An analytical method for separating antibody and peptide composite preparations

Abstract

The present invention discloses an analytical method for separating antibody and polypeptide composite preparations, which is to perform liquid chromatography analysis using a chromatographic column connected in series with an ion exchange chromatographic column and a SEC chromatographic column. Generally, by distribution determination, it is necessary to first purify the polypeptide and then analyze and determine it, which is time-consuming and labor-intensive. The tandem method of the present invention can meet the determination requirements by a single HPLC exchange separation.

Description

Technical Field

[0001] The invention relates to an analytical method for separating a composite preparation of an antibody and a polypeptide, and belongs to the technical field of biological macromolecule characterization analysis. Background Art

[0002] As biotechnology matures, more and more peptide drugs and antibody drugs are being developed and applied in clinical practice. The synthesis technology of peptide drugs is mature, and it has the advantages of easy synthesis, modification and optimization combination, and its medicinal value can be quickly determined; antibody drugs have high targeting, and can specifically act on disease-related receptors, cell surface molecules or cytokines, reducing the impact on normal cells, improving the safety of drug treatment, and have small toxic side effects, good biocompatibility, and are not easy to cause immune response and rejection. Due to these advantages, antibody drugs and peptide drugs have made remarkable achievements in the field of medicine and have received more and more attention and research. At present, these two drugs have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, hepatitis, diabetes, and AIDS, and have the advantages of wide adaptability, high safety and significant efficacy.

[0003] With the continuous development of science and technology, compound preparations (including compound antibody drugs) have an increasingly important position in the field of medicine. Compound preparation drugs refer to drugs made by mixing two or more therapeutic drugs in a certain proportion or method. Compared with single drugs, this drug may have a wider and more synergistic therapeutic effect in the treatment of diseases. Compound preparation drugs can be a mixture of drugs of different categories, or a mixture of different types of drugs of the same category. Through reasonable formulation, this drug can achieve better therapeutic effects, reduce interactions between drugs, and improve patient compliance. For example, in the treatment of heart disease, it may be necessary to control blood pressure, lower lipids and anticoagulate at the same time. At this time, multiple different types of drugs can be made into compound preparations, so that patients can obtain multiple therapeutic effects in one medication. Similarly, in anti-cancer treatment, compound preparation drugs also have important applications. Drugs targeting different targets can be used in combination to increase the effectiveness of treatment and reduce adverse reactions of drugs. In general, the emergence of compound preparation drugs has enriched treatment options and can better meet the treatment needs of various diseases.

[0004] The advantages of compound preparations are particularly prominent, so quality control is crucial. Only by strictly controlling each production link can the quality of compound preparations be stable, safe and effective. For compound preparations of some antibodies and peptides with strong electrostatic forces, it is usually necessary to purify and separate them step by step, first separating one of the preparations and then measuring them separately, which is time-consuming, labor-intensive and complicated to operate. Therefore, a more direct and convenient analysis method needs to be developed. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a simple and easy-to-operate analytical method for separating antibody and polypeptide complex preparations, and to determine the situation of polypeptide aggregates in the complex preparations through a one-step analysis.

[0006] Technical solution: In order to solve the above technical problems, the present invention provides an analytical method for separating antibody and polypeptide composite preparations, which uses a chromatographic column in which an ion exchange chromatographic column and a SEC chromatographic column are connected in series for liquid chromatography analysis.

[0007] Wherein, the ion exchange chromatography column includes Antibodix WCX NP5; the SEC chromatography column includes SRT-CSEC-300.

[0008] The sample first passes through an ion exchange column and then passes through a SEC column.

[0009] Wherein, the mobile phase A of the liquid chromatography analysis is: 20mM Tris; mobile phase B is: 20mM Tris+0.5M NaCl; mobile phase C is: ACN.

[0010] Wherein, the pH of the mobile phase of the liquid chromatography analysis is 6.0-8.0. The pI of the polypeptide sample is about 5.4, and it is negatively charged in the mobile phase above pH 6.0, and there is no retention on the cation chromatography column (the filler is negatively charged in the system above pH 6.0). The pI of the antibody is about 9.2. The sample is positively charged in the mobile phase system below pH 8.0 and can be retained on the cation chromatography column. Furthermore, the two can be separated by the cation chromatography column, and then separated by SEC. Therefore, the mobile phase range is set within this range.

[0011] Wherein, the elution gradient of the liquid chromatography analysis is 0-20min, the volume fraction of mobile phase A is 80%, the volume fraction of mobile phase B is 0%, and the volume fraction of mobile phase C is 20%; 20-21min, the volume fraction of mobile phase A changes from 0→40%, the volume fraction of mobile phase B changes from 0→40%, and the volume fraction of mobile phase C is 20%; 21-70min, the volume fraction of mobile phase A is 40%, the volume fraction of mobile phase B is 40%, and the volume fraction of mobile phase C is 20%; 70-71min, the volume fraction of mobile phase A changes from 40→80%, the volume fraction of mobile phase B changes from 40→0%, and the volume fraction of mobile phase C is 20%; 71-90min, the volume fraction of mobile phase A is 80%, the volume fraction of mobile phase B is 0%, and the volume fraction of mobile phase C is 20%.

[0012] The detection conditions of the liquid chromatograph are as follows: flow rate: 180 cm / h; detection wavelength: 280 nm; column temperature: 25-35°C.

[0013] Among them, the molecular weight of the antibody in the antibody and polypeptide complex preparation is 80-150kD, and the pI is greater than 8.0; the molecular weight of the polypeptide is 2-4KD, and the pI is less than 7.0. SEC uses molecular weight or size to separate samples, which has an exclusion limit. The SRT-C SEC-300 exclusion limit is , and the SRT-C SEC-300 in the invention can be used to measure aggregates and fragments. Although the polypeptide has a small molecular weight, it is chain-shaped and has a large rotation radius, so it can be better separated on the chromatographic column. The range of pI use is mainly based on the fact that antibodies with larger pI (pI greater than 8.0) are positively charged in the system within the pH range (2-8) that conventional SEC can tolerate, which will form electrostatic effects with the silicon hydroxyl groups (negatively charged) on the surface of the chromatographic column, and there is adsorption, which interferes with the determination of other samples. In the above claim 5, the pH range of 6.0-8.0 is defined. The antibody is positively charged in this pH range and the filler is negatively charged in this pH range by using pI greater than 8. The cation exchange column will retain the antibody sample on the column, which can be washed down by a high salt system later to avoid affecting the identification of other components. Therefore, samples with pI greater than 8 that will produce electrostatic effects are more suitable for this method. The isoelectric point of the polypeptide should be lower than 6.0. In the above claim 5, the pH range of 6.0-8.0 is defined. In order to ensure that the polypeptide and the Antibodix WCX NP5P column are not retained, the isoelectric point of the polypeptide needs to be lower than 6.0. Because the mobile phase pH is higher than 6.0, the polypeptide sample will be negatively charged and repel the ion exchange column (filler is negatively charged), so that the sample can be measured normally on the SEC column.

[0014] Principle of the present invention: Determine the fragmentation of polypeptides in a mixed preparation of antibodies (antibodies with strong electrostatic forces) and polypeptides, the antibody molecular weight is 150kd, the polypeptide is 4kd, and SEC is used to analyze the mixed preparation. The antibody adsorption is strong, the peak distribution is the entire analysis time period, and it interferes with the separation of polypeptides. It is impossible to perform component analysis on the polypeptides in the mixed preparation. The antibody in the mixed preparation can be purified and the polypeptides can be analyzed separately, but the two-step operation will result in a long analysis time and complicated operation. Later, it was found through experiments that the strong adsorption of antibodies on the chromatographic column is mainly caused by the secondary effect of electrostatics. Adding a certain amount of sodium chloride behind the gradient can quickly elute the antibody. Therefore, the present invention develops a method of IEX tandem SEC, based on the slight difference in the isoelectric point of antibodies and polypeptides, the isoelectric point of antibodies is 9.2, and the isoelectric point of polypeptides is 5.4. Under the system of pH 8.0, the polypeptide is not retained on the cationic chromatographic column, and the antibody is retained on the cationic chromatographic column. The antibody can be retained on the chromatographic column by the action of IEX, the polypeptide is separated first, and then the salt system is added to promote the elution of the antibody. The separation of the two is completed.

[0015] When Antibidomix WCX NP5P was used alone, the peptides flowed through and were not retained on the column. Antibodies were retained on the column, but they were used to separate charge heterogeneous species, which is not related to the present invention and was therefore not tested.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The Antibodix ion exchange column is specially designed for the separation of antibody proteins, and the column has the characteristics of high resolution, high column efficiency and high recovery rate. The filler matrix is ​​rigid, spherical, highly cross-linked non-porous polystyrene-divinylbenzene (PS / DVB) particles. A highly hydrophilic, nanometer-thick neutral polymer thin layer is bonded to the surface of the PS / DVB resin. The hydrophobic PS / DVB resin surface is completely covered by this hydrophilic material, thereby eliminating the nonspecific adsorption of PS / DVB to antibody proteins, ensuring that it has a high separation efficiency and biological sample recovery rate.

[0017] Generally, the distribution determination requires the purification of the peptide first and then its analysis and determination, which is time-consuming and labor-intensive. The tandem method can meet the determination requirements through a single HPLC exchange separation.

[0018] The bonded stationary phase of SRT-C SEC chromatographic column is prepared by bonding a uniform nano-thick hydrophilic coating on a high-purity silica matrix with good mechanical stability using surface modification technology. The unique chemical modification technology can accurately control the thickness of the hydrophilic coating, thereby ensuring reliable reproducibility between columns. Due to the use of chemical bonding technology and complete coating coverage, SRT-C SEC has excellent stability, and the uniform surface coating ensures efficient separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The detection profiles of different samples under conventional SEC method are as follows: (a) is antibody; (b) is peptide; (c) is compound preparation; (d) is the overlay of compound preparation and single-labeled peptide and single-labeled antibody;

[0020] Figure 2 The separation effects of different samples in Example 2 are as follows: (a) is the antibody separation effect, (b) is the polypeptide separation effect, (c) is the composite preparation separation effect, and (d) is the overlay of the composite preparation and the single-labeled polypeptide and the single-labeled antibody. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0022] Example 1 Analytical spectra of polypeptide and antibody complex preparations, and single-labeled polypeptides and single-labeled antibodies in a Tris plus 20% ACN system

[0023] 1. Chromatographic conditions:

[0024] (1) Mobile phase A: 20 mM Tris (pH 8.0);

[0025] Mobile phase B: acetonitrile (ACN);

[0026] (2) Sample: humanized anti-CD20 monoclonal antibody (concentration: 2 mg / mL, pI: 9.2, molecular weight approximately 145 kD);

[0027] Human insulin (concentration: 75 mg / mL, pI 5.4, molecular weight about 4KD);

[0028] A combination of peptide and antibody (human insulin peptide: humanized anti-CD20 monoclonal antibody = 2:75, mg / mg);

[0029] (3) Chromatographic column: SRT-C SEC-300 (5 μm, 7.8×300mm);

[0030] (4) Flow rate: 180 cm / h;

[0031] (5) Detector: UV 280nm;

[0032] (6) Column temperature: RT;

[0033] (7) Sample volume: 3uL (antibody), 5uL (peptide), 20μl (peptide monoclonal antibody complex preparation);

[0034] (8) Elution gradient:

[0035] Table 1

[0036] Time / min A％ B％ 0 80 20 150 80 20

[0037] 2. Operation steps:

[0038] (1) Equilibrium: 80% 20 mM Tris (pH 8.0) + 20% ACN, residence time about 30 min;

[0039] (2) Injection: Injection test according to the above injection volume (antibody, peptide, combination of peptide and antibody);

[0040] (3) Isocratic elution: 0-150 min;

[0041] Using the SRT-C SEC-300 column, the composite preparation and its antibody single label, and the peptide single label eluted within 150 minutes. The peptide single label had good symmetry, but the antibody had strong adsorption, and the peak was widened, which caused great interference to the analysis of the peptide. The composite preparation was overlaid with the antibody single label and the peptide single label. The results showed that under this system, the antibodies and peptides in the composite preparation were difficult to separate well under conventional SEC methods ( Figure 1 ).from Figure 1 a It can be seen that in the pure SEC system, the antibody sample has a strong adsorption effect, the peak time is long, and the peak shape is poor. Figure 1 As can be seen from b, the peptide peak is symmetrical, and some aggregates and fragments can be separated, with a good separation effect. Figure 1 c It can be seen that in the composite preparation, the antibody peak interferes with the peak shape of the peptide, which will lead to inaccurate peptide test data and a larger aggregate peak area. There is no baseline separation between the peptide and antibody peaks ( Figure 1 d).

[0042] Table 2

[0043] Sample type Peptide aggregate area ratio Peptide aggregate resolution Peptide single labeling 0.38 1.88 Combination preparations 1.06 0.96

[0044] Example 2 Spectra of analysis results of polypeptide and antibody complex preparations, and single-labeled polypeptides and single-labeled antibodies on tandem chromatographic columns

[0045] 1. Chromatographic conditions:

[0046] (1) Mobile phase A: 20 mM Tris (pH 8.0);

[0047] Mobile phase B: 20 ​​mM Tris + 0.5 M NaCl (pH 8.0);

[0048] Mobile phase C: ACN;

[0049] (2) Sample: humanized anti-CD20 monoclonal antibody (concentration: 2 mg / mL, pI: 9.2, molecular weight approximately 145 kD);

[0050] Human insulin (concentration: 75 mg / mL, pI: 5.4

[0051] Molecular weight about 4KD);

[0052] A combination of peptide and antibody (human insulin peptide: humanized anti-CD20 monoclonal antibody = 2:75, mg / mg);

[0053] (3) Chromatographic column: Use peek tubing to insert SRT-C SEC-300 (5 μm, 7.8×300mm) was connected in series behind Antibodix WCXNP5 (5μm, 4.6×50mm), and the sample was first passed through the ion exchange column and then the SEC column.

[0054] (4) Flow rate: 180 cm / h;

[0055] (5) Detector: UV 280nm;

[0056] (6) Column temperature: RT;

[0057] (7) Sample volume: 3uL (antibody), 5uL (peptide), 20μL (peptide monoclonal antibody complex preparation);

[0058] (8) Elution gradient:

[0059] Table 3

[0060] Time / min A％ B％ C％ 0 80 0 20 20 80 0 20 21 40 40 20 70 40 40 20 71 80 0 20 90 80 0 20

[0061] 2. Operation steps:

[0062] (1) Equilibrium: 80% 20 mM Tris (pH 8.0) + 20% ACN, residence time about 30 min;

[0063] (2) Injection: Injection test according to the above injection volume (antibodies, peptides, and complex preparations of peptides and antibodies);

[0064] (3) Elution: 0-20 min isocratic elution with 80% 20 mM Tris (pH 8.0) + 20% ACN;

[0065] (4) Cleaning: 40% B + 20% C equalization test for 50 min;

[0066] (5) Post-equilibrium: Run for 20 min after 80% A + 20% C phase to prepare for the next injection.

[0067] The first step of elution, 0-20min is mainly to determine the separation of polypeptides. Since the isoelectric point of the polypeptide is 5.4, it is negatively charged under the condition of mobile phase pH 8.0, so it is not retained on the cation exchange column (AntibodixWCXNP5) chromatographic column; while the isoelectric point of the antibody is 9.2, it is positively charged under the mobile phase system of pH 8.0, so it can be retained on the cation exchange column (Antibodix WCXNP5), but the NaCl system is not added from 0 to 20min, so the antibody sample from 0 to 20min is still retained on the column. NaCl is introduced from 21min to 70min. As we all know, NaCl, as a commonly used elution salt for ion exchange, can competitively elute the sample hanging on the column, so the antibody peak can be eluted from 21min to 70min. Therefore, based on this separation principle, the antibody and polypeptide samples can be completely separated to complete the detection of polypeptide aggregates.

[0068] Table 4

[0069] Sample type Peptide aggregate area ratio Peptide aggregate resolution Peptide single labeling 0.37 2.36 Combination preparations 0.34 1.86

[0070] The separation results are shown in Table 3. After testing, the resolution between the two components was 2.2, and the resolution between the mixed sample polypeptide and its aggregate was 1.86.

Claims

1. An analytical method for separating a composite preparation of an antibody and a polypeptide, characterized in that: Liquid chromatography analysis was performed using a chromatographic column in which an ion exchange chromatography column and a SEC chromatography column were connected in series.

2. The analysis method according to claim 1, characterized in that: The ion exchange chromatography column includes Antibodix WCXNP5; the SEC chromatography column includes SRT-C SEC-300.

3. The analysis method according to claim 1, characterized in that: The sample first passes through an ion exchange column and then passes through a SEC column.

4. The analysis method according to claim 1, characterized in that The mobile phase A of the liquid chromatography analysis: 20 mM Tris; mobile phase B: 20 ​​mM Tris+0.5 M NaCl; mobile phase C: ACN.

5. The analysis method according to claim 1, characterized in that: The pH of the mobile phase for the liquid chromatography analysis is 6.0-8.

0.

6. The analysis method according to claim 1, characterized in that: The elution gradient of the liquid chromatography analysis is 0-20 min, the volume fraction of mobile phase A is 80%, the volume fraction of mobile phase B is 0%, and the volume fraction of mobile phase C is 20%; 20-21 min, the volume fraction of mobile phase A changes from 0 to 40%, the volume fraction of mobile phase B changes from 0 to 40%, and the volume fraction of mobile phase C is 20%; 21-70 min, the volume fraction of mobile phase A is 40%, the volume fraction of mobile phase B is 40%, and the volume fraction of mobile phase C is 20%; 70-71 min, the volume fraction of mobile phase A changes from 40 to 80%, the volume fraction of mobile phase B changes from 40 to 0%, and the volume fraction of mobile phase C is 20%; 71-90 min, the volume fraction of mobile phase A is 80%, the volume fraction of mobile phase B is 0%, and the volume fraction of mobile phase C is 20%.

7. The analysis method according to claim 1, characterized in that: The detection conditions of the liquid chromatograph are: flow rate: 180 cm / h; detection wavelength: 280 nm; column temperature: 25-35°C.

8. The analysis method according to claim 1, characterized in that: The molecular weight of the antibody in the antibody and polypeptide composite preparation is 80-150 kD, and the pI is greater than 8.0; the molecular weight of the polypeptide is 2-4 kD, and the pI is less than 7.0.

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