A reversed-phase chromatographic method for the purity of enterokinase protein

Abstract

This invention belongs to the field of chromatographic detection and analysis, and discloses a reversed-phase chromatographic analysis method for the purity of enterokinase protein. High-performance liquid chromatography (HPLC) is used to detect enterokinase in the sample solution. In this HPLC method, the chromatographic column is selected from reversed-phase chromatographic packing material C18 or C8, with a particle size of 3.5~5 μm and a pore size of 120~1000 Å. The mobile phase is selected as follows: an aqueous solution of heptafluorobutyric acid (HFA) is used as mobile phase A, and an acetonitrile solution of HFA is used as mobile phase B. This invention improves the separation of the main peak and subsequent impurities in the enterokinase stock solution by screening suitable mobile phase systems and column types. It also optimizes chromatographic parameters such as the proportion of modifier in the mobile phase, column temperature, sample loading, mobile phase gradient, and wavelength, ultimately achieving effective separation of the main peak and subsequent impurities in the enterokinase stock solution. Furthermore, the analysis time is short and the repeatability is good, providing an effective analytical method for the quality control of enterokinase stock solution.

Description

Technical Field

[0001] This invention belongs to the field of chromatographic detection and analysis, specifically relating to a reversed-phase chromatographic analysis method for the purity of enterokinase protein. Background Technology

[0002] Enteropeptidase (EP), a highly specific serine protease, functions by recognizing and cleaving a specific amino acid sequence (Asp-Asp-Asp-Asp-Lys) at the N-terminus of trypsinogen, thereby activating trypsin to initiate the digestive cascade reaction. In recombinant protein drug development, its strict substrate specificity makes it a crucial tool, and the purity of enteropeptidase directly determines the purity, activity, and safety of the drug.

[0003] Generally, enterokinase is prepared via Pichia pastoris fermentation. After activation culture in the seed culture, it is transferred to a fermenter for amplification. During the process, parameters such as temperature, pH, and dissolved oxygen are precisely controlled, and protein expression is induced. After fermentation, the cells are collected, cell walls are broken down, and the target protein is extracted. The protein is then filtered, ion-exchanged, and ultrafiltered to obtain the enterokinase stock solution. The impurities generated in this process mainly include enterokinase self-degradation fragments, misfolded inactive variants, protein aggregates, and products of excessive glycosylation modification. In addition, oxidation and deamidation reactions can also generate corresponding degradation impurities.

[0004] Currently, analytical methods for enterokinase purity mainly focus on electrophoresis and high-performance liquid chromatography (HPLC). Among these, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is cumbersome and time-consuming, and its limited resolution and semi-quantitative nature lead to poor repeatability and accuracy of the detection results. The HPLC method uses a conventional C18 column, but since enterokinase is a basic protein, it easily undergoes non-specific adsorption with the stationary phase of the column, resulting in broadening and severe tailing of the target protein peak, making it impossible to effectively separate enterokinase from other proteins.

[0005] The existing invention CN119064510A uses octadecylsilane-bonded silica gel as a filler and trifluoroacetic acid aqueous solution and trifluoroacetic acid acetonitrile solution as mobile phases to analyze the purity of enterokinase protein. However, this method has poor separation ability of impurity peaks after the main peak of enterokinase, and the single-needle analysis time is as long as 40-80 min. It is questionable whether it can be used to analyze the enterokinase stock solution obtained by Pichia pastoris fermentation.

[0006] Based on the above situation, there is an urgent need to develop a simple, quick, effective separation method with high precision and stability for the purity analysis of enterokinase proteins, so as to meet the quality control requirements in the research and development of recombinant protein drugs and the industrial production of enterokinase. Summary of the Invention

[0007] The purpose of this invention is to overcome at least one deficiency of the prior art and provide a reversed-phase chromatography method for the purity analysis of enterokinase protein.

[0008] The technical solution adopted in this invention is:

[0009] This invention provides a reversed-phase chromatographic analysis method for the purity of enterokinase protein. The method employs high-performance liquid chromatography (HPLC) to detect enterokinase in a sample solution. The HPLC column is selected from reversed-phase chromatographic packing material C18 or C8, with a particle size of 3.5–5 μm and a pore size of 120–1000 Å. The mobile phase in the HPLC method is selected as follows: an aqueous solution of heptafluorobutyric acid (HFA) is used as mobile phase A, and an acetonitrile solution of HFA is used as mobile phase B.

[0010] In some embodiments, the volume percentage of heptafluorobutyric acid in the aqueous solution of heptafluorobutyric acid is 0.05% to 0.1%.

[0011] In some embodiments, the volume percentage of heptafluorobutyric acid in the acetonitrile solution of heptafluorobutyric acid is 0.05% to 0.1%.

[0012] In some embodiments, the elution gradient used in the high-performance liquid chromatography is:

[0013] At the initial 0 min, the volume of mobile phase A was 85 ± 0.05%, and the volume of mobile phase B was 15 ± 0.05%.

[0014] At 15±0.01 min, the volume of mobile phase A was 60±0.05%, and the volume of mobile phase B was 40±0.05%.

[0015] At 30±0.01 min, the volume of mobile phase A was 48±0.05%, and the volume of mobile phase B was 52±0.05%.

[0016] At 35±0.01 min, the volume of mobile phase A was 25±0.05%, and the volume of mobile phase B was 75±0.05%.

[0017] At 37±0.01 min, the volume of mobile phase A was 25±0.05%, and the volume of mobile phase B was 75±0.05%.

[0018] At 37.1 ± 0.01 min, the volume of mobile phase A was 85 ± 0.05%, and the volume of mobile phase B was 15 ± 0.05%.

[0019] At 43±0.01 min, the volume of mobile phase A was 85±0.05% and the volume of mobile phase B was 15±0.05%.

[0020] In some embodiments, the elution gradient used in the high-performance liquid chromatography is:

[0021] At the initial stage (0 ± 0.01 min), the volume of mobile phase A was 85 ± 0.05%, and the volume of mobile phase B was 15 ± 0.05%.

[0022] At 15±0.01 min, the volume of mobile phase A was 57±0.05%, and the volume of mobile phase B was 43±0.05%.

[0023] At 30±0.01 min, the volume of mobile phase A was 50±0.05%, and the volume of mobile phase B was 50±0.05%.

[0024] At 35±0.01 min, the volume of mobile phase A was 25±0.05%, and the volume of mobile phase B was 75±0.05%.

[0025] At 35.1 ± 0.01 min, the volume of mobile phase A was 85 ± 0.05%, and the volume of mobile phase B was 15 ± 0.05%.

[0026] At 40±0.01 min, the volume of mobile phase A was 85±0.05% and the volume of mobile phase B was 15±0.05%.

[0027] In some embodiments, the injection volume of the sample solution is 10~30 μL.

[0028] In some embodiments, the column temperature in the high-performance liquid chromatography is 35~55 °C.

[0029] In some embodiments, the column length in the high-performance liquid chromatography is 50-250 mm.

[0030] In some embodiments, the detection wavelength in the high-performance liquid chromatography is 210~214 nm.

[0031] In some embodiments, the chromatographic column used in the high-performance liquid chromatography is selected from reversed-phase chromatography packing material C8.

[0032] The beneficial effects of this invention are:

[0033] This invention improves the separation of impurities following the main peak of enterokinase by screening suitable mobile phase systems and column types.

[0034] This invention also optimizes chromatographic parameters such as the proportion of modifier in the mobile phase, column temperature, sample loading, mobile phase gradient, and wavelength, ultimately achieving effective separation of the main peak and subsequent impurities in the enterokinase stock solution. At the same time, the analysis time is short and the repeatability is good, providing an effective analytical method for the quality control of enterokinase stock solution. Attached Figure Description

[0035] Figure 1 The chromatogram is for the purity analysis of the enterokinase stock solution in Example 1.

[0036] Figure 2 The chromatogram for purity analysis of enterokinase stock solution in Example 2 is shown.

[0037] Figure 3 The chromatogram for purity analysis of enterokinase stock solution in Example 3 is shown.

[0038] Figure 4 The chromatogram for purity analysis of enterokinase stock solution in Example 4 is shown.

[0039] Figure 5 The chromatogram for purity analysis of enterokinase stock solution in Example 5 is shown.

[0040] Figure 6 The chromatogram for purity analysis of enterokinase stock solution in Example 6 is shown.

[0041] Figure 7 This is a chromatogram for the specificity investigation of the purity method of enterokinase stock solution in Example 7.

[0042] Figure 8 The graph is a linear curve for the purity test of enterokinase stock solution in Example 7. Detailed Implementation

[0043] The following disclosure provides many different implementations or examples for different ways of implementing the present invention.

[0044] Although heptafluorobutyric acid, pentafluoropropionic acid, and trifluoroacetic acid all function as ion-pairing reagents in reversed-phase chromatography, increasing retention on the column by binding their negatively charged carboxyl groups to positively charged peptide residues (such as lysine and arginine residues) to form electrically neutral hydrophobic complexes, their retention capabilities differ due to variations in the length of their fluorocarbon chains. Heptafluorobutyric acid, with its longer chain, is significantly more hydrophobic, meaning its ion-pairing complex is more lipophilic and binds more firmly to the column.

[0045] Unless otherwise specified, all reagents and consumables used in this invention are commercially available. The enterokinase stock solution used in the embodiments and comparative examples of this invention was obtained by Pichia pastoris fermentation.

[0046] Example 1

[0047] This embodiment explores the specific parameters for separation using reversed-phase chromatography packing material C8. Enterokinase stock solution was analyzed according to the detection parameters in Table 1 below, and the detection chromatograms are shown in the table. Figure 1 .Depend on Figure 1It can be seen that the separation degree R between the enterokinase main peak and the subsequent impurities is about 0.91. At the same time, the hydrophilic impurity peak before the main peak is not embedded in the solvent peak, resulting in the best overall separation effect.

[0048] Table 1

[0049]

[0050] Example 2

[0051] This embodiment explores the specific parameters for separation using reversed-phase chromatography with C18 packing material. An Agilent ZORBAX 300SB-C18 column (3.5 μm particle size, 4.6 × 150 mm length) was used. Enterokinase stock solution was injected and analyzed according to the detection parameters in Table 2 below. The detection chromatograms are shown below. Figure 2 Compared with Example 1 and Figure 1 In comparison, the separation degree between the enterokinase main peak and the subsequent impurities was 0.97, indicating that the column efficiency of the two columns was comparable, but the hydrophilic impurities were embedded, and the elution gradient parameters need to be further optimized.

[0052] Table 2

[0053]

[0054] Example 3

[0055] Compared with Example 1, this embodiment adjusted the elution gradient and analyzed the enterokinase stock solution according to the detection parameters in Table 3 below. The detection chromatogram is shown in [Table 3]. Figure 3 The resolution R between the main peak and the subsequent impurities of enterokinase was 1.00, but the peak height of the main peak was about 690 mAU under the same sample loading, which was lower than 825 mAU in Example 1, making it unsuitable for the detection of low-concentration samples.

[0056] Table 3

[0057]

[0058] Example 4

[0059] Compared with Example 1, the injection volume in this embodiment was adjusted to 30 μl. The enterokinase stock solution was analyzed according to the detection parameters in Table 4 below, and the detection chromatogram is shown in [Table 4]. Figure 4 .contrast Figure 1 It can be seen that the separation degree between the main peak of enterokinase and the subsequent impurities is slightly reduced, with a separation degree R of 0.85.

[0060] Table 4

[0061]

[0062] Example 5

[0063] In this embodiment, 0.1% pentafluoropropionic acid-water solution and 0.1% pentafluoropropionic acid-acetonitrile system were used as mobile phases. Enterokinase stock solution was analyzed according to the parameters in Table 5 below. The detection chromatograms are shown below. Figure 5 Compared with Example 1 and Figure 1 In contrast, the main peak of enterokinase and the subsequent impurities showed a shoulder-like peak shape, indicating poor separation, and the 0.1% pentafluoropropionic acid-aqueous solution and 0.1% pentafluoropropionic acid-acetonitrile systems were not applicable.

[0064] Table 5

[0065]

[0066] Example 6

[0067] In this embodiment, 0.1% trifluoroacetic acid-water solution and 0.1% trifluoroacetic acid-acetonitrile system were used as mobile phases. Enterokinase stock solution was analyzed according to the detection parameters in Table 6 below. The detection chromatograms are shown in the table below. Figure 6 Compared with Example 1 and Figure 1 In contrast, the enterokinase residue was completely embedded in the main peak without separation, indicating that the 0.1% trifluoroacetic acid-water solution and 0.1% trifluoroacetic acid-acetonitrile systems are not suitable.

[0068] Table 6

[0069]

[0070] Example 7

[0071] The enterokinase stock solution and blank buffer were injected and tested using the same method and operating parameters as in Example 1, and the following experiments were conducted.

[0072] (1) Examination of method specificity

[0073] Depend on Figure 7 It can be seen that the blank buffer solution does not interfere with the elution position of enterokinase, indicating that the method specificity meets the requirements.

[0074] (2) Method repeatability test

[0075] Two samples were taken from each of the three batches of enterokinase stock solution and analyzed using the method of this invention. The retention time, purity, peak area, and peak height of the main peak of each batch of enterokinase samples were statistically analyzed, and the results are shown in Table 7. As can be seen from the results in Table 7, the RSD% values ​​of retention time, purity, peak area, and peak height of all six samples are less than 2.0%, indicating that the repeatability of the method meets the requirements.

[0076] Table 7 Summary of Repeatability Test Results

[0077]

[0078] (3) Method linearity examination

[0079] Samples with concentrations of 0.5 mg / ml, 0.7 mg / ml, 0.97 mg / ml, 1.3 mg / ml, and 1.5 mg / ml were prepared from the enterokinase stock solution. Three replicates were prepared for each concentration. The method of this invention was used for sample injection and detection. The purity and peak area of ​​the main peak of each batch of enterokinase samples were statistically analyzed. A linear fit was performed with concentration as the x-axis and peak area as the y-axis. The results are shown in Table 8. Figure 8 linear correlation coefficient R 2 The value is 0.9997, indicating good linearity.

[0080] Table 8 Summary of linearity study results for enterokinase

[0081]

[0082] (4) Examination of method accuracy

[0083] Enterokinase stock solution was used to prepare samples at concentrations of 0.5 mg / ml, 0.7 mg / ml, 0.97 mg / ml, 1.3 mg / ml, and 1.5 mg / ml, with three replicates for each concentration. The method of this invention was used for injection and detection, and the purity of the main peak of each batch of enterokinase samples was statistically analyzed. The recovery rate of the main peak purity at the 0.97 mg / ml concentration was taken as 100%. The recovery rate was calculated by dividing the purity of each concentration of enterokinase sample by the purity of the main peak at the 0.97 mg / ml concentration. The results are shown in Table 9. Table 9 shows that within the concentration range of 0.5–1.5 mg / ml, the recovery rate of the main peak purity of the enterokinase stock solution is between 98% and 104%, with an RSD% value not exceeding 2.0%, which meets the requirements.

[0084] Table 9 Summary of the accuracy test results for enterokinase

[0085]

[0086] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A reversed-phase chromatographic method for the purity analysis of enterokinase protein, characterized in that, High performance liquid chromatography was used to detect enterokinase in the sample solution. The chromatographic column used in the high performance liquid chromatography method is selected from reversed-phase chromatography packing material C18 or C8, the particle size of the chromatographic column is 3.5~5 μm, and the pore size of the chromatographic column is 120~1000 Å. The mobile phase in the high-performance liquid chromatography method is selected as follows: an aqueous solution of heptafluorobutyric acid is used as mobile phase A, and an acetonitrile solution of heptafluorobutyric acid is used as mobile phase B.

2. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The volume percentage of heptafluorobutyric acid in the aqueous solution of the heptafluorobutyric acid is 0.05% to 0.1%.

3. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The volume percentage of heptafluorobutyric acid in the acetonitrile solution of heptafluorobutyric acid is 0.05% to 0.1%.

4. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The elution gradient used in the high-performance liquid chromatography method is: At the initial 0 min, the volume of mobile phase A was 85 ± 0.05%, and the volume of mobile phase B was 15 ± 0.05%. At 15±0.01 min, the volume of mobile phase A was 60±0.05%, and the volume of mobile phase B was 40±0.05%. At 30±0.01 min, the volume of mobile phase A was 48±0.05%, and the volume of mobile phase B was 52±0.05%. At 35±0.01 min, the volume of mobile phase A was 25±0.05%, and the volume of mobile phase B was 75±0.05%. At 37±0.01 min, the volume of mobile phase A was 25±0.05%, and the volume of mobile phase B was 75±0.05%. At 37.1 ± 0.01 min, the volume of mobile phase A was 85 ± 0.05%, and the volume of mobile phase B was 15 ± 0.05%. At 43±0.01 min, the volume of mobile phase A was 85±0.05% and the volume of mobile phase B was 15±0.05%.

5. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The elution gradient used in the high-performance liquid chromatography method is: At the initial stage (0 ± 0.01 min), the volume of mobile phase A was 85 ± 0.05%, and the volume of mobile phase B was 15 ± 0.05%. At 15±0.01 min, the volume of mobile phase A was 57±0.05%, and the volume of mobile phase B was 43±0.05%. At 30±0.01 min, the volume of mobile phase A was 50±0.05%, and the volume of mobile phase B was 50±0.05%. At 35±0.01 min, the volume of mobile phase A was 25±0.05%, and the volume of mobile phase B was 75±0.05%. At 35.1 ± 0.01 min, the volume of mobile phase A was 85 ± 0.05%, and the volume of mobile phase B was 15 ± 0.05%. At 40±0.01 min, the volume of mobile phase A was 85±0.05% and the volume of mobile phase B was 15±0.05%.

6. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The injection volume of the sample solution is 10~30 μL.

7. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The column temperature in the high-performance liquid chromatography method is 35~55 ℃.

8. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The column length in the high-performance liquid chromatography method is 50~250 mm.

9. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The detection wavelength in the high-performance liquid chromatography method is 210~214 nm.

10. The reversed-phase chromatographic analysis method according to claim 1, characterized in that, The chromatographic column used in the high-performance liquid chromatography method is selected from reversed-phase chromatography packing material C8.

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