JP-7855344-B2 - Method for analyzing the degree of γ-carboxyglutamate modification of vitamin K-dependent proteins

Inventors

• 森 裕一朗
• 前村 史子

Assignees

• ＫＭバイオロジクス株式会社

Dates

Publication Date

20260508

Application Date

20211217

Claims (12)

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. An analytical method characterized by comprising at least the step of separating the Gla domain peptides according to their degree of Gla modification by reverse-phase liquid chromatography in the presence of a trivalent chromium salt .
2. The analytical method according to claim 1, further comprising adding a chelating agent and separating Gla domain peptides according to their degree of Gla modification by reverse-phase liquid chromatography.
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, it separates factors with 11 Gla modifications, factors with 10 Gla modifications, and factors 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.

Description

This invention relates to a method for analyzing the degree of γ-carboxyglutamic acid (Gla) modification of vitamin K-dependent proteins, such as vitamin K-dependent blood coagulation factors. Vitamin K is a type of fat-soluble vitamin that acts as a cofactor for γ-carboxylase, an enzyme that converts glutamic acid residues (Glu) to γ-carboxyglutamic acid residues (Gla). Proteins containing such Gla are called vitamin K-dependent proteins. For example, vitamin K-dependent blood coagulation factors such as coagulation factor X (FX) have a Gla domain containing approximately 10 Gla at their N-terminus. Although Gla is an amino acid not commonly found in proteins, in vitamin K-dependent proteins, Gla is introduced through post-translational modification during the intracellular production process (Non-Patent Literature 1). The Gla domain plays a role in coordinating Ca ions and is crucial for the physiological function of vitamin K-dependent proteins. Therefore, when purifying vitamin K-dependent proteins from human plasma or expressing and purifying them as recombinants for use as pharmaceuticals, accurately analyzing the degree of Gla modification in the Gla domain of vitamin K-dependent proteins is important, and analytical methods capable of this are needed. One such analytical method involves analyzing the total amount of Gla in a protein by alkaline hydrolysis, since Gla is broken down into glutamic acid by acid hydrolysis. However, this method only quantifies the total amount as an amino acid composition. Therefore, if the degree of Gla modification in the protein is heterogeneous, only an average value is obtained. Furthermore, even if there are multiple Gla modification sites, information about the heterogeneity of the molecular species present is not obtained. Mass spectrometry is another method for analyzing Gla modifications. However, mass spectrometry of Gla-containing peptides and proteins is difficult to perform quantitatively due to the influence of ionization caused by their negative charge and diversity. 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 practice. Furthermore, because Gla is highly negatively charged, there are reports that proteins with different Gla modification levels 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 Gla residues. When recombinant FIX protein is subjected to anion exchange chromatography, it has been shown to separate into peaks according to Gla modification level. Moreover, based on the fact that these peaks remain separated even after neuraminidase digestion of recombinant FIX protein, and the results of peptide mapping analysis, it has been reported that while human FIX contains 12 Gla residues, this recombinant FIX protein has 10-Gla, 11-Gla, and 12-Gla modified forms (Non-Patent Literature 3). However, regarding blood coagulation factor X (FX), it has been reported that anion exchange chromatography of bovine FX separates it into two peaks not based on differences in Gla modification, but on 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. Furthermore, as another analytical method for Gla modifications, site-specific analysis of Gla modifications can be estimated using peptide mapping; for example, an example of FIX analysis 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, identifying some modification sites from peptide mapping alone is difficult. 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, and the resulting FX lacking the Gla domain is purified by anion exchange chromatography. 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. K. Hansson, et al., Journal of Thrombosis and Haemostasis 2005, 3: 2633-2648K.W. Hallgren, et al., J Proteome Res 2013, 12: 2365-2374S. Gillis, et al., Protein Science 1997, 6: 185-196T. Morita, et al., J