Method for producing a controlled mixture of two or more different antibodies
Inventors
HIBBERT, Richard • de Jong, Rob • Labrijn, Aran Frank • Gerritsen, Arnout • Schuurman, Janine • Parren, Paul
Assignees
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Publication Number
US-12247065-B2
Publication Date
2025-03-11
Expiration Date
Abstract
The present invention relates to a method for controlling the composition of a mixture of two or more different antibodies, such as tow or more different monoclonal antibodies, using chromatography. The mixture is for use as a drug product and the method includes a controlled downstream process for the production of a predetermined ratio of the two or more different antibodies.
Core Innovation
The invention relates to a method for producing an output mixture of two or more different antibodies, where the antibodies differ in their amino acid sequences in a way that enables separation by chromatography. The sequence difference results in a difference in charge properties, hydrophobic properties, and/or affinity for a chromatography resin, so that the antibodies interact differently with the chromatography resin.
In the method, the two or more different antibodies are provided in an input mixture at, or essentially at, not a desired or predetermined concentration ratio. The antibodies are then separated by chromatography, and recovered in amounts required to provide the desired or predetermined concentration ratio in the output mixture.
The disclosure further describes chromatography-based downstream processing in which fractions or pools are used to achieve predefined mixture composition targets, including purity targets for pooled fractions. It also describes using chromatography assays and separability criteria, including resolution and baseline separation behavior in affinity chromatography, together with charge-based separation and affinity-based separation with orthogonal light-chain-binding affinity resins, to obtain controlled output mixture ratios and purity.
Claims Coverage
The provided content identifies one independent claim centered on using amino-acid-sequence differences that enable chromatography separation, starting from an input mixture not at the target ratio, separating by chromatography, and recovering antibodies to reach the predetermined ratio. The summarized dependent claims add quantitative constraints, separability evaluation criteria, chromatography configurations, and antibody substitutions that eliminate binding to specific affinity resins.
Chromatography separation enabled by amino-acid sequence differences that yield predetermined ratio output
A method for producing an output mixture of two or more different antibodies having a difference in their amino acid sequences, wherein the difference results in a difference in charge properties and/or hydrophobic properties and/or affinity for a chromatography resin so that the two or more antibodies interact differently with a chromatography resin, and wherein the two or more different antibodies are present at, or essentially at, a desired or predetermined concentration ratio.
Input mixture not at target ratio, chromatography separation, and recovery to predetermined concentration ratio
The method comprises providing an input mixture wherein the two or more different antibodies are not present at, or essentially at, the desired or predetermined concentration ratio; separating the two or more antibodies by said chromatography; and recovering the two or more antibodies in the amounts required to provide the output mixture.
Fraction purity and pooling at predetermined concentration ratio
Separating into different fractions, selecting fractions having at least 80% purity for one antibody, and pooling selected fractions at a predetermined concentration ratio to recover the output antibody mixture.
Controlled relative abundance and pairwise w/w ratio ranges
Characterized by having the least abundant among two or more different antibodies at least 1–10% (w/w) of the most abundant antibody and/or by having the (w/w) amount ratio between any two antibodies fall between 1:5 and 5:1.
Single chromatography step or predetermined mixture of chromatography resins
Separating the two or more antibodies using either a single chromatography step with one chromatography resin or using a predetermined mixture of chromatography resins at a specified ratio.
Separable antibodies determined using resolution criteria and chromatography assays
Determining that two or more antibodies are separable when they meet defined resolution criteria (Rs thresholds) and are evaluated using specified chromatography assays and either an ionic strength gradient or a pH gradient, or when affinity chromatography shows baseline separation in unbound versus eluting fractions.
Amino acid substitutions to eliminate binding to a specified affinity resin
Modifying antibodies by introducing specified amino acid substitutions in heavy-chain variable regions, light-chain variable regions, kappa light-chain constant regions, CH1 domains, and heavy-chain constant regions so that the resulting antibodies eliminate binding to a specified affinity resin used in chromatography.
The claims focus on producing an output mixture at a desired or predetermined concentration ratio by separating antibodies whose amino-acid-sequence differences cause differential chromatography interactions, then recovering the separated antibodies in required amounts. Additional features constrain fraction purity and abundance or ratio ranges, specify a single resin step or a fixed resin mixture, define separability by resolution and affinity baseline behavior, and describe substitutions aimed at eliminating binding to particular affinity resins.
Stated Advantages
Enables production of an output mixture of two or more antibodies at, or essentially at, a desired or predetermined concentration ratio.
Provides controlled separation and recovery of different antibodies based on sequence-enabled differences in chromatography interactions (charge properties, hydrophobic properties, and/or affinity).
Allows selection and pooling of fractions to achieve controlled purity, including at least 80% purity for one antibody in pooled fractions.
Allows separability to be determined using defined resolution criteria and/or affinity chromatography baseline separation behavior.
Documented Applications
Use of charge-based chromatography separation, including preparative CIEX and analytical CIEX verification, to resolve engineered charge-variant antibody mixtures into controlled output compositions with specified ratio behavior and controlled purity in fractions.
Use of affinity chromatography with orthogonal light-chain-binding resins, including KappaSelect, CaptureSelect KappaXL, Protein L, and LambdaFabSelect, to separate antibody mixtures with controlled mixture ratios after affinity purification.
Use of affinity-resin binding knock-out via point mutations in antibody light-chain/affinity interactions, including kappa V110D and Protein L-affinity S12P, to control which antibodies bind to specific affinity resins during chromatography.
Use of quantitative separability assessment, including analytical cation exchange chromatography and resolution-based criteria, to verify resolved peaks and purity behavior in the separated antibody outputs.
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