the use of the fusion partner Sp2/0-Ag 14.
The viability was determined by the trypan blue exclusion test. The number of apoptotic particles was counted in the Burker chamber after treatment of the cell suspension by 6 M guanidinium hydrochloride (Piacentini and Fesus, 1990). The viability index was calculated from viable cell counts according to Luan et al. (1987). The cells were grown first in T-flasks in the medium R, then transferred to the appropriate medium in a spinner flask (650 ml culture volume), and the grown inoculum was seeded in the bioreactor (280 to 310 x 103 m1-1 initial cell density). Fresh medium was added to the final volume 1.3 1. All batch cultures were performed at least in duplicate.
Cell culture Cell culture reactor
The protein-free supplement containing ferric citrate (500 gM), ethanolamine (20 p-M), ascorbic acid (20 p,M), hydrocortisone (5 nM) and salts of several trace elements (Kov~: and Fran~k, 1987) was added to various basalmedia together with Hepes (2 g/l), sodium bicarbonate (3.75 g/l), sodium pyruvate (110 mg/l) and gentamycine (50 mg/l). Composition of the basal media: R: Control basal medium RPMI 1640. RAV: RPMI 1640 supplemented with additional glutamine (300 mg]l), additional glucose (2 g/l), MEM essential amino acids, and MEM vitamins. RAVGlc: Medium R A V fortified by daily additions of glucose (2 g/l). RAVGln: Medium RAV fortified by daily additions of glutamine (300 rag/l). RAVGIcn: Medium R A V fortified by daily additions of glucose (2 g/I) and glutamine (300 mg/1). R forte: Strongly fortified medium based on RPMI 1640 according to Jo et al. (1990).
All media and extra medium components were from Sevac (Prague). The cells were counted in the Burker chamber.
The laboratory bioreactor LF-2 (Workshops of the Czechoslovak Academy of Sciences) was equipped with a blade stirrer rotating at 34 r.p.m. The values of pH, redox potential, and dissolved oxygen concentration were monitored continuously. The pH of the culture was kept automatically at 7.2 by gaseous CO2 introduced in the headspace or by sodium bicarbonate solution. Automatic control of dissolved oxygen concentration (40% air saturation) was achieved by flushing the headspace with oxygen. The temperature of the bioreactor culture was kept at 35~
Sample analyses
The antibody concentration was determined using an enzyme-linked immunosorbent assay (ELISA) in samples diluted 200 times or 500 times. Purified TSH-5.07 antibody obtained by successive liquid chromatography steps (Fran~k and Dolnfkov~i, 1991) served as IgG1 standard. Peroxidase-conjugated pig anti-mouse immunoglobulin (Sevac, Prague) was used for antibody quantitation. Concentration of glucose was measured using the enzyme analyzer EA-104E (Chemoproject, Prague).
35
Results Cell growth and viability The effect of the composition of the basal medium was investigated in batch cultures (1.3 1) carried out under standard conditions specified in detail in the section Methods. Cell growth in the control medium R was found to be rather slow (Fig. 1, Table 1). Fortification by additional essential amino acids and vitamins (medium R A V ) brought about a significant enhancement of growth and viability. On the other hand, further daily supplements of glucose and/or glutamine displayed only a moderate additional effect on specific growth rate and on maximum cell count. Supplements of glucose alone led to
marked decrease of viability beginning the third day of the culture. The growth in the medium R forte was characterized by the highest cell densities, both total and viable, even though the specific growth rate Ix was in the same range as in the media derived from R A V . W h e n the culture in R forte was kept till 165 h, the total cell density reached 2.45 • 106 m1-1, i.e. 2.19 times the maximum total cell density obtained in the medium R. The difference was even more striking with the maximum viable cell density, that was 2.95 times higher than that in the control medium. The counts of apoptotic particles showed a dramatic difference between the culture in the control medium R and in all other media. Whereas in the control culture about one third of the cell 100 I
75
+
2.5
T
R
"
x RAV [] R forte ~
,.
"
•
", \
a
RAVGIc
v
RAVGIn
o
RAVG
,
50 ,
",, u Icn
"--
.50
'T r
......,....."'
"' ..
-40
. 1.5 q~
3O
d-
[] ....
~J r~
25
",~
2.0
1.0
" -4.,I
E
......
X.,.,..
20 9
"'" " ' . . , . . J
o
I~ 0 . 5
W. . . . . .
0 O
3'0
6'0
9'0
I
120 //6 Time
~7.............. V"............ V
3'0
6'0
9'0
, h
Fig. 1. Growth and viability of TSH-5.07 hybridomacells in various basal media with protein-free supplement.
lio
910 0
0
36
Table 1. Growth and production parameters of hybridoma TSH-5.07 cultured in various media Parameter
Medium R
Specific growth rate, bt (h-1) Apoptotic particles (% of total cell count) 92 h 116 h Maximum viable cell density (x 10-3) (cell/ml)
RAV 0.009
0.023
RAVGlc 0.026
RAVGIn 0.027
RAVGlcn 0.029
0.028
26 36
Hybridoma growth and monoclonal antibody production in iron-rich protein,free medium: Effect of nutrient concentration Franti~ek Fran~k and Jana Dotm'kov~i Department o f Fundamental Cytotechnology, Institute of Molecular Genetics, Czechoslovak Academy of Sciences, CS-14220 Praha 4, Czechoslovakia Received 15 July 1991; acceptedin revised form 9 August 1991
Key words: mouse-mouse hybridoma, protein-free supplement, basal medium, apoptosis, bioreactor culture Abstract
The iron-rich (500 g M ferric citrate) protein-free supplement was added to six different basal media. Cell growth and monoclonal antibody production of a mouse-mouse hybridoma were investigated in 1.3 1 batch cultures performed in a laboratory bioreactor with automatic control of pH and dissolved oxygen concentration. RPMI 1640 served as the control medium: Fortification of the basal medium by balanced mixtures of amino acids and vitamins showed higher positive effect than daily supplementation by glucose and glutamine. Strongly fortified medium, based on RPMI 1640, was found superior to other basal media. The viability index increased by a factor of 3.04 and the total antibody production by a factor of 2.82, relative to the control.
Introduction
The development of various protein-free formulations omitting both serum and purified proteins in the culture medium represents a fundamental innovation in hybridoma culture mad in monoc!onal antibody production. Protein-free media opens the way to qualitatively higher exploration of hybridoma cell physiology and biochemistry, because the culture fluid is devoid of background external macromolecules. The employment of protein-free media in monoclonal antibody production contributes to the economy of the production process and to the biosafety of the product. The iron-rich (500 I.tM ferric citrate) proteinfree medium supplement developed in this laboratory ( K o v ~ and Fran~k, 1987) enables storage
of cells adapted to protein-free growth in frozen state. Starting from cryopreserved vials, the hybridoma clone is propagated for large-scale bioreactor culture in absence of external proteins. Adapted cells may be easily re-cloned without the necessity of adding serum or proteins. Whereas the RPMI 1640 basal medium was found sufficient for stationary cultures ( K o v ~ and Fran~k, 1987), the achievement of high cell densities in spinner flasks or in a stirred bioreactor requires basal medium of higher concentration of nutrients and vitamins. RPMI 1640 was enriched with glucose, glutamine, essential amino acids and vitamins, or combined with DMEM (1:1) (Fran~k et al., 1990; Fran~k and Dolm'kov~i, 1991). The aim of this work was to investigate the effect of increased concentrations of nutrients
34 on growth and antibody secretion in a stirred suspension culture carried out in the protein-free iron-rich medium.
Materials and methods Cell line
A mouse-mouse hybridoma TSH-5.07 producing an IgG1 kappa antibody to human thyrotropin was used in this work. This hybridoma has been generated in this laboratory with the use of the fusion partner Sp2/0-Ag 14.
The viability was determined by the trypan blue exclusion test. The number of apoptotic particles was counted in the Burker chamber after treatment of the cell suspension by 6 M guanidinium hydrochloride (Piacentini and Fesus, 1990). The viability index was calculated from viable cell counts according to Luan et al. (1987). The cells were grown first in T-flasks in the medium R, then transferred to the appropriate medium in a spinner flask (650 ml culture volume), and the grown inoculum was seeded in the bioreactor (280 to 310 x 103 m1-1 initial cell density). Fresh medium was added to the final volume 1.3 1. All batch cultures were performed at least in duplicate.
Cell culture Cell culture reactor
The protein-free supplement containing ferric citrate (500 gM), ethanolamine (20 p-M), ascorbic acid (20 p,M), hydrocortisone (5 nM) and salts of several trace elements (Kov~: and Fran~k, 1987) was added to various basalmedia together with Hepes (2 g/l), sodium bicarbonate (3.75 g/l), sodium pyruvate (110 mg/l) and gentamycine (50 mg/l). Composition of the basal media: R: Control basal medium RPMI 1640. RAV: RPMI 1640 supplemented with additional glutamine (300 mg]l), additional glucose (2 g/l), MEM essential amino acids, and MEM vitamins. RAVGlc: Medium R A V fortified by daily additions of glucose (2 g/l). RAVGln: Medium RAV fortified by daily additions of glutamine (300 rag/l). RAVGIcn: Medium R A V fortified by daily additions of glucose (2 g/I) and glutamine (300 mg/1). R forte: Strongly fortified medium based on RPMI 1640 according to Jo et al. (1990).
All media and extra medium components were from Sevac (Prague). The cells were counted in the Burker chamber.
The laboratory bioreactor LF-2 (Workshops of the Czechoslovak Academy of Sciences) was equipped with a blade stirrer rotating at 34 r.p.m. The values of pH, redox potential, and dissolved oxygen concentration were monitored continuously. The pH of the culture was kept automatically at 7.2 by gaseous CO2 introduced in the headspace or by sodium bicarbonate solution. Automatic control of dissolved oxygen concentration (40% air saturation) was achieved by flushing the headspace with oxygen. The temperature of the bioreactor culture was kept at 35~
Sample analyses
The antibody concentration was determined using an enzyme-linked immunosorbent assay (ELISA) in samples diluted 200 times or 500 times. Purified TSH-5.07 antibody obtained by successive liquid chromatography steps (Fran~k and Dolnfkov~i, 1991) served as IgG1 standard. Peroxidase-conjugated pig anti-mouse immunoglobulin (Sevac, Prague) was used for antibody quantitation. Concentration of glucose was measured using the enzyme analyzer EA-104E (Chemoproject, Prague).
35
Results Cell growth and viability The effect of the composition of the basal medium was investigated in batch cultures (1.3 1) carried out under standard conditions specified in detail in the section Methods. Cell growth in the control medium R was found to be rather slow (Fig. 1, Table 1). Fortification by additional essential amino acids and vitamins (medium R A V ) brought about a significant enhancement of growth and viability. On the other hand, further daily supplements of glucose and/or glutamine displayed only a moderate additional effect on specific growth rate and on maximum cell count. Supplements of glucose alone led to
marked decrease of viability beginning the third day of the culture. The growth in the medium R forte was characterized by the highest cell densities, both total and viable, even though the specific growth rate Ix was in the same range as in the media derived from R A V . W h e n the culture in R forte was kept till 165 h, the total cell density reached 2.45 • 106 m1-1, i.e. 2.19 times the maximum total cell density obtained in the medium R. The difference was even more striking with the maximum viable cell density, that was 2.95 times higher than that in the control medium. The counts of apoptotic particles showed a dramatic difference between the culture in the control medium R and in all other media. Whereas in the control culture about one third of the cell 100 I
75
+
2.5
T
R
"
x RAV [] R forte ~
,.
"
•
", \
a
RAVGIc
v
RAVGIn
o
RAVG
,
50 ,
",, u Icn
"--
.50
'T r
......,....."'
"' ..
-40
. 1.5 q~
3O
d-
[] ....
~J r~
25
",~
2.0
1.0
" -4.,I
E
......
X.,.,..
20 9
"'" " ' . . , . . J
o
I~ 0 . 5
W. . . . . .
0 O
3'0
6'0
9'0
I
120 //6 Time
~7.............. V"............ V
3'0
6'0
9'0
, h
Fig. 1. Growth and viability of TSH-5.07 hybridomacells in various basal media with protein-free supplement.
lio
910 0
0
36
Table 1. Growth and production parameters of hybridoma TSH-5.07 cultured in various media Parameter
Medium R
Specific growth rate, bt (h-1) Apoptotic particles (% of total cell count) 92 h 116 h Maximum viable cell density (x 10-3) (cell/ml)
RAV 0.009
0.023
RAVGlc 0.026
RAVGIn 0.027
RAVGlcn 0.029
0.028
26 36
