Antibody purification
Inventors
Freichels, Régine • Favre-Bulle, Olivier • Côte, François • Mérot, Bertrand
Assignees
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Publication Number
US-11618769-B2
Publication Date
2023-04-04
Expiration Date
Abstract
The present invention relates to methods for purifying heterologous antibodies from caprine milk, such as goat milk. The present invention includes protein A based affinity chromatography to obtain purified transgenically expressed antibodies, in particular concentrates free from endogenous caprine antibodies.
Core Innovation
The invention provides a method for purifying antibodies using caprine whey comprising a monoclonal heterologous antibody. The method contacts the caprine whey with a protein A containing matrix, separates the protein A containing solid matrix from the caprine whey, and elutes the heterologous antibody from the protein A containing matrix.
Prior to eluting the heterologous antibody, endogenous caprine antibodies are eluted at a pH higher than the pH used for eluting the heterologous antibodies. Endogenous caprine antibodies are eluted at a pH ranging from 4.0 to 5.0, and heterologous antibodies are eluted at a pH ranging from 2.0 to 4.0.
The method further includes equilibrating the protein A containing matrix and/or the caprine whey to a pH ranging from 6 to 7 before contacting the caprine whey with the protein A containing matrix. The disclosed approach is presented as a chromatography workflow that exploits differential elution conditions to separate endogenous caprine antibodies from a monoclonal heterologous antibody, with improvements in heterologous antibody purity and reduced carryover of goat IgG.
Claims Coverage
The independent claim covers a method for purifying a monoclonal heterologous antibody from caprine whey using a protein A containing matrix with a controlled, sequential pH-based elution that separates endogenous caprine antibodies from the heterologous antibody. The inventive features include six main features, with dependent claims narrowing pre-processing and chromatography format and specifying antibody class and exclusions.
Protein A capture and separation from caprine whey
Caprine whey comprising a monoclonal heterologous antibody is contacted with a protein A containing matrix, the protein A containing solid matrix is separated from the caprine whey, and the heterologous antibody is eluted from the protein A containing matrix.
Sequential elution by higher pH for endogenous caprine antibodies
Prior to eluting the heterologous antibody, endogenous caprine antibodies are eluted at a pH which is higher than the pH for eluting the heterologous antibodies.
Endogenous and heterologous elution pH ranges
Endogenous caprine antibodies are eluted at a pH ranging from 4.0 to 5.0 and heterologous antibodies are eluted at a pH ranging from 2.0 to 4.0.
Equilibrating to pH 6 to 7 before contacting protein A matrix
The protein A containing matrix and/or the caprine whey is equilibrated to a pH ranging from 6 to 7 before contacting the caprine whey with the protein A containing matrix.
Packed bed chromatography format
The chromatography is packed bed chromatography.
Heterologous antibody defined as an IgG antibody
The heterologous antibody is an IgG antibody, and is not a caprine antibody.
Across the independent claim and key dependents, the core claim distinguishes endogenous caprine antibodies from a monoclonal heterologous antibody by eluting endogenous caprine antibodies first at a higher pH and eluting heterologous antibodies at lower pH, with protein A matrix and/or whey equilibration at pH 6 to 7. Additional limitations narrow the chromatography format to packed bed and refine the heterologous antibody definition.
Stated Advantages
Reducing goat IgG carryover and enhancing heterologous antibody purity.
Improving resin utilization and throughput by limiting goat IgG binding.
Improved purity and yield are described, including high purity (up to 99%+ antibody protein content) and improved yield/purity.
Documented Applications
Protein-A affinity chromatography for purifying heterologous monoclonal antibodies from caprine/goat milk or whey using a protein A containing matrix with differential pH elution.
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