Method for analyzing the degree of γ-carboxyglutamate modification of vitamin K-dependent proteins

Abstract

To provide a method for analyzing Gla modification levels in vitamin K dependent protein.SOLUTION: An analytical method at least comprises the steps of: digesting vitamin K dependent protein with chymotrypsin, to cleave a Gla domain, to obtain Gla domain peptides; and sorting the Gla domain peptides by liquid chromatography according to the Gla modification levels. Such an analytical method enables the Gla modification levels to be determined quantitatively and qualitatively.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a method for analyzing the γ-carboxyglutamic acid (Gla) modification degree of vitamin K-dependent proteins such as vitamin K-dependent blood coagulation factors.

Background Art

[0002] Vitamin K is a kind of fat-soluble vitamin and acts as a cofactor for γ-carboxylase, an enzyme that converts glutamic acid residues (Glu) into γ-carboxyglutamic acid residues (Gla). Proteins having such Gla are called vitamin K-dependent proteins. For example, vitamin K-dependent blood coagulation factors such as factor X (FX) of blood coagulation have a Gla domain containing about 10 Gla at their N-terminus. Gla is an amino acid not found in general proteins, but in vitamin K-dependent proteins, Gla is introduced by post-translational modification during the intracellular production process (Non-Patent Document 1).

[0003] This Gla domain has a role of coordinating Ca ions and plays an important role in the physiological function of vitamin K-dependent proteins. Therefore, in order to use vitamin K-dependent proteins as pharmaceuticals, when purifying from human plasma or expressing and purifying as recombinant products, it is important to accurately analyze the Gla modification degree in the Gla domain of vitamin K-dependent proteins, and an analytical method that enables this is required.

[0004] As one such analytical method, for example, since Gla is decomposed into glutamic acid by acid hydrolysis, the total amount of Gla in a protein can be analyzed by alkaline hydrolysis. However, since only the total amount is quantified as the amino acid composition, when the Gla modification degree in the protein is non-uniform, only a numerical value as an average value can be obtained, and information regarding the heterogeneity of the existing molecular species cannot be obtained despite the presence of multiple Gla modification sites.

[0005] Mass spectrometry is another method for analyzing Gla modifications. However, mass spectrometry of Gla-containing peptides and proteins is difficult to perform quantitatively because their negative charge and diversity affect ionization. To overcome this, methylation of Gla carboxyl groups has also been attempted (Non-Patent Literature 2). However, confirming sufficient methylation of each carboxyl group is difficult and therefore not common.

[0006] Furthermore, because Gla is highly negatively charged, there are reports that proteins with different degrees of Gla modification can be separated by anion exchange chromatography (Non-Patent Literature 3). Human blood coagulation factor IX (FIX), a type of vitamin K-dependent blood coagulation factor, contains 12 residues of Gla, and it has been shown that when recombinant FIX protein is subjected to anion exchange chromatography, it separates into peaks according to the degree of Gla modification. Moreover, based on the fact that each peak remains separated even after neuraminidase digestion of recombinant FIX protein, and the results of peptide mapping analysis, it has been reported that while human FIX has 12 Gla residues, this recombinant FIX protein has 10-Gla, 11-Gla, and 12-Gla modified forms (Non-Patent Literature 3).

[0007] However, regarding blood coagulation factor X (FX), it has been reported that in anion exchange chromatography of bovine FX, the two peaks are separated not by differences in Gla modification, but by the presence or absence of sulfate group modification of the tyrosine residue (Tyr) in the activating peptide at the NH2 terminus of the FX heavy chain (Non-Patent Literature 4). Therefore, these reports indicate that not all vitamin K-dependent blood coagulation factors, including Gla, can be separated by Gla modification degree based on the negative charge of Gla using anion exchange chromatography.

[0008] Furthermore, as another analytical method for Gla modifications, site-specific analysis of Gla modifications can be estimated using peptide maps, and for example, an example of analysis of FIX is shown in Non-Patent Document 3. However, when performing site-specific analysis of Gla modifications in human FX, which has 11 Gla modification sites, it is difficult to identify some modification sites from peptide maps alone.

[0009] It has been reported that vitamin K-dependent blood coagulation factors can be digested with chymotrypsin to cleave the Gla domain and prepare vitamin K-dependent blood coagulation factors lacking the Gla domain (Non-Patent Literature 5). In this Non-Patent Literature 5, bovine FX is digested with chymotrypsin to cleave the Tyr44-Lys45 region of bovine FX, and the FX lacking the Gla domain is purified by anion exchange chromatography.

[0010] However, to the best of our knowledge, there have been no reports of using chymotrypsin-cleaved Gla domains to determine the degree of Gla modification in vitamin K-dependent proteins such as vitamin K-dependent blood coagulation factors. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] K. Hansson, et al., Journal of Thrombosis and Haemostasis 2005, 3: 2633-2648 [Non-Patent Document 2] KW Hallgren, et al., J Proteome Res 2013, 12: 2365-2374 [Non-Patent Document 3] S. Gillis, et al., Protein Science 1997, 6: 185-196 [Non-Patent Document 4] T. Morita, et al., J Biol Chem 1986, 261:4008-4014 [Non-Patent Document 5] T. Morita, et al., J Biol Chem 1986, 261:4015-4023 [Non-Patent Document 6] T. Zama, et al., Br J Haematol 1999, 106:809-811 [Non-Patent Document 7] L. Thim, et al., Biochemistry 1988, 27:7785-7793 [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] The present inventors have now discovered that by treating vitamin K-dependent proteins with chymotrypsin and analyzing the resulting Gla domain, it is possible to analyze the detailed degree of Gla modification of vitamin K-dependent proteins, and have also obtained insights into more appropriate analytical conditions. The present invention is based on these findings.

[0013] Therefore, the present invention aims to provide a novel method for analyzing the degree of Gla modification of vitamin K-dependent proteins.

[0014] Furthermore, the method for analyzing the degree of Gla modification of vitamin K-dependent proteins according to the present invention is a method for analyzing the degree of γ-carboxyglutamic acid (Gla) modification of vitamin K-dependent proteins, The process involves digesting the vitamin K-dependent protein with chymotrypsin to cleave the Gla domain and obtain a Gla domain peptide. The process involves separating the Gla domain peptides according to their degree of Gla modification by liquid chromatography. It is characterized by containing at least [a certain element]. [Effects of the Invention]

[0015] According to the method of the present invention, the degree of Gla modification of vitamin K-dependent proteins can be appropriately analyzed.

Brief Description of Drawings

[0016] [Figure 1] Amino acid sequence of the Gla domain of human vitamin K-dependent blood coagulation factors (Fig. 4(A) of Non-Patent Document 1). In the figure, FVII represents blood coagulation factor VII, FIX represents blood coagulation factor IX, FX represents blood coagulation factor X, PT represents blood coagulation factor II (prothrombin), PC represents protein C, PZ represents protein Z, and PS represents protein S. [Figure 2] Reverse phase HPLC chromatograms at a wavelength of 280 nm of the chymotrypsin digest of FX and the blank obtained in Example 1 (A) and an enlarged view of the chromatograms at wavelengths of 214 nm and 280 nm with a retention time of 35 to 40 minutes (B). [Figure 3] Superimposed C4 reverse phase HPLC chromatograms at a wavelength of 214 nm when a calcium salt or a trivalent chromium salt was added to the chymotrypsin-digested Gla domain peptide sample of FX obtained in Example 2. [Figure 4] C18 reverse phase HPLC chromatograms at a wavelength of 214 nm of the Gla domain peptide of human FX and the blank obtained in Example 3. The assignment results of each peak are shown in the figure. [Figure 5] C4 reverse phase HPLC chromatograms at a wavelength of 214 nm of the chymotrypsin digests of human FVIIa and human rFVIIa obtained in Example 4 (A) and an enlarged view of the chromatograms with a retention time of 36 to 40 minutes (B). [Figure 6] Superimposed C18 reverse phase HPLC chromatograms at a wavelength of 214 nm depending on the presence or absence of addition of a trivalent chromium salt to the Gla domain peptide sample of human FVIIa obtained in Example 5.

Modes for Carrying Out the Invention

[0017] Vitamin K-dependent proteins and Gla modification The analytical method according to the present invention targets the degree of Gla modification of vitamin K-dependent proteins. Here, vitamin K-dependent proteins are proteins in which glutamic acid residues (Glu) are converted to γ-carboxyglutamic acid residues (Gla) in a vitamin K-dependent manner. Specific examples include vitamin K-dependent blood coagulation factors such as coagulation factor II, coagulation factor VII, coagulation factor IX, and coagulation factor X; vitamin K-dependent blood coagulation regulatory factors such as protein C or protein S; and osteocalcin and protein Z.

[0018] The vitamin K-dependent proteins targeted by the analytical method according to the present invention have a Gla domain at their NH2 terminus containing approximately 10 γ-carboxyglutamic acid residues (Gla). For example, the amino acid sequence of the Gla domain of a human vitamin K-dependent blood coagulation factor is shown in Figure 1 (Figure 1 is Fig. 4(A) from Non-Patent Literature 1). In Figure 1, Gla is indicated by γ. Common amino acid residues across all blood coagulation factors are shaded. From the figure, it can be seen that the region around 40 residues from the N-terminus is rich in aromatic amino acid residues (Phe(F), Trp(W), and Tyr(Y)) that form the cleavage sites of chymotrypsin.

[0019] This Gla domain plays a role in coordinating Ca ions and is crucial for the physiological function of vitamin K-dependent proteins. Therefore, accurately analyzing the degree of Gla modification in the Gla domain of vitamin K-dependent proteins is important. According to the analytical method of the present invention, peptides with different degrees of Gla modification derived from vitamin K-dependent proteins can be separated by chromatography, and furthermore, their quantification is also possible.

[0020] When vitamin K is deficient, blood coagulation factors are produced in an immature state of Gla modification. The analytical method according to the present invention can, for example, detect the presence of such immature blood coagulation factors. Furthermore, it can determine the degree of modification not only for vitamin K-dependent proteins derived from living organisms but also for vitamin K-dependent proteins obtained through genetic recombination, and can qualitatively and quantitatively detect the presence of unmodified Gla components that are evaluated as impurities.

[0021] Regarding the qualitative and quantitative determination of specific modification levels, the following explanation can be given using vitamin K-dependent blood coagulation factor X (FX) as an example. The N-terminal Gla domain of FX, shown in Figure 1, consists of 45 amino acid residues and contains 11 Gla residues. Generally, it is possible that there exist 11-Gla modified human FX, in which all 11 residues from Gla6 (6th residue) to Gla39 (39th residue) from the NH2 terminus are modified; similarly, 10-Gla modified human FX, in which 10 residues from Gla32 (32nd residue) from the NH2 terminus are modified; and 9-Gla modified human FX, in which 9 residues from Gla29 (29th residue) are modified. On the other hand, a molecular abnormality of the Gla32 site of FX (Gla32Gln) is known as FX Tokyo, and has been reported to cause a significant prolongation of prothrombin time (Non-Patent Literature 6). Therefore, a 9-Gla modified human FX, in which Gla32 is not modified and modifications extend to Gla29, can also be considered an inactive FX. According to the analytical method of the present invention, the presence and abundance of these can be determined.

[0022] Furthermore, the qualitative and quantitative determination of specific modification levels can be explained using blood coagulation factor VII (FVII) as an example. The N-terminal Gla domain of FVII, shown in Figure 1, consists of 45 amino acid residues and contains 10 Gla residues. Generally, it is possible that there are 10Gla modified human FVII, in which all 10 residues from Gla6 (6th residue) to Gla35 (35th residue) from the NH2 terminus are modified, and similarly, 9Gla modified human FVII, in which 9 residues from Gla29 (29th residue) from the NH2 terminus are modified. In recombinant activated human blood coagulation factor VII (rFVIIa), the existence of a 9Gla modified human FVII, in which Gla35 is not modified, has been reported (Non-Patent Literature 7).

[0023] Digestion by chymotrypsin In the analytical method according to the present invention, first, a vitamin K-dependent protein is digested with chymotrypsin. As described above, vitamin K-dependent proteins have a region rich in aromatic amino acid residues (Phe, Trp, and Tyr) that serve as cleavage sites for chymotrypsin around 40 residues from the N-terminus, and in this step, these amino acid residues are used as cleavage sites. A Gla domain peptide is then obtained.

[0024] The chymotrypsin available in this invention may be an enzyme commercially available for biochemical use, and α-chymotrypsin may be used. Furthermore, chymotrypsin treated with a trypsin inhibitor, such as TLCK (tosyl-L-lysyl chloromethyl ketone), may be used to promote substrate-specific cleavage of chymotrypsin.

[0025] In the analytical method according to the present invention, the conditions for digestion of vitamin K-dependent proteins with chymotrypsin are not limited as long as cleavage is possible in the region rich in aromatic amino acid residues (Phe, Trp, and Tyr) around 40 residues from the N-terminus. For example, the weight ratio of vitamin K-dependent protein to chymotrypsin can be set to about 1 / 5 to 1 / 10000, and the reaction can be carried out at a temperature of 10 to 40°C for about 1 to 900 minutes. For example, Non-Patent Literature 5 describes a condition in which FX is cleaved by adding 1 / 700 weight ratio TLCK-treated chymotrypsin and reacting it in 0.05 mol / L Tris-HCl, 0.1 mol / L NaCl, pH 7.5 at 22°C for 50 minutes. However, the method is not limited to this example, and the Gla domain peptide can be cleaved from vitamin K-dependent proteins by using any amount of chymotrypsin under conditions in which chymotrypsin acts.

[0026] Liquid chromatography In the analytical method according to the present invention, the Gla domain peptide obtained by digestion with chymotrypsin is then subjected to liquid chromatography to separate it according to its degree of Gla modification.

[0027] According to a preferred embodiment of the present invention, the separation of the Gla domain peptide is performed by reversed-phase liquid chromatography (HPLC). The column is C1 (methyl), C4 (butyl), C8 (octyl), or C 18 The column is not limited to any reverse-phase chromatography column that has a functional group such as an octadecyl group and can be used for the separation of peptides and proteins. The mobile phase is commonly a system in which trifluoroacetic acid or formic acid is added to water and an organic solvent such as acetonitrile, respectively, and mixed using a linear gradient; however, it is not limited to this system as long as it can separate peptides and proteins. Furthermore, the eluted peptides can be detected and quantified by ultraviolet absorption at wavelengths around 210 nm or 280 nm due to the presence of aromatic amino acids. When quantifying at a wavelength of 280 nm, the extinction coefficients of each component must be considered.

[0028] In the present invention, during separation by liquid chromatography, some peptides may coordinate with metal ions thought to originate from stainless steel components in the high-performance liquid chromatography (HPLC) apparatus. Therefore, according to a preferred embodiment of the present invention, for example, a trivalent chromium salt or the like can be added to the analytical sample that has been pre-digested with chymotrypsin. Alternatively, to avoid coordination with such metal ions, for example, a chelating agent such as ethylenediaminetetraacetic acid (EDTA) salt can be added to the analytical sample that has been pre-digested with chymotrypsin. The amount added is not particularly limited, but according to one embodiment of the present invention, the final concentration of the added trivalent chromium salt is at least 0.25 mol / L, or the final concentration of the added chelating agent is at least 0.005 mol / L. More preferably, the final concentration of the added trivalent chromium salt is 0.25 mol / L to 0.5 mol / L. Furthermore, depending on the analyte, preferred amounts of additives can be found. For example, in the case of chymotrypsin-digested peptides of human blood coagulation factor X (FX), adding chromium(III) chloride (chromium trichloride) to the analytical sample at a final concentration of approximately 0.5 mol / L allows for the separation of Gla domain peptides by reverse-phase chromatography while coordinating chromium ions. Similarly, for example, adding EDTA to human activated blood coagulation factor VII (FVIIa) at a final concentration of approximately 5 mmol / L allows for digestion with chymotrypsin while suppressing the influence of calcium salts that may be present in the sample, and enables the separation of Gla domain peptides by reverse-phase chromatography without coordinating metal ions. Of course, for some peptides, it is also possible to separate Gla domain peptides by reverse-phase chromatography without adding such metal salts or chelating agents to the analytical sample. [Examples]

[0029] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0030] Example 1: (a) Digestion of human blood coagulation factor X (FX) by chymotrypsin 0.3 mg of human blood coagulation factor X (FX), purified from human plasma, was digested with bovine pancreas-derived TLCK-treated α-chymotrypsin (product number C3142, Sigma-Aldrich) in a 0.1 mol / L Tris-HCl, pH 7.5 buffer. The chymotrypsin was added at a weight ratio of 1 / 500 to the FX and reacted at room temperature for 1 hour. The reaction was then stopped by adding an acid (trifluoroacetic acid (TFA)), and this was used as the human FX chymotrypsin digest in the following steps.

[0031] (b) Separation by HPLC Human FX chymotrypsin digests were subjected to reverse-phase HPLC using a C4 column. As a result, FX lacking the Gla domain eluted at a retention time of 42 minutes. Alkaline hydrolysis of the peak fraction revealed γ-carboxyglutamic acid between retention times of 37 and 39 minutes, as indicated by the double arrows (Figure 2A). This peak was fractionated into eight fractions, A1 to D2 (Figure 2B). The analytical conditions for the reverse-phase HPLC were as follows. • Column: 214TP5410, 4.6 mm × 10 cm, manufactured by Vydac ·Mobile phase A: 0.05% TFA ·Mobile phase B: 0.05% TFA, 80% CH3CN [Table 1]

[0032] (c) Peak identification Each peak fraction of Gla-containing peptides was analyzed by flow injection ESI-MS. A single quadrupole ESI-MS instrument, LCMS-2020 (Shimadzu Corporation), was used, and 60% CH3CN was delivered as the mobile phase at a flow rate of 0.3 mL / min to inject the separated peptide samples. Both positive and negative ions were scanned in the m / z range of 300 to 2000. When Gla is modified, an m / z 44 greater than that of a Glu residue is observed for each modification site. Based on the observed m / z, each peak was assigned as shown in Table 2. By chymotrypsin digestion, the human FX Gla domain peptide was cleaved at the COOH terminus of Trp41 or Tyr44. Peak fractions C1, C2, D1, and D2 showed m / z 52 greater than peak fractions A1, A2, B1, and B2, respectively, suggesting the possibility of peptides coordinated with chromium ions. [Table 2]

[0033] Example 2 (a) Analysis of Gla domain peptide of human blood coagulation factor X (FX) 0.5 mg of FX sample was digested with 1 / 500 volume of TLCK-treated α-chymotrypsin in 0.1 mol / L Tris-HCl, pH 7.5 (room temperature, 1 hour). After digestion, 1 / 40 volume of 10% TFA was added to stop the reaction. Then, the Gla domain peptide fraction of human FX was separated by HPLC according to Example 1(b) and collected together.

[0034] (b) Separation by HPLC The Gla domain peptide fraction of human FX was subjected to HPLC analysis in the same manner as in Example 1(b), after adding calcium chloride or chromium(III) chloride to an amount equivalent to 20 or 10 μg of FX.

[0035] The resulting chromatogram is shown in Figure 3. When the human FX Gla domain peptide fraction was analyzed by HPLC with calcium chloride at a final concentration of 100 mmol / L, the peak shape of the human FX Gla domain peptide fraction did not change significantly. Although not shown in the figures, the peak shape did not change when nickel(II) sulfate, magnesium chloride, or zinc chloride was added.

[0036] Next, when chromium(III) chloride was added to the Gla domain peptide fraction of human FX, the A1, A2, B1, and B2 peaks decreased in a concentration-dependent manner, while the C1, C2, D1, and D2 peaks increased. Furthermore, when the same concentration of chromium(III) chloride was added to a relatively small amount of the human FX Gla domain peptide fraction, the C1, C2, D1, and D2 peak content increased further. When a final concentration of 250 mmol / L of chromium(III) chloride was added, the A1, A2, B1, and B2 peaks almost disappeared and were converted into C1, C2, D1, and D2 peaks (Figure 3). Therefore, peaks C1, C2, D1, and D2 were considered to be Gla domain peptides coordinated with chromium ions.

[0037] Furthermore, when comparing the retention time range from peaks A1 to B2 with that of peaks C1 to D2, the latter range of retention times was larger, suggesting that coordinating chromium ions could potentially improve the separation of each peak.

[0038] Example 3 (a) Analysis of the degree of Gla modification in human FX 15 μg of human blood coagulation factor X (FX) was digested in 0.1 mol / L Tris-HCl, pH 7.5 with TLCK-treated α-chymotrypsin at a weight ratio of 1 / 500 for 1 hour at room temperature. Then, 10% TFA was added in an amount of 1 / 40 of the reaction volume to stop the reaction. Next, chromium(III) chloride was added to a final concentration of 0.5 mol / L to the reaction mixture. This sample was then subjected to the following conditions C 18 The sample was subjected to reverse-phase HPLC. • Column: 5C18AR300, 4.6 mm × 25 cm, manufactured by Nacalai Tesuque. ·Mobile phase A: 0.05% TFA ·Mobile phase B: 0.05% TFA, 80% CH3CN

[0039] [Table 3]

[0040] The resulting chromatogram is shown in Figure 4.

[0041] (c) Determination of Gla modification ESI-MS analysis was performed according to Example 1(c), and each peak was assigned as shown in Figure 4. From the peak area ratio results, it was found that human FX mainly consists of 80% 11-Gla modified peptides and 20% 10-Gla modified peptides. This was consistent with the abundance ratio of Gla39-containing peptides and Glu39-containing peptides in the Lys-C peptide map of human FX. It was also found that a 41-residue-length 9-Gla modified peptide eluted at the position indicated by the arrow in Figure 4, but almost no 9-Gla modified peptides were observed in FX derived from human plasma. The generation of two peptides with different peptide lengths, 44-residue and 41-residue, was thought to be due to the amount of chymotrypsin used and the degree of Gla modification. From the above, the degree of Gla modification of human FX could be determined from the peak area ratio of HPLC.

[0042] Example 4 (a) Digestion of human activated blood coagulation factor VII (FVIIa) by chymotrypsin Activated human blood coagulation factor VII (FVIIa) was purified from human plasma (manufactured by KM Biologics, Inc.). Recombinant activated human blood coagulation factor VII (rFVIIa) was purchased from Novo Nordisk. 0.27 mg of FVIIa and 0.16 mg of rFVIIa were each reacted in a buffer of 0.1 mol / L Tris-HCl, 0.005 mol / L EDTA, pH 7.5 with a final concentration of 10 mmol / L of p-amidinophenylmethanesulfonyl fluoride hydrochloride at room temperature for 30 minutes to inhibit the autolysis of FVIIa and rFVIIa. Next, 1 / 500 by weight of TLCK-treated α-chymotrypsin was added and the reaction was allowed to proceed at room temperature for 1 hour. Subsequently, 1 / 40 of the reaction volume of 10% TFA was added to stop the reaction, and these were used as chymotrypsin digests of FVIIa and rFVIIa in the following steps.

[0043] (b) Separation by HPLC The chymotrypsin digests of FVIIa and rFVIIa were subjected to C4 reverse-phase HPLC according to Example 1(b). As a result, FVIIa and rFVIIa lacking the Gla domain were eluted at a retention time of 48 minutes. Alkaline hydrolysis of the peak fraction revealed γ-carboxyglutamic acid between retention times of 37 and 40 minutes, as indicated by the double arrows (Figure 5A). This peak was fractionated into four fractions a1 to b2 (Figure 5B).

[0044] (c) Determination of Gla modification Each peak fraction of the Gla-containing peptide was analyzed by ESI-MS using the flow injection method according to Example 1. Based on the observed m / z, each peak was assigned as shown in Table 4. Upon chymotrypsin digestion, the Gla domain peptides of FVIIa and rFVIIa were cleaved at the COOH terminus of Leu39 or Phe40. It is also well known that chymotrypsin cleaved the COOH terminus of Leu.

[0045] The results of the peak area ratio revealed that FVIIa consists almost entirely of 10Gla modifiers. Furthermore, rFVIIa was thought to consist of approximately 30% 10Gla modifiers and 70% 9Gla modifiers. In recombinant activated human blood coagulation factor VII (rFVIIa), the existence of a 9Gla-modified form lacking Gla35 modification has been reported based on NH2-terminal amino acid sequence analysis, but its abundance has not been quantitatively determined, and it has been indicated as approximately half the amount (Non-Patent Literature 7). Furthermore, in the Lys-C peptide map of FVIIa, peptides containing Gla35 are remarkably hydrophilic, making it impossible to separate these peptides by reverse-phase HPLC, and therefore the degree of Gla35 modification could not be evaluated. However, this method allowed the degree of Gla modification of FVIIa and rFVIIa to be determined from the peak area ratio in HPLC.

[0046] [Table 4]

[0047] Example 5 The Gla domain peptide peaks a1 and b1 of FVIIa were isolated, and chromium(III) chloride was added to a portion of each peak fraction to a final concentration of 0.5 mol / L. The results were then compared according to Example 3, with or without the addition of chromium(III) chloride. 18 The samples were subjected to reverse-phase HPLC. As a result, the Gla domain peaks a1 and b1 of FVIIa were altered by the addition of chromium(III) chloride, respectively. 18 It eluted more slowly on reverse-phase HPLC (Figure 6).

Claims

1. A method for analyzing the degree of γ-carboxyglutamic acid (Gla) modification of vitamin K-dependent proteins, The process involves digesting the vitamin K-dependent protein with chymotrypsin to cleave the Gla domain and obtain a Gla domain peptide. The Gla domain peptide is separated according to its degree of Gla modification by reverse-phase liquid chromatography in the presence of a trivalent chromium salt. An analytical method characterized by comprising at least [a certain element].

2. The analytical method according to claim 1, further comprising adding a chelating agent and separating Gla domain peptides by reverse-phase liquid chromatography according to their degree of Gla modification.

3. The analytical method according to claim 1, wherein the vitamin K-dependent protein is a vitamin K-dependent blood coagulation factor or a vitamin K-dependent blood coagulation regulatory factor.

4. The analytical method according to claim 3, wherein the vitamin K-dependent blood coagulation factor is selected from the group consisting of blood coagulation factor II, blood coagulation factor VII, blood coagulation factor IX, and blood coagulation factor X.

5. The analytical method according to claim 3, wherein the vitamin K-dependent blood coagulation regulatory factor is protein C or protein S.

6. The analytical method according to claim 1, wherein the vitamin K-dependent protein is osteocalcin or protein Z.

7. The analytical method according to claim 1, wherein the vitamin K-dependent protein is an activated form of a vitamin K-dependent blood coagulation factor or a vitamin K-dependent blood coagulation regulatory factor.

8. The analytical method according to claim 1, wherein the final concentration of added trivalent chromium salt is at least 0.25 mol / L.

9. The analytical method according to claim 2, wherein the final concentration of the chelating agent added is at least 0.005 mol / L.

10. The analytical method according to claim 8, wherein the final concentration of added trivalent chromium salt is 0.25 mol / L to 0.5 mol / L.

11. The analytical method according to any one of claims 1 to 10, wherein, when the vitamin K-dependent blood coagulation factor is blood coagulation factor X, the vitamin K-dependent factor is separated into those with 11 Gla modifications, those with 10 Gla modifications, and those with 9 Gla modifications.

12. The analytical method according to claim 11, wherein the Gla-modified substance is 44 residues from the N-terminus of blood coagulation factor X, or 41 residues from the N-terminus thereof.

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