A method for detecting charge heterogeneity of recombinant human follicle stimulating hormone

Abstract

The application discloses a detection method of charge heterogeneity of recombinant human follicle stimulating hormone, which comprises the following steps: component separation of the recombinant human follicle stimulating hormone is carried out by using HPLC; and the chromatographic conditions are as follows: a Proteomix SAX-NP5 is used as a chromatographic column; a Tris buffer is used as a mobile phase A; a Tris buffer containing NaCl is used as a mobile phase B; a gradient elution program is as follows: during 0 min to 36-45 min, the mobile phase A is reduced from 100% to 0%-20% and then is increased to 100%; and the mobile phase B is increased from 0% to 75%-100% and then is decreased to 0%. The method can realize effective separation of charge heterogeneity of the complex protein of the recombinant human follicle stimulating hormone, and has the advantages of simple operation, convenient fraction collection, low cost and convenience for separation and identification of charge heterogeneity of the recombinant human follicle stimulating hormone and subsequent activity research and analysis.

Description

Technical Field

[0001] This invention belongs to the field of bioproduct analysis technology, specifically relating to a method for detecting the charge heterogeneity of recombinant human follicle-stimulating hormone. Background Technology

[0002] During the production process of recombinant protein drugs, such as cell culture, purification, formulation preparation, storage, and use, the charge properties of the drugs undergo heterogeneous changes, which can lead to the generation of different charge isomers in the same protein product.

[0003] The presence of charge heterogeneity can affect product stability and solubility, leading to changes in efficacy and pharmacokinetics, complete inactivation, and enhanced immunogenicity. The degree of charge heterogeneity control reflects the consistency of drug manufacturing processes and is a key quality attribute of recombinant protein drugs. Article 35 of Section 2, "Drug Marketing Authorization," of Chapter 3, "Drug Marketing Registration," of the *Drug Registration Management Measures* stipulates that generic drugs should be consistent with the reference formulation in quality and efficacy. Related drug regulations also point out the need to steadily improve the safety, efficacy, and accessibility of drugs, continue to promote the consistency evaluation of generic drug quality and efficacy, improve the technical guidance principles, consistency evaluation policies, and technical standards system related to generic drugs, and further promote the improvement of generic drug quality. Therefore, research and development of charge heterogeneity analysis for recombinant protein drugs is also highly valuable in the research and development of biosimilars, consistency evaluation studies of original drugs, and process quality control.

[0004] Recombinant human follicle-stimulating hormone (FSH) is a heterodimeric (α- and β-subunits) glycoprotein with a complex glycoform. Currently, common methods for detecting its charge heterogeneity include isoelectric focusing electrophoresis (IEF) and capillary isoelectric focusing (iCIEF). IEF can detect the isoelectric point of a sample, but it cannot accurately analyze each charge variant. iCIEF can analyze each charge variant, but due to the lack of a fraction collector and the instrument's sample capacity being only nanoliters, it cannot be scaled up like chromatographic methods. Therefore, separating and collecting acid-base variants for identification, subsequent characterization, and activity studies is very difficult. After iCIEF separation, traditional methods such as IEX (ion-exchange chromatography), IEF, or FFE (free-flow electrophoresis) must be used to collect the observed protein charge variants. Because IEX, IEF, or FFE differ from iCIEF in separation mechanism and resolution, the collected fractions often do not match the results well with iCIEF. Prolonged operation and the use of strong chemical reagents often lead to protein denaturation during fraction collection. Free-flow electrophoresis (FFE) is complex, cumbersome, and expensive for collecting charge variants; IEX has limited separation capabilities, failing to effectively separate many charge isomers, especially trace amounts. Furthermore, the separation and analysis of charge isomers often involve high temperatures and extreme pH treatments, leading to the generation of additional charge isomers that interfere with the detection and analysis of charge heterogeneity. Existing research, CN108333261A, discloses the detection of charge variants in target proteins using pH-IEC ion exchange chromatography; however, this method is only suitable for detecting simple structures such as monoclonal antibodies and cannot be used for detecting charge heterogeneity in complex structures such as recombinant human follicle-stimulating hormone. CN114113450A discloses an ion-exchange chromatography method using isocratic elution to separate charge heterogeneous substances in protein products. This method can only separate and detect simple proteins, and cannot detect charge heterogeneity of complex structures such as recombinant human follicle-stimulating hormone.

[0005] Given the limitations of existing analytical methods in detecting the charge heterogeneity of recombinant human follicle-stimulating hormone (rFSH) and the stringent requirements for drug quality and safety, it is urgent to develop a method that is simple to operate, can effectively and accurately separate and analyze complex charge variants like rFSH, and allows for easy fraction collection to meet the needs of subsequent fraction characterization and activity studies. Summary of the Invention

[0006] In view of the shortcomings of existing recombinant protein charge heterogeneity analysis, this invention provides an HPLC analytical method for detecting the charge heterogeneity of recombinant human follicle-stimulating hormone (FSH). This method can effectively separate charge heterogeneities of complex proteins such as FSH, and is simple to operate, convenient for fraction collection, and low in cost, making it highly suitable for the separation and identification of charge heterogeneity in FSH and subsequent activity studies.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for detecting the charge heterogeneity of recombinant human follicle-stimulating hormone (FSH), comprising the following steps: separating the components of recombinant human FSH using HPLC; the chromatographic conditions are as follows: Column: Proteomix SAX-NP5 Mobile phase: Tris buffer was used as mobile phase A, and Tris buffer containing NaCl was used as mobile phase B; The gradient elution program is as follows: during the period from 0 min to 36-45 min, mobile phase A decreases from 100% to 0%-25% and then increases back to 100%; mobile phase B increases from 0% to 75%-100% and then decreases back to 0%.

[0008] In some specific embodiments of the present invention, the chromatographic column is a Proteomix SAX-NP5, 2.1*250mm*5μm or a Proteomix SAX-NP5, 4.6*250mm*5μm.

[0009] In certain specific embodiments of the present invention, when the chromatographic column is a Proteomix SAX-NP5, 2.1*250mm*5μm, the gradient elution program is as follows:

[0010] In certain specific embodiments of the present invention, when the chromatographic column is a Proteomix SAX-NP5, 2.1*250mm*5μm, the gradient elution program is as follows:

[0011] In certain specific embodiments of the present invention, when the chromatographic column is a Proteomix SAX-NP5, 4.6*250mm*5μm, the gradient elution program is as follows:

[0012] In certain specific embodiments of the present invention, when the chromatographic column is a Proteomix SAX-NP5, 4.6*250mm*5μm, the gradient elution program is as follows:

[0013] In some specific embodiments of the present invention, the concentration of the Tris buffer is 10~30 mmol / L, preferably 20 mmol / L.

[0014] In some specific embodiments of the present invention, the pH value of the Tris buffer solution is 5-6, preferably 5.8.

[0015] In certain specific embodiments of the present invention, the NaCl concentration in mobile phase B is 300 mmol / L-800 mmol / L; preferably, the NaCl concentration in mobile phase B is 300 mmol / L, 400 mmol / L, 500 mmol / L, 600 mmol / L, 700 mmol / L, or 800 mmol / L; more preferably, the NaCl concentration in mobile phase B is 300 mmol / L, 500 mmol / L, or 800 mmol / L; more preferably, the NaCl concentration in mobile phase B is 500 mmol / L.

[0016] In some specific embodiments of the present invention, the chromatographic conditions further include: a UV detection wavelength of 200nm-300nm; preferably, the chromatographic column is a Proteomix SAX-NP5, with a wavelength of 215nm when the column is 2.1*250mm*5μm, and a wavelength of 280nm when the column is 4.6*250mm*5μm. And / or: the flow rate is 0.2 ml / min ~ 0.6 ml / min; preferably, the column is Proteomix SAX-NP5, with a flow rate of 0.2 ml / min when the column is 2.1*250mm*5μm, and the flow rate is 0.5 ml / min when the column is 4.6*250mm*5μm. And / or: the injection volume is 10 μl to 50 μl. Preferably, the chromatographic column is Proteomix SAX-NP5, and the injection volume is 10 μl when the column is 2.1*250mm*5μm, and 50 μl when the column is 4.6*250mm*5μm. And / or: Column temperature is 20~40℃.

[0017] The beneficial effects of this invention are: This invention provides a method for detecting the charge heterogeneity of recombinant human follicle-stimulating hormone. By investigating key factors affecting the separation effect, such as liquid chromatography column, mobile phase salt concentration, and elution program, a method suitable for the analysis and detection of charge heterogeneity of complex recombinant proteins such as recombinant human follicle-stimulating hormone is constructed.

[0018] By using the method of this invention, at least the following drawbacks of existing recombinant human follicle-stimulating hormone (FSH) charge isoform detection methods are addressed: The method of this invention enables effective separation of each charge isoform of FSH. Compared with the current industry gold standard iCIEF method, it not only maintains essentially the same separation effect of acid-base peaks and peak area percentage for charge isoforms, but also overcomes the difficulty of fraction collection in the iCIEF method. Furthermore, the detection method provided by this invention is simple to operate, relatively convenient for fraction collection, and yields high-purity fractions, facilitating subsequent analysis and research on the physicochemical properties and biological activities of the relevant charge isoforms.

[0019] The detection method involved in this invention is the result of the inventor's painstaking efforts. This method has universal applicability; the number of peaks and separation effect remain consistent when the injection volume is 10 μl, 20 μl, 30 μl, 40 μl, and 50 μl. The method is highly specific; the blank control solution (containing only the excipient solution of the test sample) has no interference with the peak position. It exhibits good repeatability; parallel tests of five groups show that the RSD of the percentage of the main peak area of ​​the test sample is 0.1%, higher than the industry requirement (not higher than 5%); the RSD of the percentage of the acidic peak area is 0%, higher than the industry requirement (not higher than 10%); and the RSD of the percentage of the basic peak area is 2.2%, higher than the industry requirement (not higher than 10%). Furthermore, the method has high precision; under different experimental instruments and different chromatographic columns, the RSD of the percentage of the main peak area of ​​the test sample is 0.1-0.2%, the RSD of the percentage of the acidic peak area is no more than 0%-1%, and the RSD of the percentage of the basic peak area is no more than 2.2%-2.6%. The separated fraction has high purity and can be directly used for the identification of acid-base peak products and activity studies, providing a valuable reference for the optimization of the production process and quality control of recombinant human follicle-stimulating hormone protein.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 Initial condition test spectrum Figure 2 Table 3. Gradient elution program test spectra Figure 3Table 4. Gradient elution program test spectra Figure 4 Table 5. Gradient elution program test spectra Figure 5 Table 6. Gradient elution program test spectra Figure 6 Table 7. Gradient elution program test spectra Figure 7 Spectrum of NaCl at a concentration of 0.5 mol / L Figure 8 Molecular weight map Detailed Implementation Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0023] 1. Source of experimental instruments and reagents: instrument: Reagents: 2. Preparation of test sample: Take the recombinant human follicle-stimulating hormone stock solution into the injection bottle and inject it directly.

[0024] Example 1 Initial test conditions Recombinant human follicle-stimulating hormone stock solution was used as the test sample. A Proteomix SAX-NP5 (2.1*250mm*5μm) strong anion chromatography column was selected for the experiment. The column temperature was 40℃, the injection volume was 10μl, the UV detection wavelength was 215nm, the mobile phase A was 20mmol / L Tris buffer, and the mobile phase B was "mobile phase A, 0.3mol / L NaCl" solution.

[0025] Adjust the pH of mobile phase A to 4.8, 5.8, or 6.8 using hydrochloric acid: see Table 1 for specific elution conditions.

[0026] Table 1 Gradient elution table The detection results are shown in Table 2. Mobile phase A showed better peak separation at pH 5.8; however, while the main peak could be separated from the acid and base peaks, the resolution was poor and unsuitable for detecting and collecting charge heterogeneity. The chromatogram at pH 5.8 is shown below. Figure 1 .

[0027] Table 2 Summary of peak area results for different mobile phases at pH Example 2: Examination of the elution process The analysis time was fixed at 35-36 min, the pH of mobile phase A was 5.8, the elution conditions were as shown in Table 3-7, and other conditions were the same as in Example 1. Relevant experimental results are as follows: Figure 2-6 .

[0028] Table 3 Gradient Elution Table Table 4 Gradient elution table Table 5 Gradient Elution Table Table 6 Gradient Elution Table Table 7 Gradient Elution Table The results are analyzed and summarized as follows: Table 8 Summary of Peak Area Results NA: indicates that they are not separated.

[0029] The separation results for each peak are shown in the table below: Table 9. Statistical table of peak separation. NA: indicates that they are not separated.

[0030] The elution program, as shown in Table 6, showed good separation of each peak. Due to the low flow rate, mobile phase A was kept at 100%, and the running time was extended from 35 min to 40 min for further study.

[0031] Example 3: Investigation of Injection Volume To facilitate the collection of each fraction and to examine the effect of injection volume on sample separation, injection volumes of 20 μl and 30 μl were selected, and it was found that the number of peaks and separation effect were consistent.

[0032] Example 4: Investigation of mobile phase salt ion concentration The separation effect of NaCl concentrations of 0.1 mol / L, 0.5 mol / L, 0.8 mol / L, and 1.0 mol / L in mobile phase B was then investigated. The elution conditions are as shown in Table 10.

[0033] Table 10. Summary of Peak Area Results The study found that the salt ion concentration in the mobile phase significantly affects the separation of charged heteroparticles from the main peak. When the salt concentration in mobile phase B is 0.5 mol / L, the chromatographic peak separation is good. Decreasing or increasing the salt concentration in mobile phase B leads to overlapping alkaline peaks that cannot be effectively separated. The test results for a NaCl concentration of 0.5 mol / L in mobile phase B are shown in the spectrum. Figure 7 .

[0034] Example 5: Investigation of the chromatographic column To further improve the efficiency of acid-base peak collection, a larger inner diameter Proteomix SAX-NP5 column (4.6*250mm*5μm) was selected, the UV detection wavelength was changed from 215nm to 280nm, the flow rate was changed from 0.2 ml / min to 0.5 ml / min, the injection volume was 50μl, the column temperature was room temperature, mobile phase A was 20mmol / L Tris buffer, pH 5.8; mobile phase B was "mobile phase A, 0.5mol / L NaCl solution", and the elution conditions are shown in Table 11.

[0035] Table 11 Gradient Elution Table Table 12. Statistical analysis of peak area results for different methods Under the above conditions, the number of each main peak measured in the experiment is consistent with the currently publicly used iCIEF method, and the separation between each peak is good, which can achieve efficient collection of the fraction.

[0036] The following experimental examples further illustrate the beneficial effects of the present invention. Experimental Example 1: Methodological Validation Using the experimental conditions determined in Example 6, namely Proteomix SAX-NP5 (4.6*250mm*5μm), UV wavelength of 280nm, flow rate of 0.5 ml / min, injection volume of 50μl, column temperature of room temperature, mobile phase A of 20mmol / L Tris buffer, pH 5.8, and mobile phase B of "mobile phase A, 0.5mol / L NaCl solution", and elution conditions as shown in Table 13, the specificity and precision studies were carried out as follows.

[0037] Table 13 Gradient Elution Table 1. Specificity research The recombinant human follicle-stimulating hormone (FSH) test sample and the blank control solution (finished blank excipient solution) were tested under the optimized conditions of Example 6. The results showed that the blank control solution (finished blank excipient solution) had no interfering peaks at the sample elution position, indicating that the method has good specificity.

[0038] 2. Precision Study 2.1 Repeatability Prepare 5 samples in parallel, inject each sample continuously with one injection, and statistically analyze the results of 5 experiments. The RSD of the main peak area percentage should not be higher than 5%, and report the RSD of the area percentage of each acid and base peak.

[0039] Table 14. Statistics of peak area results for repeatability The RSD of the percentage of the main peak area of ​​the five parallel-prepared test samples was 0.07%, which is higher than the industry requirement (not higher than 5%); the RSD of the percentage of the acidic peak area was 0.10%, which is higher than the industry requirement (not higher than 10%); and the RSD of the percentage of the alkaline peak area was 0.11%, which is higher than the industry requirement (not higher than 10%).

[0040] 2.2 Intermediate Precision Five samples were prepared in parallel using both Thermo U3000 and Agilent 1260 chromatographs. Each sample was injected once consecutively, and the results of the five experiments were statistically analyzed. The RSD of the percentage area of ​​each acid and basic peak was reported. In the ten samples, the RSD of the percentage area of ​​the main peak of the test sample was 0.10%; the RSD of the percentage area of ​​the acidic peak was 0.11%; and the RSD of the percentage area of ​​the basic peak was 0.10%, indicating that the detection method has good reproducibility.

[0041] Meanwhile, under the following experimental conditions: Proteomix SAX-NP5 (2.1*250mm*5μm), UV wavelength 215nm, flow rate 0.2 ml / min, injection volume 10μl, column temperature room temperature, mobile phase A 20mmol / L Tris buffer, pH 5.8; mobile phase B "mobile phase A, 0.5mol / L NaCl solution", and elution conditions as shown in Table 15, specificity and repeatability were verified.

[0042] Table 15 Gradient Elution Table The study results showed that the blank control solution (finished blank excipient solution) had no interfering peaks at the sample elution positions, indicating good specificity of this method. The RSD of the percentage of the main peak area of ​​the five parallel-prepared test samples was 0.05%, the RSD of the percentage of the acidic peak area was 0.07%, and the RSD of the percentage of the basic peak area was 0.10%, all of which are higher than the industry requirements.

[0043] 3. Collection and identification of acid-base peaks 3.1 Collection based on charge heterogeneity The recombinant human follicle-stimulating hormone (FSH) sample and the original drug Gonal-F were desalted and excipient-free before injection. The acid peak, basic peak, and main peak of both samples were collected using the detection method of this invention. The peak spectra of the collected sample were basically consistent with those of the original drug Gonal-F. However, both the sample and Gonal-F contained a high proportion of the main peak in their basic peaks after collection, suggesting a conversion process from basic to main peaks during sample collection.

[0044] 3.2 Identification of molecular weight by charge heterogeneity property spectrometry The test sample and the original drug Gonal-F were injected for testing. The results showed that the molecular weight profiles of the test sample (recombinant human follicle-stimulating hormone) and the original drug Gonal-F were similar. The main components of the α and β chains of the charge heterogeneous components were similar, and the molecular weights were basically the same. Related results can be found in... Figure 8 .

[0045] 4. Analysis and Discussion: The analytical method of this invention exhibits strong specificity; the blank control solution (finished product blank excipient solution) shows no interfering peaks at the sample elution position. It demonstrates good repeatability, with the RSD of the percentage peak area of ​​the main peak of the test sample being 0.05%-0.07%, far exceeding industry requirements (not exceeding 5%). The RSD of the percentage peak area of ​​acidic peaks is 0.07%-0.10%, far exceeding industry standards (not exceeding 10%), and the RSD of the percentage peak area of ​​basic peaks is 0.10%-0.11%, also exceeding industry standards (not exceeding 10%). It also demonstrates good precision; using different detection instruments, the RSD of the percentage peak area of ​​the main peak of the test sample is 0.10%, the RSD of the percentage peak area of ​​acidic peaks is 0.11%, and the RSD of the percentage peak area of ​​basic peaks is 0.10%. Furthermore, it provides excellent separation, effectively separating both acidic and basic peaks from the main peak, and obtaining highly pure separated components. Furthermore, this method is very convenient and simple to operate for collecting charged heterogeneous particles, and the collected fraction has high purity. It also provides a very valuable reference for the subsequent optimization of the production process and quality control of recombinant human follicle-stimulating hormone protein.

Claims

1. A method for detecting the charge heterogeneity of recombinant human follicle-stimulating hormone, characterized in that: The steps include: injecting recombinant human follicle-stimulating hormone stock solution directly into a sample vial; separating the components of the recombinant human follicle-stimulating hormone stock solution using HPLC; the chromatographic conditions are as follows: Column: Proteomix SAX-NP5; Mobile phase: Tris buffer was used as mobile phase A, and Tris buffer containing NaCl was used as mobile phase B; The concentration of the Tris buffer solution is 10-30 mmol / L, and the pH value is 5.

8. The concentration of NaCl in the mobile phase B is 500 mmol / L; The chromatographic column was a Proteomix SAX-NP5, 2.1*250mm*5μm, with a wavelength of 215nm, and the gradient elution program was as follows: The chromatographic column was a Proteomix SAX-NP5, 4.6*250mm*5μm, with a wavelength of 280nm, and the gradient elution program was as follows:

2. The detection method according to claim 1, characterized in that: The concentration of the Tris buffer solution was 20 mmol / L.

3. The detection method according to claim 1, characterized in that: The chromatographic conditions also include a flow rate of 0.2 ml / min to 0.6 ml / min.

4. The detection method according to claim 3, characterized in that: When the column is a Proteomix SAX-NP5 with a diameter of 2.1*250mm*5μm, the flow rate is 0.2ml / min; when the column is a Proteomix SAX-NP5 with a diameter of 4.6*250mm*5μm, the flow rate is 0.5ml / min.

5. The detection method according to claim 1, characterized in that: The chromatographic conditions also include an injection volume of 10 μL to 50 μL.

6. The detection method according to claim 5, characterized in that: When the column is a Proteomix SAX-NP5, 2.1*250mm*5μm, the injection volume is 10μL; when the column is a Proteomix SAX-NP5, 4.6*250mm*5μm, the injection volume is 50μL.

7. The detection method according to claim 1, characterized in that: The chromatographic conditions also include a column temperature of 20~40℃.

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