WO1990001333A1

WO1990001333A1 - METHOD FOR PREPARING tPA COMPOSITIONS

Info

Publication number: WO1990001333A1
Application number: PCT/US1989/003301
Authority: WO
Inventors: William R. Tolbert; Christopher P. Prior
Original assignee: Invitron Corp
Current assignee: Invitron Corp
Priority date: 1988-08-02
Filing date: 1989-07-31
Publication date: 1990-02-22
Anticipated expiration: 1991-02-02
Also published as: AU4187389A

Abstract

Tissue plasminogen activator (tPA) is efficiently produced in perfusion cell cultures using HSA in serum-free media. The resulting conditioned media may be purified using SepharoseR to provide a composition suitable for injection. The composition may be further purified to yield Type I and Type II tPA in high yield. Surprisingly, culture in serum-free media containing HSA provides a tPA product enriched in the more active Type II tPA.

Description

METHOD FOR PREPARING tPA COMPOSITIONS

Description

Technical Field

This invention relates to methods for pre¬ paring pharmaceutical compositions of active polypep- tides. More specifically, this invention relates to a method for preparing compositions of tPA and an excipient protein by co-purification.

Background of the Invention

Human tissue plasminogen activator (tPA) is a serine protease that converts plasminogen into plasmin. Plasmin degrades the fibrin matrix of blood clots, thereby restoring the hemodynamic condition of an open vascular system after an internal vascular accident has produced thrombosis or thromboembolism. Vascular dis¬ ease states involving partial or total blockage of blood vessels which are amenable to treatment with plasminogen activators include, stroke, pulmonary embolism, myocar- dial infarction, as well as deep vein and peripheral artery obstructions. tPA is one of two immunologically distinct types of plasminogen activators found in human plasma and other body fluids. It has an M of 68,000, and its activity is potentiated by fibrin. The enzyme acts at the site of a thrombus, and demonstrates a higher affin¬ ity for fibrin than does the other (urokinase-type) plasminogen activator. Thus, tPA is considered to be the physiologically relevant thrombolytic agent and is considered to have significant potential as a therapeu¬ tic agent.

Due to the extremely low concentration of tPA in blood and tissue extracts, other sources and means of producing this preferred thrombolytic agent have been sought. Preferred methods of production for commercial utilization involve growing cells capable of producing tPA in culture, including cells transformed with re- combinant vectors which encode this protein. A pre¬ ferred method for producing tPA, including that ex¬ pressed from recombinant DNA vectors, is in mammalian cell culture systems. Mammalian cells have the capabil¬ ity to synthesize human proteins with the proper con¬ figuration, correct disulfide bonding, and arrays of carbohydrate side chains which all result in a protein which mimics the naturally occurring protein in terms of activity and lack of immunogenicity.

A DNA sequence encoding tPA and the amino acid sequence of tPA is disclosed by Pennica et al, Nature, (1983) 301:214; Ny et al, Proc Nat Acad Sci USA (1984) 8J.:5355; and patent GB 2,119,804 B. tPA has been syn¬ thesized in a variety of cells, including those trans¬ formed with recombinant expression vectors containing sequences which encode tPA and its derivative analogs. See, for example, Van Brunt, Biotechnology (1986) :505.

A great diversity of procedures and apparatus for in vitro cell culture production of biomolecules of interest has been utilized heretofore. Generally, the procedures for cell culture utilizing the different ap¬ paratus may be classified as batch procedures, semi- batch procedures, and continuous or perfusion proce¬ dures. In batch procedures, the cells are grown in a fixed amount of nutrient and harvested after significant growth ceases; the desired product is isolated from the cell culture after the first round of growth. Semi- batch procedures are similar to batch procedures; how¬ ever, after harvest the cells are submitted to repeated rounds of growth and expression by resuspension in fresh medium_,. In perfusion processes the cells are maintained in the culture vessel, and fresh medium is perfused in while spent medium (also called conditioned medium) is withdrawn in a continuous or semi-continuous process. Cell propagation processes have been reviewed in Van Brunt supra; Reuveny et al, J Immunol Meth (1986), j3 :61; and J. Feder and W.R. Tolbert Am Biotech Lab (1983), 3_:24; and Tolbert et al (1985), "Large-Scale Mammalian Cell Culture" (J. Feder and W.R. Tolbert, eds . , Academic Press. Inc.) p. 97.

Once expression of the protein has been achieved, purification may prove to be a substantial problem. Expression in bacterial hosts generally results in the presence of large amounts of inflammatory and immunogenic bacterial proteins, which must be re¬ moved to provide a pharmaceutically-acceptable product. Further, tPA exhibits a pronounced affinity for glass¬ ware, and measures must be taken to ensure that the entire production yield does not remain bound to the fermentation and purification apparatus. The final goal, once these problems are overcome, is generally tPA of high specific activity. Such material, however, is typically too concentrated for direct administration. Collen et al, U.S. Pat. No. 4,752,603 dis¬ closes a process for preparing a tPA-like protein from human melanoma cell cultures, and a process for its pu¬ rification. Collen disclosed purification using a zinc chelate agarose ligand exchange column, followed by affinity chromatography on lectin agarose, optionally followed by gel filtration, preferably using cross- linked dextran particles. Collen also disclosed addi¬ tion of aprotinin to the various solutions during puri¬ fication (except for the last step) to inhibit proteo- lysis of the tPA during purification. Collen reported a yield of 46% of tPA having a specific activity of about 500,000 IU/ g, based on WHO First International Refer¬ ence Preparation of tPA.

Michio et al, EPO 217,379 disclosed formu¬ lations designed to increase the low aqueous solubility of tPA- These formulations include large quantities of arginine, optionally NaCl, and may include additional, proteins as excipients. Michio disclosed an exemplary formulation containing 5x10 IU tPA, 52.5 mg arginine HC1, and 20 mg HSA. tPA can be extracted from a variety of tissues and has been purified from pig heart (P. Wallen et al, Biochim Biophys Acta (1982) 1 :318-328). A recent review of tPA and its characterization is found in A. Klausner, Biotechnology (1986) :706-71. An inter¬ national standard of human tPA having an activity of 1,000 units per vial has been prepared and is used as the primary standard in assessing tPA activity. This standard purified tPA was prepared from a Bowes melanoma cell conditioned media and purified to a specific activ¬ ity of about 500 x 10 IU/mg. A variety of assays is available for tPA, and the international unit is defined in terms of a comparison of the tested preparation to the activity shown by this primary standard in the same assayl The apparent activity of the preparation will vary in absolute terms, but not relative to the stand¬ ard, depending on the precise conditions of the assay. For example, in one standard in vitro assay as described by J.H. Verheijen et al, Thrombosis Res (1982) 27:377- 385, several factors are known to influence activity. This assay involves the catalyzed conversion of plasmin¬ ogen to plasmin and measurement of the plasmin formed colorimetrically. The activity of all tPA preparations in this assay can be enhanced by addition of cyanogen bromide fragments of fibrinogen, by the addition of fi- brinogen itself, or by the addition of fibrin. Other useful in vitro assay systems include the direct meas¬ urement of lysis of fibrin clots as described by P. Wallen et al, Biochim Biophys Acta (1982) (supra) . In vivo assays are also available.

It has been demonstrated that tPA isolated from Bowes melanoma cells is separable into two "types", Type I. and Type II, by fractionation with arginine- or lysine-conjugated Sepharoses. These types of tPA differ in glycosylation pattern; Type I tPA is glycosylated at each of three canonical asparagine glycosylation sites; Type II has glycosylation at only two of these sites.

Cloning of the gene encoding Bowes melanoma tPA, and its sequencing and deduction of amino acid sequence (D. Pennica et al, Nature (1983) 301:214-221) shows that there are four potential glycosylation sites involving asparagines at positions 117, 184, 218 and 448 in the 527-residue tPA chain. Apparently, asparagine- 218 is not used as a glycosylation site; in Type I tPA, all three of the remaining sites are occupied; in Type II, only positions 117 and 448 are bound to carbo¬ hydrates. The difference in carbohydrate composition therefore resides in the A chain; when tPA is cleaved, typically in the presence of plasmin, cleavage occurs at the proteolytic site between arg₂₇₅-ile_₇₆ (G. Pohl et al, EMBO Workshop on Plasminogen Activators, Amalfi, Italy, (October 1985)). In addition, P. Wallen et al, Eur J Biochem (1983) 132:681-684 reported that purified Bowes tPA, obtained in a three-step purification pro¬ tocol involving adsorption onto an affinity column of antibodies to porcine tPA, arginine-Sepharose , and Sephadexs- G-150, had a specific activity of about 220 x 10 IU/mg, and showed a molecular weight of about 72 kd, but when hydrolyzed into the two-chain (A and B) form gave A chains differing in molecular weight (37 kd and 40 kd) , a difference which was putatively ascribed to glycosylation pattern.

Subsequently, full-length Type I and Type II tPA have been reported as partially separated on arginine-Sepharoses or lysine Sepharoses. M. Ranby et al, FEBS Lett (1982) 146:239-242 used arginine- Sepharoses- and obtained two overlapping peaks of un- reported specific activity. Ranby also showed that the differences in molecular weights in these two peaks reside in the A chain, and cited the observation of C. Komanger et al, Thromb and Hemat (1981) 4_6:602-603 that these two types occur in a variety of mammalian tissues. A subsequent publication by D.C. Rijken, Thromb and Hemost (1985) 5_4:788-791 reported the separation of the two types of tPA on lysine-Sepharoses- and concluded from comparison studies with tPA preparations which had been secreted in the presence of tunicamycin, an inhibitor of N-glycosylation, that glycosylation at the asparagine residues had no influence on the enzymatic activity of tPA.

Finally, M. Einarsson et al, Biochim Biophys Acta (1985) 830: 1-10 reported separation of Type I and Type II on lysine Seρharose&. Although their data showed only partial separation, they indicated that lengthening the column permitted complete separation of the two forms. Their results were asserted to be supe¬ rior to those previously reported by Ranby. Specific activities in the range of 150 x 10 IU/mg for Type I and 225 x 10 IU/mg for Type II were obtained using the World Health Organization International reference preparation of urokinase (established 1975, 66, 46).

Commonly-owned copending US patent application 178,392, filed 7 April 1988 and incorporated herein by reference in full, disclosed that the combination of properly configured lysine Sepharose® columns with supe¬ rior preliminary purification procedures can yield Type I and Type II tPA in specific activities and purities comparable to those previously obtained for the tPA mix¬ ture. As Type I and Type II tPA have differential ac¬ tivities according to the conditions of the assays em¬ ployed, it is apparent that the spectrum of behavior to be exhibited in vivo by each of these molecules will differ. Accordingly, it is advantageous to obtain highly purified forms of each of these two biologically distinct types.

Disclosure of the Invention

We have now invented a method for preparing tPA compositions which -overcomes the problems set forth above, and others. In our method, tPA is co-purified with an excipient protein such as human serum albumin (HSA), to provide directly a composition of suitable concentration for administration to mammals, including humans. By co-purifying, tendency for tPA to bind to glassware and apparatus is inhibited, and bulk yields are greatly enhanced. Although a high specific activity (^500,000 IU/mg) is never attained, we achieve a compo¬ sition having an active amount of tPA without the loss of material associated with purification to such levels.

Further, we have invented an improved process for preparing type I and II tPA in pure form with enhanced yield (compared to the process of USSN 178,392). Surprisingly, we have found that our process further provides an improved ratio of Type II to Type I tPA (Type II is more active) .

Modes of Carrying Out The Invention

A. Definitions

"Control sequence" refers to DNA sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending on the host organ¬ ism; in eukaryotes, generally, such control sequences include promoters, terminators, and in some instances, enhancers. The term "control sequences: is intended to include_., at a minimum, all components whose presence is necessary for expression, and may also include addi¬ tional components whose presence is advantageous to the expression of the desired gene.

"Operably linked" refers £0 a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

"Expression system" refers to a DNA segment which contains at least one "expression cassette", which cassette contains a coding sequence to be expressed and the control sequences, operably linked to it, which enable its expression.

"Effluent" refers to cell culture medium con¬ taining tPA synthesized by cells contained in a cell culture vessel. The terms "effluent" and "conditioned medium" are used interchangeably herein.

The term "support matrix" refers to a gen¬ erally inert medium which can be derivatized as desired. Support matrices within the scope of this invention include, without limitation, agarose, Sepharoses, Sephadexs>, Sephacol?, polyacrylamide, cellulose (and cellulose derivatives), and membrane-type supports, such as nylon, polystyrene, and the like, including hollow fibers .

The term "negatively-charged ionic group" refers to a radical which bears a negative formal charge under the pH conditions used herein, and which does not react irreversibly with the tPA or HSA to be eluted. Preferred negatively-charged ionic groups are easily attached to support matrices, or are commercially avail¬ able in such form. Exemplary negatively-charged ionic groups include without limitation sulfonic, phospho, carboxy, carboxymethyl, and the like. Sulfonic groups are presently preferred.

The term "basic amino acid" refers to an a ino acid having a side chain which accepts protons at the pHs used herein. Exemplary basic amino acids include lysine and arginine, preferably lysine. Histidine may also be employed.

The term "mammalian serum albumin" (MSA) refers to the serum albumin from any convenient mammal, for example human, bovine, ovine, equine, murine, etc. Human serum albumin (HSA) is presently preferred, par¬ ticularly for compositions intended for administration to humans. However, as the serum albumin is purified away from tPA in the process of preparing purified Type I and Type II tPA, serum albumin from other species may be used in these processes.

The terms "serum-free media" and "substan¬ tially serum-free" refer to cell culture media which contain serum in amounts less than about 1%, preferably less than about 0.1%, and most preferably contain no serum at all. Serum-free media sufficient for basal cell culture are commercially available. In the prac¬ tice of the instant invention, the medium is preferably supplemented with insulin and transferrin (in addition to MSA, preferably HSA) , and additionally contains an antibiotic such as methotrexate. As used herein, "tPA" refers to the 527 amino acid protein having the primary structure disclosed by Pennica et al, (supra) and its naturally occurring variants, including allelic variations. Type I tPA is defined as the foregoing primary structure which con¬ tains glycosylation at three sites which correspond to positions 117, 184 and 448 in the sequence described by Pennica. Type II tPA is defined as tPA containing glycosylation only at sites corresponding to positions 117 and 448 of the Pennica structure.

. A "substantially pure mixture" of HSA and tPA refers to a composition which contains essentially only tPA (as Type I and/or Type II) and human serum albumin. Other proteins (e.g., from the cell culture medium) may be present in small amounts, not exceeding about 3% for any given protein. At least 95% of the composition will be HSA and tPA. The substantially pure mixture will also be acceptable for administration to humans by parenteral means. The mixture has a specific activity of about 50,000-400,000 IU/mg protein, preferably 100,000-300,000 IU/mg protein.

The term "high yield" as used herein means a yield of pharmaceutically acceptable tPA in excess of 50%, preferably greater than 60%, based on the amount of tPA present in the crude conditioned media. Previous reports of tPA purification fail to recover even 50% of the tPA initially present in conditioned media; yields in the range of 35-46% are the maximum reported. The purification process of the instant invention provides purified tPA in excess of 50% yield.

It has been shown that both Type I and Type II tPA occur in extracts from a variety of mammalian tissue sources. While it is known that the glycosylation of the molecule varies in the structure of the carbohydrate moieties attached at these positions depending on the cells producing the protein, it is nevertheless the case that the distribution of glycosylation with respect to these canonical sites is the same regardless of source. Thus, tPA recombinantly produced in mammalian cells, such as Chinese hamster ovary (CHO) cells or mouse cells; native tPA obtained from colon cells, uterine cells, or melanoma cells, etc. exhibits both Type I and Type II variants. The glycosylation differences among tPA prepared from these various sources reside in the structures of the carbohydrate chains themselves.

"Specific activity" in IU/mg is defined herein by comparison to the International Standard tPA obtained from the National Institute of Biological Standards, England. The International tPA standard is a lyo- philized preparation of tPA Type I and Type II, unsepar- ated, having a total of 1000 IU per vial. The absolute activity of tPA is highly dependent on the conditions of the assay, for example in the in vitro assay measuring conversion of plasminogen to plasmin referenced above, addition of various materials increases the apparent activity. However, the specific activity relative to the standard will not change by virtue of this varia¬ tion. That is, the standard tPA preparation exhibits the same variation with regard to conditions, and thus the activity relative to it remains the same unless the inherent character of the protein has been changed. Thus, differences in the specific activity of Type I and Type II preparations are normalized with regard to the mixture — i.e., the standard.

Other standards for measuring tPA activity have also been used. For example, much of the earlier literature is based on comparison to a WHO International Standard which is, in fact, urokinase. The activities measured by Einarsson, M. et al. (supra) were, for example, obtained with reference to this urokinase standard. A convenient assay used in defining the above parameters is the fibrin-stimulated microtiter tPA assay. In this assay, plasminogen is converted to plasmin by tPA in the presence of fibrin as a stimula¬ tor, and the plasmin formed is assayed using the syn¬ thetic substrate S-2251.

Immulon II 96-well microtiter plates are used. Into each well are placed 200 ul of 10 ug/ml solution in water of fibrinogen (from which plasminogen had been removed by lysine Sepharoses- column) . The plates are allowed- to air dry overnight. To obtain clotting, 200 ul/well bovine thrombin is added (Sigma) supplied as a 1 NIH unit/ml solution in PBS containing 0.1% BSA and 1 mM CaCl- . The plates are left 45 minutes at room tem¬ perature, and then rinsed with saline Tweens, followed by two 30 minute washes with saline Tweens. The final wash is shaken out and the plate is again air dried. The dried plates can be stored refrigerated.

For the assay itself, 100 ul of tPA standard dilution or appropriately diluted unknown are pipetted into each well. The covered plate is incubated for 15 minutes at 37 C, and the reaction is started by the addition of 100 ul of a plasrainogen/S-2251 mixture which contains two parts 10 mM S-2251, one part plasminogen solution (1 ug/ml) and seven parts assay buffer. The mixture is prepared immediately prior to use. (Assay buffer is Tris saline prepared by dissolving 605 mg Tris in 800 ml distilled water and adjusting the pH to 8.8 with 6 N HC1, then dissolving 5.84 g NaCl and 0.1 g Triton X-100 into the buffer and diluting to 1 L.)

The absorbance at 405 nm is then measured at 20, 40 and 60 minutes, and standard curves are plotted using absorbance values of 1 or less. A tPA standard formulation is used for reference. B. General Method

The practice of the present invention employs, unless otherwise indicated, conventional techniques of cell culture, molecular biology, microbiology, recombi¬ nant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., "Animal Cell Culture" (R.K. Freshney, ed. 1986); Maniatis et al, "Molecular Cloning: A Laboratory Manual" (1982); "DNA Cloning", Vol I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" (B.D. Ha es and S.J. Higgins, eds 1984); B. Perbal, "A Practical Guide To Molecular Cloning" (1984); the series, "Methods In Enzymology" (Academic Press, Inc.), "Gene Transfer Vectors For Mammalian Cells" (J.H. Miller, and M.P. Calos, eds., 1987, Cold Spring Harbor Laboratory) , "Large-Scale Mammalian Cell Culture" (J. Feder and W.R. Tolbert, eds., Academic Press, Inc., N.Y.), and "Animal Cell Biotechnology" (G.B. Griffiths, ed. , Academic Press, London, in press) . All patents and publications mentioned herein, both supra and infra, are hereby incorporated by refer¬ ence .

In the invention, the types of cells used are any cells which are capable of producing tPA at levels suitable for commercial production, i.e., preferably at least about 1 ug tPA/10 cells/day, and which secrete the product into the cell culture medium. A variety of cells which are capable of expressing tPA are known in the art. For example, rat prostate adenocarcinoma cells (U.S. Pat. No. 4,661,453), and a human melanoma cell line (Bowes, EPO 41,766, published Dec. 16, 1981); Rijken and Collen (1981), and Kluft et al (1983)) have been shown to synthesize tPA.

The cells expressing tPA may also be cells transformed with recombinant vectors containing a nucleotide sequence encoding tPA operably linked to control sequences which allow its expression and secre¬ tion. Transformed cells of this type include bacterial cells, for example, transformed E. coli cells, trans¬ formed B. subtilis cells, and the like. Also included are fungal cells, for example, yeast cells. See, for example, EPO No. 85306432.7. Mammalian cells may also be transformed with a recombinant vector which contains an expression system which encodes tPA. For example, as discussed below, the synthesis of tPA may be by Chinese hamster ovary cells (CHO) transformed with a vector pSTH-MDH, which contains the tPA sequences and an ampli- fiable DHFR gene. The construction of pSTH-MDH and the CHO host cell line are described in commonly owned USSN 010,871; examples of other vectors which may also be used to transform mammalian cell^'lines are also de¬ scribed in this application, and include pSV-tPA17, and pSTH-SDH. Another vector which may be used to transform CHO cells is that described by R.J. Kaufman et al, J Mol Biol (1982) , 159:601.

The cell culture may be grown in any accept¬ able system, for example, perfusion, batch, and semi- batch. Perfusion systems are presently preferred. In perfusion culture, medium is perfused through the reactor at a rate proportional to the number of cells and their metabolic characteristics. Process parameters can be maintained by introducing reagents and gases directly into the vessel or into the medium as it is being fed to the vessel. Effluent is removed from the perfusion chamber on a continuous or semi-continuous basis. Examples of perfusion systems are described in Tolbert et al. (1985), Tolbert et al (1987), and compo¬ nent parts in U.S. Pat. Nos.: 4,166,768; 4,178,209; 4,184,916; 4,537,860; and 4,335,215.

Mammalian cells which grow in suspension cul¬ ture may be used to produce tPA in a perfusion culture system, as described in U.S. Pat. Nos. 4,166,768; 4,178,209; and 4,184,916. In this system, a constant environment is maintained in the growth vessel by con¬ tinually adding fresh nutrients and removing waste prod¬ ucts at a rate proportional to the number of cells in the vessel. Under these conditions, the system can operate for prolonged periods of time with cell densi¬ ties from 10 to 30 times the maximum cell density in a typical batch reactor. A gentle "sail" agitator system is used to prevent cell damage at these high cell densi¬ ties. The filtration unit separates the cells from the product-containing cell conditioned medium and returns the cells to the growth vessel as fresh medium is added. Dissolved oxygen and carbon dioxide, as well as pH levels, are continually monitored and maintained throughout the production run. The effluent from the reactor contains waste products and the desired protein products secreted by the cells. Product reservoirs may be removed periodically for further processing by con¬ centration and other conventional protein purification techniques.

When prolonged cell growth is required for production of desired protein products, the suspension culture perfusion system maintains an optimal cell den¬ sity in the growth vessel by transferring excess cells to the harvest vessel. After the optimal cell density has been attained in the growth vessel, the serum con¬ centration in the fresh medium can be significantly reduced. Preferably, the cells are cultured in defined media which does not contain serum. We have found that by culturing cells in serum-free media containing a mam¬ malian serum albumin (preferably HSA), the resulting tPA is produced with an enhanced Type II/Type I ratio. By using serum-free HSA media, we have obtained tPA compo¬ sitions containing >50% Type II, as compared with the products of serum-containing media which typically pro¬ vide tPA that is about 40% Type II.

The amount any type of tPA in the effluent may be determined by any method which conveniently distin¬ guishes the species, for example, by the difference in molecular weight on gel filtration or upon gel electro- phoresis using Laemmli gels under non-reducing condi¬ tions. The presence of tPA in the fractions obtained by molecular weight sizing materials can be detected by procedures known in the art, for example, by determi¬ nation -of. the enzymic tPA activity, or by immunoassay- An Elisa assay for tPA is described in P.G. Abrams et al, J Immunol (1984), 132:1611, and kits providing the reagents for Elisa assays of tPA are commercially avail¬ able.

In vivo, and usually in vitro, wild-type mono- meric tPA is synthesized as a single chain proenzyme of approximate molecular weight of 70 kd, which can be cleaved by plasmin or trypsin, without disrupting the disulfide linked two-chain molecular structure. Upon reduction, tPA dissociates into a heavy and a light chain, with approximate molecular weights of 35,000. Wild type tPA contains two kringles linked by a hexapep- tide linker sequence. A kringle is a triple disulfide- linked sequence of amino acids which form a loop. These kringles are believed to be responsible for binding of tPA to fibrin (Thorsen, Biochim Biophys Acta (1975), 3_92:55) .

The invention provides highly purified forms of Type I and Type II tPA through separation of a puri¬ fied tPA/HSA mixture. By using the simplified purifica¬ tion process of the invention, higher final yields of purified tPA are attained.

In general, the conditioned media are cooled from about 37 C (or the cell culture temperature) to about 4 C and the pH lowered to 5.0 with dilute acetic acid. The media is then applied to a cationic exchange resin, having a negatively-charged ionic group bound to a support matrix. Suitable ionic groups include sulfate and carboxymethyl, preferably sulfate. Suitable support matrices include agarose, Sepharoses>, Sephadexs, Sephacolϊ', polyacrylamide, cellulose, membranes, and the like. Presently preferred is Fast-Flow S Sepharoses. The resin is pre-equilibrated with 50 mM NaOAc/100 mM NaCl, pH 5.0 to adsorb the tPA and HSA.

The adsorbed tPA/HSA is eluted with 20 mM Tris/1 M NaCl pH 8.0. At this point, the resulting material comprises a substantially pure mixture of HSA and tPA, having an activity of about 50,000 to about 400,000 IU/mg protein (preferably about 100,000 to 300,000 IU/mg). This is a suitable concentration for administration to mammals, including humans. The prod¬ uct at this point may be purified further to provide pure Type I and Type II tPA. Alternatively, the compo¬ sition may be sterile-filtered, dialyzed (optionally lyophilized) , and packaged for administration. Option¬ ally, one may further purify the tPA/HSA mixture prior to sterile filtration, e.g., by anionic ion exchange (e.g., Fast-Flow Q Sepharoses^*) , followed by gel filtra¬ tion, to provide a tPA/HSA mixture having substantially the same specific activity, but containing fewer con¬ taminating materials.

If completely purified material is desired, the eluted tPA/HSA is concentrated, for example by pre¬ cipitation with salts, ultrafiltration, etc. Presently preferred is precipitation with ammonium sulfate at ap¬ proximately 550 g/1. A clean separation into Type I and Type II tPA is then made using a support matrix conju¬ gated to a basic amino acid, preferably a lysine- Sepharosei- column.

This process differs from the process dis¬ closed in copending USSN 178,392 in that it eliminates the steps of PABA-Sepharoses> adsorption and Sephacryls filtration, and results in enhanced yield of tPA without reduction in specific activity.

The tPA produced by the processes of the invention may be used for the purposes already described for tPA, including therapy for disorders related to blood clots. In such cases the product will be formu¬ lated in pharmacologically acceptable compositions, and administered in therapeutically acceptable doses, as described in commonly-owned copending U.S. application 130,901, filed 10 December 1987 and specifically incorporated herein by reference in full.

Effective dosages and administration regimens are known in the art. The precise dosage administered will depend upon the species, age, size, and general condition of the subject, as well as the exact nature and severity of the condition to be treated. Thus, a precise effective dose cannot be specified in advance. However, as a general guide, it is expected that an ef¬ fective dose for treating thrombosis (for example, myo- cardial infarction, deep vein thrombosis, etc.) in a human will range from about 0.1-5 x 10 IU/dose. This dose is preferably administered by bolus injection i.v., but may alternatively be accomplished by slower i.v. infusion.

C Examples

The examples presented below are provided as a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the invention in any way.

Polyacrylamide gel electrophoresis is carried out as described by Laemmli (1970) in the presence or absence of sulfhydryl reducing reagents.

Analysis of tPA enzymic activity is performed using a direct amidolytic synthetic substrate assay, es- sentially as described by P. Friberger et al, Prog in Chem Fibr & Thromb IV (1979), (Davidson, J.A.F., ed) , p. 149; and P. Friberger, Thesis Scand J Clin Lab Invest (1982), 4_2 Suppl. 162, p. 58; in the presence of plasmin and soybean trypsin inhibitor, and using as sub¬ strate S-2288. One can distinguish between Type I and Type II by using synthetic substrate S-2251 in a similar assay method, as described by J.H. Verheijen et al, Tromb Res (1982) 2_7:377-85.

Immunoblotting techniques and Elisa assays for the' detection and/or quantitation of tPA are performed as described by Abrams supra, using as antibody a mouse IgG. monoclonal antibody which recognizes an epitope in the A chain of human tPA.

Cell density is determined as packed cell volume (PCV) as described by W.R. Tolbert et al, Ann Biochem (1980), 106:109 ■

Example 1 (Production of tPA in Cell Culture) (A) CHO cells transformed with the vector pSTH-MDH, as described in commonly owned USSN 010,871, are grown in any suitable, commercially available basal growth media, preferably a medium which does not contain serum. Suitable media include, without limitation, Ham's F-12, DMEM, IMDM and the like. Mammalian serum albumin, preferably HSA is added to the medium at a con¬ centration of about 0.1% to about 0.2%, and further sup¬ plemented with methotrexate, insulin, and transferrin.

Cells are grown in a 100 L perfusion system as described in commonly-owned copending application. USSN 092,502, filed 3 September 1987, incorporated herein by reference in full. The rate of perfusion is balanced to the PCV in an effort to deliver nutrients and remove waste products at a rate compatible with cell me¬ tabolism. Under these conditions, which are analogous to an in vivo environment, the cells are allowed to grow to a PCV of about 10-20 ml/L, to maximize production of tPA. The effluent is immediately transferred to 4°C upon collection.

(B) A control group of cells are cultured under the conditions described in part (A) above, but replacing the HSA added to the medium with 5% fetal calf serum.

Example 2 (Preparation of Injectible Composition)

(A) The crude conditioned medium obtained from Example 1 is purified by the following procedure. The buffers used are first prepared as follows:

#1 50 mM sodium acetate, 100 mM NaCl, pH 5.0; #2 20 mM Tris, 1.0 M NaCl, pH 8.0; #3 PBS, pH 8.0, 0.1 M KSCN; #4 PBS, pH 8.0, 0.25 M KSCN, 0.6 M arginine.

A 5-liter S-Sepharoses- Fast-Flow bioprocess column (Pharmacia; 25.2 cm x 10 cm) is equilibrated with five column volumes of buffer #1 at 150 1/h. Cell cul¬ ture conditioned media are pH adjusted to 5.0 with dilute acetic acid. After adjustment, the media are applied to the column at the same flow rate. The pH and conductivity of the media are such that tPA and HSA bind selectively to the column electrostatically while the bulk of the serum proteins and non-proteinaceous material (nucleic acids, phospholipids, etc.) do not bind to the resin and are removed in the column flow- through. The tPA/HSA mixture is eluted fro the column with about 4 column volumes of buffer #2.

The resulting composition consists essentially of tPA (as a mixture of Type I and II) and HSA, having an activity of 50-400 x 10 IU/mg (preferably 100,000- 300,000 IU/mg). (B) The composition is in a form suitable for packaging, and may if desired be injected. I.e., this composition is in stable form, and does not require the addition of supplementary proteins to enhance stability and bulk, etc. Optionally, one may further purify the mixture without substantially altering the specific activity by treatment with an additional ion exchange resin (preferably Fast-Flow Q Sepharoses) and gel fil¬ tration. If desired, the composition may be suspended in saline or other suitable injection carriers for intravenous administration. The gel filtration step may conveniently be used to change buffers to one acceptable for immediate administration.

Example 3 (Separation of Type I and Type II tPA)

The composition prepared in Example 2(A) is concentrated by precipitation with ammonium sulfate (550 g/1) at 4°C for a minimum of 4 hours. The precipitate is collected by centrifugation (5000 rpm for ap¬ proximately 30 minutes). The pellets may be stored at - 70°C, if necessary. The pellet is then dissolved in buffer #3, and clarified by centrifugation and/or fil¬ tration.

The purified tPA thus obtained is then sep¬ arated cleanly into Type I and Type II tPA using a 2.6 x 100 cm column of lysine Sepharose® to selectively adsorb the tPA. The column is pre-equilibrated with buffer #3 and the tPA loaded onto the column in amounts ranging from about 50-800 mg. Elution is effected by applica¬ tion of a gradient of buffer #3 and #4 which results in a clean separation of Type I and Type II tPA in each case.

The recovery of Type I and Type II tPA, and the quality of the separation, is independent of the amount loaded onto the column over a ten fold range. This result is unexpected, and the upper limit is yet to be found.

Each of the peaks containing Type I and Type II tPA is assayed according to the procedure set forth above using the International Standard of tPA obtained from the National Institute of Biological Standards as specified above, or a secondary standard compared thereto. In each case, the protein eluted as Type I tPA shows an activity of 420 x 10 IU/mg, within experimen¬ tal error; that eluted as Type II tPA shows a specific activity of about 550 x 10 IU/mg within experimental error. The yield obtained is superior to the yields reported in the prior art (including copending USSN 178,392). This may be due in part to the simplification of the purification process; however, the fact that intermediate steps in purification may be omitted with¬ out adverse impact on the final purity of the product was unexpected.

Further, it is found that the tPA elaborated in serum-free media containing serum albumin contains a greater ratio of Type II to Type I tPA than the tPA elaborated in media containing FCS. The control (FCS) tPA generally exhibits a ratio of II/I of about 40/60, whereas the process of the invention yields a ratio of about 60/40 or greater. Although the reason for this improvement is unknown, we believe that it is due to the change in media, from serum-containing media to serum- free media containing serum albumin (particularly HSA) . This improved culture condition should generally provide tPA that is greater than 50% Type II, preferably 60% or greater Type II.

Example 4 (Formulations) Formulations suitable for administering tPA to a subject may be prepared as follows: (A) Approximately 50 mg of purified tPA (Type

I or II) in the elution buffer remaining from Example 3 is transferred via gel filtration to a solution of 150 mM arginine, 50 mM sodium citrate, 20 mg/ml HSA, pH 6.0,

7 to form a solution having about 2.5 x 10 IU/ l. This represents approximately one unit dose for an adult human, in a form ready for immediate administration.

The solution may be sterile filtered if desired, and packaged for storage.

(B) The solution prepared in part (A) above is then^' lyophilized using standard sterile freeze drying techniques (as used for other glycoproteins at similar concentrations) to form a dry powder. This powder may be reconstituted into solution for parenteral (prefer¬ ably intravenous) administration. The dry powder may be stored indefinitely, preferably under an inert atmos¬ phere (e.g., dry nitrogen). Refrigeration may extend the shelf life.

Claims

WHAT IS CLAIMED:

1. A pharmaceutical composition suitable for administration to a mammal, which composition comprises: a substantially pure mixture of human serum albumin and tPA, having a specific activity of about 50,000 IU/mg to about 400,000 IU/mg.

2. The composition of claim 1 wherein said specific activity is about 100,000 IU/mg to about 300,000^' IU/mg.

3. The composition of claim 1 wherein said tPA comprises at least 50% Type II tPA.

4. A process for preparing a pharmaceutical tPA composition in high yield, which process comprises: culturing tPA-producing cells in a culture medium containing human serum albumin to provide a conditioned medium containing tPA and HSA; and chromatographing said conditioned medium using a support matrix conjugated with negatively-charged ionic groups to provide a composition consisting essentially of tPA and HSA, having a specific tPA activity of about 50,000 IU/mg to about 400,000 IU/mg protein.

5. The process of claim 4 wherein said specific activity is about 100,000 IU/mg to about 300,000 IU/mg.

6. The process of claim 4 wherein said support matrix conjugated with negatively-charged ionic groups comprises S Sepharoseϊ' Fast Flow.

7. The process of claim 4 wherein said culture medium is substantially free of serum.

8. The process of claim 7 wherein said tPA is at least 50% Type II tPA.

9. A method for treating a mammal for thrombosis, which method comprises: administering an aqueous composition consisting essentially of tPA and mammalian serum albumin, having a tPA activity of about 50,000 IU/mg. o about 400,000 IU/mg protein.

10. The method of claim 9 wherein said specific activity is about 100,000 IU/mg to about 300,000 IU/mg.

11. A process for preparing Type I tPA in high yield, which process comprises: culturing tPA-producing cells in a culture medium containing mammalian serum albumin to provide a conditioned medium containing tPA and MSA; chromatographing said conditioned medium using a support matrix conjugated with negatively-charged ionic groups to provide a composition consisting essentially of tPA and MSA, having a specific tPA activity of about 50,000 IU/mg to about 400,000 IU/mg protein; concentrating said composition; suspending said concentrate and chromato¬ graphing said suspension using support matrix conjugated with basic amino acid; and collecting substantially pure Type I tPA.

12. The process of claim 11 wherein said support matrix conjugated with negatively-charged ionic groups comprises Fast Flow S Sepharoses, and said mammalian serum albumin is HSA.

13. The process of claim 11 wherein said specific activity is about 100,000 IU/mg to about 300,000 IU/mg.

14. The process of claim 11 wherein said concentration comprises precipitation with ammonium sulfate.

15. The process of claim 11 wherein said support matrix conjugated with basic amino acid is lysine-Sepharoses 4B.

16. A process for preparing Type II tPA in high yield, which process comprises: culturing tPA-producing cells in a culture medium containing mammalian serum albumin to provide a conditioned medium containing tPA and MSA; chromatographing said conditioned medium using a support matrix conjugated with negatively-charged ionic groups to provide a composition consisting essentially of tPA and MSA, having a specific tPA activity of about 50,000 IU/mg to about 400,000 IU/mg protein; concentrating said composition; suspending said concentrate and chromato¬ graphing said suspension using a support matrix ' conjugated with basic amino acid; and collecting substantially pure Type II tPA.

17. The process of claim 16 wherein said media is substantially serum-free.

18. The process of claim 17 wherein said tPA in said tPA/MSA composition is at least 50% Type II tPA.

19. The process of claim 18 wherein said support matrix conjugated with negatively-charged ionic groups comprises Fast Flow S Sepharoses, and said mammalian serum albumin is HSA.

20. The process of claim 18 wherein said specific activity is about 100,000 IU/mg to about 300,000 IU/mg.

21. The process of claim 18 wherein said concentration comprises precipitation with ammonium sulfate.

22. The process of claim 18 wherein said support matrix conjugated with basic amino acid is lysine-Sepharoseδ 4B.

23. Type I tPA, having a specific activity of about 420,000 IU/mg.

24. Type II tPA, having a specific activity of about 550,000 IU/mg.