The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed:

FIELD OF THE INVENTION

The present invention relates generally to the use of novel fermentation and chromatographic procedures separately and jointly for the production of Recombinant TNK-tPA, a tissue plasminogen activator, in biologically active form from fluids, especially mammalian host cell culture supernatants. The invention relates primarily to a new way of recovering higher yield of purified Recombinant Tenecteplase by using bio analytical techniques such as but not limited to chromatographic procedures.

BACKGROUND AND PRIOR ART OF THE INVENTION

In past, various media and methods were used for the cell culture manufacturing of recombinant glycoprotein or monoclonal antibody. Commonly employed bioreactor process includes; batch, semi fed-batch, fed-batch, perfusion and continuous fermentation. The ever- increasing demand of monoclonal antibody and other recombinant proteins in properly glycosyalted forms have increased the prospects of cell culture process development. In addition the regulatory hurdles imposed on the serum containing process has led to the development of cell culture process in a completely chemically defined environment.

Numerous techniques have in the past been applied in preparative separations of biochemically significant materials. Commonly employed preparative separatory techniques include: ultrafiltration, column electrofocusing, fiatbed electrofocusing, gel filtration, electrophoresis, isotachophoresis and various forms of chromatography. Among the commonly employed chromatoghraphic techniques are ion exchange and adsorption chromatography. The extensive application of recombinant methodologies to large-scale purification and production of eukaryotic protein has increased the prospect of obtaining the molecule in required quantity using simplified purification procedures.

TNK-tPA (TNKase®, tenecteplase) is a plasminogen activator produced by recombinant DNA technology using the genetically modified Chinese hamster ovary cells. TNK-tPA binds to fibrin and converts plasminogen to plasmin. This conversion to plasmin is increased relative to the presence of fibrin. TNK-tPA is 80-fold more resistant to plasminogen activator inhibitor-1 than t-PA. This leads to a decrease in plasminogen activation in the systemic The TNK-tPA molecule was genetically engineered from wild-type recombinant tissue plasminogen activator (rtPA) to preserve the full fibrinolytic activity of wild-type t-PA while also producing specific clinical characteristics. It includes three mutations, which enhance fibrin specificity, prolong its half-life, and greatly increase resistance to plasminogen activator inhibitor 1 (PAI-1) compared to t-PA. TNK-tPA possess fibrin specificity 14 fold greater than that of wild-type t-PA1. This activity is desirable for a fibrinolytic because it permits targeting of the clot in the infarct-related artery while conserving fibrinogen and minimizing systemic plasminogen activation.

The name "TNK" reflects the three domains of the wild type t-PA molecule (the T, N, and K domains) that have been altered.

It is produced by a mammalian cell (Chinese Hamster Ovary) [CHO] suspension culture in a nutrient medium containing the antibiotic Gentamicin. Gentamicin is not detectable in the final product. TT^K-tPA is packaged as a sterile, white to pale yellow, preservative-free lyophilized powder for intravenous (IV) administration.

The plasminogen activator t-PA is a fibrin specific activator of plasminogen. The fibrin specificity is a result of the interaction of the kringle-2 domain of t-PA with specific lysine residues on fibrin. Tissue type plasminogen activator (t-PA) a glycosylated serine protease is produced as an active single chain molecule with a molecular weight of about 70 kDa. The native single-chain t-PA (sc-tPA) is converted by plasmin to a two-chain t-PA (tc-tPA). Two types of native sc-tPA are known. Type I sc-tPA is fully glycosylated, while type II lacks glycosylation at Asn-184. Plasma levels of t-PA are approximately 5-10 ng/ml. The majority of t-PA in circulation is present as a complex with PAI-1 (plasminogen activator inhibitor type 1).

The t-PA molecule contains five domains that have been designated as: finger region (F) which includes amino acids 1 to about 44 growth factor region (G) which stretches from amino acids 45 to 91 kringle one (K1) stretching from 92 to 173 amino acids kringle two (K2) from about amino acid 180 to 261 serine protease domain (P) from amino acids 264 to the C-terminal end.

These domains are situated generally adjacent to one another or are separated by short "linker" regions and account for the entire 527 amino acid sequence of the mature form of t- PA.

The growth factor region (G) has been associated with cell surface binding activity while the kringle 2 (K2) region has been strongly associated with fibrin binding and with the ability of fibrin to stimulate the activity of t-PA. The serine protease domain is responsible for the plasminogen activating activity of t-PA. (IgGl).

Tenecteplase is indicated for use in the reduction of mortality associated with acute myocardial infarction (AMI).

OBJECTIVES OF THE INVENTION

The main object of the present invention is to use novel fermentation and chromatographic procedures for rapid and efficient recovery of Recombinant Tenecteplase, a monoclonal antibody to human TNF-alpha, from cell culture supernatant. This invention is directed to a process for facilitating maximum product (recombinant Tenecteplase) recovery.

SUMMARY OF THE INVENTION

The present invention relates to the use of novel fermentation process for the over expression of Recombinant TNK-tPA, a thrombolytic, protein in CHO cells.

The present invention also relates to the use of novel chromatographic procedures separately and jointly for the production of Recombinant TNK-tPA, a thrombolytic, protein, in biologically active form from fluids, especially mammalian host cell culture supernatants.

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1: Process chromatogram of cation chromatography: Chromatographic profile of the first step of recovery of the target protein from the cat ion exchange chromatography. This is the purification chromatogram from a supernatant containing Tenecteplase as explained in example 1 (Supernatant derived from a harvested media containing Tenecteplase after clarification). The blue line represents the UV absorbance at 280nm. Majority of the other protein comes out in the flow through as can be observed in the figure.

Figure 2: Process chromatogram of anion chromatography: Chromatographic profile of the second step of recovery of the target protein from the anion exchange chromatography. This figure shows a chromatogram of purification from the eluent of the cat ion exchange chromatography as explained in the example 1 (Start material on to this column was the buffer exchanged elute containing the Tenecteplase from the cat ion exchange column). It is clear from the figure that the majority of the load has bound and come in eluent mode.

Figure 3: Electrophoretic pattern of Drug substance: showing comparable molecular weight with RMP where Lane No. 1: Molecular weight Marker, Lane No. 2: RMP and Lane No. 3: Formulated Drug Substance. Electrophoretic profile reveals the purity and identity of the target protein with the Originator. The electrophoretic diagram depicts the stability of the drug substance and also the purity of the process close to homogeneity and comparability of the drug substance with that of the Originator. RMP refers to Reference Medicinal Product - TNKase®

Fig 4: Western blot analysis: Test material and RMP are transferred on to the membrane and incubated with Mouse anti-tPA monoclonal antibody and probed with Goat anti Mouse IgG ALP Conjugate (1:5000 dilution) Antibody. Drug substance showing clear corresponding signal with RMP where Lane No. 1: Molecular weight Marker, Lane No. 2: RMP and Lane No. 3: Formulated Drug Substance. This clearly reflects the specificity and identity of the test sample compared to that of the originator.

Fig 5: Zymography analysis: Test material and RMP are loaded on to the 12% SDS-PAGE containing 0.1% casein (Img/ml) and 10ug/ml plasminogen (combined act as substrate) was stained with coomassie. Showing a zone of clearance indicting that the tPA has used the substrate in the gel to digest the casein. Drug substance showing clear corresponding signal with RMP where Lane No. 1: Molecular weight Marker BSA alone. Lane No. 2: RMP, Lane No. 3: blank and Lane No. 4: Formulated Drug Substance. It is clear from the figure that the functionality of the protein in question is comparable with that of the originator.

Fig 6: RP HPLC profile of Drug substance showing comparable profile with RMP. It is very clear from the figure that the retention time of both the originator and the test sample are matching and the charge of the molecule is very much comparable with the originator as one can see from the profile of the RP HPLC of both the test sample and the originator.

DETAILED DESCRIPTION OF THE INVENTION

This present invention relates to the rapid and efficient recovery of Recombinant TNK-tPA from cell culture supernatant from Cell culture fluid by means of Ion exchange chromatography. This chromatographic step is used for capture of TNK-tPA. This separation involves in selective binding of the desired compound to resin based on the pi and then elation with elution buffer. Culture supernatants are clarified before chromatographic treatment. TNK-tPA eluent fractions are enriched with biologically active material, but they will be subjected to further processing by Ion exchange chromatographic step. These processes are used for removal of process related impurities like host cell protein and host cell DNA. The present invention also relates to the recombinant TNK-tPA recovery procedure involving serial application different chromatographic techniques as mentioned previously. All different steps, conditions and compositions are disclosed in the invention.

Example 1:

Clarification of the cell culture harvest was carried out by using a cellulose disposable filter with 650 - 1000 cm effective filtration area and with an operating pressure of not more than 30 psi. The filfrate was checked for turbidity and target protein content. Clarified filtrate diafiltered against 25 mM sodium acetate, pH 5.0. Cation-exchange chromatography was used in binding and elution mode + 0.3 M arginine+0.25mM NaC1 as equilibration buffer. After the sample is loaded on to the column, it is washed with equilibration buffer followed by 50 mM sodium acetate, pH 5.0 + 0.3 M arginine+0.25niM NaC1. The protein of interest was eluted with 50 mM sodium acetate, pH 5.0 + 0.3 M arginine+1M NaC1 (Fig 1). The eluate was diafilterd aginst 50mM Tirs pH 8.0 using a 30kDa TFF membrane. Anion exchange chromatography in binding mode was carried out. The column was equilibrated with This buffer pH 7.8 - 8.2. Coloumn was washed with the equilibrating buffer. Protein of interest was eluted using 50 mM sodium acetate, pH 4.0. This step was used for the removal of process related impurities like leach ate protein A, host cell DNA and host cell protein. (Fig 2). Neutralized eluate was filtered for virus removal using viral removal filter. The filtrate was buffer exchanged using a 30 kDa TFF membrane. The drug substance was characterized as per the specifications. The Drug Substance (Active Pharmaceutical Ingredient) was formulated using the ingredients mentioned below. They are 0.55 g L-arginine, 0.17 g phosphoric acid, and 4.3 mg polysorbate 20, yields a solution containing 50 mg of TNK- tPA.

Example 2:

The formulated material was characterized as per the specifications set by product development specification. A 10% SDS PAGE under reducing condition was studied for the sample derived from the PAGE showed a clear corresponding band with RMP (Fig 3). Western blot analysis was carried out where in Drug substance showing clear corresponding signal with RMP (Fig 4). Functional activity was assed by zymograpy wherein Drug substance showing the zone of clearance corresponding with the RMP (Fig 5), RP HPLC profile showed for test molecule, which was very much comparable with the RMP (Fig 6).

CLAIMS

We claim:

1. A process for the recovery and purification of recombinant Monoclonal antibody to TNK-tPA„ a thrombolytic agent comprising steps of: contacting culture supernatant(s) with resin(s) for selective adsorption of compound(s); eluting the adsorbed compound and subjecting the enriched product to series of separation techniques.

2. The process as claimed in claim 1, where elution of adsorbed compound is performed by affinity chromatography.

3. The process as claimed in claim 1, where monoclonal antibody to TNK-tPA„ a thrombolytic agent containing eluent fractions are purified using ion exchange chromatography.

4. The process as claimed in claim 1, wherein supernatant is obtained from cell fermentation.

5. The process as claimed in claim 1, wherein said supernatant is mammalian host cell culture supernatant.

6. The process as claimed in claim 1, wherein said supernatant is cell culture-derived fluid.

7. The process as claimed in claim 1, wherein said supernatant is mammalian cell culture derived fluid

8. The process as claimed in claim 1, wherein the process buffer 10-80 mM sodium acetate, , pH 4.0-5.0 + O.IM -0.5M M arginine+0.5-2.0M NaC1 is used for the recovery of target protein from culture supernatant.

9. The process as claimed in claim 1, wherein the process buffer is particularly 20-60 mM sodium acetate, pH 4.0-5.0 + O.IM -0.4M M arginine+0.5-2.0M NaC1 is used for the recovery of target protein from culture supernatant.

10. The process as claimed in claim 1, wherein said culture supernatant(s) are concentrated and clarified before contacting resins.

11. The process as claimed in claim 1, where eluent is held for 45 - 60 min at acidic pH at room temperature for virus inactivation.

12. The process as claimed in claim 1, where recombinant Monoclonal antibody recovered and purified is TNK-tPA.