Method for producing pharmaceutically effective recombinant human low molecular weight urokinase

Abstract

This document describes the CHO cell line and the manufacturing process for low molecular weight recombinant human urokinase (rh-LMW-uPA) using this cell line. This process enables the production of low molecular weight urokinase with high productivity and improved characteristics in terms of safety, quality, and stability.

Description

[Technical Field]

[0001] This invention relates to a method for producing low molecular weight recombinant human urokinase (rh-LMW-uPA), particularly stable and active glycosylated recombinant human low molecular weight urokinase, from eukaryotic cell lines using recombinant DNA technology. This process makes it possible to produce physiologically active (mature) glycosylated recombinant human low molecular weight urokinase in culture medium.

[0002] The technical aspect of the present invention is the production of glycosylated recombinant human low molecular weight urokinase by applying genetic engineering techniques to eukaryotic cells of the CHO strain. [Background technology]

[0003] Fibrinolysis is the process of dissolving blood clots to prevent vascular occlusion. The fibrinolytic system is primarily regulated by proteases and protease inhibitors. The key enzyme in this system is plasmin, which is mainly produced from inactive plasminogen via its direct activators—tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA)—which are counteracted by specific inhibitors [Wun, T.-C., Schleuning, W.-D. & Reich, E. Isolation and Characterization of Urokinase from Human Plasma. The Journal of Biological Chemistry 257, 3276-3283 (1982)]. Plasminogen is converted to active plasmin by uPA, which dissolves fibrin clots and produces fibrin degradation products [Lin, H. et al. Therapeutics targeting the fibrinolytic system. Experimental & Molecular Medicine 2020 52:3 52, 367-379 (2020)].

[0004] Human uPA is an important serine protease that has been used clinically for many years as a thrombolytic agent [Gurewich, V. Fibrinolytic Mechanisms of tPA, prouPA, Mutant prouPA and Their Implications for Therapeutic Thrombolysis. Cardiovascular Engineering and Technology 2013 4:4 4, 328-338 (2013); Tomasi, S., Sarmientos, P., Giorda, G., Gurewich, V. & Vercelli, A. Mutant Prourokinase with Adjunctive C1-Inhibitor Is an Effective and Safer Alternative to tPA in Rat Stroke. PLoS One 6, (2011)]. This is pro-uPA (sc-uPA, 411 amino acids, MV 54kDA) [Gurewich, V. Pro-urokinase: physiochemical properties and promotion of its fibrinolytic activity by urokinase and by tissue It is secreted into the bloodstream from various tissues in the form of a single-chain glycosylation enzyme called a plasminogen activator with which it has a complementary mechanism of action. [Semin Thromb Hemost 14, 110-115 (1988)], and has very low amid-degrading activity [Hedstrom, L. Serine protease mechanism and specificity. Chem Rev 102, 4501-4523 (2002)], and is activated by plasmin to produce an α chain (residues 1-158) and a β chain (residues 159-411). 158 -I 159It is activated by cleaving the peptide bond between them [Spraggon, G. et al. The crystal structure of the catalytic domain of human urokinase-type plasminogen activator. Structure 3, 681-691 (1995)]. The fully active dimeric protein uPA (tc-uPA) thus produced is also known as high molecular weight urokinase (HMW-uPA) [Primary structure of single-chain pro-urokinase - PubMed.https: / / pubmed.ncbi.nlm.nih.gov / 2931434 / ; Magill, C., Katz, BA & MacKman, RL Emerging therapeutic targets in oncology: urokinase-type plasminogen activator system.http: / / dx.doi.org / 10.1517 / 14728222.3.1.1093, 109-133 (2005); Spraggon, G. et al. The crystal structure of the catalytic domain of human urokinase-type plasminogen activator.Structure 3, 681-691 (1995)]. It converts plasminogen to plasmin and promotes the proteolysis of fibrinogen to produce fibrin.Conversion to dimers increases interdomain flexibility [Behrens, MA et al. Activation of the Zymogen to Urokinase-Type Plasminogen Activator Is Associated with Increased Interdomain Flexibility. J Mol Biol 411, 417-429 (2011)], which makes substrate interactions more efficient and increases urokinase enzyme activity by approximately 100 times [Fleuryso, V., Lijnenl, HR & Anglb-Canosji, E Mechanism of the Enhanced Intrinsic Activity of Single-chain Urokinase-type Plasminogen Activator during Ongoing Fibrinolysis*. Journal of Biological Chemistry 268, 18554-18559 (1993)].

[0005] Lys 135 -Lys 136 (Ricin 135 -Ricine 136Further cleavage at ) generates an amino-terminal fragment (ATF, amino acids 1-135), which contains an EGF-like domain, i.e., a growth factor domain (GFD) (residues 1-49 of the human uPA sequence) and a kringle domain (amino acids 50-131) (Figure 1).The remaining 136-158 residue fragment is called a linked peptide (CP), a low molecular weight (LMW-UK, amino acids 136-411, MW 33kDa) that is bound to the catalytic domain at the carboxyl terminus via a disulfide crosslink [Gurewich, V Pro-urokinase: physiochemical properties and promotion of its fibrinolytic activity by urokinase and by tissue plasminogen activator with which it has a complementary mechanism of action. Semin Thromb Hemost 14, 110-115 (1988); Stepanova, V. v. & Tkachuk, VA Urokinase as a Multidomain Protein and Polyfunctional Cell Regulator. Biochemistry (Moscow) 2002 67:1 67, 109-118 (2002)], which is known to maintain its catalytic activity and has been used as a therapeutic agent [Gates, J. & Hartnell, GG When urokinase was gone: commentary on another year of thrombolysis without urokinase. J Vasc Interv Radiol 15, 1-5 (2004); Gurewich, V. Pro-urokinase: physiochemical properties and promotion of its fibrinolytic activity by urokinase and by tissue plasminogen activator with which it has a complementary mechanism of action. Semin Thromb Hemost 14, 110-115 (1988)].

[0006] HMW-uPA and LMW-uPA have similar catalytic activities against plasminogen [Sato, S. et al. High-affinity urokinase-derived cyclic peptides inhibiting urokinase / urokinase receptor-interaction: effects on tumor growth and spread. FEBS Lett 528, 212-216 (2002)].

[0007] In urokinase isolated from urine, the high molecular weight form is more abundant, while in urokinase obtained from cultured kidney cells, the low molecular weight form is more abundant [Aditiviya & Khasa, Y. P. The evolution of recombinant thrombolytics: Current status and future directions.https: / / doi.org / 10.1080 / 21655979.2016.12297188, 331-358 (2016)].

[0008] In vivo, after secretion, pro-uPA binds to uPAR (urokinase-type plasminogen activator receptor, uPAR, or CD87) and is converted to Lys 158 -Ile 159 (isoleucine 159The bond between pro-uPA and the receptor is cleaved, converting it to an active dimer state (i.e., tc-uPA or uPA). Pro-uPA bound to the receptor is activated more rapidly by plasmin than when it is free in plasma [Irigoyen, JP, Munoz-Canoves, P., Montero, L., Koziczak, M. & Nagamine, Y. The plasminogen activator system: biology and regulation. Cellular and Molecular Life Sciences CMLS 1999 56:1 56, 104-132 (1999)]. Activated uPA converts plasminogen bound to the adjacent membrane into plasmin.

[0009] The amino-terminal fragment of uPA (ATF, residues 1-135) contains all the binding sites necessary for interaction with the receptor [Barinka, C. et al. Structural Basis of Interaction between Urokinase-type Plasminogen Activator and its Receptor. J Mol Biol 363, 482-495 (2006)], a glycosylphosphatidylinositol (GPI)-anchored membrane protein. The resulting complex has been shown to be involved in two distinct biological cascades: (a) plasminogen activation, which results in proteolytic activity, and (b) signal transduction, which controls cell adhesion and mitogenesis. Indeed, uPAR has multiple functional roles related to tumor progression, such as tumor growth, apoptosis, metastasis, angiogenesis, multidrug resistance (MDR), prognosis, etc. High expression of uPAR is detected in various cancer cells but is at very low levels in normal cells. This suggests that uPAR levels in tumor tissues are closely related to the malignancy of tumors and the prognosis of cancer patients [Zhai, B. T. et al. Urokinase-type plasminogen activator receptor (uPAR) as a therapeutic target in cancer. Journal of Translational Medicine vol. 20 Preprint at https: / / doi.org / 10.1186 / s12967-022-03329-3 (2022)].

[0010] Since rh-LMW-uPA does not contain the ATF fragment, it cannot bind to the urokinase receptor (uPAR), avoiding the activation of the signal transduction cascade by uPAR-uPA binding.

Summary of the Invention

[0011] The present invention arose from the need to utilize the plasminogen activator of dimeric recombinant human glycosylated low molecular weight urokinase (rh-LMW-uPA) as an alternative to urokinase extracted from urine and used for catheter cleaning, which is one of the most widely used thrombolytic agents for central venous catheter occlusion in the UK [Kumwenda, M. J., Dougherty, L., Jackson, A. & Hill, S. Prospective Audit to Study urokinase use to restore Patency in Occluded central venous catheters in hematology and oncology patients (PASSPORT 2). https: / / doi.org / 10.1177 / 1129729820950997 22, 568-574 (2020)].

[0012] For this purpose, a CHO cell line was developed, deposited with the Culture Collection of Switzerland AG (CCOS), and identified with the number CCOS 2068.

[0013] The structure of human rh-LMW-uPA described here consists of two polypeptide chains, an α-chain and a β-chain, which are linked by an interchain disulfide bond between Cys 13 and Cys 121 and this bond is surprisingly activated by proteolysis of the Lys 23 -Ile 1 peptide bond in the culture medium of the CHO cell line identified above. The β-chain contains Cys 31 -Cys 47 、Cys 39 -Cys 110 、Cys 135 -Cys 204 、Cys 167 -Cys 183 、Cys 194 -Cys 222It contains five intrachain disulfide bonds. Furthermore, the β chain has a fully active catalytic domain, as well as the consensus sequence Asn 144 -It has an N-glycosylation site within the Ser-Tyr chain and an O-glycosylation site within the α-chain.

[0014] A significant number of currently approved protein-based drugs require appropriate glycosylation to achieve optimal therapeutic effects. This is because glycosylation affects various physiological processes at both the cellular and protein levels. uPA is O- and N-glycosylated. Fucose residues are linked to Thr-18 in the epidermal growth factor-like domain of uPA from cultured, urinary, and recombinant human kidney cells via O-glycosylation [Kentzer, EJ, Buko, A., Menon, G. & Sarin, VK Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase. Biochem Biophys Res Commun 171, 401-406 (1990); Buko, AM et al. Characterization of a posttranslational fucosylation in the growth factor domain of urinary plasminogen activator. Proc Natl Acad Sci USA 88, 3992-3996 (1991)]. The position of fucosylated Thr-18 in this domain suggests its importance in specific receptor / ligand binding systems that carry out various biological functions [The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 3031025 / ].Furthermore, N-glycosylation of human uPA occurs with asparagine 302 (Asn-302) [Bansal, V. & Roychoudhury, PK Production and purification of urokinase: A comprehensive review. Protein Expr Purif 45, 1-14 (2006); Lenich, C., Pannell, R., Henkin, J. & Gurewich, V. The influence of glycosylation on the catalytic and fibrinolytic properties of pro-Urokinase. Thromb Haemost 68, 539-544 (1992)]. Occurs in the protease domain [Steffens, GJ, Gnnzler, WA, otting, F., Frankus, E. & Flohe, L. The complete amino acid sequence of low molecular mass urokinase from human urine. Hoppe Seylers Z Physiol Chem 363, 1043-1058 (1982); Irigoyen, JP, Munoz-Canoves, P., Montero, L., Koziczak, M. & Nagamine, Y. The plasminogen activator system: biology and regulation. Cellular and Molecular Life Sciences CMLS 1999 56:1 56, 104-132 (1999)].N-glycans also contain Man (mannose), Gal (galactose), Fuc (fucose), GlcNac (sodium glucosyl), Neu5Ac (neurosyl 5-oxoglucosyl), and GalNAc (sodium galactosyl) residues [Steffens, GJ, Gunzler, WA, otting, F., Frankus, E. & Flohe, L. The complete amino acid sequence of low molecular mass urokinase from human urine. Hoppe Seylers Z Physiol Chem 363, 1043-1058 (1982); McLellan, WL, Vetterlein, D. & Roblin, R. The glycoprotein nature of human plasminogen activators. FEBS Lett 115, 181-184 (1980)]. The biological behavior of recombinant but nonglycosylated sc-uPA has been demonstrated to be similar to that of glycosylated urinary recombinant proteins [Li, XK, Lijnen, HR, Nelles, L., Hu, MH, & Collen, D. Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator. Biochim Biophys Acta 1159, 37-43 (1992); The influence of glycosylation on the catalytic and fibrinolytic properties of pro-urokinase - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 1455401 / ].However, studies have shown that recombinant, non-glycosylated sc-uPA exhibits higher plasmin-mediated cleavage efficiency, higher proteolytic activity, and faster inactivation by plasminogen inhibitors compared to its recombinant glycosylated counterpart [The influence of glycosylation on the catalytic and fibrinolytic properties of pro-urokinase - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 1455401 / ]. Furthermore, studies have shown that the sialic acid content of recombinant sc-uPA negatively impacts its clearance in vivo [Henkin, J., Dudlak, D., Beebe, DP & Sennello, L. High sialic acid content slows prourokinase turnover in rabbits. Thromb Res 63, 215-225 (1991)].

[0015] In rabbits, recombinant prourokinase (pro-uPA) containing 2.5 to 3 sialic acid molecules per protein has a significantly shorter half-life compared to pro-uPA with a lower degree of sialation. In the range of 0 to 1.5 sialic acid residues per pro-uPA molecule, the metabolic rate of the protein is relatively insensitive to the sialic acid content.

[0016] The present invention relates to the consensus sequence Asn of the catalytic domain (Figure 2). 144 (Asparagine) 144 This relates to the preparation process of N-glycosylated human rh-LMW-uPA within ). 144 As already described in the literature, Asn contains mannose, galactose, fucose, sodium glucosyl, and neurosyl 5-oxoglucosyl residues. 302 It has been identified as the (pro-uPA structure). Glycosylated Asn 302uPA is suggested to be more susceptible to plasmin-mediated activation and more resistant to inhibitors [The influence of glycosylation on the catalytic and fibrinolytic properties of pro-urokinase - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 1455401 / ]. Furthermore, 2.2 sialic acid residues were identified per rh-LMWUK molecule, and surprisingly, O-glycans derived from core 1 AS, core 1 MS, and core 1 DS were found to be present on the α-chain of the linked peptide (Figure 2).

[0017] Currently, the commercialization of recombinant uPA has not yet been approved by the FDA or EMA. However, various attempts are being made to produce uPA based on recombinant DNA technology [Aditiviya & Khasa, YP The evolution of recombinant thrombolytics: Current status and future directions. https: / / doi.org / 10.1080 / 21655979.2016.12297188, 331-358 (2016); Mican, J., Toul, M., Bednar, D. & Damborsky, J. Structural Biology and Protein Engineering of Thrombolytics. Comput Struct Biotechnol J 17, 917 (2019)]. This confirms, on the one hand, the clinical relevance of this molecule, and on the other hand, the need for such an approach from the standpoint of safety and purity.

[0018] Unlike commercially available urine-extracted HMW urokinases, the activated recombinant LMW type prepared according to the method of the present invention overcomes the potential risk of transmission of infectious or contaminant substances [The Case of Abbokinase and the FDA: The Events Leading to the Suspension of Abbokinase Supplies in the United States - Journal of Vascular and Interventional Radiology. https: / / www.jvir.org / article / S1051-0443(07)61798-9 / fulltext; Hartnell, GG & Gates, J. The case of Abbokinase and the FDA: The events leading to the suspension of abbokinase supplies in the United States. Journal of Vascular and Interventional Radiology 11, 841-847 (2000)], and, unlike streptokinases, is non-antigenic thanks to a human-derived sequence secreted by eukaryotic cells [Mican, J., Toul, M., Bednar, D. & Damborsky, J. Structural Biology and Protein Engineering of Thrombolytics. Comput Struct Biotechnol J 17, 917 (2019); Ouriel, K. Safety and Efficacy of the Various Thrombolytic Agents. Reviews in Cardiovascular Medicine 2002, 3(S2), 17-24 3, 17-24 (2002)].Mammalian urine contains only trace amounts of urokinase (10-15 ng / mL) [Vetterlein, D. & Calton, GJ Purification of urokinase from complex mixtures using immobilized monoclonal antibody against urokinase light chain. Thromb Haemost 49, 24-27 (1983)], making the purification of this enzyme a cumbersome and expensive procedure. Furthermore, the multiple steps in the extraction process generally result in low yields, further contributing to the high cost of this enzyme [Rouf, SA, Moo-Young, M. & Chisti, Y. Tissue-type plasminogen activator: Characteristics, applications and production technology. Biotechnol Adv 14, 239-266 (1996)].On the other hand, Bernik and Kwaan [Bernik, MB & Kwaan, HC Plasminogen activator activity in cultures from human tissues. An immunological and histochemical study. J Clin Invest 48, 1740-1753 (1969)] reported that cultured kidney cells secrete 50-100 ng / mL of uPA in vitro [Roychoudhury, PK, Khaparde, SS, Mattiasson, B. & Kumar, A. Synthesis, regulation and production of urokinase using mammalian cell culture: A comprehensive review. Biotechnol Adv 24, 514-528 (2006); Bansal, V. & Roychoudhury, PK Production and purification of urokinase: a comprehensive review. Protein Expr Purif 45, 1-14 (2006)]. Because the concentration of urokinase in human urine is extremely low, eukaryotic cells cultured in vitro are an excellent alternative for the production and purification of urokinase. By using the genetically modified CHO cell line of the present invention, stable human recombinant glycosylated low molecular weight urokinase can be produced at a concentration of approximately 600,000 ng / mL using the process disclosed herein.

[0019] Several attempts to produce recombinant urokinase have been reported in the literature: - Lys 158 Gly 158 (rscu-PA-Gly158) or Glu 158These mutants were generated and expressed in pro-uPA CHO cell line mutants substituted with (rscu-PA-Glu158). These mutants had lower specific activity than wild-type rscu-PA and did not undergo plasmin-mediated conversion to the double-chain form [Nelles, L., Lijnen, HR, Collens, D. & Holmes, WE The Journal of Biological Chemistry Characterization of Recombinant Human Single Chain Urokinase-type Plasminogen Activator Mutants Produced by Site-specific Mutagenesis of Lysine 158". Journal of Biological Chemistry 262, 5682-5689 (1987)]. - Recombinant pro-uPA and its deletion mutants were expressed in Saccharomyces cerevisiae using the GAL7 promoter and the rennin precursor peptide sequence of Mucor pusillus, leading to intracellular accumulation in the endoplasmic reticulum. However, they were inactive in their native state and required solubilization and refolding to obtain biological activity [Hiramatsu, R., Horinouchi, S. & Beppu, T. Isolation and characterization of human pro-urokinase and its mutants accumulated within the yeast secretory pathway. Gene 99, 235-241 (1991)]. - Glycosylated and non-glycosylated forms of prourokinase were produced in the yeast Pichia pastoris. The non-glycosylated form was less stable due to proteolysis, but showed catalytic activity comparable to recombinant pro-uPA of mammalian origin. However, glycosylation of prourokinase by P. pastoris inhibited its fibrinolytic activity [Wang, P., Zhang, J., Sun, Z., Chen, Y. & Liu, JN Glycosylation of Prourokinase Produced by Pichia pastoris Impairs Enzymatic Activity but Not Secretion. Protein Expr Purif 20, 179-185 (2000)]. - Subculture of human umbilical vein endothelial cells produced a single-chain uPA with low fibrinolytic activity, no fibrin specificity, but high affinity for plasminogen [Booyse, FM, Lin, PH, Traylor, M. & Bruce, R. The Journal of Biological Chemistry. Purification and Properties of a Single-chain Urokinase-type Plasminogen Activator Form Produced by Subcultured Human Umbilical Vein Endothelial Cells*. Journal of Biological Chemistry 263, 15139-15145 (1988)]. - Prourokinase (r-scuPA, pro-uPA) salpras is a non-glycosylated form of recombinant scu-PA (411 amino acids) produced in E. coli. It was developed as a fibrinolytic agent. Unlike tcu-PA, which lacks fibrin specificity and consequently increases the risk of major bleeding after administration, scu-PA can mediate specific clot lysis in the presence of fibrin [Roychoudhury, PK, Khaparde, SS, Mattiasson, B. & Kumar, A. Synthesis, regulation and production of urokinase using mammalian cell culture: A comprehensive review. Biotechnol Adv 24, 514-528 (2006)]. Furthermore, it is cleaved in vivo into a dimeric urokinase form, producing plasmin [Moser, M. & Bode, C. Pharmacology and clinical trial results of saruplase (scuPA) in acute myocardial infarction. Expert Opin Investig Drugs 8, 329-335 (1999)]. - Currently, urokinase (Avokinase®, quinicyl) TMThis drug is commercially produced using human neonatal kidney cells. It contains low molecular weight uPA as its active ingredient. This drug was first approved by the FDA in 1978. However, due to concerns about viral contamination [The Case of Abbokinase and the FDA: The Events Leading to the Suspension of Abbokinase Supplies in the United States - Journal of Vascular and Interventional Radiology. https: / / www.jvir.org / article / S1051-0443(07)61798-9 / fulltext; Hartnell, GG & Gates, J. The case of Abbokinase and the FDA: The events leading to the suspension of abbokinase supplies in the United States. Journal of Vascular and Interventional Radiology 11, 841-847 (2000)], the FDA has suspended the use of this drug since December 1998 due to deviations from Good Manufacturing Practices (GMP). Since 2002, its use in cases of pulmonary embolism has been re-approved. However, these cells can only proliferate for a limited period (30-40 generations). Extensive screening and testing of kidney donors are necessary to restore these primary cells. - Abbott Laboratories has developed recombinant prourokinase known as Prolyse®, which is being tested in the UK as a treatment for various thromboembolic conditions [Comparison of safety and efficacy of the various thrombolytic agents - PubMed. https: / / pubmed.ncbi.nlm.nih.gov / 12556739 / .50; Ouriel, K. et al. Thrombolysis or peripheral arterial surgery: phase I results. TOPAS Investigators. J Vasc Surg 23, 64-75 (1996)]. ruPA is derived from genetically engineered mouse hybridoma cell lines and is purified from the culture medium through a series of chromatographic steps in aqueous solution, thus being fully glycosylated. The lyophilized therapeutic product was reconstituted with sterile water for injection. Over 90% of ruPA is in the high molecular weight form, and its specific activity, as measured by blood clot dissolution tests, was approximately 170,000 IU / mg [Ouriel, K., Veith, FJ & Sasahara, AA Thrombolysis or peripheral arterial Phase I results surgery]. Pharmacokinetic studies in monkeys revealed that ruPA has a short half-life of 7 minutes, shorter than that of the low molecular weight form. However, despite these differences, the clinical effects of both drugs are similar. However, this drug has not yet received FDA approval [Roychoudhury, PK, Khaparde, SS, Mattiasson, B. & Kumar, A. Synthesis, regulation and production of urokinase using mammalian cell culture: A comprehensive review. Biotechnol Adv 24, 514-528 (2006)].

[0020] The applicant had previously developed a process for preparing recombinant urokinase in culture medium for genetically modified eukaryotic cells. This process, described in EP 1 245 681, required the use of an alkanoic acid as an activator and resulted in a product of mixed low and high molecular weight uPA.

[0021] The applicant has now discovered and developed an improved process for the production of recombinant human low molecular weight urokinase (rh-LMW-uPA) in eukaryotic cell line medium (CCOS 2068). This process does not require the use of alkanates and yields only LMW-uPA as the product, thus eliminating the need to separate a mixture of LMW-uPA and HMW-uPA. [Brief explanation of the drawing]

[0022] [Figure 1] Structures of pro-uPA and uPA. Pro-uPA, which contains a growth factor domain (GFD), a kringle domain (KD), and a catalytic serine protease domain, is secreted as a single-chain precursor and undergoes catalytic cleavage between the peptide bonds of Lys158 and Ile159 to produce a double-chain uPA. A second protein cleavage further cleaves the double-chain uPA between Lys135 and Lys136, forming an inactive amino-terminal fragment (ATF) and a catalytically active low-molecular-weight uPA (LMW-uPA) [Mahmood, N., Mihalcioiu, C. & Rabbani, SA Multifaceted role of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR): Diagnostic, prognostic, and therapeutic applications. Frontiers in Oncology vol. 8 Preprint at https: / / doi.org / 10.3389 / fonc.2018.00024 (2018)]. [Figure 2] Structure of low molecular weight recombinant human urokinase obtained by the process of the present invention. [Modes for carrying out the invention]

[0023] Therefore, the object of the present invention is a process for producing rh-LMW-uPA in culture medium for the CHO cell line (CCOS 2068).

[0024] The general characteristics of the process that is the objective of this invention are shown below. 1. Upstream process (production stage of recombinant protein via CHO) The master cell bank (MCB) and working cell bank (WCB) are stored in gaseous nitrogen. Each cryovial contains 1 mL of concentrated cell culture medium, preferably 10 × 10⁶, with an antifreeze, preferably DMSO, added at a concentration of 5-10% v / v, preferably 7.5% v / v. 6 Contains cells / mL. The cells are thawed in a 125 mL flask (Spinner Flask Corning, catalog number 3152) using preheated chemically defined culture medium, preferably CD OptiCHO (Gibco, catalog number 12681011). The culture is performed at 0.2–0.4 × 10⁶ cells. 6 Cells / mL, preferably 0.3 × 10⁶ 6 Inoculate the cells at a concentration of cells / mL and culture at 37°C, 8.0% CO2, and saturated rH (preferably 85% or more) under stirring at 40 rpm. Cell cultures are checked regularly, and VCD and viability are measured by cell counting using trypan blue staining. Culture density of at least 1.0 × 10 6 Cells / mL, preferably 1.0 × 10⁶ 6 From 2.0 × 10 6 Subculturing is performed when the cell count reaches between 0.2 and 0.4 × 10⁶ cells / mL. The subculturing procedure involves diluting the culture medium with a new chemically defined medium, preferably CD OptiCHO, and subculturing to 0.2–0.4 × 10⁶ cells / mL. 6 cells / mL, preferably 0.3 × 10⁶ 6 This involves reinoculating the bacteria at a cell / mL concentration. The cells are grown in a 1 L flask (Corning spinner flask, part number 3561) by subculturing as described above. Inoculation of the bioreactor should be 0.1-0.4 × 10 6 Cells / mL, preferably 0.3 × 10⁶ 6 The inoculation should be performed in cells / mL. The inoculation volume can be 1:3 to 1:8 relative to the final inoculation volume. The bioreactor culture settings are as follows: - pH: 7.0~7.2, preferably 7.05 - Dissolved oxygen: 40-60%, preferably 50% - Temperature: 35~37.5℃, preferably 37.0℃ - Stirring speed: 0.15~0.45 m / s (angular velocity), preferably 0.35 m / s - Gas flow rate: 0.02~0.2 vvm, preferably 0.075 vvm The cells are cultured for 2-4 days, preferably 3 days. After this culture period, the proliferated cells can be used for inoculation into larger bioreactors or for production purposes. In this case, the culture conditions are changed as follows: - pH: 6.90~7.05, preferably 6.90 - Dissolved oxygen: 40-50%, preferably 40% - Temperature: 35~37.5℃, preferably 37.0℃ - Stirring speed: 0.15~0.45 m / s (peak speed), preferably 0.35 m / s - Gas flow rate: 0.02~0.2 vvm, preferably 0.075 vvm Continuous addition of Ex-Cell Advanced Feed 1G (SAFC catalog number #24368C-10L) should be started at a flow rate of 2-8% / day (preferably 4%) of the initial volume. When using the cultured material for propagation, the above procedure should be followed for the production stage. The culture is continued while maintaining a glucose concentration of 1.0 g / L or higher, preferably 2 to 11 g / L. Harvest 12 to 15 days after inoculation, preferably on the 14th day. At the end of the culture, the cell supernatant is collected by centrifugation or deep filtration. The latter method is performed using Millistak+ Pro HC or Millistak+ HC deep filter (Millistak+ Pro HC is preferred). The cell culture is filtered with 100 LMH, and the culture load (culture volume) is 2-10 g / m² in size. 2 (Wet biomass) 90-680 L / m³ 2 Then, the bacterial load is reduced by filtration. 2. Downstream process (purification of recombinant protein) Slowly add 5% (v / v) acetic acid and 100 mM sodium chloride to the supernatant to adjust the pH to 5.4-8.0 (preferably 5.5). It is desirable to perform the pH correction within 20 minutes. After pH correction, the supernatant is filtered through a double-layer filter (0.5-0.2 μm), preferably using a Merck Express SHC 0.45-0.2 μm filter at a flow rate range of 250-1700 LMH. The supernatant is loaded onto a resin conjugated with pABA (p-aminobenzamidine) ligand at a linear load flow rate range of 90-150 cm / h, preferably 100 cm / h. The resin is then buffered in a suitable buffer solution with a pH of 5.5-8.0, preferably 100 mM sodium acetate and 10 mM CaCl. 2、 The solution must be pre-equilibrated with a pH 5.5 buffer. Due to the high binding specificity between the p-ABA ligand and the protein, based on the active site of the serine protease, this chromatographic step is considered the first step in viral reduction. After the loading process, resin washing is required, which should be carried out within a linear load flow velocity range of 90-150 cm / h, preferably 100 cm / h for 2.5 CV. For washing, a suitable buffer solution with a pH of 5.5-8.0, preferably 100 mM sodium acetate, 10 mM CaCl2, and a pH 5.5 buffer solution should be used. Subsequently, a washing step is carried out at a linear load flow rate range of 90 to 150 cm / h for 2.5 CV, preferably 100 cm / h. For this, an appropriate high ionic strength buffer with pH 5.5 to 8.0, preferably 100 mM sodium acetate buffer, 10 mM CaCl2, 900 mM sodium chloride, and pH 5.5 is used. After the high ionic strength washing step, re-equilibrium is performed in a linear load flow velocity range of 90 to 150 cm / h, preferably at 100 cm / h for 2.5 CV, using an appropriate buffer solution with a pH of 5.5 to 8.0, preferably 100 mM sodium acetate buffer, 10 mM CaCl2, and pH 5.5. The purified intermediate sample is eluted under acidic conditions of pH 2.7–4.0 using glycine hydrochloride or acetate buffer, preferably 100 mM glycine hydrochloride buffer pH 2.7. Up to 400 mM sodium chloride can be added to these buffers. The expected yield at this stage is 60% or higher and is evaluated based on enzyme activity. The column's efficiency can be restored with 1M acetic acid and 20% v / v ethanol, and the column can be stored in 76mM sodium chloride and 24% v / v ethanol. The intermediate eluted from the pABA column is further adjusted to a pH of 3.4-4.0, preferably 4.0. pH correction is performed by adding a strong acid or strong base, preferably 500 mM NaOH or 5% v / v HCl, over 20 minutes. This method, combining acid elution and storage time, is used as a secondary reduction of viral contamination. Virus inactivation at low pH can be carried out at 4°C to 25°C for 2 to 20 hours, preferably at 4°C. The intermediate sample is further purified using a strong cation exchange resin, preferably Fractogel EMD SO3- (Merck, catalog number 1.16882). This is done after equilibration with a suitable buffer at pH 5.0–7.4, preferably 20 mM sodium phosphate buffer at pH 6.0, at a linear flow rate of 100–200 cm / h, preferably 200 cm / h. A washing step is required and should be carried out with a linear flow rate range of 100-200 cm / h, preferably 200 cm / h per 2.5 CV, using a suitable buffer solution with a pH of 5.0-7.4, preferably 20 mM sodium phosphate buffer solution with a pH of 7.4. The intermediate product is eluted at 2.5 CV with a suitable high ionic strength buffer at pH 5.0–7.4, preferably 20 mM sodium phosphate buffer, 350 mM sodium chloride, pH 7.4, at a linear flow rate range of 100–200 cm / h, preferably 200 cm / h. The yield at this stage is 80% or higher, based on enzyme activity. Column efficiency is restored with 20 mM sodium phosphate, 1 M sodium chloride, and pH 7.4. Columns should be washed in-place with 500 mM NaOH, and stored in a 150 mM sodium chloride, 20% v / v ethanol solution. The intermediate eluted by cation exchange is filtered to remove the virus as a third step to reduce viral contamination. For filtration, Viresolve shield (Merck part number VPPS101NB1) and Viresolve pro (Merck part number VPMD101NB1) were used at a constant pressure of 1.8 to 2.2 bar, preferably 2.0 bar. Alternatively, constant pressure filtration at 1.8 to 2.2 bar, preferably 2.0 bar, is possible using Sartopore 0.1 μm (Sartorius, part number 5443538M8M7FFA) followed by Planova 20N (Asahi Kasei part number 20NZ-300). 3. Formulation The intermediate after filtration for virus removal is further formulated by tangential flow filtration (TFF) at a feed flow rate range of 200-400 LMH, preferably 360, while maintaining an intermembrane pressure of 0.8-1.2 bar, preferably 1.0. Regenerated cellulose membranes can be used. The cutoff should be less than 10 kDa, preferably less than 5 kDa. Typically, seven times the volume of buffer is required to complete the formulation. The buffer should be sodium phosphate or sodium acetate with a pH of 4.0 to 7.0, with up to 2.5 mM EDTA and up to 6% (w / v) mannitol added as needed. The final concentration of the pharmaceutical substance can be achieved in the range of 0.5 to 10 g / L, and it should be stored at 4° to -80°C. 4. Specifications of formulation raw materials The protein concentration should be between 0.5 and 10 g / L. The specific activity of the final pharmaceutical substance is 200,000 to 300,000 IU / mg. The monomer purity measured by HPLC-SEC is 98.0% or higher. The total sialic acid content is 5% or less, the HCPs content is less than 100 ppm, and the residual DNA content is less than 10 ppm. Comparing the essential features of the process objectives of the present invention with the process described in EP 1 245 681, the advantages of the process according to the present invention become clear. - The use of novel cell lines enables the production of low molecular weight urokinases only, resulting in improved product safety, quality, and stability. - The enzyme that converts sc-uPA to tc-uPA does not require the addition of alkanates to the culture medium for activation. Activation is possible even in the presence of proteases, such as uPA itself. - Productivity increases significantly (7000 IU / mL vs. 120000 IU / mL). - The resulting product has a high sialic acid content, improving its stability in the patient's body. The present invention will be described in detail by the following embodiments, however, these are not limiting. Examples

[0025] List of abbreviations used in the examples: MCB: Master cell bank WCB: Working cell bank DMSO: Dimethyl sulfoxide CHO: Chinese Hamster Ovary Cells rH: Relative humidity VCD: Viable Cell Count DO: Dissolved Oxygen LMH: liters per square meter per hour CV: Column Volume CIP: Cleaning in place TMP:Transmembrane Pressure HCPs:Host cell proteins material JPEG2026516228000002.jpg41153

[0026] Example 1 Thaw one vial of MCB or WCB and add 0.3x10 to CD Opti CHO. 6 Cells were inoculated at a concentration of cells / mL and cultured at 37.0°C, 140 rpm, 85% relative humidity, and CO2 8.0. VCD and viability were monitored regularly during culture. VCD values ​​ranged from 1.0 to 3.0 × 10⁶. 6 When the cell count was at a certain level (cells / mL), the culture was split by dilution with fresh medium. The seeding concentration after splitting was 0.3 × 10⁶. 6 The concentration was cells / mL. After increasing the cell culture medium to the level of the bioreactor inoculation solution, add 0.3 × 10 Multifors MUF 2C Pack to CD Opti CHO. 6 Cells were inoculated at a concentration of cells / mL. CHO cells were cultured for 3 days at 37.0°C, 50% DO, pH 7.05, vibrational angular velocity of 0.29 m / s, and pressure of 0.075 vvm. On the third day, we began feeding the cultured cells with Ex-Cell advanced feed 1G at a rate of 4% (of the initial volume) per day. Simultaneously with the start of feeding, the culture conditions were changed to 37.0°C, 50% DO, pH 7.05, oscillation angular velocity of 0.31 m / s, and 0.100 vvm. Culture was continued for 14 days, and the supernatant was collected at the end of the period.

[0027] Example 2 Thaw one vial of MCB or WCB and add 0.3 × 10¹⁶ to CD Opti CHO. 6 Cells were seeded at 1 / mL and cultured at 37.0°C, 140 rpm, 85% relative humidity, and CO2 8.0. The culture was regularly monitored for VCD and viability. VCD was 1.0–3.0 × 10⁶. 6 The culture was split by dilution with fresh medium when the cell concentration was at a level of 0.3 × 10⁶ cells / mL. The concentration of the inoculated solution after splitting was 0.3 × 10⁶. 6 The concentration was cells / mL. After increasing the cell culture medium to the inoculation volume for the bioreactor, add Multifors MUF 2C Pack 0.3 × 10 6 CHO cells were inoculated at a concentration of cells / mL into CD Opti CHO. The CHO cells were cultured for 3 days at 37.0°C, 50% DO, pH 7.05, vibrational angular velocity of 0.29 m / s, and 0.075 vvm. On the third day, the culture was fed with Ex-Cell advanced feed 1G at a rate of 4% per day (of the initial volume). After the feeding phase was completed, the culture conditions were changed to 37.0°C, 50% DO, pH 6.90, oscillation angular velocity of 0.31 m / s, and 0.075 vvm. The culture was continued for 14 days, and the supernatant was collected at the end of the period.

[0028] Example 3 The supernatant was acidified to pH 6.5 and loaded onto pABA resin for affinity chromatography. The column resin was equilibrated with 20 mM sodium phosphate buffer, 400 mM sodium chloride, pH 6.5 before loading the protein. The linear flow rate was set to 90 cm / h. After the loading stage, washing with 20 mM sodium phosphate buffer, 400 mM sodium chloride, pH 6.5 was required. Products bound to the resin were further eluted with 100 mM glycine, pH 2.7. CIP and column storage were performed according to the supplier's instructions.

[0029] Example 4 The supernatant was basicized to pH 8.0 and loaded onto pABA resin for affinity chromatography. Before protein loading, the column resin needed to be equilibrated with 20 mM sodium phosphate buffer and 400 mM sodium chloride at pH 6.5. The linear flow rate was set to 30 cm / h. After the loading stage, washing with 20 mM sodium phosphate buffer and 100 mM sodium chloride at pH 6.5 was required. The product bound to the resin was further eluted with 100 mM acetic acid and 100 mM sodium chloride at pH 4.0. CIP and column storage were performed according to the supplier's instructions.

[0030] Example 5 The intermediate obtained in the capture step was adjusted to pH 5.0 with 0.5 M sodium hydroxide solution, loaded onto a strong cation exchange resin, and subjected to ion exchange chromatography. The column resin was equilibrated with 20 mM sodium phosphate buffer, pH 6.0 before protein loading. The linear flow rate was set to 100 cm / h. After the loading step, the column was washed twice with 20 mM sodium phosphate buffer, pH 6.0, followed by washing with 20 mM sodium phosphate buffer, pH 7.4. The product was further eluted with 20 mM sodium phosphate buffer, 350 mM sodium chloride, pH 6.0. CIP and column storage were performed according to the manufacturer's instructions.

[0031] Example 6 The intermediate obtained in the capture step was adjusted to pH 5.0, then loaded onto a strong cation exchange resin and subjected to ion exchange chromatography. Before protein loading, the column resin was equilibrated with 20 mM sodium phosphate buffer, pH 6.0. The linear flow rate was set to 100 cm / h. After the loading step, one wash was performed with 20 mM sodium phosphate buffer, pH 6.0. The product was eluted with 20 mM sodium phosphate buffer, 350 mM sodium chloride, pH 7.0. CIP and column storage were performed according to the manufacturer's instructions.

[0032] Example 7 The eluate from ion exchange chromatography was eluted into a regenerated cellulose TFF membrane module with a 10 kDa cutoff at 200 g / m². 2 The system was loaded with the specified load. The recirculation flow rate was set to 300 LMH, and the TMP was set to 1.0 bar. For the final formulation, seven times the volume of 50 mM sodium acetate pH 5.5 formulation buffer was used. Diafiltration can be performed in the protein concentration range of 5-10 g / L. The raw materials for the final formulation were concentrated to 5.0 g / L and frozen at -20°C or below.

[0033] Example 8 The eluate from ion exchange chromatography is transferred to a 5 kDa cutoff regenerated cellulose TFF membrane module at a rate of 50 g / m². 2 The system was loaded with the specified load. The recirculation flow rate was set to 360 LMH, and the TMP was set to 1.0 bar. For the final formulation, seven times the volume of formulation buffer containing 10 mM sodium phosphate, 2.5 mM EDTA, and 6% v / v pH 7.0 was used. Diafiltration can be performed when the protein concentration is in the range of 5 to 10 g / L. The final formulation raw material was concentrated to 0.5 g / L and frozen at -20°C or below.

Claims

1. This CHO cell line is deposited with the Culture Collection of Switzerland AG (CCOS) and identified by the number CCOS 2068.

2. A process for producing low molecular weight recombinant human urokinase (rh-LMW-uPA) in the culture medium of the cell line CCOS2068 according to claim 1, comprising the steps of cell culture, recovery of the supernatant by centrifugation or deep filtration, purification of the obtained urokinase with resin, and formulation by tangential flow filtration.

3. The process according to claim 2, wherein cell culture is carried out under the following conditions. - pH is 6.90 to 7.05, preferably 6.90 - Dissolved oxygen is 40-50%, preferably 40% - The temperature should be 35 to 37.5°C, preferably 37.0°C. - The stirring speed is 0.15 to 0.45 m / s (tip speed), preferably 0.35 m / s - Gas flow rate is 0.02 to 0.2 vvm, preferably 0.075 vvm Furthermore, the glucose concentration is maintained at 1.0 g / L or higher, preferably 2 to 11 g / L.

4. The supernatant was collected by filtration with 100 LMH, and the culture load was 2–10 g / m² in size. 2 (Wet biomass) 90-680 L / m³ 2 The process according to claim 2.

5. The process according to claim 2, wherein, before purification with resin, the pH of the supernatant is adjusted to 5.4 to 8.0, preferably 5.5, by slowly adding 5% v / v acetic acid and 100 mM sodium chloride, preferably over 20 minutes.

6. The supernatant is purified on a resin conjugated with pABA (p-aminobenzamidine) ligand, where the resin is pH 5.5 to 8.0, preferably pH 5.5, containing 100 mM sodium acetate and 10 mM CaCl. 2 The process according to claim 2, wherein the buffer is pre-equilibriumized.

7. The process according to claim 2, wherein purified urokinase is eluted from the resin under acidic conditions of pH 2.7 to 4.0 using glycine hydrochloride or acetate buffer, preferably 100 mM glycine hydrochloride at pH 2.7, and the eluted product is further adjusted to pH 3.4 to 4.0, preferably 4.0, by adding an alkaline solution, preferably 500 mM NaOH, for 20 minutes.

8. The process according to claim 6, wherein after purification with a pABA ligand-binding resin, the urokinase is further purified with a strong cation exchange resin by washing with a low ionic strength buffer at pH 7.4, preferably 20 mM sodium phosphate buffer, and eluting at 2.5 CV in a linear flow rate range of 100 to 200 cm / h, preferably 200 cm / h, using a high ionic strength buffer at pH 5.0 to 7.4, preferably 20 mM sodium phosphate, 350 mM sodium chloride, and pH 7.

4.

9. The process according to claim 2, wherein formulation by tangential flow filtration (TFF) is carried out with a supply flow rate range of 200 to 400 LMH, preferably 360, maintaining an intermembrane pressure of 0.8 to 1.2 bar, preferably 1.0, and a cutoff of less than 10 kDa, preferably less than 5 kDa.

10. The process according to claim 9, wherein a sodium phosphate or sodium acetate buffer with a pH of 4.0 to 7.0 is used, and EDTA up to 2.5 mM and mannitol up to 6% (w / v) are added as needed.

11. The specifications of the obtained product are: - Protein concentration: 0.5–10 g / L, Storage temperature: 4°C–-80°C - Specific activity is 200,000–300,000 IU / mg - Monomer purity >98.0% by HPLC-SEC The process according to claim 2.

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