The disclosure provides methods for the isolation, separation, and purification of antibodies. The method comprises an affinity chromatography capture step, anion exchange chromatography polishing step, and cation exchange chromatography polishing step.

The invention relates to a method of isolating an immunoglobulin, comprising the steps of: a) providing a separation matrix comprising at least 15 mg/ml multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the porous support comprises cross-linked polymer particles having a volume-weighted median diameter (d50,v) of 56-70 micrometers and a dry solids weight of 55-80 mg/ml; b) contacting a liquid sample comprising an immunoglobulin with the separation matrix; c) washing the separation matrix with a washing liquid; d) eluting the immunoglobulin from the separation matrix with an elution liquid; and e) cleaning the separation matrix with a cleaning liquid comprising at least 0.5 M NaOH.

The present invention relates to a multimodal adsorption medium, in particular a multimodal chromatography medium, a method for its production, as well as use of the adsorption medium according to the invention or an adsorption medium produced according to the invention for the purification of biomolecules.

The present invention relates to a method for separating at least one soluble protein fraction from an aggregated casein-containing material, the method comprises the steps of: (i) providing the aggregated casein-containing material; (ii) Contacting the aggregated casein-containing material with a chromatographic support allowing one or more soluble protein(s) present in the aggregated casein-containing material to be retained by the chromatographic support; (iii) Obtaining a permeate fraction from the chromatographic support comprising aggregated casein; (iv) Optionally washing the chromatographic support; (v) Subjecting the chromatographic support to at least one elution buffer obtaining at least one soluble protein fraction from the chromatographic support; and wherein the chromatographic support comprises one or more mixed-mode ligands capable of binding the soluble proteins from the aggregated casein-containing material.

This invention relates to the application of hydroxyapatite chromatography to the purification of at least one antibody from a preparation containing high molecular weight aggregates. Further, this invention relates to an integration of ceramic hydroxyapatite chromatography into a combination chromatographic protocol for the removal of high molecular weight aggregates from an antibody preparation.

Herein is reported a method for obtaining a polypeptide in monomeric form comprising the steps of a) providing a solution comprising the polypeptide m monomeric form and m aggregated form, wherein the ratio of monomeric to aggregated form is 4:1 or less as determined by size exclusion chromatography, b) performing a mixed-mode chromatography in bind-and-elute mode, or a hydrophobic interaction chromatography in flow-through mode, or a size-exclusion chromatography, and c) performing a weak cation exchange chromatography in bind-and-elute mode or flow-through mode, and thereby obtaining the polypeptide m monomeric form.

The invention describes a method of purifying polysaccharide protein conjugates using mixed mode chromatography. The method involves contacting a crude polysaccharide protein conjugate with a mixed mode resin comprising an inert porous shell and an activated core under conditions of low conductivity that allow binding of the contaminants and collecting the unbound polysaccharide protein conjugate in a flowthrough.

Aspects of the disclosure relate to methods of purifying complexes comprising a protein (e.g., antibody) covalently linked to an oligonucleotide. In some embodiments, complexes comprising a protein covalently linked to an oligonucleotide are purified and isolated from unlinked oligonucleotide using an mixed-mode resin that comprises positively-charged metal sites and negatively charged ionic sites, e.g., hydroxyapatite resin. In some embodiments, complexes comprising a protein covalently linked to an oligonucleotide are purified from a mixture comprising the complexes, unlinked protein, and unlinked oligonucleotide using a purification step involving hydrophobic interaction chromatography resin followed by a purification step involving mixed-mode resin.

The subject invention pertains to proteins are purified by a mixed-mode chromatography system formed by attaching a ligand with cation exchange and hydrophobic 1,3-droxoisoindolin-2-yl group functionalities to a large-pore support matrix, the only linkage between the ligand and the support matrix being a chain having a backbone of one, two, three, four, or five atoms between the hydrophobic group and the support matrix.

This tool utilizes orthogonality concepts used for analytical chromatography and apply them to chromatography for downstream processing applications utilizing a small set of product-agnostic, optimally orthogonal resins with which most separations (capture or polishing) can be accomplished. Libraries of components for separation mediums are provided. The library of components is administered to the separation mediums and combination of the separation mediums at varying pHs, and the separability and orthogonality of each is quantified. The separability is a measure of a probability that the separation mediums will separate a pair of components, whereas the orthogonality is a measure of the enhancement in separability upon addition of another separation medium. By identifying those combinations of separation mediums that not only provide advantageous separability, but also high orthogonality, sets of separation mediums can be more easily provided or wholly designed for use in processing applications.