A novel method for rapid separation of charge heterogeneous compounds based on isocratic elution

Abstract

The present invention discloses a novel method for rapid separation of charged heterogeneous substances in protein products. The method uses isocratic elution ion exchange chromatography to separate charged heterogeneous substances in protein products, that is, simple and convenient isocratic elution is used to replace the commonly used gradient elution. The advantages are: significantly accelerating the separation speed and improving the analysis efficiency; simplifying the experimental process and reducing the manpower and material costs; and having wide applicability. The same method can be used for the separation and analysis of charged heterogeneous substances in a variety of similar protein products.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the separation and analysis of charged heterogeneous forms of protein products, and specifically refers to an ion exchange chromatography method based on isocratic elution for rapid separation of charged heterogeneous forms of protein products. Background Art

[0002] During the production (including cell culture, purification, preparation production, etc.), storage, transportation and use of recombinant protein products, various factors may cause modification and degradation of the protein surface, resulting in differences in surface charge and charge heterogeneity of the protein; in addition, impurities produced by the host cells themselves and introduced during the production process can also cause protein heterogeneity. However, the structure and drug properties of recombinant protein drugs will be affected to varying degrees by charge heterogeneities. Therefore, controlling charge heterogeneities is an important factor in evaluating the production process and quality of recombinant protein products.

[0003] Ion exchange chromatography (IEC) is an effective and widely used method for separating and analyzing charge heterogeneities. At the same time, this chromatography can also perform quantitative and qualitative analysis of protein characteristics. Researchers used ion exchange chromatography to detect and collect charge heterogeneities of PD-1 monoclonal antibodies, and combined with other characterization methods to study the structure and biological function of anti-PD-1. Ion exchange chromatography mostly uses gradient elution, including salt gradient and pH gradient; however, whether it is salt gradient or pH gradient, the gradient elution method requires workers to explore the conditions of gradient change, such as gradient change time, ratio, etc., and then continuously optimize to achieve a good separation effect, which is bound to consume a lot of manpower, material resources and financial resources, especially for enterprises or institutions developing multiple different projects. Therefore, it is imperative to improve and optimize the ion exchange chromatography detection method. Summary of the invention

[0004] The object of the present invention is to provide a novel separation method of charge heterogeneous substances based on isocratic elution and its application. The ion exchange chromatography method for separating charge heterogeneous substances of protein products is based on the difference in binding force between charged proteins and oppositely charged stationary phases, thereby achieving the purpose of separation. The ion exchange chromatography method based on isocratic elution is simple to operate, has high separation efficiency, good repeatability, high accuracy and wide applicability.

[0005] The method for separating charge heterogeneous species in a protein product based on isocratic elution provided by the present invention comprises the following steps: using ion exchange chromatography with isocratic elution to separate charge heterogeneous species in the protein product.

[0006] The protein product may be a recombinant protein product, which includes a PD-1 antibody. The PD-1 antibody may be a PD-1 monoclonal antibody or a PD-1 polyclonal antibody. The PD-1 monoclonal antibody may be selected from any one of the following: HL02 and HL03.

[0007] The HL02 is trastuzumab produced by Roche, with the trade name Herceptin; the HL03 is rituximab produced by Roche, with the trade name MabThera.

[0008] The protein product may be protein or a processed protein product.

[0009] The protein may be an active protein or an inactivated protein.

[0010] The protein may be a natural protein or an artificial protein.

[0011] As a preferred technical solution of the present invention, the mobile phase used is 90-110mM KH2PO4 solution, and the specific value is 100mM.

[0012] Preferably, NaOH is used to adjust the pH value of the mobile phase to 6.00-6.20, specifically 6.10.

[0013] Preferably, the flow rate of the mobile phase is 0.45-0.55 mL / min, and the specific value is 0.5 mL / min.

[0014] As a preferred technical solution of the present invention, the ion exchange chromatography column used in the ion exchange chromatography is a cation exchange chromatography column with a carboxylic acid group, and the specific cation exchange chromatography column can be BioLC ProPac WCX-10, 4×250 mm, 10 μm.

[0015] As a preferred technical solution of the present invention, the detector used in the ion exchange chromatography is an ultraviolet detector with a detection wavelength of 200-300 nm, specifically 280 nm.

[0016] Preferably, the temperature of the chromatographic column used in the ion exchange chromatography is 30-40°C, specifically 35°C.

[0017] Preferably, the injection volume of the sample in the ion exchange chromatography is 25-35uL, and the specific value is 30uL.

[0018] Preferably, the injection concentration of the sample in the ion exchange chromatography is 2 mg / mL.

[0019] The present invention uses isocratic elution to perform ion exchange to separate the charged heterogeneous species of protein products. Compared with gradient elution, the present invention has the following advantages:

[0020] (1) Fast separation speed and significantly shortened peak elution time;

[0021] (2) Good separation effect, with comparable separation results to gradient elution;

[0022] (3) It has a wide range of applications and is also applicable to the separation and detection of charged heterogeneous species of other PD-1 products.

[0023] The present invention adopts a new idea to perform ion exchange chromatography on monoclonal antibodies, that is, replacing the traditional IEC gradient elution with isocratic elution. This method significantly shortens the detection time and improves work efficiency. Compared with gradient elution, the use of isocratic elution can not only achieve a comparable separation effect, but also shorten the peak time by half; at the same time, it also simplifies the experimental preparation process and saves certain configuration resources. In addition, this method is also applicable to the separation of charged heterogeneous compounds in other PD-1 product projects, which means that isocratic elution ion exchange chromatography has the potential for wide application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the chromatogram of the PD-1 protein product (HL03) after isocratic elution;

[0025] Figure 2 This is the chromatogram of the PD-1 protein product (HL03) after gradient elution;

[0026] Figure 3 This is the chromatogram of the PD-1 protein product (HL02) after isocratic elution;

[0027] Figure 4 This is the chromatogram of the PD-1 protein product (HL02) after gradient elution. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0029] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0030] The PD-1 protein product HL02 used in the following examples is produced by Roche, with the trade name Herceptin and the production batch number N7282B06; HL03 is produced by Roche, with the trade name MabThera and the production batch number H0191B10.

[0031] Embodiment 1:

[0032] (1) Sample preparation: HL02 and HL03 were diluted to 2 mg / mL with ultrapure water. The diluted samples were filtered through a 0.22 um filter membrane and waited for loading;

[0033] (2) Preparation of mobile phase: prepare 100 mM KH2PO4 solution, and accurately adjust the pH value of the solution to 6.10 with NaOH, filter through a 0.22 um filter membrane, and label it for later use;

[0034] (3) Using the mobile phase in step (2), the sample in step (1) is separated and analyzed by high performance liquid chromatography, and the chromatographic conditions are shown in the following table:

[0035] Table 1 HPLC chromatographic parameters - isocratic elution

[0036] High performance liquid phase system Agilent 1260HPLC system Chromatographic columns BioLC ProPac WCX-10,4×250mm,10μm Flow rate 0.5mL / min UV detector wavelength 280nm Injection volume 30μL Column temperature 35℃ Sample tray temperature 8-12℃ Mobile phase 100 mM potassium dihydrogen phosphate, pH = 6.10 Elution method Isocratic elution Elution time 40min

[0037] Figure 1 Table 2 shows the chromatogram of HL03 after isocratic elution. Figure 2 Figure 3 is the chromatogram of HL03 after gradient elution, and Table 4 is the data comparison of HL03 after two elution methods.

[0038] Table 2 HPLC chromatographic parameters - gradient elution

[0039]

[0040]

[0041] Table 3 Comparison of the results of HL03 separation by isocratic elution and gradient elution

[0042]

[0043] From Table 3, we can see that the peak area % of the acid peak, main peak and base peak obtained by the two elution methods are basically the same (in cation exchange chromatography, the area before the main peak is defined as the acid peak, and the area after the main peak is defined as the base peak); in addition, the isocratic elution method can significantly shorten the peak elution time from 15.75min of gradient elution to 6.35min, which shows that the isocratic elution method can not only separate the charge heterogeneous products well, but also greatly reduce the peak elution time, and has the advantage of rapid separation.

[0044] At the same time, the isocratic elution ion exchange chromatography of the present invention is not only applicable to specific protein products. In addition to HL03, this method can also be applied to the separation and analysis of charge isomers of HL02 monoclonal antibody products. Similarly, the charge isomers of HL02 are separated and analyzed according to the isocratic elution chromatography parameters in Table 1 and the gradient elution chromatography parameters in Table 2. Figure 3 This is the chromatogram of HL02 after isocratic elution. Figure 4 is the chromatogram of HL02 after gradient elution, and Table 4 is the comparison of the results of HL02 after isocratic elution and gradient elution.

[0045] Table 4 Comparison of the results of HL02 separation by isocratic elution and gradient elution

[0046]

[0047] Obviously, the ion exchange chromatography using isocratic elution can also be used for the detection and analysis of charge isomers of HL02 and HL03. The above data further confirm that compared with the common gradient elution, the new method of isocratic elution in ion exchange chromatography has the characteristics of rapid separation, which cannot be ignored in biomedical analysis technology; in addition, the isocratic elution ion exchange chromatography provided in the present invention is suitable for the separation of various PD-1 product charge isomers.

[0048] In summary, the ion exchange chromatography used in the present invention changes the commonly used gradient elution mode to isocratic elution, which has the advantages of simplicity, high efficiency, wide applicability, etc.

[0049] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for separating charged heterogeneous species in a protein product, comprising the following steps: separating the charged heterogeneous species in the protein product using isocratic elution ion exchange chromatography; The mobile phase used in the ion exchange chromatography is 90-110 mM KH2PO4 solution; The mobile phase is adjusted to a pH value of 6.00-6.20 using NaOH; The ion exchange chromatography column used in the ion exchange chromatography is a cation exchange chromatography column with a carboxylic acid group; The protein product is a recombinant protein product, and the recombinant protein product includes PD-1 monoclonal antibody; The PD-1 monoclonal antibody is selected from trastuzumab and / or rituximab.

2. The method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.45-0.55 mL / min.

3. The method according to claim 1 or 2, characterized in that: The column temperature of the ion exchange chromatography column used in the ion exchange chromatography is 30-40°C.

4. The method according to claim 1 or 2, characterized in that: The detector used in the ion exchange chromatography is an ultraviolet detector with a detection wavelength of 200-300nm.

5. The method according to claim 1 or 2, characterized in that: The injection volume of the sample in the ion exchange chromatography is 25-35uL.

6. The method according to claim 1 or 2, characterized in that: The injection concentration of the sample in the ion exchange chromatography was 2 mg / mL.

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