Method for purifying antibody coupling medicine

Abstract

The invention discloses a purification method of an antibody coupling medicine. The method comprises the following specific steps: 1) balancing a hydrophobic charge-induced chromatographic column; and 2) loading the crude product of the antibody-coupled drug. And 3) loading the hydrophobic charge-induced chromatographic column, and washing. And (4) recovering an antibody coupling drug product. And 5) regenerating the chromatographic column. The invention is characterized in that a rapid and convenient purification method for the antibody coupling drug is developed. The key point of the method is that the product is directly captured from the antibody-coupled drug crude product in the hydrophobic charge-induced chromatographic column, the high-purity product is recovered by reducing the pH elution buffer solution, and the method has the advantages of simple operation steps and stable process, and is beneficial to quickly obtaining the high-purity antibody-coupled drug product.

Description

Technical Field

[0001] The invention relates to a method for purifying an antibody-coupled drug. Background Art

[0002] Antibody-Drug Conjugate (ADC) is a type of targeted biological agent composed of a specific monoclonal antibody and a specific small molecule cytotoxic drug. By taking advantage of the specific binding characteristics of monoclonal antibodies to target antigens, small molecule cytotoxic drugs are targeted and delivered to the target tumor cell site using monoclonal antibodies as carriers, thereby playing a role in killing tumors. It is known as a "biological missile". ADC drugs combine the advantages of high targeting of antibodies and high anti-tumor activity of cytotoxic drugs. While retaining the tumor-killing properties of small molecule cytotoxic drugs, they selectively reduce the off-target side effects of small molecule cytotoxic drugs, effectively improving the benefit-risk ratio of anti-tumor treatment. In recent years, ADC drugs have been one of the hot research directions in the field of precision tumor treatment. At present, many ADC drugs have been approved for marketing worldwide. With the research on more tumor-specific antigen targets and the in-depth understanding of the release mechanism of tumor cytotoxic drugs, ADC has shown an explosive development trend in the field of tumor treatment.

[0003] ADC also faces some challenges and constraints in its application. ADC has complex composition, difficult process development, low impurity control level, poor drug stability, and extremely high requirements for large-scale production, which also limits its promotion and application in clinical practice. After the antibody is reduced and coupled and small molecule cytotoxic drugs are coupled to prepare ADC, the molecular hydrophobicity of ADC tends to increase, and the molecular stability will also deteriorate, such as easy aggregation and degradation. Due to the high activity of small molecule cytotoxic drugs, the residual small molecule cytotoxic drugs in the coupling production process need to be reduced to an extremely low level in the final product to ensure the safety of the product. The traditional ADC process often uses ultrafiltration steps to remove small molecule cytotoxic drugs, but the ultrafiltration removal efficiency is low, and a large amount of organic solvents are required for cleaning, which is time-consuming, and the removal is often not thorough, and it is also easy to cause product aggregates. There are also reports on the use of hydrophobic chromatography or cationic chromatography to purify ADC, but since both of these chromatography methods cannot avoid the use of solutions with higher salt concentrations, many ADCs are sensitive to salt concentrations and are prone to aggregation or even precipitation.

[0004] Hydrophobic charge induction chromatography is a new type of bio-separation and purification method based on the principle of hydrophobic interaction and electrostatic interaction. Under neutral conditions, hydrophobic interaction can bind the target protein, while under acidic conditions, electrostatic repulsion can achieve elution. This mixed mode chromatography technology avoids the use of high salt concentrations to regulate and control adsorption and elution, and can effectively purify biomacromolecules that are unstable to salt.

[0005] The present invention aims at antibody-drug conjugates, especially antibody-drug conjugates that are unstable at high salt concentrations, and residual small molecule cytotoxic drugs that are difficult to remove, and adopts hydrophobic charge induction chromatography to purify crude antibody-drug conjugates. The hydrophobic charge chromatography column can bind to target molecules at neutral or near-neutral pH and can quickly elute and recover target products by lowering the pH, thereby realizing rapid and efficient purification of antibody-drug conjugates. Summary of the invention

[0006] The purpose of the present invention is to provide a method for purifying an antibody-drug conjugate.

[0007] A method for purifying an antibody-drug conjugate comprises the following steps:

[0008] 1) Washing the hydrophobic charge induction chromatography column with equilibration buffer for 3 to 8 column volumes;

[0009] 2) adjusting the crude antibody-drug conjugate product to a pH of 5 to 7 to obtain a loading solution;

[0010] 3) Loading the sample solution onto a hydrophobic charge induction chromatography column with a loading amount of 20 to 60 g / L filler;

[0011] 4) flushing the chromatography column with equilibration buffer for 3 to 8 column volumes;

[0012] 5) using an elution buffer to elute and recover the product;

[0013] 6) Regenerate the column using regeneration buffer.

[0014] The balance buffer is citric acid or acetate buffer with a pH value of 5 to 7; the loading solution is a crude antibody-drug conjugate; the hydrophobic charge-induced chromatography column is a chromatography column filled with ABI-Bestarose; and the elution buffer is citric acid or acetate buffer with a pH value of 3.0 to 4.5.

[0015] The present invention aims at the difficulties in purifying antibody-drug conjugates, such as easy aggregation and precipitation, and difficulty in removing residual small molecule cytotoxic drugs. The hydrophobic charge induction chromatography technology is used to make full use of the advantage of the hydrophobic charge induction chromatography column that no high concentration of salt needs to be added. The target molecule can be adsorbed by adjusting the pH to a neutral or near-neutral pH, and the high-purity product can be recovered by lowering the pH. The advantages of the present invention are: 1) high adsorption capacity; 2) convenient operation; 3) easy to scale up; 4) no need to add high concentration of salt; 5) conducive to protecting product stability; 6) high purity of the recovered product. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawing is a spectrum of the SEC purity detected by high performance liquid chromatography of the eluted product of the present invention. DETAILED DESCRIPTION

[0017] The present invention provides a method for purifying an antibody-drug conjugate. A hydrophobic charge-induced chromatography column is washed with a balance buffer; a crude antibody-drug conjugate is adjusted to obtain a loading liquid; the loading liquid is loaded onto a hydrophobic charge-induced chromatography column; the chromatography column is washed with a balance buffer; and the product is eluted and recovered with an elution buffer. The method of the present invention does not require the addition of high-concentration salt, avoids product aggregation or precipitation, and the product purity is higher than 97%.

[0018] A method for purifying an antibody-drug conjugate comprises the following steps:

[0019] 1) Washing the hydrophobic charge induction chromatography column with equilibration buffer for 3 to 8 column volumes;

[0020] 2) adjusting the crude antibody-drug conjugate product to a pH of 5 to 7 to obtain a loading solution;

[0021] 3) Loading the sample solution onto a hydrophobic charge induction chromatography column with a loading amount of 20 to 60 g / L filler;

[0022] 4) flushing the chromatography column with equilibration buffer for 3 to 8 column volumes;

[0023] 5) using an elution buffer to elute and recover the product;

[0024] 6) Regenerate the column using regeneration buffer.

[0025] The balance buffer is citric acid or acetate buffer with a pH value of 5 to 7; the loading solution is a crude antibody-drug conjugate; the hydrophobic charge-induced chromatography column is a chromatography column filled with ABI-Bestarose; and the elution buffer is citric acid or acetate buffer with a pH value of 3.0 to 4.5.

[0026] The present invention will be further described below by way of examples:

[0027] Example 1

[0028] Take an ABI-Bestarose chromatography column with a column volume of 6.5 mL, and rinse the chromatography column with 50 mM citric acid equilibration buffer at pH 7.0 for 3 column volumes. Take a slurry containing 0.39 g of antibody-drug conjugates, adjust the pH to 7.0 with Tris buffer, and obtain a loading slurry. Load the loading slurry onto the chromatography column with a loading amount of 60 g / L filler. Rinse the chromatography column with the above-mentioned citric acid equilibration buffer for 8 column volumes. Use 50 mM citric acid elution buffer at pH 3.0 to elute and recover the product, collect the eluted components, and obtain an antibody-drug conjugate product with a SEC purity of 97.7%, and the residual small molecule cytotoxic drug content is below the detection limit.

[0029] Example 2

[0030] Take an ABI-Bestarose chromatography column with a column volume of 6.5 mL, and rinse the chromatography column with a 50 mM acetic acid equilibration buffer at pH 5.5 for 5 column volumes. Take a slurry containing 0.13 g of antibody-drug conjugates, adjust the pH to 5.5 with Tris buffer, and obtain a loading slurry. Load the loading slurry onto the chromatography column with a loading amount of 20 g / L filler. Rinse the chromatography column with the above-mentioned acetic acid equilibration buffer for 3 column volumes. Use a 50 mM acetic acid elution buffer at pH 4.5 to elute and recover the product, collect the eluted components, and obtain an antibody-drug conjugate product with a SEC purity of 98.5%, and the residual small molecule cytotoxic drug content is below the detection limit.

[0031] Example 3

[0032] Take an ABI-Bestarose chromatography column with a column volume of 18.2 mL, and rinse the chromatography column with a 50 mM acetic acid equilibration buffer at pH 5.0 for 3 column volumes. Take a feed solution containing 0.73 g of antibody-drug conjugates, and adjust the pH to 5.5 with Tris buffer to obtain a loading solution. The loading solution is loaded onto the chromatography column, and the loading amount is 40 g / L filler. Rinse the chromatography column with the above-mentioned acetic acid equilibration buffer for 5 column volumes. Use a 50 mM acetic acid elution buffer at pH 4.0 to elute and recover the product, collect the eluted components, and obtain an antibody-drug conjugate product with a SEC purity of 98.2%, and the residual small molecule cytotoxic drug content is below the detection limit.

[0033] Example 4

[0034] Take an ABI-Bestarose chromatography column with a column volume of 18.2 mL, and rinse the chromatography column with 50 mM citric acid equilibration buffer at pH 6.5 for 6 column volumes. Take a slurry containing 0.91 g of antibody-drug conjugates, adjust the pH to 6.5 with Tris buffer, and obtain a loading slurry. Load the loading slurry onto the chromatography column, and the loading amount is 50 g / L filler. Rinse the chromatography column with the above-mentioned citric acid equilibration buffer for 6 column volumes. Use 50 mM citric acid elution buffer at pH 3.5 to elute and recover the product, collect the eluted components, and obtain an antibody-drug conjugate product with a SEC purity of 98.0%, and the residual small molecule cytotoxic drug content is below the detection limit.

[0035] Example 5

[0036] Take an ABI-Bestarose chromatography column with a column volume of 40.2 mL, and rinse the chromatography column with a 50 mM citric acid equilibrium buffer at pH 6.0 for 5 column volumes. Take a slurry containing 1.61 g of antibody-drug conjugates, adjust the pH to 6.0 with Tris buffer, and obtain a loading liquid. Load the loading liquid onto the chromatography column with a loading amount of 40 g / L filler. Rinse the chromatography column with the above-mentioned citric acid equilibrium buffer for 6 column volumes. Use a 50 mM citric acid elution buffer at pH 3.8 to elute and recover the product, collect the eluted components, and obtain an antibody-drug conjugate product with a SEC purity of 98.2%, and the residual small molecule cytotoxic drug content is below the detection limit.

[0037] Example 6

[0038] Take an ABI-Bestarose chromatography column with a column volume of 40.2 mL, and rinse the chromatography column with a 50 mM acetic acid equilibrium buffer at pH 5.8 for 7 column volumes. Take a slurry containing 2.0 g of antibody-drug conjugates, adjust the pH to 5.8 with Tris buffer, and obtain a loading liquid. Load the loading liquid onto the chromatography column, and the loading amount is 50 g / L filler. Rinse the chromatography column with the above-mentioned citric acid equilibrium buffer for 6 column volumes. Use a 50 mM acetic acid elution buffer at pH 4.2 to elute and recover the product, collect the eluted components, and obtain an antibody-drug conjugate product with a SEC purity of 98.7%, and the residual small molecule cytotoxic drug content is below the detection limit.

[0039] Example 7

[0040] Take an ABI-Bestarose chromatography column with a column volume of 155 mL, and rinse the chromatography column with a 50 mM citric acid equilibrium buffer at pH 5.8 for 4 column volumes. Take a slurry containing 3.9 g of antibody-drug conjugates, adjust the pH to 5.8 with Tris buffer, and obtain a loading liquid. Load the loading liquid onto the chromatography column with a loading amount of 25 g / L filler. Rinse the chromatography column with the above-mentioned citric acid equilibrium buffer for 8 column volumes. Use a 50 mM citric acid elution buffer at pH 4.2 to elute and recover the product, collect the eluted components, and obtain an antibody-drug conjugate product with a SEC purity of 98.5%, and the residual small molecule cytotoxic drug content is below the detection limit.

[0041] Example 8

[0042] Take an ABI-Bestarose chromatography column with a column volume of 155 mL, and rinse the chromatography column with a 50 mM citric acid equilibrium buffer at pH 6.8 for 5 column volumes. Take a slurry containing 4.8 g of antibody-drug conjugates, adjust the pH to 6.8 with Tris buffer, and obtain a loading slurry. Load the loading slurry onto the chromatography column, and the loading amount is 31 g / L filler. Rinse the chromatography column with the above-mentioned citric acid equilibrium buffer for 8 column volumes. Use a 50 mM citric acid elution buffer at pH 3.3 to elute and recover the product, collect the eluted fractions, and obtain an antibody-drug conjugate product with a SEC purity of 98.0%, and the residual small molecule cytotoxic drug content is below the detection limit.

[0043] Example 9

[0044] Take an ABI-Bestarose chromatography column with a column volume of 390 mL, and rinse the chromatography column with 50 mM acetic acid equilibration buffer at pH 5.5 for 6 column volumes. Take a slurry containing 11.7 g of antibody-drug conjugates, adjust the pH to 5.5 with Tris buffer, and obtain a loading slurry. Load the loading slurry onto the chromatography column, and the loading amount is 30 g / L filler. Rinse the chromatography column with the above-mentioned citric acid equilibration buffer for 8 column volumes. Use 50 mM acetic acid elution buffer at pH 3.7 to elute and recover the product, collect the eluted components, and obtain an antibody-drug conjugate product with a SEC purity of 99.0%, and the residual small molecule cytotoxic drug content is below the detection limit.

Claims

1. A method for purifying an antibody-drug conjugate, characterized in that The steps include: 1) Washing the hydrophobic charge induction chromatography column with equilibration buffer for 3 to 8 column volumes; 2) adjusting the crude antibody-drug conjugate product to a pH of 5 to 7 to obtain a loading solution; 3) Loading the sample solution onto a hydrophobic charge induction chromatography column with a loading amount of 20 to 60 g / L filler; 4) flushing the chromatography column with equilibration buffer for 3 to 8 column volumes; 5) using an elution buffer to elute and recover the product; 6) Regenerate the column using regeneration buffer.

2. The method for purifying an antibody-drug conjugate according to claim 1, characterized in that The equilibrium buffer is a citric acid or acetic acid buffer with a pH value of 5-7.

3. The method for purifying an antibody-drug conjugate according to claim 1, characterized in that The loading liquid is a crude product of antibody-drug conjugate.

4. The method for purifying an antibody-drug conjugate according to claim 1, characterized in that The hydrophobic charge induction chromatography column is a chromatography column filled with ABI-Bestarose (agarose hydrophobic charge induction chromatography filler with 5-aminobenzimidazole as a functional ligand).

5. The method for purifying an antibody-drug conjugate according to claim 1, characterized in that The elution buffer is citric acid or acetate buffer with a pH value of 3.0 to 4.5.

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