WO1993013196A2

WO1993013196A2 - Method for increasing recombinant protein production

Info

Publication number: WO1993013196A2
Application number: PCT/US1992/011188
Authority: WO
Inventors: Debra A. Barngrover; John Mcpherson
Original assignee: Genzyme Corporation
Priority date: 1991-12-24
Filing date: 1992-12-23
Publication date: 1993-07-08
Also published as: WO1993013196A3

Abstract

Methods and compositions for increasing the production of recombinant proteins are provided. In one aspect, eukaryotic expression systems are cultured in media containing an appropriate amount of transforming growth factor-β. In another aspect, eukaryotic expression systems are cultured in glutamate-containing, essentially glutamine-free media.

Description

METHODS FOR INCREASING RECOMBINANT PROTEIN PRODUCTION

Description

Background of the Invention

Recombinant proteins are produced by introducing cloned DNA into an appropriate host cell line using an appropriate cloning vector, culturing the host cells, and recovering the expressed protein. Mitotically competent eukaryotic host cells (i.e. continuous cell lines) are typically utilized, because most bacterial cells can not perform the necessary folding and post-translational modifications (e.g. glycosylation. phosphorylation and proteolytic cleavage) that are required to produce most eukaryotic proteins.

Eukaryotic expression systems can be used to produce quantities of proteins that are normally available only in limited amounts from natural sources. To this end, it is desirable to employ techniques which optimize recombinant protein production. One technique for increasing the production of recombinant proteins from eukaryotic expression systems, is by including an enhancer element in the cloning vector. The presence of an enhancer increases transcription (i.e. increases the copy number of messenger RNA (mRNA)), and thereby increases the amount of recombinant protein expressed. This approach is described in U.S. Patent No. 4,663,281 , which issued May 5, 1987 to Gillies. A similar technique, described in

European Patent Publication No. EP 0 307 248 by Levinson and Mather, increases transcription in a eukaryotic expression system by incorporating an oncogene into the cloning vector.

in a further technique, known as "gene amplification", a drug resistance marker (e.g. the gene encoding dihydrofolate reductase (dhfr)) is included in the cloning vector, which is used to transfect a eukaryotic host cell line with a gene encoding a desired recombinant protein, or the gene encoding the desired recombinant protein and the drug resistance marker are cotransfected into host cells. When the cells carrying the two genes are exposed to increased concentrations of the appropriate drug (e.g. methotrexate), sublines in which the number of copies of the drug resistance marker and the foreign gene are greatly amplified, can be selected and cultured for the production of increased quantities of recombinant protein. (See for example, U.S. Patent No. 4,656, 134 issued April 7,

1987 to Ringold; U.S. Patent No.4,740-461 issued U.S. Patent No. 4.766.067 issued August 23. 1988 to Biswas; and U.S. Patent No.4.956,288 issued September 11. 990 to Barsoum).

Although use of the above-described methods have resulted in increased yields of recombinant proteins, there is a need to further optimize the production of recombinant proteins from eukaryotic expression systems.

Summary of the Invention

The invention relates to improved processes for increasing the production of recombinant products from eukaryotic expression systems. In one aspect, the improvement comprises culturing the eukaryotic expression system which encodes the desired recombinant product in the presence of transforming growth factor β (TGF-β). In the presence of TGF-β, recombinant products are produced from eukaryotic expression systems at a greater level than the level produced in the absence of TGF-β and the productivity does not decrease as rapidly overtime.

In one embodiment, an appropriate amount of TGF-β is added to media in which a eukaryotic expression system is cultured during the production phase of recombinant cell culture. In another embodiment, TGF-β is encoded in the eukaryotic expression system and is expressed into production media along with the desired recombinant product.

In α preferred embodiment, the eukaryotic expression system is a recombinant Chinese Hamster Ovary (CHO) cell and the recombinant product is glucocerebrosidase. Recombinant CHO cells cultured in production media, which includes TGF-β produce 50-100% more glucocerebrosidase than recombinant CHO cells cultured in production medium, which does not include TGF-β.

The invention further relates to media containing TGF-β. which is suitable for use in the production phase of recombinant protein production.

In another aspect, the improvement comprises culturing a eukaryotic expression system which encodes a desired recombinant product in glutamate- containing, essentially glutamine-free medium.

According to one embodiment of this aspect of the invention, an increased yield of a recombinant product is obtained by culturing a eukaryotic expression system encoding the recombinant product in glutamate-containing, essentially glutamine-free media, allowing production and secretion of the recombinant product, and purifying the recombinant product from the culture media. In a preferred embodiment, the eukaryotic expression system is a recombinant Chinese Hamster Ovary (CHO) cell and the recombinant product is β-glucocerebrosidase.

The invention also relates to recombinant products produced by the disclosed process and to glutamate-containing. essentially glutamine-free media. In addition to its utility in increasing recombinant protein production, such glutamate- containing, glutamine-free media is more stable and therefore can be stored longer than glutamine-containing media.

Brief Description of the Drawings

Figure 1 is a graph illustrating the yield of recombinant glucocerebrosidase

(GCR) obtained overtime from an expression system cultured in medium containing either lOng/ml (closed square). 1 ng/ml (open triangle). 0.1 ng/ml (closed circle) or no (open circle) TGF-β - . Figure 2 is α graph illustrating the yield of GCR obtained over time from an expression system cultured in glutamate-containing 925 media alone (open triangle), glutamate-containing 925 media to which 1 ng/ml TGF-β i has been added during the production phase (closed square), or glutamine-containing 925 media alone (open circle).

Figure 3 is a graph illustrating the yield of GCR obtained over time from an expression system cultured in glutamate-containing 925 media alone (open square), glutamate-containing 925 media to which 1 ng/ml TGF-β2 has been added during the production phase (closed square), or glutamate-containing 925 media to which

10 ng/ml TGF-β2 has been added during the production phase (closed circle).

Figure 4 is a graph illustrating the yield of GCR obtained over time from an expression system cultured in glutamate-containing 925 media alone (open square), glutamate-containing 925 media into which 10 ng/ml TGF-β 1 has been added during the growth and production phases (closed circle); glutamate-containing 925 media into which 10 ng/ml TGF-β 1 has been added only during the production phase (closed square).

Figure 5 is a graph illustrating the yield of GCR obtained over time from expression systems cultured in the presence of TGF-β2 (open square) or in the absence of TGF-β2(closed square), in 40-liter and 160-liter bioreactors.

Figure 6 is a graph illustrating the yield of recombinant glucocerebrosidase (r- GCR) over time obtained from recombinant CHO cells cultured in medium containing glutamate compared to cells cultured in medium containing glutamine.

Detailed Description of the Invention

in one aspect, the subject invention is based on the surprising finding that culturing a eukaryotic expression system in production media which includes transforming growth factor-β (TGF-β), results in increased yields of a recombinant product (e.g. protein, poiypeptide, peptide). Therefore, an appropriate amount of TGF-β can be added to the media in which eukaryotic expression systems are cultured during the production phase of recombinant protein production, to obtain increased yields of the product. Alternatively, a gene encoding TGF-β can be incorporated into a eukaryotic expression system, in a manner such that TGF-β is expressed into the production medium in conjunction with the desired recombinant product.

For use in the subject invention, TGF-β can be obtained commercially (e.g., from Genentech, South San Francisco, CA). Alternatively, TGF-β can be produced in recombinant cell culture, for example, as described in U.S. Patent No. 4,886,747; or the protein can be purified, for example, as described in Frolick. C.A., et. al., Proc

Natl. Acad. Sci. USA 80:3676-3680 (1983); Childs. C.B., et. a Proc. Ngtl. Acad. Sci. USA 79:5312-5316 (1982); Assoian. R.K., J. Bjol. Chem. 258:7155-7160 (1983); and U.S. Patent No. 4,774,322. The contents of the above-cited references are expressly incorporated herein by reference. At the present time, five homologous forms of TGF-β have been identified, known as TGF-βs 1-5. Reference to TGF-β herein is meant to reference any one of the identified forms, as well others identified in the future, including TGF-β derivatives, analogues, and peptides which are useful in the method described herein.

In another aspect, the subject invention relates to the finding that eukaryotic expression systems cultured in media which contains glutamate in place of glutamine produce increased amounts of recombinant protein. This result is surprising, since glutamine is known to be both a major source of energy (accounting for about 30-50% of a cell's energy needs) and a requisite building block in the biosynthesis of proteins. As a result, most tissue culture media contains about 0.8 to

4mM glutamine, which is higher than the concentration of any other amino acid.

Based on the finding that eukaryotic expression systems cultured in media which contains glutamate in place of glutamine produce increased amounts of recombinant protein the invention provides an improved process for producing a recombinant product. According to the process of the invention, a eukaryotic expression system, which encodes α desired recombinant product, is cultured in medium containing an appropriate amount of glutamate. but essentially no glutamine. The cultured expression system is then allowed to produce and secrete recombinant product, which is purified from the culture media.

As used herein, the phrase "eukaryotic expression system" refers to a eukaryotic cell, which is capable of expressing a desired recombinant product (e.g., a protein, poiypeptide or peptide). A eukaryotic expression system is made by introducing cloned genetic material (i.e. DNA or RNA) into an appropriate, mitotically competent eukaryotic host cell, using an appropriate vector (e.g. a pfasmid or viral vector). Several permanent animal cell lines have been employed to produce recombinant proteins on a manufacturing scale. These include Chinese Hamster Ovary (CHO) cells. Baby Hamster Kidney (BHK21) cells, myeloma cells and mouse C127 cells. Enhancer elements, selective markers and/or amplifiable markers. which serve to increase the production of recombinant proteins can be included in the cell lines using techniques which are well-known. Cell lines which most efficiently express protein may be selected by expressing the protein in a selective medium, and examining the levels of intracellular protein. mRNA or secreted protein.

In order to grow, produce and secrete recombinant proteins, eukaryotic expression systems may first need to be adapted for growth in glutamate-containing, essentially glutamine-free media. Adaptation (which can be accomplished either before or after cloned genetic material is introduced into a eukaryotic host cell) is necessary, because the gene encoding glutamine synthetase (the enzyme, which catalyzes the conversion of glutamate to glutamine) is generally repressed in cells which have been cultured in glutam.ne-containing culture media.

In general, adaptation can be accomplished by initially culturing cells in a medium containing a high level of glutamate and no glutamine, continually passing the cells in this medium, and as their growth rate increases (signalling adaptation), gradually reducing the glutamate level. Adapted cells can then be used to make stock cultures according to well-known techniques. Adaptation is maintained by continually culturing cells in glutamate-containing, glutamine-free medium.

Recombinant CHO cells were adapted by initially being grown in media containing 20mM glutamate, but no glutamine. The cells were then cultured in T- flasks and when the culture vessel was confluent or when the saturation density was reached, the cells were split or diluted according to standard methods. The growth rate of cells grown in 20mM glutamate-containing media was then measured and compared to the growth rate of cells grown in glutamine-containing medium. Initially the growth rate in glutamate-containing media was slow, as the cells were adapting to the absence of glutamine. The cells were then passed in the 20mM glutamate medium until the growth rate and saturation density were similar to that seen in glutamine-containing medium. At that time, the glutamate level was lowered to 16mM and the adaptation process repeated. As the cells adapted, the level was dropped to 8mM and finally 4mM.

Once a suitable eukaryotic expression system has been developed, it can be cultured by any of a number of techniques which are well-known (e.g. suspension, microcarrier, roller bottle, or hollow fiber cultures). Growth of cells on microcarriers (e.g. collagen coated dextran. DEAE-substituted dextran, gelatin or polystyrene) is preferred, because it allows for easy separation of the medium from the cells and can be scaled up to large volumes.

Initially, eukaryotic expression systems should be cultured in media, which allows cells to grow to a high density, preferably greater than 5x10 ° cells/ml of medium used. The optimization of each individual component of a growth medium ensures that one is using the best medium for a particular expression system.

However, as a good first approximation, it is relatively easy to screen a number of commercially available media, such as Dulbecco's modified Eagle's medium. (See Freshney, R.I.. Culture of Animal Cells. A Manual of Basic Technique (Alan R. Liss, New York (1987); and Gibco Catalogue, chapter 1. "Cell Culture Media and Balanced Salt Solutions') ). Particularly suitable media for the culturing of eukaryotic cells according to the process of the invention include: mammalian cell culture media such as Dulbecco's Modified Eagle's Medium (DMEM). DMEM/F12, RPMI 1640, MCDB 100-400. NCTC-135, Media 199.925 and Iscove's Modified Dulbecco's Medium; insect cell culture media such as Grace's and IPL-41; and amphibian culture media. Serum (e.g. Fetal Bovine Serum (FBS) and Donor Calf Serum (DCS)), hormones, and other factors, which enhance cell growth, can be included into media as needed.

Commercial media designed for the growth of frequently used eukaryotic expression systems (e.g. CHO medium, Hana Biologicals, Alameda, CA), can also be used.

Glutamate-containing, essentially glutamine-free medium (i.e. media which includes glutamate as the source of glutamine) can be produced by replacing glutamate for glutamine in the medium.

The media optimized for growth may be replaced or altered for the production phase of recombinant protein synthesis. Media optimized for production should be capable of maintaining cells in a viable, slow-growing or non-growing productive state. In addition, the production medium should accommodate secretion and stabilization of a recombinant product. For example, as described in U.S. Serial No. 07/644,159 filed January 21 , 1991. the teachings of which are incorporated herein by reference, compositions containing thiols accommodate the secretion and stabilization of recombinant glucocerebrosidase. Also production media should accommodate the harvesting of the recombinant product. For example, serum may be eliminated from media used in the production phase, so that the desired recombinant product constitutes a significant percentage of the protein in the medium.

According to one embodiment of the invention, an effective amount of TGF- β is added to the media in which eukaryotic expression systems are cultured so that it is present during the production phase of recombinant protein synthesis. TGF-β in an amount in the range of 1-20 ng/ml should be suitable for most applications. However, the optimal amount of TGF-β can vary depending on the composition of the production media. Various components of the culture media may act to either enhance or inhibit the action of TGF-β. For example, since TGF-β is sensitive to reducing agents, thiol- containing compounds, such as dithiothreitol (DTT) appear to have an inhibitory effect on TGF-β's action. Therefore, to compensate for the inhibitory effect of DTT, more TGF-β must be included in the production media. The exact amount of TGF-β to include in a particular culture media can be determined experimentally.

According to another embodiment of the subject invention, TGF-β is encoded in a eukaryotic expression system and is expressed into the production media in conjunction with the desired product. A nucleic acid sequence encoding TGF-β can be introduced into a host cell by being encoded in the same vector which encodes the desired recombinant product. Alternatively, the sequence encoding TGF-β can be separately introduced into the host cell by means of a separate cloning vector.

Recombinant products can be harvested from the production media by any method which is appropriate for the method in which the expression system was cultured. For example, if cells have been cultured by a method which allows easy separation of cells and medium (e.g. microcarrier or hollow fiber culture), separation can be performed on a regular basis and the production media containing recombinant protein can be removed and replaced with fresh production media. Alternatively, separation can be performed on a continuous basis in a perfusion mode. Once harvested, the recombinant protein can be purified using standard purification techniques (e.g. chromatography, ultrafiltration. precipitation).

The invention also relates to media containing TGF-β, which is suitable for use in the production phase of recombinant protein synthesis. TGF-β containing production media can be made by adding TGF-β to any media, which maintains the particular eukaryotic expression system in a viable, slow-growing or non-growing productive state. Preferred production media is serum-free, so that the desired recombinant product constitutes a significant percentage of the protein in the medium. Preferred media for producing recombinant proteins according to the methods of the subject invention include; mammalian cell culture media such as Dulbecco's Modified Eagle's Medium (DMEM). DMEM/F12, RPMI 1640. MCDB 100-400. NCTC-135, Media 199, 925 and Iscove's Modified Dulbecco's Medium, which contains an appropriate amount of TGF-β.

The present invention can be further illustrated by the following examples, which are not intended to be limiting in any way.

Example 1: Creation of β-qlucocerebrosidase Expression System

A β-glucocerebrosidase (GCR) expression vector was constructed from the vector pSV2-dhfr (Subramani et. al., MoL and Cell. Bio. 9: 854-864 (1981)) as follows: pSV2dhfrwas cut with Bglll, blunt-ended with T4 polymerase. recircularized with T4 ligase, cut again with Hindlll, blunt-ended, ligafed to Bglll linkers, cut with Bglll, ligated to recircular-ze. cut with Bglll and ligated with a DNA fragment encoding GCR.

The GCR vector, known as pGB20, was then transfected into CHO dihydrofolate reductase (DHFR) mutant cells DG44 (G. Urlaub et. al.,Som. Cell Molec. Genet. J£; 555-666 (1986)); and exponentially growing DG44 cells were transfected with lOug of plasmid pGB20 per 100 mm dish using the lipofectin TM transfection method as described by the manufacturer (BRL, Gaithersburg, MD).

Following transfection. the medium was removed and replaced with a non- selective medium for 24 hrs. This medium was then replaced with a selective medium alpha-MEM (Gibco), minus nucleotides. Cell colonies which grew in the selective medium were harvested and aliquots lysed with a 0.05M sodium citrate buffer, pH 6.2. containing 12.0 g/L sodium choiic acid and 12.0 ml/L of 1-butanol. The intrαcellulαr GCR levels were measured in the lysed samples using the fluorogenic substrate 4-methyl-umbelliferyl-b-D-glucoside. (Suzuki. K.. Methods in Enzymol. 50:478- 479 (1978). After 12 days in selection media, cell colonies containing high levels of intracellular GCR were expanded and stepwise amplified with increasing levels of methotrexate up to lO.OmM. One cell line. Line II-15B, was used for experiments.

Example 2: Dose-effect of TGF-B

Line II- 15B was used to inoculate four 250ml. microcarrier cultures. One of the cultures contained 925 media alone; one contained media into which 0.1 ng/ml TGF- βl was added during the production phase of recombinant protein synthesis; one contained media and 1 ng/ml TGF-β 1 , added during the production phase; and one contained media into which 10 ng/ml TGF-β 1 was added during the production phase. Daily harvests were taken and the amount of product measured by an activity assay.

Figure 1 shows the dose effect of TGF-β. Of the concentrations tested, lOng/ml of TGF-β resulted in the highest production of GCR.

Example 3: Effect of TGF-β in Culture Media Containing Glutamate n

Place of Glutamine

In order to test the effect of TGF-β in glutamate-containing media, two 250ml microcarrier cultures in a glutamate-containing 925 media, were inoculated with line II-15B. At the production phase, 1 ng/ml TGF-βl was added to one of the cultures.

As a control, one 250 ml microcarrier culture in glutamine-containing 925 media was also inoculated with line II-15B. Daily harvests were taken and the amount of product measured by an activity assay.

Figure 2 shows that TGF-β and glutamate-containing media has an additive effect. GCR expression systems cultured in glutamate-containing media which includes TGF-β. produce more product at the outset and the production increases overtime. Example 4: Effect of TGFB2

In order to determine whether other TGF-βs have the same enhancing effect on the production of recombinant proteins, three 250 ml microcarrier cultures with glutamate-containing 925 media were inoculated with line II-15B. During the production phase, 10 ng/ml TGF-β2 was added to one of the cultures, 1 ng/ml was added to the other culture and the third culture was used as a control.

Figure 3 shows that TGF-β2 also effectively increases recombinant protein production. As was seen with TGF-β 1. 10 ng/ml produced a greater effect than 1 ng/ml.

Example 5: Effect of TGF-β Present During the Growth and Production Phases of Recombinant Protein Production

Three 250 ml microcarrier cultures containing glutamate-containing 925 media were inoculated with line II-15B. 10 ng/ml of TGF-β 1 was added to one of these cultures so that it was present during both the growth and production phases. 10 ng/ml of TGF-β 1 was added to one of the other microcarrier cultures only during t e production phase. No TGF-β was added to the third microcarrier culture, which was used as a control.

Figure 4 shows that whether TGF-β is present only in the production phase or whether it is present in both the growth and production phases does not make much of a difference. As long as TGF-β is at least present in the production phase, the yield of recombinant protein produced from the expression system is increased.

Example 6: Effect of TGF-β on Protein Expression in Large Volume Bioreactors The human recombinant GCR producing cell line designated II-15B-98 was cultured in a 40L bioreactor with 925 medium supplemented with TGF-β at a concentration of 5 ng/ml. Medium from the reactor was harvested on a daily basis and expression of the recombinant protein was quantitated by use of the PNP synthetic substrate colorometric assay (Suzuki. K. Methods in Enzymol. 50:478-479 (1978)). A representative bioreactor run for production of GCR performed in the absence of TGF-β was carried out for comparison.

Figure 5 plots the average values obtained for duplicate determinations and are expressed as the percent of the expression level observed on the first day of production. Note the significantly increased stability of expression in the reactor supplemented with TGF-β.

EXAMPLE 7: Production of r-GCR from recombinant CHO cells cultured in 925 medium containing glutamate.

Recombinant CHO cells (i.e. DG44 cells (Uriaub. G., et.al., Som. Cell & Mol. Gen., 126:555-566 (1986) transfected with plasmid pGB20 (Subramani, S., et.al., Mol. & Cell. Bio., 19:854-864 (1981)), adapted to glutamate as described above, were harvested by trypsinization from a confluent roller bottle and used to inoculate four 250ml. microcarrier cultures. Two of the cultures were propagated in 925 growth medium (Genzyme, Cambridge MA) containing 4.8mM glutamate. but no glutamine. The remaining two cultures were given the same growth medium, but with 4mM glutamine and only 0.8mM glutamate. Both growth media were supplemented with 10% serum. The growth medium was exchanged every 48 hours, until the cell density reached 7 X 10⁶ cell/ml. At this point, the cultures were fed with 925 media, which was not supplemented with 10% serum and contained either 4.8mM glutamate or 4mM glutamine + O.βmM glutamate, as appropriate. Daily harvests were taken and the amount of product measured by an activity assay.

Figure 6 shows the average productivity in the two different medium conditions and demonstrates that productivity was 60% higher in the spinners without glutamine.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

What is claimed is;

1. A process for producing a recombinant product, comprising:

a. culturing a eukaryotic expression system in a production medium which includes an effective amount of transforming growth factor-β; b. allowing production and secretion of a recombinant product encoded in the eukaryotic expression system; c. removing and collecting the production medium containing the recombinant product secreted in step b; and d. purifying the recombinant product collected in step c.

2. A process according to Claim 1. wherein the transforming growth factor-β is selected from the group consisting of transforming growth factor-β 1 , transforming growth factor-β 2 and transforming growth factor-β 3.

3. A process according to Claim 1 , wherein the eukaryotic expression system is a mammalian cell expression system.

4. A process according to Claim 3, wherein the recombinant product is glucocerebrosidase.

5. In the production of a recombinant product from a eukaryotic expression system, the improvement comprising adding an effective amount of TGF-β to the media in which a eukaryotic expression system is cultured.

6. A recombinant product produced by the process of Claim 1.

7. A recombinant product of Claim 6 which is β-glucocerebrosidase.

8. A serum-free cell culture medium comprising α mammalian cell culture production media into which has been added transforming growth factor-β in an amount producing increased production of recombinant product.

9. In the production of recombinant protein from a eukaryotic expression system, the improvement of culturing the expression system in essentially glutamate-containing, glutamine-free media.

10. The improvement of Claim 9, wherein the eukaryotic expression system is a recombinant Chinese Hamster Ovary cell.

11. The improvement of Claim 9. wherein the recombinant product is β- glucocerebrosidase.

12. A process for producing a recombinant product, comprising:

a. culturing the adapted eukaryotic expression system in a glutamate- containing, glutamine-free culture medium;

b. allowing production and secretion of a recombinant product encoded in the adapted eukaryotic expression system;

c. removing and collecting the medium containing the recombinant product secreted in step b; and

d. purifying the recombinant product collected in step c.

13. A process of Claim 12, wherein the eukaryotic expression system is a recombinant Chinese Hamster Ovary cell.

14. A process of Claim 13. wherein the recombinant product is β- glucocerebrosidase .

15. A recombinant product produced by the process of Claim 12.

16. A recombinant product of Claim 15, which is recombinant β- glucocerebrosidase.