ADC (antibody-drug conjugate) cation exchange chromatographic purification method

An antibody-drug-conjugated, cation-exchange technology, applied in the field of protein separation and purification, to achieve the effects of reducing production costs, controlling risks, expanding the scope of application, and being easy to operate

Active Publication Date: 2014-12-17
BEIJING MABWORKS BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, regardless of random coupling or site-specific coupling techniques, there is a problem of protein aggregation during the modified coupling reaction.

Method used

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  • ADC (antibody-drug conjugate) cation exchange chromatographic purification method
  • ADC (antibody-drug conjugate) cation exchange chromatographic purification method
  • ADC (antibody-drug conjugate) cation exchange chromatographic purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] A cation-exchange chromatography purification method for antibody-conjugated drugs, using cation-exchange chromatography to purify antibody-conjugated drugs, controlling the small molecule / antibody coupling ratio (DAR) and polymers, and the purification method includes the following steps:

[0037] Adjust the ADC sample and equilibration buffer to pH 4-7, conductance 2-8 mS / cm, and select bind-elute mode or overload mode for purification.

[0038] Further, the loading amount of the overload mode is not less than 300 mg / mL medium; the process of the overload mode includes: equilibrating the buffer to balance the chromatography medium, and then loading the antibody-conjugated drug, and waiting for the ADC sample to be adsorbed After saturation and start of flow-through, the target protein flow-through with DAR value and multimer content meeting the requirements is collected, and impurity multimers and proteins with high DAR value are adsorbed on the chromatographic column....

Embodiment 2

[0049] Example 2 Preparation of antibody-drug conjugate (ADC)

[0050]Preparation of lysine-coupled ADC samples: Before performing the modification reaction, replace the antibody (Ab) stock solution with the modification reaction buffer solution. Add bifunctional cross-linking reagent SMCC (linker) to modify lysine, and the reaction lasts for 2-3 hours. After the modification reaction, the SMCC not linked to the antibody was removed by G25 Sephadex chromatography or tangential flow filtration (UF / DF) system, and the modified intermediate product Ab-MCC was replaced with the coupling buffer ( pH 5-6). Then add the small molecule toxin DM1, couple with the linker on the antibody, and react for 3-15 hours to obtain a spare lysine-coupled ADC sample (Ab-DM1). The DAR value measured by UV spectrophotometry is 3.7-3.8 .

[0051] Preparation of cysteine-coupled ADC samples: Replace Ab stock solution into reaction buffer (pH 7-8). Add the reducing agent TCEP to react for 1-2 hou...

Embodiment 3

[0053] Embodiment 3 DAR value is measured

[0054] The obtained ADC sample was prepared in Example 2, and the coupling ratio of small molecule drug / antibody was determined by ultraviolet (UV) spectrophotometry. According to the different extinction coefficients of antibodies and drugs at different wavelengths, the drug-antibody coupling ratio (DAR) was derived from the binary linear equation. Specifically: dilute the antibody conjugate to 0.3-0.5 mg / mL, and scan the absorbance of the diluted ADC sample at a wavelength of 230-330 nm. The maximum absorption peak of the antibody is at 280 nm, the maximum absorption peak of the small molecule DM1 is at 252 nm, and the maximum absorption peak of the small molecule MMAE is at 248 nm. For the Ab-DM1 sample, the absorbance at 252 nm and 280 nm were respectively selected, and for the Ab-MMAE sample, the absorbance at 248 nm and 280 nm were respectively selected. According to the literature (Yu Chuanfei et al. Drug in an antibody dru...

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Abstract

The invention provides an ADC cation exchange chromatographic purification method. The ADC cation exchange chromatographic purification method can be used for performing purification in a combined elution mode or an overload mode, control the DAR (drug antibody ratio) of ADC drugs within a target technology range and meanwhile achieve the effects of eliminating polymers. The ADC cation exchange chromatographic purification method is a novel ADC preparation and purification technique, reduces the production cost and risk, meanwhile, achieves control to the mole DAR of micromolecules / antibodies and polymers of the ADC drugs, is beneficial to development of ADC production processes with higher controllability, lower cost and lower risks, obtains products with higher quality and guarantees medication safety and therapeutic effects.

Description

technical field [0001] The invention belongs to the technical field of protein separation and purification, in particular, the invention relates to a cation exchange chromatography purification method of an antibody-coupled drug (ADC). [0002] technical background [0003] Antibody-drug conjugates (ADCs) are a new generation of antibody-targeted therapeutic drugs, which are mainly used in the treatment of cancer tumors. ADC drugs are composed of three parts: small molecule cytotoxic drug (Drug), antibody (Antibody) and linker (Linker) connecting antibody and chemical drug. Small molecule toxin is bound to antibody protein by chemical coupling method. Antibodies will specifically recognize and guide small-molecule drugs to cancer cell targets expressing cancer-specific antigens, and enter cancer cells through endocytosis. The linker part is broken under the action of intracellular low pH value environment or lysosomal protease, releasing small molecule cytotoxin, so as to ...

Claims

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Application Information

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IPC IPC(8): A61K47/48B01D15/36
Inventor 刘慧芳黎晓维金春阳郜富权谢晨颖李锋
Owner BEIJING MABWORKS BIOTECH
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