Depletion of light chain mispaired antibody variants by hydrophobic interaction chromatography
Inventors
von Hirschheydt, Thomas • Rueger, Petra • Weydanz, Birgit • Hertenberger, Hubert
Assignees
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Publication Number
US-11945839-B2
Publication Date
2024-04-02
Expiration Date
Abstract
The present invention relates to methods for separating multispecific CrossMab antibodies from light chain mispaired variants thereof in a solution comprising CrossMab bispecific antibodies and mispaired antibody variants thereof by hydrophobic interaction chromatography.
Core Innovation
The invention concerns separating a multispecific CrossMab antibody from a light chain (LC) mispaired variant thereof using hydrophobic interaction chromatography (HIC). The disclosed approach contacts the multispecific CrossMab antibody and the LC mispaired variant with a HIC medium and separates the multispecific CrossMab antibody by eluting it away from the LC mispaired variant.
The HIC medium is a matrix of particles substituted with ligands, with alternatives in which the particles are substituted with butyl groups, phenyl groups, or polypropylene glycol (PPG) groups. The medium includes defined average particle diameter ranges for each ligand type, and these HIC medium properties govern whether mispaired LC variants are resolved from correctly paired multispecific CrossMab antibody.
The provided examples include characterization and validation of HIC-based separation using SEC-HPLC, HIC-HPLC, SEC-MALS, CE-SDS, and ESI-MS fraction peak assignment. The results are described in terms of separation performance, depletion, yield, and byproduct reduction in product pools, and Toyopearl Hexyl 650c is described as showing breakthrough/low binding and poor separation.
Claims Coverage
The independent claims cover an HIC-based separation method for a multispecific CrossMab antibody from an LC mispaired variant, with the HIC medium defined by ligand-substituted particle matrices using three ligand/particle-size alternatives. The inventive features focus on the contacting/eluting separation scheme and the HIC medium particle size and ligand chemistry that enable depletion and separation of the mispaired LC variant.
HIC medium with butyl-substituted particles
A method using a hydrophobic interaction chromatography (HIC) medium in which the matrix of particles has an average size of 50 μm or less in diameter and the ligands are butyl groups, the method contacting the multispecific CrossMab antibody and the LC mispaired variant with the HIC medium and eluting the multispecific CrossMab antibody to separate it from the LC mispaired variant.
HIC medium with phenyl-substituted particles
A method using a hydrophobic interaction chromatography (HIC) medium in which the matrix of particles has an average size of from 35 μm to 60 μm in diameter and the ligands are phenyl groups, the method contacting the multispecific CrossMab antibody and the LC mispaired variant with the HIC medium and eluting the multispecific CrossMab antibody to separate it from the LC mispaired variant.
HIC medium with PPG-substituted particles
A method using a hydrophobic interaction chromatography (HIC) medium in which the matrix of particles has an average size of from 35 μm to 100 μm in diameter and the ligands are polypropylene glycol groups, the method contacting the multispecific CrossMab antibody and the LC mispaired variant with the HIC medium and eluting the multispecific CrossMab antibody to separate it from the LC mispaired variant.
HIC depletion of mispaired LC variant
A method comprising subjecting a solution comprising the CrossMab antibody and the LC mispaired variant thereof to a hydrophobic interaction chromatography (HIC) step, thereby obtaining the CrossMab antibody depleted of the LC mispaired variant, wherein the chromatographic medium used in the HIC step comprises a matrix of particles substituted with ligands with one of the butyl, phenyl, or polypropylene glycol (PPG) ligand/particle-size alternatives.
Overall, the claims center on performing HIC to separate, including deplete, a multispecific CrossMab antibody from an LC mispaired variant, where the HIC medium is defined by ligand-substituted particle matrices. The independent claims specify three alternatives for ligand chemistry coupled to defined particle diameter ranges.
Stated Advantages
Depleting the CrossMab antibody of the LC mispaired variant thereof.
Separating the multispecific CrossMab antibody from the LC mispaired variant thereof.
Reducing byproducts in product pools.
Documented Applications
Separating multispecific CrossMab antibodies from LC mispaired variants using HIC, with performance characterization by SEC-HPLC, HIC-HPLC, SEC-MALS, CE-SDS, and ESI-MS.
Evaluating HIC media for resolution of mispaired LC variants from correctly paired antibody, including assessment of product pool depletion, yield, and byproduct reduction, and identification of poor separation associated with breakthrough or low binding for Toyopearl Hexyl 650c.
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