Recombinant blood coagulation factor VII, expression vector, pharmaceutical composition and application

Abstract

The invention relates to a recombinant blood coagulation factor VII, an expression vector, a pharmaceutical composition and application, the recombinant blood coagulation factor VII is obtained by expression of mammalian cells, and the recombinant blood coagulation factor VII does not contain immunogenic sugar or does not contain a detectable amount of immunogenic sugar. The recombinant blood coagulation factor VII does not contain non-human protein and also does not contain immunogenic sugar Neu5Gc, that is, Neu5Gc cannot be detected in the recombinant blood coagulation factor VII, which indicates that the recombinant blood coagulation factor VII can reduce immunogenicity and improve safety, and also potentially avoids the taboo that non-human protein allergy people cannot use the recombinant blood coagulation factor VII; the recombinant blood coagulation factor VII disclosed by the invention has good stability, biological activity and therapeutic activity, so that the recombinant blood coagulation factor VII disclosed by the invention can be widely applied to treatment of hemorrhagic diseases.

Description

Technical Field

[0001] The present application relates to the field of coagulation factors, and specifically to recombinant coagulation factor VII, an expression vector, a pharmaceutical composition and applications. Background Art

[0002] Coagulation factor VII, referred to as (FVII), is an important component of the coagulation cascade. FVII has a complex structure, including: N-terminal γ-carboxyl domain (Gla), two "epidermal growth factor (EGF)-like" domains and serine protease domains, and is composed of 406 amino acids. Under autocatalysis, FVII is cut into light chain and heavy chain at 152-153, which is composed of a light chain with 152 amino acids of about 20kDa and a heavy chain with 254 amino acids of about 30kDa. The two chains are connected by a disulfide bond between 135-262. FVII is glycosylated to form activated coagulation factor VII (activated factor VII, FVIIa). FVIIa can be used in medicine to treat hemophilia or uncontrolled bleeding. Due to the complex post-translational modification of FVII, it is difficult to obtain highly active recombinant activated factor VII after recombinant expression, making it impossible to apply recombinant activated factor VII well. At present, there are two products of recombinant activated factor VII on the market. The first one is NovoSeven, which was approved by Novo Nordisk in 1999. It is expressed by BHK cells and potentially contains immunogenic sugars (α-Gal, Neu5Gc) and mouse proteins. The second one is Sevenfact, which was approved by LFB USA in 2020. It is secreted by the mammary glands of transgenic rabbits and potentially contains immunogenic sugars (α-Gal, Neu5Gc) and rabbit proteins. At present, many companies have conducted research and development on FVIIa, but due to the complexity of its structure and post-translational modifications, most of them have given up at different stages. Only NovoSeven has been approved for marketing in China, and it is expensive. New products are urgently needed to meet clinical needs.

[0003] In addition, in addition to recombinantly expressed activated coagulation factor VII, there are also extracted prothrombin complex (Feiba, etc.) and the coagulation factor IXa / X bispecific antibody Hemlibra (Emicizumab, Shuyoulile) approved by the FDA in 2017 for the treatment of hemophilia A or hemophilia B with factor VIII or IX inhibitors. Hemlibra prevents bleeding events in hemophilia A and B by simulating coagulation factor VIIIa. With the advancement of technology, the quality of blood-extracted prothrombin complex (Feiba, etc.) has been greatly improved, but the risk of viral contamination cannot be completely avoided, and the source is also greatly limited; Hemlibra is used to prevent bleeding events in hemophilia A and B, and cannot be used as a therapeutic product. Summary of the invention

[0004] In view of this, the present application proposes a recombinant coagulation factor VII, an expression vector, a pharmaceutical composition and its application, which can reduce immunogenicity while having activity and excellent viral safety.

[0005] In a first aspect, embodiments of the present application provide a recombinant coagulation factor VII, wherein the recombinant coagulation factor VII is obtained by expression of mammalian cells, and the recombinant coagulation factor VII does not contain immunogenic sugars or does not contain detectable amounts of immunogenic sugars.

[0006] In combination with the first aspect, the recombinant coagulation factor VII is cleaved into a light chain and a heavy chain at position 152-153 in the amino acid sequence shown in SEQ ID NO: 1 to form an activated recombinant coagulation factor VII.

[0007] In combination with the first aspect, the immunogenic sugar includes N-glycolylneuraminic acid Neu5Gc.

[0008] In combination with the first aspect, the recombinant coagulation factor VII does not contain non-human proteins.

[0009] In combination with the first aspect, the recombinant coagulation factor VII has a glycosylation modification pattern.

[0010] In conjunction with the first aspect, the recombinant coagulation factor VII includes at least one of the following features:

[0011] (1) The recombinant coagulation factor VII has O-glycosylation modification, and the O-glycosylation modification includes: (Ser 52 )-Glc-(Xyl) m and (Ser 60 )-Fuc, wherein m=0-2;

[0012] (2) the recombinant coagulation factor VII has an N-glycosylation modification, and the recombinant coagulation factor VII has at least one of the following glycoforms in the peptide segment of LC:N145 and the peptide segment of HC:N170: at least one of a complex terminal galactosylation glycoform, a complex terminal sialylation glycoform, a complex terminal galactosamine glycoform, and a fucosylation glycoform;

[0013] (3) The recombinant coagulation factor VII has an N-glycosylation modification, and the recombinant coagulation factor VII has at least one of the following glycoforms in the peptide segment of LC: N145 and the peptide segment of HC: N170: at least one of a complex terminal galactosylation glycoform, a complex terminal sialylation glycoform, a complex terminal galactosamine glycoform and a fucosylation glycoform, and the complex terminal sialylation glycoform accounts for more than 50% of the glycoforms in the recombinant coagulation factor VII, preferably more than 60%;

[0014] (4) The recombinant coagulation factor VII has an N-glycosylation modification, and the recombinant coagulation factor VII has at least one of the following glycoforms in the peptide segment of LC: N145 and the peptide segment of HC: N170: at least one of a complex terminal galactosylation glycoform, a complex terminal sialylation glycoform, a complex terminal galactosamine glycoform and a fucosylated glycoform, and the complex terminal galactosamine glycoform accounts for less than 50% of the glycoforms in the recombinant coagulation factor VII, preferably less than 40%.

[0015] In combination with the first aspect, the recombinant coagulation factor VII is obtained by expressing in a human cell line GEX, a cell line derived from the human cell line GEX, or a cell line homologous to the human cell line GEX.

[0016] In combination with the first aspect, the recombinant coagulation factor VII contains at least 9 γ-carboxylation modification sites among the 10 glutamic acids (Glu) in its GLA region.

[0017] In a second aspect, an embodiment of the present application provides an expression vector, wherein the expression vector contains the gene of the recombinant coagulation factor VII described in the first aspect, and the expression vector can be expressed in a host cell line GEX and has activity.

[0018] In a third aspect, an embodiment of the present application provides a pharmaceutical composition, which includes the recombinant coagulation factor VII described in the first aspect and a pharmaceutically acceptable excipient or adjuvant.

[0019] In a fourth aspect, the embodiments of the present application provide use of the recombinant coagulation factor VII described in the first aspect, the expression vector described in the second aspect, or the pharmaceutical composition described in the third aspect in the treatment of bleeding diseases and trauma hemostasis.

[0020] In conjunction with the fourth aspect, the bleeding disease includes at least one of hemophilia A, hemophilia B, factor XI deficiency, factor VII deficiency, thrombocytopenia and von Willebrand disease.

[0021] The technical solution of this application has at least the following beneficial effects:

[0022] The recombinant coagulation factor VII of the present application is obtained by expressing mammalian cells. The recombinant coagulation factor VII does not contain immunogenic sugars or does not contain detectable amounts of immunogenic sugars. In particular, the immunogenic sugar Neu5Gc and non-human proteins are not detectable in the recombinant coagulation factor VII of the present application, indicating that the recombinant coagulation factor VII of the present application can reduce immunogenicity, improve safety, and potentially avoid the contraindications for use by people who are allergic to non-human proteins. The recombinant coagulation factor VII of the present application has good stability, biological and therapeutic activity, so that the recombinant coagulation factor VII of the present application can be widely used in the treatment of bleeding diseases or traumatic hemostasis, especially for type A or type B hemophilia in the presence of factor VIII or factor IX inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions of the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is the N-sugar-free molecular weight map of the recombinant coagulation factor VII preparation sample of this application;

[0025] Figure 2 This is the TIC spectrum of the N-glycan-reducing molecular weight of the recombinant coagulation factor VII preparation sample of the present application;

[0026] Figure 3 for Figure 2 Schematic diagram of the self-cleavage of the HC chain;

[0027] Figure 4 This is the molecular weight spectrum of LC component 1 in the recombinant coagulation factor VII preparation sample of this application after N-glycan reduction;

[0028] Figure 5 The molecular weight spectrum of LC component 2 in the recombinant coagulation factor VII preparation sample of this application is reduced by N-glycan

[0029] Figure 6 This is the molecular weight spectrum of HC fragment 3 (HC chain 164-254) after N-glycan reduction in the recombinant coagulation factor MII preparation sample of this application;

[0030] Figure 7 This is the molecular weight spectrum of HC N-reduced sugar in the recombinant coagulation factor VII preparation sample of this application;

[0031] Figure 8This is the molecular weight spectrum of HC fragment 1+2 (HC chain 1-163) after N-glycan reduction in the recombinant coagulation factor VII preparation sample of the present application;

[0032] Fig. 9 This is the theoretical amino acid sequence coverage diagram of the recombinant coagulation factor VII preparation sample of this application;

[0033] Fig.10 This is the secondary ion coverage spectrum of the peptide segment (1-18) of the sample of the recombinant coagulation factor VII preparation of this application;

[0034] Fig.11 This is the secondary ion coverage spectrum of the peptide segment (19-28) of the recombinant coagulation factor VII preparation sample of this application;

[0035] Fig.12 This is the secondary ion coverage spectrum of the peptide segment (23-32) of the recombinant coagulation factor VII preparation sample of this application;

[0036] Fig.13 This is the secondary ion coverage spectrum of the peptide segment (33-38) of the recombinant coagulation factor VII preparation sample of this application;

[0037] Fig.14 Secondary ion spectrum of LC: N145 related peptide segment of the recombinant coagulation factor VII preparation sample (sample 1) of this application;

[0038] Fig.15 This is the secondary ion spectrum of the LC: N170 related peptide segment of the recombinant coagulation factor VII preparation sample (sample 2) of this application;

[0039] Fig.16 This is the N-sugar identification profile of the recombinant coagulation factor VII preparation sample of this application;

[0040] Fig.17 UPLC-FLR N-glycoprofile of the recombinant factor VII preparation sample of this application;

[0041] Fig.18 This is a schematic diagram of the O-sugar sites and sugar chains of the relevant peptide segments of the O-glycopeptide (39-62) of the recombinant coagulation factor VII preparation sample of this application;

[0042] Fig.19 This is the DSC spectrum of the recombinant coagulation factor VII preparation sample of this application. DETAILED DESCRIPTION

[0043] In order to better understand the technical solution of the present application, the embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0044] It should be clear that the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0045] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0046] It should be understood that the term "and / or" used in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0047] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0048] Coagulation factor VII (FVII) is a glycoprotein dependent on vitamin K, which participates in the coagulation process in its activated form. FVII is secreted in the form of a single peptide chain with 406 amino acid residues and a molecular weight of about 50kDa. FVII contains four special domains: the N-terminal γ-base domain (Gla), two "epidermal growth factor (EGF)-like" domains and a serine protease domain. The activation of FVII is characterized by the cleavage of the arginine 152-isoleucine 153 domain (arginine 152-isoleucine 153) connection. The activated coagulation FVII is composed of a light chain with a molecular weight of about 20kDa of 152 amino acids and a heavy chain with a molecular weight of about 30kDa of 254 amino acids, which are connected to each other by a single disulfide bond (cysteine ​​135-cysteine ​​262). Unless otherwise expressly stated. Preferably, the coagulation factor VII of the present application is human coagulation factor VII.

[0049] The host cell line GEX is derived from a human myeloid leukemia cell line and can impart complex human glycosylation characteristics to a variety of recombinant biomacromolecules such as natural proteins, antibodies, and fusion proteins. According to the present invention, the host cell line GEX can also refer to cells or cell lines derived from GEX.

[0050] Sialic acid is an acidic amino sugar with 9 carbon atoms and a pyranose structure, and its systematic name is 5-amino-3,5-dideoxy-D-glycero-D-galactosylnonone sugar. N-glycolylneuraminic acid (Neu5Gc) is an important one among them. N-glycolylneuraminic acid is a kind of immunogenic sugar in recombinant coagulation factor VII. The presence of N-glycolylneuraminic acid can cause adverse clinical reactions such as allergic reactions in the use of recombinant coagulation factor VII.

[0051] The present application provides a recombinant coagulation factor VII that can be obtained by expressing in a human host cell or a human cell line. Preferably, the recombinant coagulation factor VII can be produced in a human bone marrow cell line, preferably a human bone marrow leukemia cell line, in particular a cell line GEX or a cell line homologous to the cell line GEX. The present application found that the recombinant coagulation factor VII produced in the above-mentioned specific cell line can exhibit a specific glycosylation pattern and can produce unexpected therapeutic and pharmacological effects. The following present application expresses coagulation factor VII in the above-mentioned cell line, wherein coagulation factor VII is derived from the UniProt database, the sequence ID is P08709 (FA7_HUMAN), and the amino acid sequence of coagulation factor VII is shown in SEQ ID NO: 1. After autocatalysis, coagulation factor VII is cut into a light chain and a heavy chain at the 152-153 site to obtain an activated coagulation factor VII.

[0052] The recombinant coagulation factor VII of the present application is prepared by the following steps:

[0053] Step 1: Transfect the expression vector containing coagulation factor VII into The transfected cells were subjected to pressure screening and monoclonal cell line screening to obtain an engineered cell line that can stably express coagulation factor VII. As for the human cell line platform, those skilled in the art can obtain it through commercial channels, which contains the cell line GEX or a cell line derived from the cell line GEX or a cell line homologous to the cell line GEX.

[0054] Step 2: Resuscitate the engineered cell line, resuspend the cell pellet in culture medium, take samples for counting, confirm that the cell seeding density meets the expansion requirements, and perform subsequent expansion after culturing the cells for 72h±12h.

[0055] Step 3: After obtaining sufficient cell culture volume in step 2, the cells are expanded by suspension culture, and then the expanded cells are transferred to the bioreactor for perfusion culture. The expression level of the target protein is detected, and the target protein is harvested after reaching the concentration required for purification, and then transferred to downstream purification.

[0056] Step 4: Use affinity chromatography to perform the first purification on the harvested liquid to collect the target protein, and use ion exchange chromatography to perform refinement and purification to obtain the target protein recombinant coagulation factor VII.

[0057] The recombinant coagulation factor VII obtained by column chromatography is subjected to post-filtration, sterilization and other post-treatments to obtain a recombinant coagulation factor VII preparation with purity, impurities and activity that meet the requirements. The following characterizations are performed on the recombinant coagulation factor VII preparation prepared above (hereinafter referred to as the sample of this application):

[0058] 1. Determine the molecular weight of N-glycans.

[0059] The samples of the present application were treated with N-glycosidase F (PNGase F) for N-glycan removal, separated and detected by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), and analyzed by UNIFI (Ver 1.9) software to measure the molecular weight of the samples without N-glycans. The test results are shown in Figure 1 .

[0060] Combination Figure 1 As shown, UPLC-MS was used to determine the molecular weight of the sample of the present application, and three main molecular weights were identified, which are: Figure 1 Peaks 1, 2 and 3 are shown, and the molecular weights of peaks 1, 2 and 3 are 46045Da, 46068Da and 46095Da, respectively. The molecular weight of peak 1 is consistent with the theoretical value of 46043Da obtained by directly adding the molecular weights of γ(9), F(1)H(1)P(2) and the amino acid sequence of the above sample through chemdraw software. It is identified as γ(9), F(1)H(1)P(2) modification, H represents hexose (here refers to glucose), F represents fucose, P represents pentose (here refers to xylose), γ represents γ-carboxylated glutamic acid, and the numbers in brackets represent the number of such modification units. Peaks 2 and 3 are identified to have multiple modification combinations, and the situation is more complicated.

[0061] 2. Determination of the molecular weight of N-glycan reduction

[0062] The samples of this application were subjected to N-glycosidase F (PNGase F) and reducing agent dithiothreitol (DTT) for N-glycan removal and reduction, and then separated and detected by UPLC-MS. The LC and HC chain N-glycan reduction molecular weights of the samples were measured by UNIFI (Ver 1.9) software analysis. The results are shown in Figure 2 and Figure 3 .

[0063] like Figure 2 As shown, the TIC spectrum of the N-sugar reducing molecular weight of the sample of this application and the five component peaks are Figure 2It can be seen that after the reverse phase chromatography separation of the sample of the present application, 5 chromatographic peaks were obtained, namely 2 LC related chromatographic peaks (component 1 and component 2) and 3 HC related chromatographic peaks. HC has two self-cleavage sites, and there will be complete HC and HC fragments 1+2 and HC fragment 3 (such as Figure 3 ). Where:

[0064] The main molecular weights (peak height Top3) of the LC fragment component 1 de-N-glycan were measured to be 17570Da, 17732Da and 17996Da, which were consistent with the theoretical molecular weights of 17569Da, 17731Da and 17995Da. The modifications were γ(9), F(1), γ(9), F(1)H(1), γ(9), F(1)H(1)P(2). The results are shown in Figure 4 . Figure 4 Here, H represents hexose (glucose in this case), F represents fucose, P represents pentose (xylose in this case), γ represents γ-carboxylated glutamic acid, and the numbers in brackets represent the number of such modified units.

[0065] The main molecular weight of LC fragment component 2 (peak height Top2) was measured to be 18020Da and 18048Da, which is consistent with the theoretical molecular weight of 18019Da and 18047Da. The modifications are γ(9), F(1)H(1)P(2)+24Da, γ(10), F(1)H(1)P(2)+8Da, respectively. The results are shown in 5. Figure 5 Here, H represents hexose (glucose in this case), F represents fucose, P represents pentose (xylose in this case), γ represents γ-carboxylated glutamic acid, and the numbers in brackets represent the number of such modified units.

[0066] The main molecular weight of the sample HC fragment 3 (HC chain 164-254) without N-sugar was measured to be 10033 Da, which is consistent with the theoretical molecular weight of 10033 Da. The result is shown in 6.

[0067] The main molecular weight of the sample HC chain N-sugar was measured to be 28072Da, which is consistent with the theoretical molecular weight of 28072Da. Figure 7 .

[0068] The main molecular weight of the sample HC fragment 1+2 (HC chain I-163) without N-glycan was measured to be 18057 Da, which is consistent with the theoretical molecular weight of 18057 Da. Figure 8 .

[0069] 3. Amino Acid Sequence Coverage

[0070] After the sample of this application was denatured with 8M guanidine hydrochloride, reduced with DTT and alkylated, the solution was replaced with 100mM Tris-HCl (pH 7.5) solution by ultrafiltration centrifugation; the replaced sample was divided into two parts, one was added with Trypsin / Lys-CMix enzyme digestion to obtain sample 1, and the other was added with Chymotrypsin enzyme digestion to obtain sample 2, and formic acid was terminated. The above two samples were separated and detected by UPLC-MS, analyzed by Biopharma Finder software, and the amino acid sequence coverage of the samples was calculated, and the comprehensive amino acid sequence coverage was obtained by combining the experimental results of 4.5.4LC N-terminal glutamic acid γ-carboxylation analysis. Among them, the Trypsin / Lys-C Mix, Chymotrypsin and LC N-terminal glutamic acid γ-carboxylation analysis reduction groups of the samples used samples digested with Trypsin and Lys-C, respectively, and the Biopharma Finder software was used to attribute and calculate the sequence coverage of each peptide segment.

[0071] The results showed that the amino acid sequence coverage of the Trypsin / Lys-C Mix digested samples was 89.6%. The uncovered peptide RSCR (LC: 110-113) in LC can be confirmed by the peptide CSDHTGTKRSCRCHEGY (LC: 102-118) identified in the Chymotrypsin digested samples. That is, the comprehensive coverage of the amino acid sequence after the combined Chymotrypsin digestion was 90.6%. Because γ-carboxylated glutamic acid (represented by γ in this article) has strong system adsorption and mass spectrometry fragmentation instability, the γ-rich peptide segment has almost no response in the mass spectrometry, so the dimethyl derivatization of γ-carboxylated glutamic acid is needed to solve it. The peptides ANAFLγγLRPGSLγRγCKγγQCSFγγARγIFKDAγRTK(1-38) not covered in LC can be identified in the γ-carboxylation derivatization experimental data, such as peptides ANAFLγγLRPGSLγRγCK(1-18), γγQCSFγγAR(19-28), SFγγARγIFK(23-32), DAγRTK(33-38). In summary, the comprehensive coverage of amino acid sequences is 100%, indicating that it is completely consistent with the theoretical sequence. The results are shown in Fig. 9 .

[0072] IV. Analysis of LC N-terminal glutamic acid γ-carboxylation

[0073] Restore Group:

[0074] The sample of this application was spin-dried, derivatized, and then spin-dried again, denatured with guanidine hydrochloride, reduced with DTT, and alkylated. The solution was replaced into a 50mM Tris-HCl (pH7.5) solution by ultrafiltration centrifugation. It was divided into two parts, one of which was digested with Lys-C for 4 hours, and the other was digested with Trypsin for 4 hours, and terminated with formic acid. UPLC-MS was used for separation and detection, and the LC N-terminal γ-carboxylated glutamic acid peptides were analyzed by Biopharma Finder software.

[0075] Non-restored group:

[0076] The sample of this application was spin-dried, derivatized, and then spin-dried again, denatured with guanidine hydrochloride, blocked with 5mM NEM, and replaced with a 50mM Tris-HCl (pH7.5) solution by ultrafiltration centrifugation. Divide into two parts, one of which is digested with Lys-C for 18 hours, and the other is digested with Trypsin for 18 hours, and terminated with formic acid. UPLC-MS separation and detection were used, and the disulfide bonds in the γ-carboxylated glutamic acid peptides rich in LC N-terminal were analyzed by Biopharma Finder software. The analysis of the reduced group and the non-reduced group can identify 4 double-methyl and mono-methyl modified peptides, namely ANAFLEELRPGSLERECK (1-18), SFEEAREIFK (23-32), EEQCSFEEAR (19-28), and DAERTK (33-38). Among them:

[0077] (a) The peptide ANAFLEELRPGSLERECK (1-18) identified that E6, E7, E14 and E16 were all dimethylated, confirming the presence of γ-carboxylation modification at these four sites. The secondary ion amino acid coverage is shown in Fig.10 .

[0078] (b) Peptide EEAR (19-28) identified dimethylation at E20, E25, and E26, and monomethylation at E19 with neutral loss (a common form of -NH3 neutral loss in peptides in mass spectrometry ion transmission), confirming the presence of γ-carboxylation modification at these four sites. The secondary ion amino acid coverage map is shown in Fig.11 .

[0079] (c) Peptide SFEEAREIFK(23-32) (non-specific enzyme cleavage peptide, with partial amino acid sequence overlap with peptide 19-28) identified E25, E26, and E29 as dimethylated, confirming the presence of γ-carboxylation modification at these three sites. The secondary ion amino acid coverage map is shown in Fig.12 .

[0080] (d) The peptide DAERTK (33-38) identified that E38 was dimethylated, confirming the presence of γ-carboxylation modification at this site. The secondary ion amino acid coverage map is shown in Fig.13 .

[0081] In summary, a total of 10 γ-carboxylation modification sites were identified in the above 4 peptides, namely E6, E7, E14, E16, E19, E20, E25, E26, E29, and E35, which are consistent with the conclusions of the γ(10)-related components identified in the de-N-glycan molecular weight and de-N-glycan reducing molecular weight.

[0082] 5. Identification of post-translational modifications

[0083] Two groups of samples of the present application were denatured with guanidine hydrochloride, reduced with DTT and alkylated, and then the solution was replaced with 100mM Tris-HCl (pH 7.5) solution by ultrafiltration centrifugation. The samples were digested with Trypsin / Lys-C Mix to reduce the complexity of peptide map analysis. The cleavage site of PNGase F enzyme was the amide bond between N-acetylglucosamine (GlcNAc) and asparagine residue inside the glycoprotein to obtain samples 1 and 2. The above two groups of samples were separated and detected by UPLC-MS and analyzed by BiopharmaFinder software to retrieve various post-translational modifications and calculate their relative proportions. Among them, the modification types retrieved by Biopharma Finder software analysis were: Carbamidomethyl C, Oxidation M, Oxidation W, Deamidation N, Hydroxylation D. The relative content of translation modification was calculated as follows:

[0084]

[0085] The statistical results are shown in Table 1.

[0086] Table 1. Statistical table of post-translational modification analysis results of samples in this application

[0087]

[0088] Note: The amino acids in bold are the modification sites; Carbamidomethyl C is introduced by sample alkylation treatment and is a fixed modification for all Cs. This modification type is no longer marked in the table.

[0089] It can be seen from the contents of Table 6 that the main post-translational modifications in the samples of this application are aspartic acid hydroxylation, asparagine deamidation and methionine oxidation modification, and the post-translational modification levels are consistent.

[0090] VI. Identification of N-Glycan Sites

[0091] In the above-mentioned characterization project 5 of recombinant coagulation factor VII, sample 1 and sample 2 were respectively subjected to identification of peptides related to potential N-glycosylation site N145 on LC and potential N-glycosylation site N170 on HC.

[0092] This application was hydrolyzed by Trypsin / Lys-C Mix and PNGase F was used to remove N-sugars. At the same time, the N-glycosylated asparagine (Asn, N) was converted to aspartic acid (Asp, D). This feature was combined with Biopharma Finder software to search for peptides converted from N to D, and two peptides were identified, namely:

[0093] a) N145 of peptide RNASKPQGR(144-152) undergoes a N-to-D transition;

[0094] b) N170 of the peptide VGDSPNITEYMFCAGYSDGSK(165-185) undergoes a transformation from N to D.

[0095] Therefore, the N-sugar site on LC is N145, and the N-sugar site on HC is N170, which is consistent with the theoretical site. The identification results are shown in Table 2. The secondary ion spectrum of the sample (DS20220603-N) LC: N145 related peptide segment is shown in Fig.14 , sample (DS202209002) LC: N170 related peptide secondary ion spectrum see Fig.15 .

[0096] Table 2. Statistical table of identification results of peptides related to glycosylation sites in this application

[0097]

[0098] VII. N-Glycan Identification

[0099] Sample 1 in the above-mentioned characterization 5 was fluorescently labeled with recombinant coagulation factor VII dye to derivatize the sugar chain. The derivatized sugar chain was purified by solid phase extraction and identified by ultra-high performance liquid chromatography-mass spectrometry. The identification results are shown in Fig.16 , it is understandable that Fig.16 In the figure, when multiple components co-elute, brackets are used to identify all identified glycoforms (such as the green dashed box). When the co-eluting components are statistically analyzed, the peak area is allocated using the mass spectrum response ratio of the co-eluting components.

[0100] Because the sugar forms are complex, the sugar forms are named according to the sugar unit composition. Because there are many co-eluting sugar forms, the two sugar forms are marked on the same chromatographic peak. The same retention time will appear in the table for two sugar forms. There is no galactose-α-1,3-galactose (α-Gal) sugar form, see Table 3.

[0101] Table 3. Sample N-glycan identification results

[0102]

[0103]

[0104] In Table 3, the retention times marked with “≈” show continuous fluctuations of multiple groups of peaks of the same component due to poor separation of complex N-sugar peaks. Only representative retention times are selected to identify different glycoforms.

[0105] 9. N-Glycan Profile (UPLC-FLR)

[0106] In the above-mentioned characterization project 5, samples 1 and 2 were respectively fluorescently labeled with recombinant coagulation factor VII dye to derivatize the sugar chains. After the derivatized and labeled sugar chains were purified by solid phase extraction, UPLC-FLR was used to detect the N-sugar spectrum of the samples. When the co-eluting components were statistically analyzed, the peak area was allocated using the mass spectrometry response ratio of the co-eluting components. The results are detailed in Fig.17 and Table 4.

[0107] Table 4. Summary of N-glycan profile test results of samples in this application

[0108]

[0109]

[0110]

[0111] In Table 4, H represents hexose, which refers to mannose or galactose, N represents N-acetylglucosamine or N-acetylgalactosamine, F represents fucose, S represents N-acetylneuraminic acid, and Sg represents N-glycolylneuraminic acid. The numbers after the letters represent the number of sugar units of this type.

[0112] It can be seen from Table 4 that N-glycolylneuraminic acid is undetectable in the recombinant coagulation factor VII of the present application, indicating that the product obtained by expressing coagulation factor VII in the human cell line GEX does not contain immunogenic sugars, which can reduce immunogenicity and improve safety.

[0113] 10. O-Glycan Confirmation

[0114] Samples 1 and 2 in the above characterization project 5 were separated and detected using UPLC-MS, and the Biopharma Finder software was used to search for potential O-glycopeptides. The added sugar units included hexose (corresponding to glucose, abbreviated as Glc, marked with a blue circle), pentose (corresponding to xylose, abbreviated as Xyl, marked with an orange five-pointed star), and deoxyhexose (corresponding to fucose, abbreviated as Fuc, marked with a red triangle). Fig.18 .

[0115] The results showed that there were 8 types of O-glycopeptides in the LC peptides (39-62), including peptides without O-glycosylation, Fuc modification, Glc modification, Glc-Xyl modification, Glc and Xyl modification, Glc-Xyl-Xyl modification, Glc-Xyl and Fuc modification, and Glc-Xyl-Xyl and Fuc modification. The theoretical modification site S was obtained by in-silico analysis. 52 and S 60 Comprehensive analysis of the primary and secondary ion fragment information of various O-glycopeptides showed that the longest sugar chain at the two O-sugar sites was (Ser 52 )-Glc-Xyl-Xyl and (Ser 60 )-Fuc, the peptide statistics are shown in Table 5. From the results in Table 5, it can be seen that the types of O-sugar chains in the samples of the present application are consistent with the theory.

[0116] Table 5. Statistics of peptide segments related to O-glycopeptide (39-62)

[0117]

[0118] In summary, the recombinant coagulation factor VII of the present application is obtained by expressing coagulation factor VII in GEX cells, that is, the recombinant activated human coagulation factor VII of the present application is expressed in domesticated and screened human GEX cells, so that the post-translational modification (especially the modification of N-sugar) of the expression product is closer to the modification characteristics of humans, and does not contain immunogenic sugars (such as Neu5Gc, etc.), which potentially improves safety. At the same time, the change of sugar form also gives the product specific physical and chemical properties and improves activity.

[0119] Performance measurement:

[0120] 1. Sialic acid detection (UPLC-FLR)

[0121] Two groups of samples of the present application were taken, recorded as Example 1 and Example 2, and the above two groups of solutions were labeled after releasing sialic acid under 4M acetic acid at high temperature, and then an organic solvent was added to the labeled product to precipitate the protein, and the supernatant solution was taken after centrifugation, separated by hydrophilic interaction chromatography, and detected by a fluorescence detector. The external standard method was used to perform quantitative analysis using the peak area and sialic acid molar amount as a standard curve, and the sialic acid content in the protein was calculated by conversion with the protein amount. The results are shown in Table 6.

[0122] Table 6. Summary of sample sialic acid detection results

[0123] Sample batch number NANA (nmol / nmol) NGNA (nmol / nmol) Example 1 1.720 Not detected Example 2 1.464 Not detected

[0124] In Table 6, NANA represents N-acetylneuraminic acid, NGNA represents N-glycolylneuraminic acid, and the sialic acid content unit (nmol / nmol) means that each nmol of protein contains X nmol sialic acid; the quantification limit of NANA in this method is 0.09nmol / nmol, and the quantification limit of NGNA is 0.009nmol / nmol.

[0125] It can be seen from the data in Table 12 that the N-acetylneuraminic acid content of the solutions of Example 1 and Example 2 is consistent, and N-hydroxyacetylneuraminic acid (NGNA) is not detected, indicating that the recombinant coagulation factor VII of the present application does not contain N-hydroxyacetylneuraminic acid, further indicating that the recombinant coagulation factor VII of the present application is non-immunogenic when used to treat patients.

[0126] 2. Thermal stability analysis (DSC, Tm value) Thermostability, DSC (Tm)

[0127] The samples of Example 1 and Example 2 were respectively tested for their melting temperatures (Tm) by differential scanning calorimetry (DSC). The method comprises the following steps:

[0128] Take the sample solution, use ultrafiltration tube to concentrate the sample to a suitable detection concentration, select a suitable scanning temperature range and heating rate, use micro differential scanning calorimeter (PEAQ-DSC) to scan and detect the buffer and sample, and calculate the Tm value of the sample. The results are shown in Table 7 and Fig.19 , from Table 7 and Fig.19 The results show that the recombinant coagulation factor VII of the present application has good thermal stability.

[0129] Table 7. Sample Tm value analysis results

[0130] Sample batch number Tm(℃) Example 1 58.58 Example 2 58.59

[0131] 3. Activity measurement data:

[0132] 1. Instrument

[0133] Table 8. Instruments used for activity determination

[0134]

[0135] 2. Materials

[0136] Table 9. Instruments used for activity determination

[0137]

[0138]

[0139] 3. Sample solution

[0140] According to the kit instructions for the relative activity comparison shown in Table 9, the above-mentioned Example 1 and Example 2 were diluted into sample stock solutions S1 and S2, and linear solutions ST1-ST5 were further prepared according to the kit instructions. The configuration parameters are shown in Table 10.

[0141] Table 10. Preparation of relative active sample solutions

[0142]

[0143] 4. Test methods

[0144] The test method comprises the following steps:

[0145] Place the relative activity test kits R1 and R2 in a coagulometer in advance and preheat at 37°C for at least 15 minutes; shake the preheated reagents R1 and R2 before use to make them uniform.

[0146] Take 50 μl of the sample solutions of the experimental group (ST1-ST5) and the control group solutions (Nochi MS6F59, Nochi*LS6EC08 (batch 1), Nochi*LS6EC08 (batch 2)) into the semi-automatic coagulation test cup (three groups for each sample), add 50 μl of pre-warmed R1, and incubate in the coagulation tester at 37°C for 1 min; then add 50 μl of pre-warmed R2 solution and incubate at 37°C for 2 min; put the test cup into the test well, and then add pre-warmed 0.025 M CaCl 2 The solution was 50 μl, and the test was performed by clicking on the button at the same time. The coagulation time was recorded. The test results are shown in Table 11.

[0147] Table 11. Coagulation times of samples and controls

[0148]

[0149] According to the content of Table 3, the recombinant coagulation factor VII prepared in this application has a shorter coagulation time than the three different types of samples of Noqi, indicating that the recombinant coagulation factor VII in this application has excellent coagulation function and has excellent development prospects for trauma hemostasis or treatment of bleeding diseases. As shown in Table 12, the consumables used in this application and their sources.

[0150] Table 12. Consumables and their sources

[0151]

[0152] In summary, the recombinant coagulation factor VII of the present application was expressed in the human cell line GEX, and the product was characterized and performance tested. It was found that the recombinant coagulation factor VII of the present application not only does not contain non-human proteins, but also does not contain the immunogenic sugar Neu5Gc, that is, Neu5Gc is not detectable in the recombinant coagulation factor VII of the present application, indicating that the recombinant coagulation factor VII of the present application can reduce immunogenicity and improve safety, and also potentially avoid the contraindication of use for people who are allergic to non-human proteins. The recombinant coagulation factor VII of the present application has good stability, biological and therapeutic activity, so that the recombinant coagulation factor VII of the present application can be widely used in the treatment of bleeding diseases and trauma hemostasis.

[0153] The present application also provides a pharmaceutical composition comprising the above-mentioned recombinant coagulation factor VII, which may include the above-mentioned recombinant coagulation factor VII or a recombinant coagulation factor VII preparation, and may also include a pharmaceutically acceptable excipient or adjuvant. The excipient may be any solution, such as physiological saline, physiological, isotonic or buffered solution and any suspension, gel or powder, as well as those suitable for pharmaceutical applications and well known to those skilled in the art.

[0154] In some embodiments, the pharmaceutical composition of the present application may further comprise other pharmaceutically active agents, in particular other pharmaceutically active agents that can be used to treat bleeding diseases, such as other coagulation factors.

[0155] In some embodiments, the administration of the pharmaceutical composition of the present application can be in any conventional medical method type, such as particularly through a systemic route, such as by intravenous, intradermal, intratumoral, subcutaneous, intraperitoneal, intramuscular or intraarterial injection.

[0156] In some embodiments, the dosage of recombinant coagulation factor VII in the pharmaceutical composition of the present application can vary depending on the amount of administration, combination with other active ingredients, degree of pathological development, etc.

[0157] Based on the physicochemical properties and biological activity of the recombinant coagulation factor VII of the present application, the recombinant coagulation factor VII or the pharmaceutical composition can be used in the treatment of bleeding diseases or traumatic hemostasis.

[0158] In some embodiments, the bleeding disorder comprises at least one of hemophilia A, hemophilia B, factor XI deficiency, factor VII deficiency, thrombocytopenia and von Willebrand disease, wherein hemophilia A and hemophilia B particularly refer to hemophilia A or hemophilia B with factor VIII or factor IX inhibitors.

[0159] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

[0160]

Claims

1. A recombinant coagulation factor VII, characterized in that The recombinant blood coagulation factor VII is obtained by expressing mammalian cells, and the recombinant blood coagulation factor VII does not contain immunogenic sugar or does not contain detectable amount of immunogenic sugar.

2. The recombinant coagulation factor VII according to claim 1, characterized in that The recombinant blood coagulation factor VII is cleaved into a light chain and a heavy chain at the 152-153 position in the amino acid sequence shown in SEQ ID NO: 1 to form an activated recombinant blood coagulation factor VII.

3. The recombinant coagulation factor VII according to any one of claims 1 to 2, characterized in that The immunogenic sugar includes N-glycolylneuraminic acid Neu5Gc.

4. The recombinant coagulation factor VII according to any one of claims 1 to 2, characterized in that The recombinant coagulation factor VII does not contain non-human proteins.

5. The recombinant coagulation factor VII according to any one of claims 1 to 2, characterized in that The recombinant blood coagulation factor VII has glycosylation modification.

6. The recombinant coagulation factor VII according to claim 5, characterized in that The recombinant coagulation factor VII comprises at least one of the following characteristics: (1) The recombinant coagulation factor VII has O-glycosylation modification, and the O-glycosylation modification includes: (Ser 52 )-Glc-(Xyl) m and (Ser 60 )-Fuc, wherein m=0-2; (2) the recombinant coagulation factor VII has an N-glycosylation modification, and the recombinant coagulation factor VII has at least one of the following glycoforms in the peptide segment of LC:N145 and the peptide segment of HC:N170: a complex terminal galactosylated glycoform, a complex terminal sialylated glycoform, a complex terminal galactosamine glycoform, and a fucosylated glycoform; (3) The recombinant coagulation factor VII has an N-glycosylation modification, and the recombinant coagulation factor VII has at least one of the following glycoforms in the peptide segment of LC: N145 and the peptide segment of HC: N170: a complex terminal galactosylation glycoform, a complex terminal sialylation glycoform, a complex terminal galactosamine glycoform and a fucosylation glycoform, and the complex terminal sialylation glycoform accounts for more than 50% of the glycoforms in the recombinant coagulation factor VII, preferably more than 60%; (4) The recombinant coagulation factor VII has an N-glycosylation modification, and the recombinant coagulation factor VII has at least one of the following glycoforms in the peptide segment of LC: N145 and the peptide segment of HC: N170: a complex terminal galactosylation glycoform, a complex terminal sialylation glycoform, a complex terminal galactosamine glycoform and a fucosylation glycoform, and the complex terminal galactosamine glycoform accounts for less than 50% of the glycoforms in the recombinant coagulation factor VII, preferably less than 40%.

7. The recombinant coagulation factor VII according to claim 1, characterized in that The recombinant coagulation factor VII is obtained by expressing in a human cell line GEX, a cell line derived from the human cell line GEX, or a cell line homologous to the human cell line GEX.

8. The recombinant coagulation factor VII according to claim 1, characterized in that The recombinant coagulation factor VII contains at least 9 γ-carboxylation modification sites in the 10 glutamic acids (Glu) in its GLA region.

9. An expression vector, characterized in that The expression vector contains the gene of the recombinant coagulation factor VII according to any one of claims 1 to 8, and the expression vector can be expressed in the host cell line GEX and has activity.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the recombinant blood coagulation factor VII according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient or adjuvant.

11. Use of the recombinant coagulation factor VII according to claims 1 to 8, the expression vector according to claim 9 or the pharmaceutical composition according to claim 10 in the treatment of bleeding diseases and hemostasis of trauma.

12. The use according to claim 11, characterized in that: The bleeding disorder includes at least one of hemophilia A, hemophilia B, factor XI deficiency, factor VII deficiency, thrombocytopenia, and von Willebrand disease.

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