Method for purifying Cys-linked antibody-drug conjugates
Inventors
de Roo, Guy • VERSTEGEN, Ruud Martin • Coumans, Rudy Gerardus Elisabeth
Assignees
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Publication Number
US-10266606-B2
Publication Date
2019-04-23
Expiration Date
Abstract
The present invention relates to a method for purifying a mixture of cysteine-linked antibody-drug conjugates, wherein the amount of non-conjugated antibody is in the range of 0-40% by weight, using hydrophobic interaction chromatography (HIC). The mixture is loaded onto a preparative HIC column using a 0.2-1.5 M aqueous salt solution, in which non-conjugated antibody is collected in a flow-through fraction, followed by elution of a purified mixture of cysteine-linked antibody-drug conjugates using a 0-100 mM aqueous salt solution.
Core Innovation
The disclosed invention relates to a method for obtaining a purified mixture of cysteine-linked antibody-drug conjugates by using preparative hydrophobic interaction chromatography in aqueous salt solutions. A cysteine-linked antibody-drug conjugates mixture of formula (I), containing 10–40% by weight non-conjugated antibody, is provided in a 0.2–1.5 M aqueous salt solution and loaded onto a preparative HIC column.
Non-conjugated antibody is removed by collecting a flow-through fraction that contains non-conjugated antibody. The column is then washed with a 0.2–1.5 M aqueous salt solution while collecting the flow-through fraction, followed by elution using a 0–100 mM aqueous salt solution to obtain a purified mixture of cysteine-linked antibody-drug conjugates.
A central feature is that no organic solvent is added in steps a, b, d, and e, so purification is performed using only aqueous salt solutions. The described approach is tied to purification of cysteine-linked ADC mixtures, including formula (II) with trastuzumab in example contexts, and to characterization of hydrophobic interaction behavior and drug-to-antibody ratio changes, including reduction or removal of DAR0 and increased average DAR in the purified mixture.
Claims Coverage
The document contains one independent claim and multiple dependent claims that narrow the chromatography hardware, salt and buffer selection, pH range, specific ADC composition, and quantitative DAR constraints. The independent claim covers an HIC workflow that separates non-conjugated antibody via flow-through collection and low or zero-salt elution using only aqueous salt solutions and no added organic solvent in specified steps.
Aqueous HIC loading conditions for cysteine-linked ADC mixture
Providing a cysteine-linked antibody-drug conjugates mixture in a 0.2–1.5 M aqueous salt solution, wherein the mixture is of formula (I) and contains 10–40% by weight non-conjugated antibody.
Flow-through collection to remove non-conjugated antibody
Loading the solution onto a preparative hydrophobic interaction chromatography column and collecting a flow-through fraction that contains non-conjugated antibody.
Salt washing and low or zero-salt elution to obtain purified cysteine-linked ADC mixture
Washing the column with a 0.2–1.5 M aqueous salt solution while collecting the flow-through fraction, and eluting with a 0–100 mM aqueous salt solution to obtain a purified mixture of cysteine-linked antibody-drug conjugates.
No added organic solvent in defined process steps
No organic solvent is added in steps a, b, d, and e.
Overall claim coverage centers on preparative HIC purification of cysteine-linked ADC mixtures using specified aqueous salt concentrations for loading, washing, and low or zero-salt elution, with non-conjugated antibody removed in a flow-through fraction and with no added organic solvent in the recited steps. Dependent claims further restrict HIC column packing media, selectable salts, buffered pH ranges, specific ADC composition including trastuzumab under formula (II), and quantitative average DAR ranges.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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