IMPROVED RECOMBINANT PROTEIN PRODUCTION

Abstract: Recombinant factor VIII production is enhanced by culturing a host cell containing the gene therefor in a serum free nutrient medium supplemented with lipoprotein.

Description: Background of the Invention Hemostasis is the primary mechanism whereby excessive extravascular blood loss is prevented by the formation of fibrin clots at the site of insult. The physiological mechanism by which these fibrin clots are formed are presently being elucidated. The evidence to date suggests that the clotting mechanism is attributed to a very complex activation cascade of various blood plasma factors each responsible for the sequential activation of the successive zymogen. Proper functioning of this cascade ultimately leads to the formation of fibrin and the cessation of blood loss. Unfortunately however, various disease states have been recognized which are attributable to metabolic errors within this blood coagulation cascade either due to a deficiency or total absence of one or more of the blood plasma factors. Chief among these disease states is classical hemophilia or simply hemophilia A. Hemophilia A is a hereditary X chromosome-linked recessive trait which is manifested by an absence of antihemophilic factor A, which is designated by convention as factor VIII. Therapeutic efforts for treating hemophilia A have focused on supplementing the blood of affected individuals by infusion with concentrates of factor VIII in various pharmaceutical formulations. Such factor VIII concentrates have, in the past, been largely prepared by cryoprecipitation of factor VIII from blood plasma. However, owing to the minute amount of the factor in normal individuals, extremely large quantities of plasma must be processed in order to isolate quantities sufficient for therapy having activity levels adequate for administration in physiologically acceptable volumes of diluent. With the advent of recombinant DNA technology and the promise of the expression of large quantities of proteins of medical importance (among others), the disadvantages of prior isolation and purification techniques for factor VIII have become increasingly evident. Indeed, recent publications have reported the successful cloning and expression of the gene encoding factor VIII (see infra ). However, as can be appreciated, even with this technical accomplishment, certain limitations in the efficiency of the overall process may need to be overcome so as to optimize the benefits flowing from such technology. For example, in the specific case of factor VIII produced by recombinant DNA techniques, the transformed host cell containing the gene encoding factor VIII is cultured in a complex medium containing other serum proteins in high concentration. Thus, upon expression of the factor VIII from the culture supernatant, difficult recovery and purification procedures from a heterogeneous population of serum proteins are still encountered. However, such difficulties could be alleviated by a means of culturing the recombinant host in the absence of serum proteins in such a manner as to support cell growth without compromising the viability of the host and its ability to express factor VIII. The present invention is directed to such means and provides for the production of recombinant factor VIII by culturing a host cell carrying a gene capable of directing the expression of factor VIII in a serum free nutrient medium supplemented with lipoprotein. Description of Pertinent Art The preparation of blood coagulation factors (particularly factor VIII) by recombinant DNA techniques has been disclosed in various publications. For example, European Patent Application 160457 describes the production of functional human factor VIII from the full DNA coding sequence thereof by genetic engineering methodologies. The factor VIII is produced in quantities sufficient to prove the identification and functionality thereof free of the contaminants with which it is normally associated in its non-recombinant cellular environment, such as vonWillebrand factor. Similarly, PCT Application WO 85/01961 describes the identification and isolation of the porcine factor VIII gene and the concomitant isolation and identification of the human factor VIII gene by virtue of the homology between porcine and human factor VIII. Also disclosed is the expression of porcine and human factor VIII by recombinant DNA techniques from said genes. Likewise, European Patent Application 150735 is said to provide methods and compositions for the production of factor VIIIc (including precursors and subunits thereof) by expression in a microorganism or mammalian tissue cell. UK Patent Application GB 2,125,409 discloses substantially the whole DNA sequence encoding factor IX (the absence or deficiency of which is associated with Christmas disease) including the production thereof in a host such as Escherichia coli . The literature discloses various roles for lipoprotein in cell culture techniques. For example, in J. Cell. Physiol. , 113(3) pp. 373-384 (1982) the authors state that high density lipoprotein is an absolute requirement for the survival and growth of bovine adrenal cortical cells when maintained on an extracellular matrix. In J. Cell. Physiol. , 120 (3) pp. 354-363 (1984) low density primary cultures of rabbit costal chondrocytes proliferated on an extracellular matrix of corneal endothelial cells when the medium was supplemented with high density lipoprotein, transferrin, fibroblast growth factor, hydro-cortisone and epidermal growth factor. Similarly, bovine lens epithelial cells when passaged to serum free medium, proliferated actively on an extracellular matrix-coated dish when exposed to a mixture of Dulbecco's modified Eagle's medium supplemented with high density lipoprotein, transferrin, insulin and fibroblast growth factor ( Exp. Eye Res. , 35 (3), pp. 259-270, 1982). The use of high density lipoprotein has been reported as a substitute for serum in the process of serially adapting the human mammary carcinoma cell line MCF-7 to grow in serum free medium ( Biochem. Biophys. Res. Commun. , 133 (1), pp. 105-112, 1985). Hence, it is known that in specific instances for particular cell lines, a high density lipoprotein-supplemented medium is needed to adapt a cell line to grow in a serum free medium and may be needed for proliferation on an extracellular matrix. In either case, the effect of lipoprotein on a given cell culture is unpredictable. For example, in Cell Struct. Funct. , 8 (1), pp. 67-76 (1983), low density lipoprotein was said to promote the growth of human normal HEL cells and 4-nitroquinoline-1-oxide-transformed human fibroblast cells in the absence of serum. However, low density lipoprotein had no effect on the optimal growth of said human fibroblast cells which were alpha-ray transformed. None of the above-cited references either disclose or suggest the use of lipoprotein as taught herein. That is to say, the use of lipoprotein to supplement the growth of mammalian cells and to effect the increased expression from said cells of a transfected, heterologous gene has not been previously reported. Specifically, no such methodology has been applied to the production of recombinant factor VIII. Summary of the Invention The present invention is directed to a method for effecting an increased expression of recombinant factor VIII in a host cell carrying the gene therefor by culturing said host cell in a serum free nutrient medium supplemented with lipoprotein so as to effect expression of said factor VIII. Detailed Description of the Invention As noted previously, techniques are available for the production of large quantities of protein by recombinant DNA techniques which quantities have heretofore not been possible to obtain. Typically, the process for identifying and isolating the gene responsible for production of the protein follows a somewhat standard protocol. The protein of interest is first isolated from its naturally occurring environment and is extensively purified utilizing techniques such as immunoaffinity purification with a monoclonal antibody specific for the protein of interest. The amino acid sequence of the polypeptide may be determined by now conventional techniques and, based on the sequence information thus obtained, oligonucleotide probes may be prepared and used to identify and/or isolate the site of synthesis of the protein from the various tissues tested. Polyadenylated mRNA may then be isolated from the identified tissue which is then utilized to form single-stranded complementary DNA (cDNA) by the action of RNA-directed DNA polymerase (also referred to as "reverse transcriptase"). The reverse transcriptase products (either partial or complete copies of the mRNA template) typically exhibit short, partially double-stranded hairpin loops at their 3′ termini which act as primers for DNA polymerase. The DNA polymerase requires the presence of a DNA strand having a free 3′-hydroxyl group to which new nucleotide residues are added thereby extending the chain in a 5′ to 3′ direction. The resulting double-stranded cDNA is then treated with S1 nuclease to remove a residual single strand fragment thereby generating a "blunt-end" duplex DNA segment. Other procedures for producing cDNA libraries are available. See, for example, Chan and Whitted in Developments in Industrial Microbiology , Vol. 9, pages 171-180 (1985). Said DNA segment encoding the protein of interest is then ligated into an appropriate vector for subsequent transformation of a suitable host thereby creating an expression system which directs the synthesis of the desired product. The general procedures alluded to above are described in greater detail in Molecular Cloning, A Laboratory Manual , Maniatis et al, Cold Spring Harbor Laboratory, 1982. Details of the above-procedures as they specifically relate to the production of recombinant factor VIII may be obtained from the following: European Patent Application 160457; PCT Application Number PCT/US84/01641, published as WO 85/01961; European Patent Application 157556; and European Patent Application 150735. Thus, as used herein, the term "recombinant factor VIII" refers to that functional protein that is produced by molecular biological techniques as outlined above which protein is capable of correcting human factor VIII deficiencies. The method disclosed and claimed herein provides for the increased expression of recombinant factor VIII from host cells carrying the gene therefor. By the term "increased expression" is meant expression to an extent greater than that possible in a recombinant system which does not utilize lipoprotein and serum free conditions. By "serum free" is meant culturing the host cells carrying the gene for factor VIII in the essential absence of serum. While it is preferable to carry out the method of the present invention in the absence of serum, the presence of minor amounts of serum which do not deleteriously affect the yield of factor VIII are clearly contemplated as falling within the scope of the present invention. However, as can be appreciated by the skilled artisan, the presence of increasing amounts of serum in the nutrient medium renders more difficult the purification of factor VIII from the increased population of heterogeneous proteins present in said serum. Host cells suitable for factor VIII expression may in general be derived from multicellular organisms, mammalian cells such as VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, baby hamster kidney (BHK) cells, W-138, COS-7. and MDCK cell lines being preferred. Particularly preferred are baby hamster kidney cells, specifically those transfected with a gene capable of directing the expression of factor VIII as described in European Patent Application 160457 (including derivatives such as clonal variants and progeny thereof). Such cell line has been deposited with the American Type Culture Collection and has been given accession number ATCC CRL-8544. The desired host cell line carrying the gene encoding factor VIII is cultured by conventional techniques through the growth phase thereof in an appropriate medium containing typical amounts of serum (i.e., anywhere from about 7 to about 10 percent). The serum is maintained in the medium until the cells reach confluency and the maintenance phase of the cell cycle begins. This procedure is standard in the art. However, at the approximate point where the culture reaches the maintenance phase of the cell cycle (i.e., when the cells reach confluency) the spent nutrient medium containing the serum is replaced with a serum free nutrient medium supplemented with lipoprotein and the culture is allowed to continue in the maintenance phase, with recovery of the factor VIII from each harvest when indicated. The skilled artisan will appreciate that the serum free conditions (supplemented with lipoprotein) under which the maintenance phase of the culture is conducted allows for a greatly facilitated purification of the factor VIII from the spent nutrient medium than if serum proteins are present. It will be further appreciated that the presence of lipoprotein in the nutrient medium not only effects an increased expression of factor VIII from the host cell (as compared to culturing under serum supplemented conditions), but also sustains the viability of the cells for a prolonged period thus making increased numbers of harvests of factor VIII possible. The nutrient medium chosen for culturing the host cell line is not critical to the present invention and may be any one of, or combination of those known to the art which are suitable for the selected host cell line. Illustrative of such media are Dulbecco's Modified Eagle Medium, Eagle's Minimum Essential Medium, Eagle's Basal Medium, Ham's Medium F-10, Ham's Medium F-12, RPMI-1640 Medium, and the like. All such media are commercially available from sources such as Sigma Chemical Company, GIBCO and M.A. Bioproducts, for example. The nutrient media may also contain various buffers such as HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) and may also be supplemented with factors such as insulin, transferrin, testosterone, sodium selenite, ethanolamine, glutamine, saturated and unsaturated fatty acids and the like in addition to the lipoprotein. The amounts of such factors added to the nutrient medium are conventional in the art. For example, the quantity of insulin may range from about 5 to about 10 micrograms per milliliter (µg/ml) of nutrient medium; the quantity of transferrin may range from about 2.5 to about 25 µg/ml of nutrient medium; the quantity of glutamine may range from about 200 to about 600 µg/ml of nutrient medium; saturated and unsaturated fatty acids may range from about 1 to about 10 µg/ml of nutrient medium; and the quantity of testosterone may range from about 10 to about 50 nanograms (ng) per milliliter of nutrient medium. The lipoprotein which is used to supplement the nutrient media may be of any origin although mammalian sources such as bovine, horse, sheep, pig or human are preferred. Particularly preferred is bovine lipoprotein. Bovine lipoprotein is commercially available from various sources such as Pentex Lipoprotein Solution (Catalog No. 82-004) and Pentex Lipoprotein Cholesterol Solution (Catalog No. 82-108), both of which are available from Miles Laboratories, Inc., Elkhart, Indiana. Bovine lipoprotein can also be prepared by ultra-centrifugal flotation of freshly isolated bovine serum using standard techniques as described, infra . The lipoprotein is present in the nutrient medium in an amount capable of continuing the viability of the cells during the maintenance phase of the life cycle of said cells. Preferably, it has been found that this amount varies from 0.1 mg/ml to 2.0 mg/ml of nutrient medium. Particularly preferred is a lipoprotein content of from 0.2 mg/ml to 1.0 mg/ml of nutrient medium. In addition to conventional techniques such as roller bottle culturing, the method of the present invention is also applicable for use with micro-carrier systems such as described in Methods in Enzymology , Vol. LVIII, "Cell Culture" (1979), W. B. Jakoby and I. H. Pastan, Editors. Suitable microcarriers include, for example, the various Cytodex microcarriers (i.e., Cytodex-1, Cytodex-2, Cytodex-3, available from Pharmacia, Inc.), Gelibeads (available from K. C. Biologicals), and the like. Further, the method of the present invention is also applicable to cell suspension systems. The culture conditions are chosen with respect to standard considerations for the cell line being used. Suffice it to say that the chosen cell line carrying the gene capable of directing the expression of factor VIII may be grown under such temperatures and for such times in a serum free nutrient medium supplemented with lipoprotein as may be sufficient to effect the increased expression of recombinant factor VIII from said host. For example, for mammalian cell lines it may be desirable to conduct the culture at a temperature of from about 36 to 38°C for about 24 to about 72 hours. However, selection of these parameters is routine and well within the skill of the artisan. Following culturing of the host cell, the factor VIII may be recovered from the spent nutrient medium by standard methodologies such as centrifugation or ultrafiltration. If desired and/or necessary, the recovered factor VIII may be purified by, for example ion exchange or size exclusion chromatography, immunopurification, and the like. The following examples are provided as a means of illustrating the present invention and are not to be construed as a limitation thereon. Example 1 Bovine lipoprotein suitable for use in the present invention may be prepared as follows. To 50 ml of fresh bovine serum (obtained from a commercial slaughterhouse) was added 16 grams (g) of potassium bromide so as to adjust the density of the bovine serum to 1.21 g/ml. The serum was then centrifuged at 40,000 revolutions per minute (rpm) for 24 hours at 4°C in an ultracentrifuge (a Beckman Ti45 being preferable) resulting in a separation of phases. The top, yellow, oily lipoprotein fraction was then collected and again centrifuged at 40,000 rpm for 24 hours. The lipoprotein fraction was isolated and dialyzed extensively against NaCl:EDTA (0.15 millimolar NaCl, 1.0 millimolar EDTA, pH 7.4). The dialyzed lipoprotein was then sterilized using a 0.2µ filter (Millipore). If desired, the lipoprotein may be quantitated by a Bradford assay kit (commercially available from Biorad, Inc. under catalog Number 500-0002) and the cholesterol content may be quantitated using a kit available from Sigma Chemical Co. (catalog number 351-20). Example 2 Baby hamster kidney cells (BHK-21) transfected with a gene capable of directing the expression of factor VIII were obtained from Genentech, Inc., South San Francisco, California, U.S.A. Said cell line was prepared as described in detail in European Patent Application 160457, and was deposited with the American Type Culture Collection and given accession number ATCC CRL-8544. The BHK-21 cells containing the gene encoding factor VIII were grown to confluency (as described in detail in the above-noted European Patent Application 160457) and were subsequently trypsinized with 0.25% trypsin-EDTA (commercially available from GIBCO). The cells were then washed with Hank's balanced salt solution (HBSS) and counted under a microscope using. a hemocytometer. 20 × 10⁶ cells were seeded into a 490 centimeter² (cm)² roller bottle (Corning Glass, Inc.) to which was added 150 ml of growth medium containing Ham's Medium F-12 and Dulbecco's (low glucose) Minimum Essential Medium (50:50, by weight) and 7% fetal bovine serum. The cells were allowed to grow at 37°C for 72 hours at a roller speed of 0.3 rpm. After the cells reached confluency, the spent media was decanted and discarded and the cells were washed with 100 ml of HBSS. A serum free nutrient medium was then prepared and contained the following: Ham's Medium F-12 and Dulbecco's (low glucose) Minimum Essential Medium (50:50, by weight), bovine lipoprotein as prepared in Example 1 (0.4 mg/ml), bovine insulin (5.0 µg/ml), bovine transferrin (2.5 µg/ml) glutamine (292 µg/ml) and MgCl₂ (15 millimolar). 100 ml of this serum free nutrient medium was then added to the cells which were allowed to grow at 37°C for 48 hours at a roller speed of 0.3 rpm. The spent serum free nutrient medium was then harvested for factor VIII and 100 ml of fresh serum free nutrient medium was added to the cells and again allowed to grow at 37°C for 48 hours at a roller speed of 0.3 rpm. This process was repeated until 3 harvests of spent serum free nutrient medium containing recombinant factor VIII were obtained. Each harvest was individually assayed for factor VIII activity using a chromogenic substrate determination method. Said assay is sold commercially as a test kit known as Coatest Factor VIII and is available from Helena Laboratories, Beaumont, Texas U.S.A. (Catalog No. 5293). The procedures for use are provided by the manufacturer or are further elaborated in European Patent Application 160457. The factor VIII activity for each of the three harvests as determined with the Coatest Factor VIII kit are set forth in Table I. As can be seen from the data presented in Table I, culturing the BHK-21 cells containing the gene encoding factor VIII under serum free conditions supplemented with lipoprotein resulted in an elevated production of factor VIII. By comparison, the use of conventional serum-containing nutrient medium not supplemented with lipoprotein to culture the above-described BHK-21 cell line (i.e., ATCC CRL-8544), produced factor VIII in a quantity of from about 150 to about 200 units/liter. Further, cells not grown under serum free conditions typically died after the second harvest. Example 3 A clonal variant of the BHK-21 cell line described in Example 2 (i.e., a clonal variant of ATCC CRL-8544) was obtained from Genentech, Inc., South San Francisco, California, U.S.A. Utilizing the procedures described in Example 2, said clonal variant was cultured under serum free conditions supplemented with lipoprotein through five harvests. These data are shown in Table II. As can be seen from the data presented in Table II, culturing the clonal variant of the BHK-21 cells containing the gene encoding factor VIII under serum free conditions supplemented with lipoprotein resulted in significantly elevated production of factor VIII. By comparison, the use of conventional serum-containing nutrient medium not supplemented with lipoprotein produced factor VIII in a quantity of from about 800 to about 1200 units/liter (for the clonal variant). Further, cells not grown under serum free conditions typically detached and died after the second harvest. Example 4 In order to show that the method of the present invention is also applicable to the production of recombinant factor VIII utilizing a microcarrier system, the following was conducted. 1 gm of Cytodex-3 microcarriers (Pharmacia, Inc.) were washed and hydrated in 500 ml of phosphate buffered saline (GIBCO) for 4 hours after which the microcarriers were autoclaved at 121°C for 30 minutes. The microcarriers were washed twice in 250 ml of a growth medium containing equal parts (by weight) of Ham's F-12 Medium and Dulbecco's Modified Eagle Medium and were then suspended in 250 ml of said growth medium supplemented with 7% fetal bovine serum in a Bellco microcarrier spinner flask (Bellco Glass, Inc.). 40 × 10⁶ BHK-21 cells transfected with a gene capable of directing the expression of factor VIII (as described in Example 3) were added to the microcarriers. The resulting cell suspension was incubated at 37°C for 3 hours with intermittent stirring (at 30 rpm for 2 minutes at 30 minute intervals) after which an additional 250 ml of the above-described growth medium supplemented with 7% fetal bovine serum was added. The microcarriers were then incubated at 37°C under continuous stirring at 30 rpm. After 72 hours, the spent medium was removed and the microcarriers were washed twice with HBSS. 500 ml of the serum free nutrient medium described in Example 2 was added to the microcarriers and the system was allowed to incubate at 37°C with continuous stirring (30 rpm). At 24 hour intervals the serum free nutrient medium was harvested and replaced in the microcarrier system with 500 ml of fresh serum free nutrient medium. The culture was terminated at day 9 with a total of 6 harvests of spent serum free nutrient medium containing recombinant factor VIII. Each harvest was individually assayed for factor VIII activity using the chromogenic substrate determination method discussed in Example 2. The data are shown in Table III. As can be seen from the data presented in Table III, by utilizing a microcarrier system to culture the BHK-21 cells containing the gene encoding factor VIII under serum free conditions supplemented with lipoprotein a significantly elevated production of factor VIII was achieved. By comparison, the use of conventional serum-containing nutrient medium not supplemented with lipoprotein in a microcarrier system produced factor VIII in a quantity of from about 200 to about 300 units/liter. Further, while only six harvests were made in this particular instance, the skilled artisan can appreciate that additional harvests may be made (typically as many as 10 to about 15) since the viability of the cells is maintained to a greater extent.

Claims: A method for effecting an increased expression of recombinant factor VIII in a host cell carrying the gene therefor which comprises culturing said host cell in a serum free nutrient medium supplemented with lipoprotein so as to effect expression of said factor VIII. The method of Claim 1 wherein said host cell is a mammalian host. The method of Claim 2 wherein said mammalian host is baby hamster kidney cells. The method of Claims 1, 2 or 3 wherein said lipoprotein is from a mammalian source. The method of Claim 4 wherein said mammalian source is selected from bovine, horse, sheep, pig or human. The method of Claim 5 wherein said lipoprotein is bovine lipoprotein. The method of Claims 1-6 wherein the amount of lipoprotein present in the nutrient medium is from 0.1 mg/ml to 2.0 mg/ml. The method of Claims 1-6 wherein the amount of lipoprotein present in the nutrient medium is from 0.2 mg/ml to 1.0 mg/ml. The method of claims 1 - 8 wherein said host cell line is ATCC CRL 8544 and derivatives thereof.

Inventors: CHAN, SHAM YUEN
Applicants: MILES INC.
Application year: 1987
Main classification: C12N15
IPC codes: C12N5, C07K14, C12R1, C12N15, C12P21