Method for producing recombinant human low molecular weight urokinase with pharmaceutical activity

Abstract

The invention discloses a CHO cell line and a method for producing recombinant human low molecular weight urokinase (rh-LLW-uPA) by using the CHO cell line. The method has high productivity and enables the production of low molecular weight urokinase having improved characteristics in terms of safety, quality and stability.

Description

Technical Field

[0001] This invention relates to a method for producing recombinant human low molecular weight urokinase (rh-LMW-uPA), and more particularly to a stable and active glycosylated recombinant human low molecular weight urokinase obtained through eukaryotic cell lines using recombinant DNA technology. This method enables the production of a physiologically active (mature) form of glycosylated recombinant human low molecular weight urokinase in a culture medium.

[0002] The technical field of this invention is the production of glycosylated recombinant human low molecular weight urokinase in eukaryotic cells of the CHO cell line using genetic engineering technology. Background Technology

[0003] Fibrinolysis is the process of dissolving blood clots to prevent vascular occlusion. The fibrinolytic system is mainly regulated by proteases and protease inhibitors. The key enzyme in this system is plasmin, which is mainly derived from inactive plasminogen through its direct activators (tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA)). 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) [and is antagonized by specific inhibitors. Plasminogen is converted into active plasmin via uPA, which determines the dissolution of fibrin clots, thereby producing 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 a key serine protease that has long been used clinically 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) Human uPA is secreted into the bloodstream by various tissues in the form of a single-chain glycosylase pro-urokinase (pro-uPA) (sc-uPA, 411 amino acids, MW 54kDA). 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) It has very low amide hydrolysis activity. Hedstrom, L. Serine protease mechanism and specificity. Chem Rev 102, 4501–4523 (2002) ] and cleavage K through plasmin 158 -I 159 The peptide bonds between them generate an α chain (residues 1-158) and a β chain (residues 159-411), which activates the [ Spraggon, G. et al. The crystal 人尿激酶型纤溶酶原激活剂催化结构域的结构。结构3,681 - 691(1995年) 单链原尿激酶的一级结构 - PubMed。https: / / pubmed.ncbi.nlm.nih.gov / 2931434 / ;Magill,C., The resulting fully active double-stranded protein uPA (tc-uPA) (also known as high molecular weight urokinase (HMW-uPA)) [Katz,B. A.和MacKman,R. L.肿瘤学中新兴的治疗靶点: 尿激酶型纤溶酶原激活系统。http: / / dx.doi.org / 10.1517 / 14728222.3.1.109 3,109 - 133(2005年);Spraggon,G.等人,人尿激酶型纤溶酶原激活剂催化结构域的晶体结构。结构 Behrens,M. A.等人,酶原激活为 尿激酶型纤溶酶原激活剂与结构域间灵活性增加有关。《分子生物学杂志》411,417 - 429(2011年) Fleuryso,V.,Lijnenl,H. R.和Anglb - Canosji,E 3, 681–691 (1995) The plasminogen is converted into plasmin, which promotes the hydrolysis of fibrinogen into fibrin. The conversion to a double-stranded form increases the flexibility between domains. 单链尿激酶型纤溶酶原激活剂在持续纤维蛋白溶解过程中增强内在活性的机制 。《生物化学杂志》268,18554 - 18559(1993年) 图1 This allows it to interact more effectively with its substrate, increasing the enzyme activity of urokinase by approximately 100 times. [Gurewich,V原 尿激酶:理化性质及其纤维蛋白溶解促进作用 ​ ​ ​ ].

[0005] Further in Lys 135 -Lys 136 Site cleavage produces 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). ​ The remaining fragment 136-158 is called the linker peptide (CP), which is linked to the catalytic region at the carboxyl terminus via a disulfide bridge in its low molecular weight form (LMW-UK, amino acids 136-411, molecular weight 33 kDa). ​ ​ 尿激酶以及与其具有互补作用机制的组织纤溶酶原激活物的活性。《Semin Thromb Hemost》14卷,第110 - 115页(1988年); Stepanova, V. v. & Tkachuk, V. A. 作为多结构域蛋白和多功能细胞调节剂的尿激酶。《生物化学(莫斯科)》2002年67卷,第1期,第67页,第109 - 118页 Gates, J. & Hartnell, G. G. 当尿激酶消失时:关于无尿激酶溶栓治疗又一年的评论。《J Vasc Interv Radiol》15卷,第1 - 5页(2004年); (2002) As is well known, this low molecular weight form retains catalytic activity and is used as a therapeutic agent. Gurewich, V. 前尿激酶:理化性质以及尿激酶和组织纤溶酶原激活物对其纤溶活性的促进作用,尿激酶和组织纤溶酶原激活物与前尿激酶具有互补的作用机制。《Semin Thromb Hemost》14卷, Sato, S. et al., 高亲和力尿激酶衍生环肽抑制尿激酶 / 尿激酶受体相互作用:对肿瘤生长和扩散的影响。《FEBS Lett》528卷,第212 - Aditiviya & Khasa, Y. P. 重组溶栓剂的演变:现状与未来方向。 https: / / doi.org / 10.1080 / 21655979.2016.1229718 8卷,第331 - 358页(2016年) ​ ​ 110–115 (1988) ].

[0006] HMW-uPA and LMW-uPA exhibited similar catalytic activity towards plasminogen. ​ ​ ​ 216 (2002) ].

[0007] Urokinase isolated from urine contains a higher proportion of high molecular weight forms, while urokinase obtained from cultured kidney cells contains a higher proportion of low molecular weight forms. ​ ​ ​ ].

[0008] In vivo, after pro-uPA is secreted, it binds to uPAR (urokinase-type plasminogen activator receptor, uPAR or CD87) and is cleaved by adjacent membrane-bound plasmin or other proteases (such as kallikrein). 158 -Ile 159 The bond is converted to an active double-stranded state (i.e., tc-uPA or uPA). The receptor-bound pro-uPA is activated by plasmin more quickly than the pro-uPA in free plasma. Irigoyen, J. P., Muñoz-Cánoves, 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) Then, the active uPA converts plasminogen bound to the adjacent membrane into plasmin.

[0009] The N-terminal fragment (ATF, residues 1-135) of uPA contains all the binding sites required to interact with its receptor (a glycosylphosphatidylinositol (GPI)-anchored membrane protein) [Barinka, C. et al., Structural Basis of Interaction between Urokinase-type Plasminogen Activator and its Receptor. JMol Biol 363, 482-495 (2006)]. The obtained complex has been shown to participate in two independent biological cascade processes: (a) plasminogen activation, leading to proteolytic activity; and (b) signal transduction, determining cell adhesion and mitosis. Indeed, uPAR has multiple functional roles associated with tumor progression, including tumor proliferation, apoptosis, metastasis, angiogenesis, multidrug resistance (MDR), and prognosis. High levels of uPAR expression were detected in various cancer cells, but extremely low levels were found in normal cells, suggesting that the level of uPAR in tumor tissue is closely related to the malignancy of the tumor and the prognosis of cancer patients [Zhai, BT et al., Urokinase-type plasminogen activator receptor (uPAR) as atherapeutic target in cancer. Journal of Translational Medicine, Vol. 20, preprinted at https: / / doi.org / 10.1186 / s12967-022-03329-3 (2022)].

[0010] rh-LMW-uPA does not contain the ATF fragment, so it cannot bind to the urokinase receptor (uPAR), thus avoiding activation of the signaling cascade through uPAR-uPA binding. Summary of the Invention

[0011] This invention stems from the need to replace urokinase extracted from urine and used for catheter cleaning by utilizing the plasminogen activator of double-stranded recombinant human glycosylated low molecular weight urokinase (rh-LMW-uPA), 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] To this end, a CHO cell line was developed and deposited at the Culture Collection of Switzerland AG (CCOS) with accession number CCOS 2068.

[0013] The structure of human rh-LMW-uPA described in this article consists of two polypeptide chains, namely the α chain and the β chain, which are connected by Cys 13 and Cys 121 The interchain disulfide bonds between the chains, this structure, is due to the Lys in the culture medium of the above CHO cell lines. 23 -Ile 1 The peptide bonds, through proteolytic hydrolysis, unexpectedly become active. The β chain contains five intrachain disulfide bonds: Cys 31 -Cys 47 Cys 39 -Cys 110 Cys 135 -Cys 204 Cys 167 -Cys 183 and Cys 194 -Cys 222 Furthermore, the β chain contains a fully active catalytic domain, and also shares a common sequence Asn. 144 -Ser-Tyr contains N-glycosylation sites, and the α chain contains O-glycosylation sites.

[0014] A significant portion of currently approved protein drugs require appropriate glycosylation to achieve optimal therapeutic effects. This is because glycosylation can affect a variety of physiological processes at the cellular and protein levels. uPA can undergo O- and N-glycosylation. In the epidermal growth factor-like domain of uPA derived from cultured, urinary, and recombinant human kidney cells, Fuc residues are linked to Thr-18 via O-glycosylation. Kentzer, E. J., Buko, A., Menon, G. & Sarin, V. K. Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase. Biochem Biophys Res Commun 171, 401-406 (1990); Buko, A. M. et al., Characterization of a posttranslational fucosylation in the growth factor domain of urinary plasminogen activator. Proc Natl Acad Sci U S A 88, 3992-3996 (1991)The presence of fucosylated Thr-18 within this domain suggests its importance in specific receptor / ligand binding systems responsible for multiple 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 / ]. In addition, human uPA at Asn-302 N-glycosylation [Bansal, V. & Roychoudhury, P. K. 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) It occurs within the protease domain. Steffens, G. J., Gü nzler, W. A., ötting, F., Frankus, E. & Flohé, L. The complete amino acid sequence of low molecular mass urokinase from human urine. Hoppe Seylers Z Physiol Chem 363, 1043–1058 (1982); Irigoyen, J. P., Muñoz-Cánoves, 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 mannose (Man), galactose (Gal), fucose (Fuc), N-acetylglucosamine (GlcNAc), and N-acetylneuraminic acid (Neu5Ac), as well as N-acetylgalactosamine (GalNAc) residues. Steffens, G. J., Günzler, W. A., ötting, F., Frankus, E. & Flohé, L. The complete amino acid sequence of low molecular mass urokinase from human urine. Hoppe Seylers Z Physiol Chem 363, 1043–1058 (1982); McLellan, W. L., Vetterlein, D. & Roblin, R. The glycoprotein nature of human plasminogen 激活剂。《欧洲生物化学学会联合会快报》115卷,第181 - 184页(1980年) The biological behavior of recombinant but non-glycosylated sc-uPA has been shown to be similar to that of glycosylated recombinant urinary proteins. 李,X.K.,利嫩,H.R.,内勒斯,L.,胡,M. H.,& 科伦,D. 非糖基化单链尿激酶型纤溶酶原激活剂重组突变体的生化特性。《生物化学与生物物理学报》1159卷,第37 - 43页(1992年);糖基化对尿激酶原催化和纤溶特性的影响 - 美国国立医学图书馆。https: / / pubmed.ncbi.nlm.nih.gov / 1455401 / 糖基化对尿激酶原催化和纤溶特性的影响 - 美国国立医学图书馆。https: / / pubmed.ncbi.nlm.nih.gov / 1455401 / 亨金, However, it has been shown that recombinant unglycosylated sc-uPA is more readily cleaved by plasminogen lysates than its recombinant glycosylated counterpart, exhibits higher proteolytic activity, and is more rapidly inactivated by plasminogen inhibitors. J.,杜德拉克,D.,比贝,D.P. & 森内洛,L. 高唾液酸含量减缓兔体内尿激酶原的周转。《血栓研究》63卷,第215 - 225页(1991年) 图2 1455401 / Furthermore, it was confirmed that the sialic acid content in recombinant sc-uPA had a negative impact on in vivo clearance. 糖基化对尿激酶原催化 和纤溶特性的影响 - 美国国立医学图书馆。https: / / pubmed.ncbi.nlm.nih.gov / 1455401 / ].

[0015] In rabbits, the recombinant prourokinase (pro-uPA) containing 2.5–3 sialic acid molecules per protein had a significantly shorter half-life than pro-uPA with a lower degree of sialylation. The metabolic rate of this protein was relatively insensitive to sialic acid content, ranging from 0 to 1.5 sialic acid residues per pro-uPA molecule.

[0016] This invention relates to a method for preparing N-glycosylated human rh-LMW-uPA, wherein N-glycosylation has a common intra-sequence Asn in the catalytic domain. 144 place ( 图2 This has been described in previous literature and identified as Asn. 302(The structure of pro-uPA) contains the following residues: Man, Gal, Fuc, GlcNac, and Neu5Ac. Glycosylated Asn has been shown. 302 uPA is more easily activated by plasmin and is more resistant to inhibitors. 阿迪蒂维亚 & 卡萨,Y.P. 重组溶栓剂的演变:现状与未来方向。https: / / doi.org / 10.1080 / ​ ​ Furthermore, 2.2 sialic acid residues were identified in each rh-LMWUK molecule, and unexpectedly, O-glycans derived from core 1 AS, core 1 MS, and core 1 DS were found on the α chain of the linker peptide. ​ ).

[0017] Currently, commercial sales of recombinant uPA have not been approved by the FDA or EMA. However, various attempts to produce uPA based on recombinant DNA technology exist. ​ ​ 21655979.2016.1229718 8, 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 not only confirms the clinical relevance of the molecule, but also demonstrates the need for such methods for safety and purity reasons.

[0018] Unlike conventional commercially available urine-derived HMW urokinase, the active recombinant LMW form prepared according to the method of the present invention overcomes the potential risk of spreading infectious pathogens and contaminants. 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, G. G. & 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) Furthermore, unlike streptokinase, it is a human-derived sequence secreted by eukaryotic cells and therefore lacks antigenicity. 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) The concentration of urokinase in mammalian urine is extremely low (10-15 ng / mL). Vetterlein, D. & Calton, G. J. Purification of urokinase from complex mixtures using immobilized monoclonal antibody against urokinase light chain. Thromb Haemost 49, 24-27 (1983) This makes the enzyme purification process lengthy and expensive. Furthermore, the multi-step extraction process typically results in low yields, making the enzyme expensive. Rouf, S. A., 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, M. B. & Kwaan, H. C. Plasminogen activator activity in cultures from human tissues. An immunological and histochemical study. J Clin Invest 48, 1740- 1753 (1969) Reports indicate that cultured kidney cells can secrete 50-100 ng / mL of uPA. Roychoudhury, P. K., Khaparde, S. S., 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, P. K. Production and purification of urokinase: a comprehensive review. Protein Expr Purif 45, 1-14 (2006) Due to the extremely low concentration of urokinase in human urine, in vitro cultured eukaryotic cells provide an excellent alternative for the production and purification of urokinase. Using the engineered CHO cell line of this invention, the method disclosed herein can produce approximately 600,000 ng / mL of stable recombinant human glycosylated low molecular weight urokinase.

[0019] Several attempts to produce recombinant urokinase have been described in the literature:

[0020] - The pro-uPA mutant was generated and expressed in the CHO cell line, in which Lys 158 residues were Gly 158 (rscu-PA-Gly) 158 ) or Glu 158 (rscu-PA-Glu) 158 These mutants have lower specific activity than wild-type rscu-PA and cannot be converted to double-stranded form by plasmin. Nelles, L., Lijnen, H. R., Collens, D. & Holmes, W. E. 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) ].

[0021] - Recombinant pro-uPA and its deletion mutants were expressed in *Saccharomyces cerevisiae* using the GAL7 promoter and the pro-rennet sequence of *Mucor pusillus*, resulting in their accumulation in the endoplasmic reticulum within the cells. However, they are inactive in their native state and must be dissolved and refolded to become biologically active. 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) ].

[0022] - Glycosylated and non-glycosylated variants of prourokinase have been generated in Pichia pastoris. The non-glycosylated form is less stable due to proteolytic activity, but its catalytic activity is comparable to that of recombinant pro-uPA from mammalian sources. However, glycosylation of prourokinase in Pichia pastoris interferes with its fibrinolytic activity. Wang, P., Zhang, J., Sun, Z., Chen, Y. & Liu, J. N. Glycosylation of Prourokinase Produced by Pichia pastoris Impairs Enzymatic Activity but Not Secretion. Protein Expr Purif 20, 179-185 (2000) ].

[0023] - Passaged human umbilical vein endothelial cells produce single-chain uPA, which has low fibrinolytic activity and no fibrin specificity, but has a high affinity for plasminogen. Booyse, F. M., Lin, P. H., 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) ].

[0024] Saruplase (prourokinase (r-scuPA, pro-uPA)) is the non-glycosylated form of recombinant scu-PA (411 amino acids) produced by *E. coli*. It has been developed as a fibrinolytic because scu-PA can mediate specific thrombolysis in the presence of fibrin, unlike tcu-PA, which lacks fibrin specificity and therefore increases the risk of bleeding after use. Roychoudhury, P. K., Khaparde, S. S., 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 can be cleaved in vivo into the form of double-stranded urokinase, thereby producing plasmin. Moser, M. & Bode, C. 急性心肌梗死中沙芦普酶(scuPA)的药理学及临床试验结果。《专家意见:药物研究》8, 329 - 335 (1999) 阿伯激酶与美国食品药品监督管理局的案例:导致美国阿伯激酶供应暂停的事件 - 《血管与介入放射学杂志》。https: / / www.jvir.org / article / S1051 - 0443(07) ].

[0025] - Currently, urokinase (Abbokinase) ® Kinlytic TM This drug is commercially produced using human neonatal kidney cells. It contains a low molecular weight form of uPA as its active ingredient. It was first approved by the FDA in 1978. However, due to viral contamination issues... 61798 - 9 / fulltext; 哈特内尔, G. G. & 盖茨, J. 阿伯激酶与美国食品药品监督管理局的案例:导致美国阿伯激酶供应暂停的事件。《血管与介入放射学杂志》11, 841–847 各种溶栓药物安全性与有效性的比较 - 美国国立医学图书馆。https: / / pubmed.ncbi.nlm.nih.gov / 12556739 / .50; 奥里尔, K. 等,溶栓治疗或外周动脉手术:I期结果。 TOPAS研究组。《血管外科杂志》23, 64 - 75 (1996) 奥里尔, K., 维思, F. J. & 萨萨哈拉, A. A. 溶栓治疗或外周动脉手术I期结果 (2000) The FDA suspended the use of this drug in December 1998 due to violations of Good Manufacturing Practices (GMP). Since 2002, the drug has been reapproved for the treatment of pulmonary embolism. However, these cells can only proliferate for a limited period (30-40 passages). Restoring these primary cells requires extensive screening and testing of kidney donors.

[0026] Abbott Laboratories developed a product called Prolyse ® Recombinant prourokinase has been tested in the UK for the treatment of various thromboembolic diseases. 罗伊乔杜里, P. K., 卡帕尔代, S. S., 马蒂亚松, B. & 库马尔, A. 合成, ​ ​ ​ ruPA is fully glycosylated because it is derived from a genetically engineered mouse hybridoma cell line and purified from the culture medium via a series of chromatographic steps in aqueous solution. The lyophilized therapeutic product is reconstituted with sterile water for injection. Over 90% of ruPA is in the high molecular weight form, with a specific activity of approximately 170,000 IU / mg as determined by a thrombolysis assay. ​ ​In pharmacokinetic studies on monkeys, ruPA had a half-life of 7 minutes, shorter than its lower molecular weight counterpart. However, despite these differences, the two drugs showed similar clinical efficacy. However, the drug has not yet received FDA approval. ​ Regulation and production of urokinase using mammalian cell culture: A Comprehensive review. Biotechnol Adv 24, 514–528 (2006) ].

[0027] The applicant had previously developed a method for preparing recombinant urokinase in a culture medium of genetically modified eukaryotic cells. This method is described in European Patent EP1245681; however, it requires the use of alkyl acids as activators and produces a mixture of low and high molecular weight uPA.

[0028] The applicant has now discovered and developed an improved method for producing recombinant human low molecular weight urokinase (rh-LMW-uPA) in eukaryotic cell line culture medium (CCOS 2068), which eliminates the need for the use of alkyl acids and the separation of the mixture of LMW-uPA and HMW-uPA, as the method yields only LMW-uPA as the product. Attached Figure Description

[0029] Figure 1 The structures of pro-uPA and uPA. Pro-uPA, containing a growth factor domain (GFD), a kringle domain (KD), and a catalytic serine protease domain, is secreted as a single-chain precursor and is expressed in Lys. 158 and Ile 159 Catalytic cleavage occurs between peptide bonds, generating a double-chain form of uPA. Through a second proteolytic cleavage, the double-chain uPA can be further processed in Lys... 135 and Lys 136 The amino-terminal fragment (ATF) cleaves between the two molecules, forming an inactive amino-terminal fragment and a catalytically active low molecular weight form of uPA (LMW-uPA). Mahmood, N., Mihalcioiu, C. & Rabbani, S. A. Multifaceted role of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR): Diagnostic, prognostic, and therapeutic applications. Frontiers in Oncology Volume 8, preprint at https: / / doi.org / 10.3389 / fonc.2018.00024 (2018) ].

[0030] Figure 2 The structure of recombinant human low molecular weight urokinase obtained by the method of the present invention. Detailed Implementation

[0031] Therefore, the object of the present invention is to provide a method for producing rh-LMW-uPA in CHO cell line (CCOS 2068) culture medium.

[0032] The general features of the method for which the present invention aims will now be described.

[0033] 1. Upstream (the stage of producing recombinant proteins via CHO)

[0034] Both the master cell bank (MCB) and the working cell bank (WCB) are stored in a gaseous nitrogen atmosphere. Each cryovial contains 1 mL of concentrated cell culture, preferably at a concentration of 10 × 10⁻⁶. 6 Cells / mL, and containing a cryoprotectant (preferably DMSO) at a concentration of 5-10% v / v, preferably 7.5% v / v.

[0035] Thaw cells in preheated, chemically determined culture medium (preferably CD OptiCHO, Gibco, catalog number 12681011) in 125 mL culture flasks (Spinner Flask Corning, catalog number 3152). The inoculum concentration should be 0.2–0.4 × 10⁻⁶ cells / mL. 6 Cells / mL, preferably 0.3 × 10⁻⁶. 6 Cells / mL were cultured at 37°C, 8.0% CO2, saturated rH (preferably >85%), and stirred at 40 rpm.

[0036] Cell cultures were examined regularly, and cell counts were performed using trypan blue staining exclusion method to determine VCD and cell viability.

[0037] When the culture density is at least 1.0 × 10⁻⁶ 6 Cells / mL, preferably 1.0 × 10⁻⁶ 6 Up to 2.0×10 6 When the culture reaches a cell / mL concentration, it is passaged. The passaged culture procedure includes diluting the culture in a fresh, chemically defined medium (preferably CDOptiCHO) at a concentration of 0.2–0.4 × 10⁻⁶ cells / mL. 6 Cells / mL, preferably 0.3 × 10⁻⁶. 6 Re-seedling was performed at a density of cells / mL.

[0038] Cells were expanded in 1L culture flasks (Spinner Flask Corning, product number 3561) by passage culture as described above.

[0039] The bioreactor operates at a concentration of 0.1-0.4 × 10⁻⁶. 6 Cells / mL, preferably 0.3 × 10⁻⁶. 6 The inoculum is seeded at a density of [number] cells / mL. The ratio of inoculum volume to final post-inoculation volume can be 1:3 to 1:8. The bioreactor culture setup is as follows:

[0040] - pH is 7.0-7.2, preferably 7.05;

[0041] - Dissolved oxygen is 40%-60%, preferably 50%;

[0042] - The temperature is 35℃-37.5℃, preferably 37.0℃;

[0043] - The stirring speed is 0.15-0.45 m / s, preferably 0.35 m / s (angular velocity);

[0044] - The gas flow rate is 0.02-0.2 vvm, preferably 0.075 vvm.

[0045] Cells are cultured for 2 to 4 days, preferably 3 days. After this culture period, the grown cells can be used as inoculum for larger bioreactors, or adapted for production purposes by changing the culture settings, as follows:

[0046] - pH is 6.90-7.05, preferably 6.90;

[0047] - Dissolved oxygen is 40%-50%, preferably 40%;

[0048] - The temperature is 35-37.5℃, preferably 37.0℃;

[0049] - The stirring speed is 0.15-0.45 m / s, preferably 0.35 m / s (peak speed);

[0050] - The gas flow rate is 0.02-0.2 vvm, preferably 0.075 vvm.

[0051] Continued addition of Ex-Cell Advanced Feed 1 G (SAFC part number: 24368C-10L) should begin at a flow rate of 2-8% of the initial volume, preferably 4% / day.

[0052] If the culture is to be used for amplification, proceed as described in the production stages above.

[0053] Continue culturing, maintaining a glucose concentration >1.0 g / L, preferably 2-11 g / L.

[0054] Harvest on days 12-15 after inoculation, preferably on day 14.

[0055] At the end of the culture, the cell supernatant is recovered by centrifugation or deep filtration. Deep filtration is performed using a Millistak+ Pro HC or Millistak+ HC deep filter (Millistak+ Pro HC preferred). The cell culture should be filtered at 100 LMH, with a culture loading of 90-680 L / m² and a sizing of 2-10 g / m² (wet biomass). The bacterial load is then reduced by filtration.

[0056] 2. Downstream (Purification stage of recombinant proteins)

[0057] The pH of the supernatant was adjusted to 5.4-8.0, preferably 5.5, by slowly adding a 5% v / v acetic acid solution containing 100 mM NaCl. pH correction was preferably completed within 20 minutes.

[0058] After pH correction, the supernatant is filtered through a 0.5-0.2µm double-layer filter membrane (preferably Merck Express SHC 0.45-0.2µm) at a flow rate of 250-1700 LMH.

[0059] The supernatant is loaded onto a resin coupled with pABA (p-aminobenzoic acid) ligand at a linear loading rate of 90-150 cm / h, preferably 100 cm / h. The resin should be pre-equilibrated with a suitable buffer solution at pH 5.5-8.0 (preferably a buffer solution of 100 mM sodium acetate, 10 mM CaCl2, pH 5.5). Due to the high binding specificity between the p-ABA ligand and the protein (based on the active site of serine proteases), this chromatographic stage is considered the first step in reducing the virus.

[0060] After the sample loading step, resin washing is required. A suitable buffer solution with pH 5.5-8.0, preferably 100 mM sodium acetate, 10 mM CaCl2, and pH 5.5, should be used for 2.5 column volumes at a linear loading rate of 90-150 cm / h, preferably 100 cm / h.

[0061] Subsequently, a washing step should be performed, in which a suitable high ionic strength buffer with pH 5.5-8.0, preferably 100 mM sodium acetate, 10 mM CaCl2, 900 mM NaCl, pH 5.5 buffer, is used for 2.5 cycles at a linear loading rate of 90-150 cm / h, preferably 100 cm / h.

[0062] Following the high ionic strength washing step, reequilibration is performed using a suitable buffer solution with pH 5.5-8.0, preferably 100 mM sodium acetate, 10 mM CaCl2, and pH 5.5, at a linear loading rate of 90-150 cm / h, preferably 100 cm / h for 2.5 cycles.

[0063] The purified intermediate sample was eluted using a glycine-HCl or acetate buffer, preferably 100 mM glycine-HCl, pH 2.7, at an acidic pH of 2.7–4.0. Up to 400 mM sodium chloride could be added to these buffers. The expected yield for this stage is >60%, and is evaluated based on enzyme activity.

[0064] Column efficiency can be restored using 1M acetic acid and 20% v / v ethanol. The column can be stored in 76mM NaCl and 24% v / v ethanol.

[0065] The intermediate eluted from the pABA column is further adjusted to pH 3.4–4.0, preferably 4.0. pH correction can be performed by adding a strong acid or base (preferably 500 mM NaOH or 5% v / v HCl) within 20 minutes. Combining acid elution with storage time can be used to further reduce viral contamination.

[0066] Virus inactivation at low pH can be carried out at 4°C to 25°C (preferably 4°C) for 2-20 hours.

[0067] The intermediate sample was further purified on a strong cation exchange resin (preferably Fractogel EMD SO3- (Merck, catalog number 1.16882)), wherein the resin was equilibrated with a suitable buffer at pH 5.0-7.4 (preferably a buffer of 20 mM sodium phosphate at pH 6.0) before purification at a linear flow rate of 100-200 cm / h, preferably 200 cm / h.

[0068] A washing step is required, and the washing should be performed as follows: using a suitable buffer solution with pH 5.0-7.4, preferably 20 mM sodium phosphate, pH 7.4 buffer solution, at a linear flow rate of 100-200 cm / h, preferably 200 cm / h for 2.5 cycles.

[0069] The intermediate product was eluted using a suitable high ionic strength buffer (pH 5.0-7.4), preferably 20 mM sodium phosphate, 350 mM sodium chloride, pH 7.4, at a linear flow rate of 100-200 cm / h, preferably 200 cm / h, for 2.5 CV cycles. The yield at this stage, based on enzyme activity assessment, was >80%.

[0070] The column efficiency was restored using a solution of 20 mM sodium phosphate, 1 M sodium chloride, and pH 7.4.

[0071] The column can be cleaned in situ with 500mM NaOH and stored using a 150mM sodium chloride and 20% v / v ethanol solution.

[0072] The intermediates eluted by cation exchange are filtered to remove viruses; this is the third step in reducing viral contamination. Constant pressure filtration is performed using a Viresolve shield (Merck, VPPS101NB1) and a Viresolve pro (Merck, VPMD101NB1) at a pressure of 1.8–2.2 bar, preferably 2.0 bar. Alternatively, constant pressure filtration can be performed using a 0.1µm Sartopore filter (Sartorius, 5443538M8M7FFA) followed by a Planova 20N filter (Asahi Kasei, 20NZ-300) at a pressure of 1.8–2.2 bar, preferably 2.0 bar.

[0073] 3. Formulation

[0074] The intermediate, after virus removal filtration, is further formulated via tangential flow filtration (TFF) at a feed flow rate of 200-400 LMH, preferably 360 LMH, maintaining a transmembrane pressure of 0.8-1.2 bar, preferably 1.0 bar. Regenerated cellulose membranes can be used. The molecular weight cutoff should be less than 10 kDa, preferably less than 5 kDa.

[0075] Formulation typically requires 7 times the volume. The buffer should be sodium phosphate or sodium acetate with a pH of 4.0-7.0, and may contain up to 2.5 mM EDTA and up to 6% (w / v) mannitol.

[0076] The final concentration of the drug is 0.5 to 10 g / L, and it should be stored at 4°C to -80°C.

[0077] 4. Specifications of the active pharmaceutical ingredient (API)

[0078] The protein concentration should be between 0.5 and 10 g / L.

[0079] The final specific activity of the drug was 200,000 to 300,000 IU / mg.

[0080] The monomer purity, as determined by HPLC-SEC, is >98.0%.

[0081] Total sialic acid content ≤5%, HCP content <100ppm, residual DNA <10ppm.

[0082] Comparing the main features of the method of the present invention with those described in EP1245681, the advantages of the method of the present invention are obvious:

[0083] - Using new cell lines enables the production of only low molecular weight urokinase, thereby improving the product's safety, quality, and stability.

[0084] Enzyme activation for the conversion of sc-uPA to tc-uPA does not require the addition of alkyl acids to the culture medium. Activation can occur in the presence of a protease, such as uPA itself.

[0085] - Productivity was significantly improved (120,000 IU / mL compared to 7,000 IU / mL).

[0086] - The resulting product has a high content of sialic acid, which improves the product's stability in patients.

[0087] The present invention will be described in more detail in the following embodiments, but these embodiments should not be construed as limiting the present invention.

[0088] Example

[0089] List of abbreviations used in the examples:

[0090] MCB: Master Cell Bank

[0091] WCB: Working Cell Bank

[0092] DMSO: Dimethyl sulfoxide

[0093] CHO: Chinese hamster ovary cells

[0094] rH: Relative humidity

[0095] VCD: Live Cell Count

[0096] DO: Dissolved oxygen

[0097] LMH: Liters per square meter per hour

[0098] CV: Column volume

[0099] CIP: In-situ cleaning

[0100] TMP: Transmembrane pressure

[0101] HCP: Host cell protein

[0102] Material

[0103]

[0104] Example 1

[0105] Thaw a small vial of MCB or WCB with 0.3 × 10⁻⁶ water. 6 Cells were seeded at a density of [number] cells / mL in CD Opti CHO medium and cultured at 37.0℃, 140 rpm, 85% rH, and 8.0 CO2. The volume density (VCD) and viability of the culture were monitored regularly. When the VCD was 1.0–3.0 × 10⁻⁶ cells / mL... 6At a cell / mL concentration, the culture was split by dilution with fresh culture medium. The seeding density after splitting was 0.3 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0106] The cell culture volume was expanded to the bioreactor inoculation scale, and then the Multifors MUF 2C Pack was inoculated at 0.3 × 10⁻⁶. 6 Cells were seeded at a density of 10 cells / mL in CD Opti CHO. The CHO cells were cultured for 3 days at 37.0°C, 50% DO, pH 7.05, a shaking angular velocity of 0.29 m / s, and 0.075 vvm.

[0107] On the third day, the culture was fed using Ex-Cell advanced feed 1G at a rate of 4% (of the initial volume) per day.

[0108] At the start of feeding, the culture parameters were adjusted to: 37.0℃, 50% DO, pH 7.05, shaking angular velocity of 0.31 m / s, and vvm of 0.100. The culture lasted for a maximum of 14 days, and the supernatant was collected at the end.

[0109] Example 2

[0110] Thaw a small vial of MCB or WCB with 0.3 × 10⁻⁶ water. 6 Cells were seeded at a density of [number] cells / mL in CD Opti CHO and cultured at 37.0 °C, 140 rpm, 85% rH, and 8.0 CO2. The volume density (VCD) and viability of the culture were monitored periodically. When the VCD was between 1.0 and 3.0 × 10⁻⁶ cells / mL... 6 At a cell / mL concentration, the culture was split by dilution with fresh culture medium. The seeding density after splitting was 0.3 × 10⁻⁶ cells / mL. 6 Cells / mL.

[0111] The cell culture volume was expanded to the bioreactor inoculation scale, and then the Multifors MUF 2C Pack was inoculated at 0.3 × 10⁻⁶. 6 Cells were seeded at a density of 10 cells / mL in CD Opti CHO. The CHO cells were cultured for 3 days at 37.0°C, 50% DO, pH 7.05, shaking angular velocity of 0.29 m / s and 0.075 vvm.

[0112] On the third day, the culture was fed using Ex-Cell advanced feed 1G at a rate of 4% (of the initial volume) per day.

[0113] After the feeding phase, the culture parameters were changed and set to: 37.0℃, 50% DO, pH 6.90, shaking angular velocity of 0.31 m / s, and vvm of 0.075. The culture lasted for a maximum of 14 days, and the supernatant was collected at the end.

[0114] Example 3

[0115] The supernatant was acidified to pH 6.5 and loaded onto pABA resin for affinity chromatography. Before loading the protein onto the column, the column resin was equilibrated with 20 mM sodium phosphate buffer (400 mM NaCl, pH 6.5). The linear flow rate was set to 90 cm / h. After the loading phase, the column was washed with 20 mM sodium phosphate buffer (400 mM NaCl, pH 6.5). The resin-bound product was further eluted with 100 mM glycine (pH 2.7). CIP and column storage were performed according to the supplier's instructions.

[0116] Example 4

[0117] The supernatant was alkalized to pH 8.0 and loaded onto pABA resin for affinity chromatography. Before protein loading, the column resin was equilibrated with 20 mM sodium phosphate buffer (400 mM NaCl, pH 6.5). The linear flow rate was set to 30 cm / h. After loading, the column was washed with 20 mM sodium phosphate buffer (100 mM NaCl, pH 6.5). Products bound to the resin were further eluted with 100 mM acetic acid (100 mM NaCl, pH 4.0). CIP and column storage were performed according to the supplier's instructions.

[0118] Example 5

[0119] The intermediate obtained from the capture step was pH-corrected to 5.0 with 0.5M sodium hydroxide solution and then loaded onto a strong cation exchange resin for 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 phase, the column was washed twice with 20 mM sodium phosphate buffer (pH 6.0) followed by 20 mM sodium phosphate buffer (pH 7.4). The product was further eluted with 20 mM sodium phosphate buffer (350 mM NaCl, pH 6.0). CIP and column storage were performed according to the manufacturer's instructions.

[0120] Example 6

[0121] The intermediate obtained from the capture step was pH-corrected to 5.0 and then loaded onto a strong cation exchange resin for 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 phase, the column was washed with 20 mM sodium phosphate buffer (pH 6.0). The product was eluted with 20 mM sodium phosphate buffer (350 mM NaCl, pH 7.0). CIP and column storage were performed according to the manufacturer's instructions.

[0122] Example 7

[0123] The ion-exchange chromatography eluent was loaded onto a regenerated cellulose TFF membrane module with a molecular weight cutoff of 10 kDa and a loading of 200 g / m³. 2 The circulation flow rate was set at 300 LMH and the TMP was set at 1.0 bar. The final formulation was prepared using 7 volumes of 50 mM sodium acetate (pH 5.5) formulation buffer. Percolation was performed at protein concentrations of 5 to 10 g / L. The final active pharmaceutical ingredient was concentrated to 5.0 g / L and frozen at below -20°C.

[0124] Example 8

[0125] The ion-exchange chromatography eluent was loaded onto a regenerated cellulose TFF membrane module with a molecular weight cutoff of 5 kDa and a loading of 50 g / m³. 2 The circulation flow rate was set at 360 LMH, and the TMP was set at 1.0 bar. The final formulation was prepared using 7 volumes of 10 mM sodium phosphate (2.5 mM EDTA, 6% v / v pH 7.0) formulation buffer. Percolation was performed at protein concentrations of 5 to 10 g / L. The final active pharmaceutical ingredient was concentrated to 0.5 g / L and frozen at below -20°C.

[0126]

[0127] PCT / RO / 134 form

Claims

1. A CHO cell line deposited at the Swiss Collection for Microorganisms and Cell Cultures (CCOS) under the accession number CCOS 2068.

2. A process for the production of recombinant human low molecular weight urokinase (rh-LMW-uPA) in the culture medium of the cell line CCOS 2068 of claim 1, comprising the following steps: cell culture; recovery of the supernatant by centrifugation or depth filtration; purification of the resulting urokinase on a resin; and formulation by tangential flow filtration.

3. The method of claim 2, wherein, The cell culture is carried out under the following conditions: - pH 6.90-7.05, preferably 6.90; - dissolved oxygen 40-50%, preferably 40%; - temperature 35-37.5°C, preferably 37.0°C; - stirring speed 0.15-0.45 m / s, preferably 0.35 m / s (tip speed); - gas flow 0.02-0.2 vvm, preferably 0.075 vvm; and the glucose concentration is maintained > 1.0 g / L, preferably 2-11 g / L.

4. The method of claim 2, wherein, The recovery of the supernatant was performed by filtration at 100 LMH and the culture loading was 90-680 L / m 2 , with a loading of 2-10 g / m 2 (wet biomass).

5. The method of claim 2, wherein, Before purification on the resin, the pH of the supernatant is adjusted to 5.4-8.0, preferably 5.5, by slow addition of 5% v / v acetic acid and 100 mM NaCl, preferably over 20 minutes.

6. The method of claim 2, wherein, The supernatant is purified on a resin coupled with the pABA (p-aminobenzamidine) ligand, previously equilibrated with a buffer at pH 5.5-8.0, preferably 100 mM sodium acetate, 10 mM CaCl2, buffer at pH 5.

5.

7. The method of claim 2, wherein, The purified urokinase is eluted from the resin at an acidic pH of 2.7-4.0 using a glycine-HCl or acetate buffer, preferably 100 mM glycine-HCl, buffer at pH 2.7; the eluate is further adjusted to a pH of 3.4-4.0, preferably 4.0, by addition of a basic solution, preferably 500 mM NaOH, over 20 minutes.

8. The method of claim 6, wherein, After purification on the resin coupled with the pABA ligand, the urokinase is further purified on a strong cation exchange resin by washing with a low ionic strength buffer at pH 7.4, preferably 20 mM sodium phosphate buffer, and elution at 2.5 CV with a high ionic strength buffer at pH 5.0-7.4, preferably 20 mM sodium phosphate, 350 mM sodium chloride, buffer at pH 7.4, at a linear flow rate of 100-200 cm / h, preferably 200 cm / h.

9. The method of claim 2, wherein, The formulation by tangential flow filtration (TFF) is carried out using a feed flow rate of 200-400 LMH, preferably 360 LMH, maintaining a transmembrane pressure of 0.8-1.2 bar, preferably 1.0 bar, with a molecular weight cut-off of less than 10 kDa, preferably less than 5 kDa.

10. The method of claim 9, wherein, A sodium phosphate or sodium acetate buffer at pH 4.0-7.0 is used, optionally with the addition of up to 2.5 mM EDTA and up to 6% (w / v) of mannitol.

11. The method of claim 2, wherein, The specifications of the resulting product are as follows: - protein concentration 0.5-10 g / L, stored at 4°C to -80°C; - specific activity of 200,000 - 300,000 IU / mg; - monomer purity > 98.0% as determined by HPLC-SEC.

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