Cytotechnology 9: 231-236, 1992. 9 1992Kluwer Academic Publishers. Printed in the Netherlands.
Process development for the production of recombinant antibodies using the glutamine synthetase (GS) system M.E. Brown, G. Renner, R.P. Field and T. Hassell Celltech Ltd. 216 Bath Road, Slough, Berks. SL1 4EN, UK Received 2 April 1992; accepted in revised form 22 October 1992
Key words: recombinant antibodies, glutamine synthetase, process development, cell line stability
Introduction Recombinant antibodies are becoming increasingly useful therapeutic agents. The antigenic determinants of murine antibodies can be chimerised or grafted on to human constant regions in an effort to reduce the antigenicity or immunogenicity of the molecule without loss of binding (Sahag a n e t al., 1986). Antibody fragments can in addition be constructed to further enhance in vivo functionality (Skerra et al., 1991) and the overall technology so employed can be used to manipulate the re-expression of routine or human antibodies into alternative host cells. This might be to improve the product yield or to utilise a host cell type that is more acceptable to the regulatory authorities. Invertebrate cell types, bacteria, the bakers' yeast S. cerevisiae and mammalian cells have been used for the expression of recombinant antibodies, although the latter are currently the method of choice since post translational modifications such as glycosylation, fully functional in mammalian cells, are believed to be important for in vivo. activity (Goochee and Monica, 1990). A variety of expression systems have been employed to create mammalian cell lines producing recombinant proteins. A double transfection system was developed where the light chain gene, coupled to the gene for G418 resistance, was first
transfected into the host cells. Transfectants were selected in the presence of G418 and screened for light chain expression. In a second step, positive lines were further transfected with the gene for the antibody heavy chain coupled to the gene encoding for resistance to mycophenolic acid (MPA). The resultant co-transfectants expressed intact functional antibody. Developments to improve product titres have included gene amplification with the dhfr system wood et al., 1990). Target genes are ligated to the gene for dihydrofolate reductase and transfected into dhfr negative cell types. Transfectants are selected in the presence of the specific dhfr inhibitor, methotrexate (MTX). Increased levels of MTX result in amplification of the dhfr sequence and associated target genes located in the host cell chromosome. Increased production of the target gene is then reported (Page and Sydenham, 1991).
The GS expression system Gene amplification has more recently been achieved through the glutamine synthetase (GS) system (Cockett et al., 1990). Glutamine synthetase catalyses the synthesis of glutamine in the presence of Mg 2+, from glutamate and ammonia. The natural function of this gene is to provide
232 glutamine for protein biosynthesis (Meister, 1980) and the enzyme is specifically inhibited by methionine sulphoximine (MSX). The system consists of a GS expression vector cassette containing the DNA encoding both the antibody heavy and light chains driven by the powerful hCMV promoter regions. The gene for glutamine synthetase, derived from Chinese Hamster Ovary (CHO) cells, is also inserted on the vector under the control of the weak SV40 promotor to act both as a selectable marker and the means to achieve gene amplification. Certain murine myeloma cell types, NS0 or Agl4, are phenotypically auxotrophic for glutamine - they cannot ~ o w in glutamine deficient medium. When transfected by vectors containing GS genes however, glutamine independence is conferred and thus transfectants can be selected in glutamine-free medium. In contrast, CHO cells possess endogenous glutamine synthetase activity which must be specifically inhibited by low levels of MSX (20 p.M) to ensure that any transfectants that emerge on glutamine deficient medium possess the target genes of interest. Gene amplification can be achieved by further application of MSX - usually at levels between 100-500 laM for CHO cells, 10-100 p.M for the NS0 cell type. Unlike other amplification systems such as dhfr, a single round of amplification is sufficient to achieve efficient expression - further rounds do not seem to result in higher product titres. Copy number typically increases from 20 to approximately 200 copies in CHO cells and from 1 to 4-10 copies in NS0 cells (Bebbington et al., 1992). The use of GS systems can therefore rapidly achieve efficient expression and high product titres. Timescales to produce new antibodies are short, typically from cDNA to cell lines expressing 200--300 mg/1 in shake flask culture in 4-5 months.
duction of human therapeutics, a key point to consider early on in the process development phase is the stability of product accumulation. Appropriate stability needs to be demonstrated for at least 60 generations (cell doublings) beyond the Manufacturers Working Cell Bank to ensure sufficient time for inoculum expansion, the production process and at least a 20 generation margin for safety. In addition it is desirable to study stability both in the presence and absence of the selective drug thereby permitting greater flexibility in the eventual production process design. Elimination of the drug would reduce manufacturing costs, simplify the overall production process and alleviate the need to assay in final product. Experiments were constructed to study the genetic stability of recombinant antibody accumulation by serially passaging both CHO and NS0 cell lines over extended periods in glutamine free culture medium. The results are presented in Figs. 1 and 2. With the presence of 250 pM MSX in the culture medium, the CHO cell line stably secreted product for 35 generations although subsequent experiments have determined stability in excess of 60 generations thereby meeting production criteria. In contrast, cells grown in culture medium without added MSX lost the ability to
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Cell Generations
m
Stability of production In order to develop recombinant antibody processes effectively to a scale suitable for the pro-
With 250~M
MSX
Seleclion
Wilhout
MSX
Selection
Fig. 1. The effect of cell generation no. on product accumulation in GS-CHO cells. (Courtesy of Celltech Ltd.)
233
Product yield m
m
[..., o
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t
,o
r
i
,
2o Cell (~eneratr i
W i t h 10p.M M S X S e l e c t i o n
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+ Wilhoul MSX Selection
Fig. 2. The effect of cell generation no. on product accumulation
in GS-NS0 cells. (Courtesy of Celltech Ltd.) accumulate product after only 2 0 - 2 5 generations. It is presumed in this instance, that the presence of endogenous GS activity in the CHO cell line reduced the reliance on vector derived GS and hence encouraged instability. NS0 cells transfected with the gene for the production o f the same recombinant antibody were likewise subjected to serial passage over extended periods. In both the presence and absence of the selective drug, accumulation o f product was found to be stable over at least 60 generations in glutamine-free medium. Thus for this product, and for all others analysed to date in NS0 cell lines, the manufacturing process can be operated entirely in the absence of the selective drug MSX.
High product yields are critical to minimise production costs. Until recently the m a x i m u m titres obtained for recombinant antibodies were only in the range 5 0 - 1 0 0 mg/1 (Field e t al., 1990). This highlighted the need to improve titres to at least those achievable from murine hybridoma lines ( 1 5 0 - 5 0 0 mg/1). In addition with batch culture systems, it is also important to combine high product titre with minimal production times so as to ensure that production plant is under optimal usage. Table 1 summarises a comparative study into the yield potential of a number of different expression and culture systems for the production of a chimeric antibody. Broadly similar levels of effort were invested in developing optimal fed batch production processes for each system and to the selection of the highest producing cell clone. These values were compared with a murine hybridoma expressing the murine version o f the antibody in a similarly optimised fed-batch fermentation system. Initial work utilised the separate transfection o f heavy and light chain using G418 and M P A selection into the CHO-K1 host cell line. Such a system achieved maximal titres of 90 mg/1 at harvest compared with 200 mg/1 from the murine hybridoma. Calculations involving overall process times and known manufacturing plant turnround times determined the overall maximal process yield from such a system to be in the range 2 - 3 kg o f unprocessed product per 2000 litre fermenter each year. Assuming major
Table 1. Effect of expression system on yield of recombinant antibody in suspension culture
Expression system
Product
Maximum product titre (mg/I)
Process productivity (kg/2000 l/annum) prior to purification
Murine hybridoma CHO (G418/MPA) CHO (GS nonamplified) CHO (GS amplified) NS0 (GS amplified)
Murine antibody Chimeric antibody Chimeric antibody Chimeric antibody Chimeric antibody
200 90 110 195 585
7-9 2-3 3-5 7-9 15-20
234 Table 2. Yield of recombinant antibody from GS NS0 cell lines in suspension culture
Product
Amplification
Scale
Titre (mg/l)
Rec Mab 1
Amplified Amplified Amplified Amplified Nonamplified Nonamplified Nonamplified Amplified Amplified Nonamplified Amplified
Flask Fed batch fermenter Flask Fed batch fermenter Flask Batch fermenter Batch fermenter Batch fermenter Flask Flask Flask
300 585 503 895 398 440 180 1026 350 189 242
Rec Mab 3 Rec Mab 4 Rec Mab 5 Rec Mab 6 Rec Mab 7
influence on overall production costs to be fixed rather than readily variable, this expression system would potentially result in significantly higher unit costs when c o m p a r e d with production of the murine version ( 7 - 9 kg/2000 1/annum potential). Such a cost penalty might limit the c o m m e r cial utility of therapeutic antibodies. Expression of chimeric antibody in an unamplified GS vector transfectant into C H O - K 1 cells resulted in marginally i m p r o v e d titles o f 1 I0 rag/1 although an overall reduction in process duration resulted in the potential to perform a greater n u m b e r o f production runs per annum. Thus the potential overall process productivity for the chimeric antibody rose significantly to 3 - 5 kg per 2000 1 fermenter per annum. Amplification of the GS sequences using M S X further increased process titres to 195 mg/1 and an overall potential productivity of 7 - 9 kg/2000 l/annum - broadly equivalent to that of the murine antibody. The utilisation of a GS amplified vector in the murine m y e l o m a N S 0 resulted in a fed batch process capable o f producing nearly 600 mg/1 at harvest. Process productivity in the range 1 5 - 2 0 kg/2000 1 per a n n u m is thus possible - exceeding previous recombinant technology and rivalling or i m p r o v i n g upon unit costs o f the original hybridom a derived antibody. Product titres for r e c o m b i n a n t antibodies in excess of 1000 mg/l have n o w been observed for
amplified G S - N S 0 cultures (Table 2) and 560 mg/1 for amplified C H O culture.
Process scale up In order to achieve the a b o v e productivities in practice, the manufacturing process must scale up in a m a n n e r consistent with small scale operation without significant loss in product titre. Figure 3 depicts data f r o m scale up studies performed with G S - a m p l i f i e d N S 0 cell line producing a C D R grafted antibody.
7
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> 0
,
1
,00 u
Shake Flask 396 m ~
Time
i
rs
,t*
A
Development A L F
200 L P r o d u c t i o n A L F
484 rag/1
444 mgtl
Fig. 3. Scale up of GS-NS0 culture producing recombinant
antibody 4. (Courtesy of Celltech Ltd.)
235 In serum-flee shake flask culture, cell numbers of approximately 2 • 106 cells/ml were observed with peak product titre of 400 mg/1 reached in 300-350 hours. The identical process was scaled up to firstly a 100 1development airlift fermenters (ALF) and secondly a 200 1 production vessel within the GMP manufacturing plant. Essentially similar cell concentrations, process lengths and overall product titres were observed at all scales. These data would suggest that further scale up to 2000 1 would not result in significant problems or product losses. Kilogram quantities of recombinant antibodies at commercially useful costs are therefore a practical proposition. FQ. 4. Process improvements for GS-NS0 producing chimeric antibody. (Courtesy of Celltech Ltd.)
Process optimisation for GS-NSO The high product titres achieved from GS-NS0 culture are not just believed to be the result of the intrinsic productivity of the cell line but also the result of extensive process optimisation. The process optimisation campaign was initiated by removal and replacement of the foetal bovine serum. The medium is further analysed and tailored to match the principal energy and other metabolic requirements of the cell in an iterative cycle of improvements. Such optimisation resulted in substantial improvements in process titres (Fig. 4). Upon initial amplification, expression of chimeric antibody from a GS-NS0 in GDMEM + 10% FBS resulted in titres of only 40-50 mg/1 in 250-300 hours. An iterative series of medium development resulted in a third major reformulation and product titres of over 300 mg/1 achieved within a 300-hour fermentation time. This represented a 6-7 fold improvement over the initial results after transfection. The utilisation of fed batch culture techniques (where further small volumes of flesh nutrients were supplied to the culture during the growth phase) resulted in further improvements in titre. Values of 560-585 mg/l were observed, again in 300 hours, representing a 13-14 fold improvement over the initial results.
Fig. 5. The effect of batch feeding on product titre in GS amplified NSO cultures. A) batch culture: B) fed-batch culture. (Courtesy of Celltech Ltd.)
236 The effect of process feeding is illustrated in detail in Fig. 5. In un-fed culture, peak product accumulation of 300 rag/1 occurred in the decline phase of the culture whilst cultures that were fed with nutrients in the growth phase achieved significantly higher biomass levels and accumulated product titres. Importantly, the increased titre was obtained at the same elapsed time as the un-fed culture and therefore represented a real 1.9 fold improvement in manufacturing plant productivity.
Conclusions GS technology has been successfully utilised for the expression of recombinant antibodies. The vectors are capable of stable product accumulation over the extended periods required for large scale manufacture. Large scale processes have been developed giving extremely high antibody yields up to, and in one instance in excess of 1 J l . It is clear that such systems represent a significant advance over previous technology and will speed the commercial application of such therapeutic products.
References Bebbington CR, Renner G, Thomson S, King D, Abrams D and Yarranton GT (1992) High level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/Technology 10: 169-175. Cockett MI, Bebbington CR and Yarranton GT (1990) High level expression of tissue inhibitor of metalloproteinases in chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technology 8: 662-667. Field RP, Brand H, Renner GL, Robertson HA, and Boraston RC (1990) Production of a chimeric antibody for tumour imaging and therapy from chinese hamster ovary (CHO) and myeloma cells. In: Production of Biologicals from Animal Cells in Culture. (pp. 742-744) Spier RE, Griffith JB and Meignier B. (Eds.), Butterworth-Heinemann, Oxford. Goochee CF and Monica T (1990) Environmental effects on protein glycosylation. Bio/Technology 8: 421-426. Meister A (1980) Enzymology and regulation In: Glutamine: Metabolism, Enzymology and Regulation. (pp 1-40). Mora J and Palacios R (Eds.), Academic Press, New York. Page MJ and Sydenham MA (1991) High level expression of the humanised monoclonal antibody campath-1H in chinese hamster ovary cells. Bio/Technology 9: 64-68. Sahagan BG, Dorai H, Saltzgaber-Muller J, Toneguzzo F, Guindon CA, Lilly SP, McDonald KW, Morrissey DV, Stone BA. Davis GL, Mclntosh PK and Moore GP (1986) A genetically engineered murine/human chimeric antibody retains specificity for human turnout-associated antigen. J. Immunol. 137: 1066-1074. Skerra A, Pfitzinger I and Pluckthun A (1991) The functional expression of antibody Fv fragments in E. coli: Improved vectors and a generally applicable purification technique. Bio/Technology 9: 273-276. Wood CR, Domer AJ, Morris GE, Alderman EM, Wilson D, O'Hara RM and Kaufman R (1990) High level synthesis of immunoglobulins in chinese hamster ovary cells. J. lmmunol. 145:3011-3016.
Process development for the production of recombinant antibodies using the glutamine synthetase (GS) system M.E. Brown, G. Renner, R.P. Field and T. Hassell Celltech Ltd. 216 Bath Road, Slough, Berks. SL1 4EN, UK Received 2 April 1992; accepted in revised form 22 October 1992
Key words: recombinant antibodies, glutamine synthetase, process development, cell line stability
Introduction Recombinant antibodies are becoming increasingly useful therapeutic agents. The antigenic determinants of murine antibodies can be chimerised or grafted on to human constant regions in an effort to reduce the antigenicity or immunogenicity of the molecule without loss of binding (Sahag a n e t al., 1986). Antibody fragments can in addition be constructed to further enhance in vivo functionality (Skerra et al., 1991) and the overall technology so employed can be used to manipulate the re-expression of routine or human antibodies into alternative host cells. This might be to improve the product yield or to utilise a host cell type that is more acceptable to the regulatory authorities. Invertebrate cell types, bacteria, the bakers' yeast S. cerevisiae and mammalian cells have been used for the expression of recombinant antibodies, although the latter are currently the method of choice since post translational modifications such as glycosylation, fully functional in mammalian cells, are believed to be important for in vivo. activity (Goochee and Monica, 1990). A variety of expression systems have been employed to create mammalian cell lines producing recombinant proteins. A double transfection system was developed where the light chain gene, coupled to the gene for G418 resistance, was first
transfected into the host cells. Transfectants were selected in the presence of G418 and screened for light chain expression. In a second step, positive lines were further transfected with the gene for the antibody heavy chain coupled to the gene encoding for resistance to mycophenolic acid (MPA). The resultant co-transfectants expressed intact functional antibody. Developments to improve product titres have included gene amplification with the dhfr system wood et al., 1990). Target genes are ligated to the gene for dihydrofolate reductase and transfected into dhfr negative cell types. Transfectants are selected in the presence of the specific dhfr inhibitor, methotrexate (MTX). Increased levels of MTX result in amplification of the dhfr sequence and associated target genes located in the host cell chromosome. Increased production of the target gene is then reported (Page and Sydenham, 1991).
The GS expression system Gene amplification has more recently been achieved through the glutamine synthetase (GS) system (Cockett et al., 1990). Glutamine synthetase catalyses the synthesis of glutamine in the presence of Mg 2+, from glutamate and ammonia. The natural function of this gene is to provide
232 glutamine for protein biosynthesis (Meister, 1980) and the enzyme is specifically inhibited by methionine sulphoximine (MSX). The system consists of a GS expression vector cassette containing the DNA encoding both the antibody heavy and light chains driven by the powerful hCMV promoter regions. The gene for glutamine synthetase, derived from Chinese Hamster Ovary (CHO) cells, is also inserted on the vector under the control of the weak SV40 promotor to act both as a selectable marker and the means to achieve gene amplification. Certain murine myeloma cell types, NS0 or Agl4, are phenotypically auxotrophic for glutamine - they cannot ~ o w in glutamine deficient medium. When transfected by vectors containing GS genes however, glutamine independence is conferred and thus transfectants can be selected in glutamine-free medium. In contrast, CHO cells possess endogenous glutamine synthetase activity which must be specifically inhibited by low levels of MSX (20 p.M) to ensure that any transfectants that emerge on glutamine deficient medium possess the target genes of interest. Gene amplification can be achieved by further application of MSX - usually at levels between 100-500 laM for CHO cells, 10-100 p.M for the NS0 cell type. Unlike other amplification systems such as dhfr, a single round of amplification is sufficient to achieve efficient expression - further rounds do not seem to result in higher product titres. Copy number typically increases from 20 to approximately 200 copies in CHO cells and from 1 to 4-10 copies in NS0 cells (Bebbington et al., 1992). The use of GS systems can therefore rapidly achieve efficient expression and high product titres. Timescales to produce new antibodies are short, typically from cDNA to cell lines expressing 200--300 mg/1 in shake flask culture in 4-5 months.
duction of human therapeutics, a key point to consider early on in the process development phase is the stability of product accumulation. Appropriate stability needs to be demonstrated for at least 60 generations (cell doublings) beyond the Manufacturers Working Cell Bank to ensure sufficient time for inoculum expansion, the production process and at least a 20 generation margin for safety. In addition it is desirable to study stability both in the presence and absence of the selective drug thereby permitting greater flexibility in the eventual production process design. Elimination of the drug would reduce manufacturing costs, simplify the overall production process and alleviate the need to assay in final product. Experiments were constructed to study the genetic stability of recombinant antibody accumulation by serially passaging both CHO and NS0 cell lines over extended periods in glutamine free culture medium. The results are presented in Figs. 1 and 2. With the presence of 250 pM MSX in the culture medium, the CHO cell line stably secreted product for 35 generations although subsequent experiments have determined stability in excess of 60 generations thereby meeting production criteria. In contrast, cells grown in culture medium without added MSX lost the ability to
R III U
I
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0 + 0
n
+mR
++
++ t~+ ~
n +
9
n
o
o
n
II
"
a o
"U
n
§ + +
+
t
I
10
§
20
i 30
.
Cell Generations
m
Stability of production In order to develop recombinant antibody processes effectively to a scale suitable for the pro-
With 250~M
MSX
Seleclion
Wilhout
MSX
Selection
Fig. 1. The effect of cell generation no. on product accumulation in GS-CHO cells. (Courtesy of Celltech Ltd.)
233
Product yield m
m
[..., o
.,~
o
_ _ 1
t
,o
r
i
,
2o Cell (~eneratr i
W i t h 10p.M M S X S e l e c t i o n
~,
,0
+ Wilhoul MSX Selection
Fig. 2. The effect of cell generation no. on product accumulation
in GS-NS0 cells. (Courtesy of Celltech Ltd.) accumulate product after only 2 0 - 2 5 generations. It is presumed in this instance, that the presence of endogenous GS activity in the CHO cell line reduced the reliance on vector derived GS and hence encouraged instability. NS0 cells transfected with the gene for the production o f the same recombinant antibody were likewise subjected to serial passage over extended periods. In both the presence and absence of the selective drug, accumulation o f product was found to be stable over at least 60 generations in glutamine-free medium. Thus for this product, and for all others analysed to date in NS0 cell lines, the manufacturing process can be operated entirely in the absence of the selective drug MSX.
High product yields are critical to minimise production costs. Until recently the m a x i m u m titres obtained for recombinant antibodies were only in the range 5 0 - 1 0 0 mg/1 (Field e t al., 1990). This highlighted the need to improve titres to at least those achievable from murine hybridoma lines ( 1 5 0 - 5 0 0 mg/1). In addition with batch culture systems, it is also important to combine high product titre with minimal production times so as to ensure that production plant is under optimal usage. Table 1 summarises a comparative study into the yield potential of a number of different expression and culture systems for the production of a chimeric antibody. Broadly similar levels of effort were invested in developing optimal fed batch production processes for each system and to the selection of the highest producing cell clone. These values were compared with a murine hybridoma expressing the murine version o f the antibody in a similarly optimised fed-batch fermentation system. Initial work utilised the separate transfection o f heavy and light chain using G418 and M P A selection into the CHO-K1 host cell line. Such a system achieved maximal titres of 90 mg/1 at harvest compared with 200 mg/1 from the murine hybridoma. Calculations involving overall process times and known manufacturing plant turnround times determined the overall maximal process yield from such a system to be in the range 2 - 3 kg o f unprocessed product per 2000 litre fermenter each year. Assuming major
Table 1. Effect of expression system on yield of recombinant antibody in suspension culture
Expression system
Product
Maximum product titre (mg/I)
Process productivity (kg/2000 l/annum) prior to purification
Murine hybridoma CHO (G418/MPA) CHO (GS nonamplified) CHO (GS amplified) NS0 (GS amplified)
Murine antibody Chimeric antibody Chimeric antibody Chimeric antibody Chimeric antibody
200 90 110 195 585
7-9 2-3 3-5 7-9 15-20
234 Table 2. Yield of recombinant antibody from GS NS0 cell lines in suspension culture
Product
Amplification
Scale
Titre (mg/l)
Rec Mab 1
Amplified Amplified Amplified Amplified Nonamplified Nonamplified Nonamplified Amplified Amplified Nonamplified Amplified
Flask Fed batch fermenter Flask Fed batch fermenter Flask Batch fermenter Batch fermenter Batch fermenter Flask Flask Flask
300 585 503 895 398 440 180 1026 350 189 242
Rec Mab 3 Rec Mab 4 Rec Mab 5 Rec Mab 6 Rec Mab 7
influence on overall production costs to be fixed rather than readily variable, this expression system would potentially result in significantly higher unit costs when c o m p a r e d with production of the murine version ( 7 - 9 kg/2000 1/annum potential). Such a cost penalty might limit the c o m m e r cial utility of therapeutic antibodies. Expression of chimeric antibody in an unamplified GS vector transfectant into C H O - K 1 cells resulted in marginally i m p r o v e d titles o f 1 I0 rag/1 although an overall reduction in process duration resulted in the potential to perform a greater n u m b e r o f production runs per annum. Thus the potential overall process productivity for the chimeric antibody rose significantly to 3 - 5 kg per 2000 1 fermenter per annum. Amplification of the GS sequences using M S X further increased process titres to 195 mg/1 and an overall potential productivity of 7 - 9 kg/2000 l/annum - broadly equivalent to that of the murine antibody. The utilisation of a GS amplified vector in the murine m y e l o m a N S 0 resulted in a fed batch process capable o f producing nearly 600 mg/1 at harvest. Process productivity in the range 1 5 - 2 0 kg/2000 1 per a n n u m is thus possible - exceeding previous recombinant technology and rivalling or i m p r o v i n g upon unit costs o f the original hybridom a derived antibody. Product titres for r e c o m b i n a n t antibodies in excess of 1000 mg/l have n o w been observed for
amplified G S - N S 0 cultures (Table 2) and 560 mg/1 for amplified C H O culture.
Process scale up In order to achieve the a b o v e productivities in practice, the manufacturing process must scale up in a m a n n e r consistent with small scale operation without significant loss in product titre. Figure 3 depicts data f r o m scale up studies performed with G S - a m p l i f i e d N S 0 cell line producing a C D R grafted antibody.
7
5 '~ _.o
> 0
,
1
,00 u
Shake Flask 396 m ~
Time
i
rs
,t*
A
Development A L F
200 L P r o d u c t i o n A L F
484 rag/1
444 mgtl
Fig. 3. Scale up of GS-NS0 culture producing recombinant
antibody 4. (Courtesy of Celltech Ltd.)
235 In serum-flee shake flask culture, cell numbers of approximately 2 • 106 cells/ml were observed with peak product titre of 400 mg/1 reached in 300-350 hours. The identical process was scaled up to firstly a 100 1development airlift fermenters (ALF) and secondly a 200 1 production vessel within the GMP manufacturing plant. Essentially similar cell concentrations, process lengths and overall product titres were observed at all scales. These data would suggest that further scale up to 2000 1 would not result in significant problems or product losses. Kilogram quantities of recombinant antibodies at commercially useful costs are therefore a practical proposition. FQ. 4. Process improvements for GS-NS0 producing chimeric antibody. (Courtesy of Celltech Ltd.)
Process optimisation for GS-NSO The high product titres achieved from GS-NS0 culture are not just believed to be the result of the intrinsic productivity of the cell line but also the result of extensive process optimisation. The process optimisation campaign was initiated by removal and replacement of the foetal bovine serum. The medium is further analysed and tailored to match the principal energy and other metabolic requirements of the cell in an iterative cycle of improvements. Such optimisation resulted in substantial improvements in process titres (Fig. 4). Upon initial amplification, expression of chimeric antibody from a GS-NS0 in GDMEM + 10% FBS resulted in titres of only 40-50 mg/1 in 250-300 hours. An iterative series of medium development resulted in a third major reformulation and product titres of over 300 mg/1 achieved within a 300-hour fermentation time. This represented a 6-7 fold improvement over the initial results after transfection. The utilisation of fed batch culture techniques (where further small volumes of flesh nutrients were supplied to the culture during the growth phase) resulted in further improvements in titre. Values of 560-585 mg/l were observed, again in 300 hours, representing a 13-14 fold improvement over the initial results.
Fig. 5. The effect of batch feeding on product titre in GS amplified NSO cultures. A) batch culture: B) fed-batch culture. (Courtesy of Celltech Ltd.)
236 The effect of process feeding is illustrated in detail in Fig. 5. In un-fed culture, peak product accumulation of 300 rag/1 occurred in the decline phase of the culture whilst cultures that were fed with nutrients in the growth phase achieved significantly higher biomass levels and accumulated product titres. Importantly, the increased titre was obtained at the same elapsed time as the un-fed culture and therefore represented a real 1.9 fold improvement in manufacturing plant productivity.
Conclusions GS technology has been successfully utilised for the expression of recombinant antibodies. The vectors are capable of stable product accumulation over the extended periods required for large scale manufacture. Large scale processes have been developed giving extremely high antibody yields up to, and in one instance in excess of 1 J l . It is clear that such systems represent a significant advance over previous technology and will speed the commercial application of such therapeutic products.
References Bebbington CR, Renner G, Thomson S, King D, Abrams D and Yarranton GT (1992) High level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Bio/Technology 10: 169-175. Cockett MI, Bebbington CR and Yarranton GT (1990) High level expression of tissue inhibitor of metalloproteinases in chinese hamster ovary cells using glutamine synthetase gene amplification. Bio/Technology 8: 662-667. Field RP, Brand H, Renner GL, Robertson HA, and Boraston RC (1990) Production of a chimeric antibody for tumour imaging and therapy from chinese hamster ovary (CHO) and myeloma cells. In: Production of Biologicals from Animal Cells in Culture. (pp. 742-744) Spier RE, Griffith JB and Meignier B. (Eds.), Butterworth-Heinemann, Oxford. Goochee CF and Monica T (1990) Environmental effects on protein glycosylation. Bio/Technology 8: 421-426. Meister A (1980) Enzymology and regulation In: Glutamine: Metabolism, Enzymology and Regulation. (pp 1-40). Mora J and Palacios R (Eds.), Academic Press, New York. Page MJ and Sydenham MA (1991) High level expression of the humanised monoclonal antibody campath-1H in chinese hamster ovary cells. Bio/Technology 9: 64-68. Sahagan BG, Dorai H, Saltzgaber-Muller J, Toneguzzo F, Guindon CA, Lilly SP, McDonald KW, Morrissey DV, Stone BA. Davis GL, Mclntosh PK and Moore GP (1986) A genetically engineered murine/human chimeric antibody retains specificity for human turnout-associated antigen. J. Immunol. 137: 1066-1074. Skerra A, Pfitzinger I and Pluckthun A (1991) The functional expression of antibody Fv fragments in E. coli: Improved vectors and a generally applicable purification technique. Bio/Technology 9: 273-276. Wood CR, Domer AJ, Morris GE, Alderman EM, Wilson D, O'Hara RM and Kaufman R (1990) High level synthesis of immunoglobulins in chinese hamster ovary cells. J. lmmunol. 145:3011-3016.
