An analysis method for determining concentration of histidine-tagged protein based on IMAC-HPLC and application thereof

Abstract

The application provides an analysis method for determining histidine tag protein concentration based on IMAC-HPLC and application thereof, and the analysis method comprises the following steps: (1) preparing reference solution, quality control solution and sample solution to be detected, and performing IMAC-HPLC detection; (2) taking the sample amount of the reference solution as the abscissa X and the peak area of the histidine tag protein as the ordinate Y, fitting a linear equation to obtain a standard curve; (3) bringing the peak area of the histidine tag protein in the quality control solution into the standard curve to calculate the measured sample amount of the histidine tag protein in the quality control solution, and the ratio of the measured sample amount to the theoretical sample amount is the recovery rate; and bringing the peak area of the histidine tag protein in the sample solution to be detected into the standard curve to calculate the measured sample amount of the histidine tag protein in the sample to be detected, and the ratio of the measured sample amount to the sample injection volume is the histidine tag protein concentration.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical analysis, specifically relating to an analytical method for determining the concentration of histidine-tagged proteins based on IMAC-HPLC and its application. Background Technology

[0002] The production process of biopharmaceutical bulk solutions is generally divided into upstream processes, primarily based on cell culture, and downstream processes, mainly involving multi-step purification steps. Upstream process research focuses on the impact of operating conditions on cell growth characteristics, metabolic levels, and the target product; however, it cannot provide timely information on the precise content of the target protein. Following cell lysis and lysate harvesting, the solution is clarified and centrifuged before proceeding to the downstream purification process. Downstream processes primarily rely on a combination of various chromatographic methods to remove and control product-related or process-related impurities.

[0003] To separate a small amount of target protein from a large pool of proteins, researchers often attach purification tags to the target protein, facilitating its harvesting and enrichment through chromatography. Histidine tags are the simplest and most widely used, typically consisting of 6-10 consecutive histidine residues. They can form coordinate bonds with immobilized transition metal ions. This property allows for the purification of histidine-tagged proteins; during chromatography, proteins separate due to their different affinities to the metal ions. Chromatographic methods, including affinity chromatography, require precise control of the protein loading volume. Underloading or overloading can lead to sample loss or increased impurities; therefore, loading control is crucial for the chromatographic process. Common loading methods in production include volumetric loading and mass loading. Volumetric loading involves controlling the loading volume, but in scale-up production, the target protein concentration may differ significantly from that in small-scale trials, making volumetric loading prone to underloading or overloading. Mass loading avoids this problem, but requires precise knowledge of the target protein concentration before loading. For complex samples such as cell lysates, it is difficult to accurately determine the concentration of the target protein containing histidine tags due to the interference of numerous other proteins.

[0004] In conclusion, it is of great significance to seek a simple, reliable, and easy-to-operate analytical method to accurately and rapidly guide the affinity chromatography loading of complex samples such as cell lysates in the research and development and production of commercial proteins. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an analytical method for determining the concentration of histidine-tagged proteins based on IMAC-HPLC and its application. This analytical method can accurately quantify histidine-tagged proteins, providing rapid guidance for process development and production.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an analytical method for determining the concentration of histidine-tagged proteins based on IMAC-HPLC, the analytical method comprising:

[0008] (1) Prepare reference solution, quality control solution and test sample solution, and perform IMAC-HPLC detection;

[0009] (2) Using the amount of the reference solution loaded as the abscissa X and the peak area of ​​the histidine-tagged protein as the ordinate Y, a linear equation was fitted to obtain the standard curve.

[0010] (3) Substitute the peak area of ​​histidine-tagged protein in the quality control solution into the standard curve to calculate the measured amount of histidine-tagged protein in the quality control solution. The ratio of the measured amount of histidine-tagged protein to the theoretical amount of histidine-tagged protein is the recovery rate. Substitute the peak area of ​​histidine-tagged protein in the test sample solution into the standard curve to calculate the measured amount of histidine-tagged protein in the test sample. The ratio of the measured amount of histidine-tagged protein to the sample injection volume is the concentration of histidine-tagged protein.

[0011] This invention is the first to combine immobilized metal ion affinity chromatography (IMAC) with HPLC technology to guide the research and development and production of commercial proteins. It can accurately and quickly guide the research and development and production of commercial proteins, including but not limited to cell lysate affinity chromatography loading, reduce the occurrence of target protein overload or underload, and greatly save research and development and production costs.

[0012] Preferably, in step (1), the reference solution and the quality control solution are prepared by the following steps:

[0013] Dissolve a histidine-tagged protein reference with an extinction coefficient greater than 0.6 in water to prepare a reference solution or quality control solution of 1.0 ± 0.2 mg / mL, such as 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, or 1.2 mg / mL.

[0014] Dissolve a histidine-tagged protein reference with an extinction coefficient less than 0.6 in water to prepare a 7.0±0.2 mg / mL reference solution or quality control solution, such as 6.8 mg / mL, 6.9 mg / mL, 7.0 mg / mL, 7.1 mg / mL, or 7.2 mg / mL.

[0015] Preferably, the solvent is selected from water or a dilution buffer, wherein the dilution buffer is 15-25mM HEPES + 450-550mM NaCl, and the pH is 7.3-7.7.

[0016] Among them, "15-25mM HEPES" can be, for example, 15mM, 17mM, 19mM, 20mM, 21mM, 23mM or 25mM, etc.

[0017] "450-550mM NaCl" can be, for example, 450mM, 470mM, 490mM, 500mM, 510mM, 530mM, or 550mM.

[0018] "pH is 7.3-7.7" can be, for example, 7.3, 7.4, 7.5, 7.6 or 7.7, etc.

[0019] In this invention, an ultraviolet detector is used. When the extinction coefficient k of the histidine tag protein is ≥0.6, the histidine tag protein exhibits good linearity in the range of 5-50 μg; when the extinction coefficient k of the histidine tag protein is <0.6, the histidine tag protein exhibits good linearity in the range of 35-350 μg.

[0020] In this invention, if the sample concentration is below the lower limit of quantitation, an appropriate ultrafiltration centrifuge tube and centrifugation conditions are selected based on the properties of the target protein to perform concentration pretreatment on the sample. If the sample concentration is above the upper limit of quantitation, the sample is diluted to the quantitation range using dilution buffer or ultrapure water.

[0021] Preferably, in step (1), the amount of reference solution loaded is controlled by changing the injection volume of the reference solution in the IMAC-HPLC detection.

[0022] Preferably, in step (1), the chromatographic column used for IMAC-HPLC detection is filled with Ni-chelated material. 2+ The preferred chromatographic column packing material is the Polar MC30-Ni Excel column.

[0023] The packing material structure of the chromatographic column is shown below:

[0024]

[0025] In this invention, immobilized metal ion affinity chromatography is used for the quantitative detection of target proteins carrying histidine tags. Immobilized metal ion affinity chromatography involves chelating transition metal ions onto a solid matrix using ligands. These positively charged transition metal ions attract the locally negatively charged histidine tags, thus specifically binding to the target protein to form a relatively stable complex. Imidazole is then used for competitive elution to enrich, separate, and analyze the target protein; the elution order is related to the binding affinity of the target protein. Zn is a commonly used transition metal ion for the separation, purification, and analysis of histidine-tagged proteins. 2+ Ni 2+ Cu 2+Co 2+ The key consumable material used in this invention is the chromatographic column packing material, which is chelated Ni. 2+ Polar MC-IMAC Excel is a metal affinity chromatography packing material with polymethacrylate microspheres as the matrix, a particle size of 30 μm, and a pore size of [missing information]. It exhibits high physical and chemical stability. In addition, Polar MC-IMACExcel is highly hydrophilic, which can minimize non-specific adsorption to biological samples, making it very suitable for enriching, separating, and analyzing histidine-tagged proteins.

[0026] Preferably, in step (1), the IMAC-HPLC detection uses a gradient elution method to separate the sample.

[0027] Preferably, in step (1), the mobile phase detected by IMAC-HPLC consists of mobile phase A and mobile phase B, wherein mobile phase A is 15-25mM HEPES + 450-550mM NaCl; and mobile phase B is 15-25mM HEPES + 450-550mM NaCl + 450-550mM imidazole.

[0028] Among them, "15-25mM HEPES" can be, for example, 15mM, 17mM, 19mM, 20mM, 21mM, 23mM or 25mM, etc.

[0029] "450-550mM NaCl" can be, for example, 450mM, 470mM, 490mM, 500mM, 510mM, 530mM, or 550mM.

[0030] "450-550mM imidazole" can be, for example, 450mM, 470mM, 490mM, 500mM, 510mM, 530mM or 550mM, etc.

[0031] Preferably, the pH of the mobile phase A is 7.3-7.7, for example, it can be 7.3, 7.4, 7.5, 7.6 or 7.7.

[0032] Preferably, the pH of the mobile phase B is 7.3-7.7, for example, it can be 7.3, 7.4, 7.5, 7.6 or 7.7.

[0033] Preferably, in step (1), the flow rate of the IMAC-HPLC detection is 0.8±0.2 mL / min, for example, it can be 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1.0 mL / min, etc.

[0034] Preferably, in step (1), the gradient elution procedure for IMAC-HPLC detection is as follows:

[0035] 0-4 min: Mobile phase A is 100%, Mobile phase B is 0%;

[0036] 4-7 min; Mobile phase A is 0-100%; Mobile phase B is 0-100%;

[0037] 7-9 min; Mobile phase A is 0%; Mobile phase B is 100%.

[0038] 9-12 min; Mobile phase A is 0-100%; Mobile phase B is 0-100%;

[0039] 12-30 min, mobile phase A is 100%, mobile phase B is 0%.

[0040] Preferably, in step (1), the gradient elution procedure for IMAC-HPLC detection is as follows:

[0041] At 0 min, mobile phase A is 100% and mobile phase B is 0%.

[0042] 4 min; Mobile phase A is 100%; Mobile phase B is 0%.

[0043] 7 min: Mobile phase A is 0%, Mobile phase B is 100%;

[0044] At 9 min, mobile phase A was 0% and mobile phase B was 100%.

[0045] 12 min; Mobile phase A is 100%; Mobile phase B is 0%.

[0046] After 30 minutes, mobile phase A was 100% and mobile phase B was 0%.

[0047] Under the gradient elution procedure of this invention, histidine-tagged target proteins can be well separated from other contaminating proteins, which is beneficial for accurate quantification of the target protein. In addition, the gradient elution time is moderate, which can analyze complex samples in a short time and quickly guide the development and production of commercial proteins, such as affinity chromatography loading of cell lysate.

[0048] Preferably, in step (1), the column temperature for IMAC-HPLC detection is set to 25±5℃, for example, it can be 20℃, 22℃, 24℃, 25℃, 26℃, 28℃ or 30℃, etc.

[0049] As a preferred embodiment of the present invention, the analysis method includes:

[0050] (1) Prepare reference solution, quality control solution and test sample solution, and perform IMAC-HPLC detection;

[0051] Histidine-tagged protein references with an extinction coefficient greater than 0.6 were dissolved in a solvent to prepare a 1.0±0.2 mg / mL reference solution or quality control solution.

[0052] For histidine-tagged proteins with an extinction coefficient k ≥ 0.6 at 280 nm, the injection volumes of the reference solution are 5 μL, 10 μL, 20 μL, 30 μL and 50 μL, respectively; the corresponding loading amounts at each point are 5 μg, 10 μg, 20 μg, 30 μg and 50 μg, respectively.

[0053] Histidine-tagged protein references with an extinction coefficient less than 0.6 were dissolved in a solvent to prepare a 7.0±0.2 mg / mL reference solution or quality control solution.

[0054] For histidine-tagged proteins with an extinction coefficient k < 0.6 at 280 nm, the injection volumes of the reference solution were 5 μL, 10 μL, 20 μL, 30 μL and 50 μL, respectively; the corresponding loading amounts at each point were 35 μg, 70 μg, 140 μg, 210 μg and 350 μg, respectively.

[0055] Chromatographic conditions for IMAC-HPLC detection:

[0056] An Agilent 1260 Infinity II liquid chromatography system was used, employing a Sepax Technologies Polar MC30-Ni Excel column with dimensions of 7.8 mm × 250 mm and a diameter of 30 μm. The column temperature was 25±5℃; the method flow rate was 0.8±0.2 mL / min; mobile phases A and B were 15-25 mM HEPES + 450-550 mM NaCl, pH 7.3-7.7 and 15-25 mM HEPES + 450-550 mM NaCl + 450-550 mM imidazole, pH 7.3-7.7, respectively; the default injection volume was 20 μL, and if not suitable for the sample, the injection volume should be adjusted accordingly; the detector wavelength was 280 nm; and the injection plate temperature was 5±3℃.

[0057] The gradient elution procedure for IMAC-HPLC detection is as follows:

[0058]

[0059] (2) Using the amount of the reference solution loaded as the abscissa X and the peak area of ​​the histidine-tagged protein as the ordinate Y, a linear equation was fitted to obtain the standard curve.

[0060] (3) The peak area of ​​histidine-tagged protein in the quality control solution is substituted into the standard curve to calculate the measured amount of histidine-tagged protein in the quality control solution. The ratio of the measured amount to the theoretical amount is the recovery rate. The peak area of ​​histidine-tagged protein in the test sample solution is substituted into the standard curve to calculate the measured amount of histidine-tagged protein in the test sample. The ratio of the measured amount to the sample injection volume is the concentration of histidine-tagged protein.

[0061] The recovery rate of quality control samples is calculated using the following formula:

[0062]

[0063] The concentration of histidine-tagged proteins is calculated using the following formula:

[0064]

[0065] Secondly, the present invention provides the application of the analytical method for determining the concentration of histidine-tagged proteins based on IMAC-HPLC described in the first aspect in the protein process development and / or production process.

[0066] Compared with existing methods that rely on column volume and experience to guide protein process development and production, this invention is convenient to operate, highly specific, stable and reliable, and is not affected by personnel or instruments. It has significant application value in protein process development and / or production.

[0067] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of brevity, this invention will not exhaustively list the specific point values ​​included in the range.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] (1) This invention is the first to combine immobilized metal ion affinity chromatography (IMAC) with HPLC technology to guide the research and development and production of commercial proteins. It can accurately and quickly guide the process of research and development and production of commercial proteins, including but not limited to cell lysis buffer affinity chromatography loading, reduce the occurrence of target protein overload or underload, and greatly save research and development and production costs.

[0070] (2) Compared with existing methods that guide protein process development and production by column volume and experience, this invention is convenient to operate, highly specific, stable and reliable, and is not affected by personnel and instruments.

[0071] (3) The present invention has a wide range of applications and can be used for the accurate quantification of most histidine-tagged proteins with extinction coefficient (k≥0.22). Attached Figure Description

[0072] Figure 1 This is the original IMAC-HPLC chromatogram of the blank control solution (mobile phase A) in Example 1.

[0073] Figure 2 This is an enlarged IMAC-HPLC chromatogram of the blank control solution (mobile phase A) in Example 1.

[0074] Figure 3 This is an overlay of the original chromatogram of the histidine-tagged protein reference sample from Example 1.

[0075] Figure 4 This is an enlarged overlay of the chromatogram of the histidine-tagged protein reference in Example 1.

[0076] Figure 5 This is the standard curve of the histidine-tagged protein reference in Example 1.

[0077] Figure 6 This is the original chromatogram of the histidine-tagged protein control sample from Example 1.

[0078] Figure 7 This is the original IMAC-HPLC chromatogram of sample 1 from Example 1.

[0079] Figure 8 This is an overlay of the original chromatogram of the histidine-tagged protein reference sample from Example 2.

[0080] Figure 9 This is an enlarged overlay of the chromatogram of the histidine-tagged protein reference in Example 2.

[0081] Figure 10 This is the standard curve of the histidine-tagged protein reference in Example 2.

[0082] Figure 11 This is the original chromatogram of the histidine-tagged protein control sample from Example 2.

[0083] Figure 12 This is the original IMAC-HPLC chromatogram of sample 2 from Example 2.

[0084] Figure 13 This is an overlay of the original chromatogram of the histidine-tagged protein reference sample from Example 3.

[0085] Figure 14 This is an enlarged overlay of the chromatogram of the histidine-tagged protein reference in Example 3.

[0086] Figure 15 This is the standard curve of the histidine-tagged protein reference in Example 3.

[0087] Figure 16 This is the original chromatogram of the histidine-tagged protein control sample from Example 3.

[0088] Figure 17 This is the original chromatogram of the cell lysis buffer sample from Example 3.

[0089] Figure 18 This is an enlarged chromatogram of the cell lysis buffer sample from Example 3. Detailed Implementation

[0090] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0091] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0092] Example 1

[0093] This embodiment provides an analytical method for determining the concentration of histidine-tagged proteins in a sample based on IMAC-HPLC. The specific steps are as follows:

[0094] A. Preparation of standard curve solution and quality control solution

[0095] Histidine-tagged protein (His-tag) was a lyophilized powder with an extinction coefficient of 1.35, greater than 0.6. A 1.0 mg / mL reference solution was prepared using ultrapure water as a diluent. Two replicates were prepared, one for the standard curve solution and the other for the quality control solution. The quality control solution was injected in 20 μL. By varying the injection volume of the reference solution, the Histidine-tagged protein loading amounts at each point on the standard curve were 5 μg, 10 μg, 20 μg, 30 μg, and 50 μg. The injection volumes of the standard curve solution and the loading amounts at each point are shown in Table 1.

[0096] Table 1

[0097] name Injection volume (μL) Sample loading amount (μg) STD-1 5 5 STD-2 10 10 STD-3 20 20 STD-4 30 30 STD-5 50 50

[0098] B. Sample solution preparation

[0099] Transfer a sufficient amount of sample solution into the injection bottle and wait for injection.

[0100] C. Preparation of blank solution

[0101] Transfer a sufficient amount of mobile phase A into the injection vial and wait for injection.

[0102] D. IMAC-HPLC detection method

[0103] (1) Mobile phase A is 20mM HEPES + 0.5M NaCl (pH 7.5), and mobile phase B is 20mM HEPES + 0.5M NaCl + 0.5M imidazole (pH 7.5).

[0104] (2) Chromatographic conditions: Agilent 1260 Infinity II liquid chromatography system; Sepax Technologies Polar MC30-Ni Excel column, column dimensions 7.8 mm × 250 mm, 30 μm. The column temperature was 25±5℃; the flow rate was 0.8mL / min; the detector wavelength was 280nm; the injection plate temperature was 5±3℃; and the default injection volume was 20μL.

[0105] (3) The gradient elution procedure is as follows:

[0106]

[0107] E. Establishment of Standard Curve

[0108] The standard curve is obtained by fitting a linear equation with the loading amount of the reference histidine-tagged protein as the x-axis and the peak area as the y-axis.

[0109] F. Calculation of Quality Control Product Recovery Rate

[0110] By substituting the peak area of ​​the histidine-tagged protein in the quality control sample into the standard curve equation, the measured loading amount of the histidine-tagged protein in the quality control sample is calculated, and the recovery rate of the quality control sample is calculated according to the following formula.

[0111]

[0112] G. Calculation of histidine-tagged protein concentration in samples

[0113] Substitute the peak area of ​​the histidine-tagged protein in the sample into the standard curve equation to obtain the loading amount of histidine-tagged protein in the sample, and calculate the concentration of histidine-tagged protein according to the following formula.

[0114]

[0115] The test results are as follows:

[0116] (1) The IMAC-HPLC chromatogram of the blank control solution is shown below. Figures 1-2 As shown. Among them Figure 1 This is the original IMAC-HPLC chromatogram of the blank control solution of this invention; Figure 2This is an enlarged chromatogram of the blank control solution obtained by IMAC-HPLC in this invention. It can be seen that there are no impurity peaks interfering with the elution of the histidine-tagged protein, indicating that this method has good specificity.

[0117] (2) The histidine-tagged protein reference IMAC-HPLC chromatogram of this embodiment is shown below. Figures 3-4 As shown, Figure 3 This is an overlay of the original chromatogram of the histidine-tagged protein reference. Figure 4 This is an enlarged overlay of the chromatogram of the histidine-tagged protein reference; the loading amount and peak area fitting results of the histidine-tagged protein in the standard curve solution are shown in Table 2, and the standard curve established accordingly is shown in [reference image]. Figure 5 .

[0118] Table 2

[0119]

[0120] (3) The IMAC-HPLC chromatogram of the histidine-tagged protein control sample in this embodiment is shown below. Figure 6 As shown in Table 3, the peak areas and quantitative results of the quality control solution are presented.

[0121] Table 3

[0122]

[0123] (4) Sample IMAC-HPLC chromatogram as shown Figure 7 As shown in Table 4, the concentration results of histidine-tagged proteins in the samples are presented in Table 4.

[0124] Table 4

[0125] name Retention time (min) Peak area (μV*sec) Concentration (mg / mL) sample 20.279 1397520 0.39

[0126] The above results show that when the extinction coefficient k of the histidine-tagged protein is ≥0.6, the protein loading amount of the method of the present invention has good linearity in the range of 5-50 μg, and can be used for the accurate determination of the concentration of histidine-tagged protein in the sample. This is of great significance for guiding the research and development and production process of commercial protein.

[0127] Example 2

[0128] This embodiment provides an analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC. The difference between Example 2 and Example 1 is that the histidine-tagged protein used in Example 2 is Recombinant Protein A (His-tag). The specific steps are as follows:

[0129] A. Preparation of standard curve solution and quality control solution

[0130] In this embodiment, the histidine-tagged protein reference standard (Recombinant Protein A, His-tag) was a lyophilized powder with an extinction coefficient of 0.22, which is less than 0.6. A 7.0 mg / mL reference standard solution was prepared using ultrapure water as a diluent. Two parallel solutions were prepared, one for the standard curve solution and the other for the quality control solution. The quality control solution injection volume was 20 μL. By varying the injection volume of the reference standard solution, the histidine-tagged protein loading amounts at each point on the standard curve were 35 μg, 70 μg, 140 μg, 210 μg, and 350 μg, respectively. The standard curve solution injection volume and loading amounts at each point are shown in Table 5.

[0131] Table 5

[0132] name Injection volume (μL) Sample loading amount (μg) STD-1 5 35 STD-2 10 70 STD-3 20 140 STD-4 30 210 STD-5 50 350

[0133] B. Sample solution preparation

[0134] Transfer a sufficient amount of sample solution into the injection bottle and wait for injection.

[0135] The preparation of blank solutions, the IMAC-HPLC detection method, the establishment of standard curves, the calculation of quality control product recovery rates, and the calculation of histidine-tagged protein concentrations in samples were consistent with those in Example 1.

[0136] The test results are as follows:

[0137] (1) The histidine-tagged protein reference IMAC-HPLC chromatogram of this embodiment is shown below. Figures 8-9 As shown in Table 6, the loading amount of histidine-tagged protein and the fitting results of the peak area in the standard curve solution are presented. The standard curve established based on this is shown in Table 6. Figure 10 .

[0138] Table 6

[0139]

[0140] (2) The IMAC-HPLC chromatogram of the histidine-tagged protein control sample in this embodiment is shown below. Figure 11 As shown in Table 7, the peak areas and quantitative results of the quality control solution are presented.

[0141] Table 7

[0142]

[0143] (3) Sample IMAC-HPLC chromatogram as shown Figure 12 As shown in Table 8, the concentration results of histidine-tagged proteins in the samples are presented in Table 8.

[0144] Table 8

[0145] name Retention time (min) Peak area (μV*sec) Concentration (mg / mL) sample 20.236 1306246 2.57

[0146] The above results indicate that when the extinction coefficient of the histidine-tagged protein is 0.22 ≤ k < 0.6, the protein loading amount of the method of the present invention exhibits good linearity in the range of 35-350 μg.

[0147] The experimental results from Examples 1 and 2 show that the method of the present invention has a very wide range of applications and can be used for the accurate quantification of most histidine-tagged proteins with extinction coefficients (k≥0.22).

[0148] Example 3

[0149] This embodiment demonstrates the practical application of the IMAC-HPLC-based analytical method for determining histidine-tagged protein concentration described in this invention in the protein process development process. It combines IMAC and HPLC technologies to determine the concentration of histidine-tagged proteins in cell lysates, guiding the chromatographic loading steps in protein process development. The specific steps are as follows:

[0150] A. Preparation of standard curve solution and quality control solution

[0151] The histidine-tagged protein had an extinction coefficient >0.6 and a concentration of 1.49 mg / mL. The reference standard was diluted to 1.0 mg / mL with 20 mM HEPES, 0.5 M NaCl, and pH 7.5 solution. Two parallel aliquots were prepared, one for the standard curve solution and one for the quality control solution. The quality control solution was injected in 20 μL. By varying the injection volume of the reference solution, the histidine-tagged protein loading amounts at each point on the standard curve were 5 μg, 10 μg, 20 μg, 30 μg, and 50 μg. The injection volumes of the standard curve solution and the loading amounts at each point are shown in Table 1.

[0152] B. Sample solution preparation

[0153] Transfer a sufficient amount of cell lysis buffer sample solution into the injection vial and wait for injection.

[0154] The preparation of blank solutions, the IMAC-HPLC detection method, the establishment of standard curves, the calculation of quality control product recovery rates, and the calculation of histidine-tagged protein concentrations in cell lysate samples were consistent with those in Example 1.

[0155] The test results are as follows:

[0156] (1) Chromatogram of the histidine-tagged protein reference IMAC-HPLC is shown below. Figures 13-14 As shown in Table 9, the loading amount of histidine-tagged protein and the fitting results of the peak area in the standard curve solution are presented. The standard curve established based on this is shown in Table 9. Figure 15 .

[0157] Table 9

[0158]

[0159]

[0160] (2) The IMAC-HPLC chromatogram of the histidine-tagged protein control sample is shown below. Figure 16 As shown in Table 10, the peak areas and quantitative results of the quality control solution are presented.

[0161] Table 10

[0162]

[0163] (3) Since the default injection volume is not suitable for cell lysis buffer samples, the sample injection volume was adjusted to 50 μL in this embodiment. The IMAC-HPLC chromatogram of the cell lysis buffer sample is shown below. Figures 17-18 As shown, where Figure 17 This is the original chromatogram of the cell lysis buffer sample. Figure 18 This is a magnified view of the chromatogram of the cell lysate sample. The concentration results of histidine-tagged proteins in the cell lysate sample are shown in Table 11.

[0164] Table 11

[0165] name Retention time (min) Peak area (μV*sec) Concentration (mg / mL) Cell lysate samples 20.278 1372073 0.44

[0166] The above results indicate that combining IMAC with HPLC technology can be used to accurately determine the concentration of histidine-tagged proteins in cell lysates of complex samples, which is of great significance for guiding the research and development and production of commercial proteins.

[0167] Example 4

[0168] Using the same experimental conditions as in Examples 1-3, a methodological study was conducted on the determination of histidine-tagged protein concentration in cell lysates based on IMAC-HPLC.

[0169] Test results

[0170] (1) This method obtains the peak area response at each point of the standard curve by changing the injection volume of the reference solution. The linear fitting results of Examples 1 and 3 are shown in Tables 2 and 9, respectively. It can be seen that when the extinction coefficient k of the histidine-tagged protein reference is ≥0.6, the linear equation correlation coefficient R is within the range of 5-50 μg. 2 ≥0.99, indicating good linearity. Table 6 shows the linear fitting results for Example 2. It can be seen that when the extinction coefficient k of the histidine-tagged protein reference is <0.6, the linear equation correlation coefficient R is good when the histidine-tagged protein loading amount is in the range of 35-350 μg. 2 ≥0.99, indicating good linearity.

[0171] (2) The precision results of the system in Examples 1-3 are shown in Table 12. The relative standard deviations of the retention time and peak area of ​​the histidine tag protein are both less than 2.0%, indicating that the precision of the IMAC-HPLC analytical method of the present invention is good.

[0172] Table 12

[0173]

[0174] (3) The accuracy results of the standard curves in Examples 1-3 are shown in Tables 3, 7 and 10, respectively. It can be seen that the recovery rate of the quality control solution is greater than 90.0%, indicating that the standard curves established by this method can be used to accurately determine the concentration of histidine tag protein in the sample.

[0175] Comparative Example 1

[0176] This comparative example provides an analytical method for determining the concentration of histidine-tagged proteins in a sample based on IMAC-HPLC. The only difference between this method and Example 1 is that the gradient elution procedure is as follows:

[0177]

[0178] The test results are as follows:

[0179] (1) This gradient elution procedure has problems such as excessive analysis time and large baseline signal interference, and cannot achieve rapid quantification of histidine-tagged target proteins;

[0180] (2) Under this gradient elution program, the separation between the target protein in the sample and the impurity peak on the right is poor, which affects the accurate quantification of histidine-tagged protein.

[0181] Comparative Example 2

[0182] This comparative example provides an analytical method for determining the concentration of histidine-tagged proteins in a sample based on IMAC-HPLC. The only difference between this method and Example 1 is that the mobile phase for IMAC-HPLC detection consists of mobile phase A and mobile phase B. Mobile phase A is 20 mM HEPES, 500 mM NaCl, pH 7.0; mobile phase B is 20 mM HEPES, 500 mM NaCl, 500 mM imidazole, pH 7.0.

[0183] The test results are as follows:

[0184] (1) When the pH of mobile phase A and mobile phase B is adjusted to 7.0, the baseline signal interference is too large, making it impossible to accurately identify the peak position of the target protein and affecting the accurate quantification of histidine-tagged protein.

[0185] Comparative Example 3

[0186] This comparative example provides an analytical method for determining the concentration of histidine-tagged proteins in a sample based on IMAC-HPLC. The only difference between this method and Example 1 is that the mobile phase for IMAC-HPLC detection consists of mobile phase A and mobile phase B. Mobile phase A is 20 mM HEPES, 0.5 M NaCl, pH 8.0; mobile phase B is 20 mM HEPES, 0.5 M NaCl, 0.5 M imidazole, pH 8.0.

[0187] The test results are as follows:

[0188] (1) When the pH of mobile phase A and mobile phase B is adjusted to 8.0, the target protein and impurity peaks cannot be well separated, which affects the accurate quantification of histidine-tagged proteins.

[0189] Comparative Example 4

[0190] This comparative example provides an analytical method for determining the concentration of histidine-tagged proteins in a sample based on IMAC-HPLC. The only difference between this method and Example 1 is that the IMAC-HPLC uses a Sepax Technologies Polar MC30-Ni Excel column with dimensions of 7.8 mm × 150 mm and a diameter of 30 μm.

[0191] The test results are as follows:

[0192] (1) Use specifications of 7.8mm×150mm, 30μm, The chromatographic column resulted in a low peak response for the histidine-tagged target protein in the sample, affecting the accurate quantification of the histidine-tagged protein.

[0193] The comparative experimental results of the above examples and Comparative Examples 1-4 show that the elution program time setting of the present invention is more suitable, enabling rapid quantification. Under the above elution program, the separation degree between the main peak and the impurity peak meets the requirements, which is beneficial for accurate quantification. Meanwhile, the pH value of the mobile phase also affects the detection results. When the pH value is outside the range of 7.3-7.7, it affects the accurate quantification of histidine-tagged proteins. The chromatographic column used in the present invention is also suitable for the detection of histidine-tagged proteins. Replacing with other chromatographic columns results in a lower peak response of the histidine-tagged target protein in the sample, affecting the accurate quantification of histidine-tagged proteins.

[0194] In summary, this invention is the first to combine immobilized metal ion affinity chromatography with HPLC technology to guide the research and development and production of commercial proteins. It can accurately and rapidly guide the research and development and production processes of commercial proteins, including but not limited to cell lysis buffer affinity chromatography sample loading, reducing the occurrence of target protein overload or underload, and significantly saving research and development and production costs. This invention provides an analytical method for determining the concentration of histidine-tagged proteins based on IMAC-HPLC. This analytical method can accurately quantify histidine-tagged proteins and has significant application value in protein process research and development and production.

[0195] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An analytical method for determining the concentration of histidine-tagged proteins based on IMAC-HPLC, characterized in that, The analytical method includes: (1) Prepare reference solution, quality control solution and test sample solution, and perform IMAC-HPLC detection; the chromatographic column used for IMAC-HPLC detection is filled with Ni-chelated Ni. 2+ The chromatographic column packing material is used; the IMAC-HPLC detection employs a gradient elution method to separate the samples; the mobile phase for IMAC-HPLC detection consists of mobile phase A and mobile phase B, wherein mobile phase A is 15-25 mM HEPES + 450-550 mM NaCl, with a pH of 7.3-7.7; and mobile phase B is 15-25 mM HEPES + 450-550 mM NaCl + 450-550 mM imidazole, with a pH of 7.3-7.

7. The gradient elution procedure for IMAC-HPLC detection is as follows: 0 min Mobile phase A is 100% Mobile phase B is 0%; 4 min mobile phase A is 100% mobile phase B is 0%; At 7 min, mobile phase A was 0% and mobile phase B was 100%. At 9 min, mobile phase A was 0% and mobile phase B was 100%. At 12 min, mobile phase A is 100% and mobile phase B is 0%. 30 min: Mobile phase A is 100%, Mobile phase B is 0%; (2) Using the amount of the reference solution loaded as the abscissa X and the peak area of ​​the histidine-tagged protein as the ordinate Y, a linear equation was fitted to obtain the standard curve. (3) Substitute the peak area of ​​histidine-tagged protein in the quality control solution into the standard curve to calculate the measured amount of histidine-tagged protein in the quality control solution. The ratio of the measured amount of histidine-tagged protein to the theoretical amount of histidine-tagged protein is the recovery rate. Substitute the peak area of ​​histidine-tagged protein in the test sample solution into the standard curve to calculate the measured amount of histidine-tagged protein in the test sample. The ratio of the measured amount of histidine-tagged protein to the sample injection volume is the concentration of histidine-tagged protein.

2. The analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to claim 1, characterized in that, In step (1), the reference solution and the quality control solution are prepared by the following steps: Histidine-tagged protein references with an extinction coefficient greater than 0.6 were dissolved in a solvent to prepare a 1.0 ± 0.2 mg / mL reference solution or quality control solution. Histidine-tagged protein references with an extinction coefficient less than 0.6 were dissolved in a solvent to prepare a 7.0±0.2 mg / mL reference solution or quality control solution.

3. The analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to claim 2, characterized in that, The solvent is selected from water or a dilution buffer, wherein the dilution buffer comprises 15-25 mM HEPES + 450-550 mM NaCl by concentration and has a pH of 7.3-7.

7.

4. The analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to claim 1, characterized in that, In step (1), the amount of reference solution loaded is controlled by changing the injection volume of the reference solution in the IMAC-HPLC detection.

5. The analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to claim 1, characterized in that, In step (1), the chromatographic column used for IMAC-HPLC detection is a Polar MC30-Ni Excel column.

6. The analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to claim 1, characterized in that, In step (1), the pH of the mobile phase A is 7.4-7.

6.

7. The analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to claim 1, characterized in that, In step (1), the pH of the mobile phase B is 7.4-7.

6.

8. The analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to claim 1, characterized in that, In step (1), the flow rate of the IMAC-HPLC detection is 0.8 ± 0.2 mL / min.

9. The analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to claim 1, characterized in that, In step (1), the column temperature for the IMAC-HPLC detection is set to 25±5℃.

10. The application of the analytical method for determining the concentration of histidine-tagged protein based on IMAC-HPLC according to any one of claims 1-9 in the protein process development and / or production process.

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