NOVEL PURIFICATION OF ANTIBODIES USING HYDROPHOBIC INTERACTION CHROMATOGRAPHY
Disclosed herein are compositions and methods for purifying antibody products from a sample matrix. In particular, the present invention relates to compositions and methods for purifying antibody products employing hydrophobic interaction chromatography media. In certain embodiments, the invention provides a method for reducing process-related impurities (e.g., host cell proteins), as well as product-related substances, including molecular weight variants (e.g., aggregates and fragments of the antibody product).
1 . A method for producing a preparation comprising a process-related impurity-reduced and/or product-related substance-reduced composition comprising a protein of interest from a sample mixture comprising a protein of interest and at least one process-related impurity and/or product-related substance, said method comprising:
(a) subjecting said sample mixture to a hydrophobic interaction media and collecting the unbound fraction;
(b) contacting the said hydrophobic interaction media after loading with a solution that is substantially similar in hydrophobic interaction functionality to the load sample and collecting the wash;
wherein the flow through and/or wash fractions constitute the process-related impurity-reduced and/or product-related substance-reduced composition comprising a protein of interest preparation
2 . The method of claim 1 , wherein prior to subjecting said sample mixture to a hydrophobic interaction chromatography media the sample mixture is subjected to an affinity chromatographic media.
3 . The method of claim 2 , wherein the affinity chromatographic media is a Protein A, G, A/G, or L media.
4 . The method of claim 2 , wherein the affinity chromatographic media is MabSuRe Protein A media.
5 . The method of claim 1 , wherein prior to subjecting said sample mixture to a hydrophobic interaction chromatography media the sample mixture is subjected to an ion exchange chromatography media.
6 . The method of any one of claim 5 , wherein the ion exchange media is selected from a cation exchange media and an anion exchange media.
7 . The method of claim 6 , wherein the ion exchange media is an anion exchange media selected from media comprising diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) group ligands.
8 . The method of claim 6 , wherein the ion exchange media is a cation exchange media selected from media comprising carboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P) and sulfonate(S) ligands.
9 . The method of claim 1 , wherein prior to subjecting said sample mixture to a hydrophobic interaction chromatography media the sample mixture is subjected to a mixed mode chromatography media.
10 . The method of claim 9 , wherein the mixed mode media is CaptoAdhere resin.
11 . The method of claim 1 wherein said antibody is a human antibody, humanized antibody, a chimeric antibody, or a multivalent antibody, or an antigen-binding portion thereof.
12 . The method of claim 1 , wherein said HIC media comprises at least one hydrophobic ligand.
13 . The method of claim 12 , wherein said at least one hydrophobic ligand is selected from the group consisting of alkyl-, aryl-ligands, and combinations thereof.
14 . The method of claim 12 , wherein at least one hydrophobic group is selected from the group consisting of butyl, hexyl, phenyl, octyl, or polypropylene glycol ligands.
15 . The method of claim 12 , wherein said HIC comprises a column.
16 . The method of claim 16 , wherein said column is selected from the group consisting of: Capto Phenyl; Phenyl Sepharose™ 6 Fast Flow with low or high substitution; Phenyl Sepharose™ High Performance; Octyl Sepharose™ High Performance; Fractogel™ EMD Propyl; Fractogel™ EMD Phenyl; Macro-Prep™ Methyl; Macro-Prep™ t-Butyl columns; WP HI-Propyl (C3)™; Toyopearl™ ether, phenyl or butyl; ToyoScreen PPG; ToyoScreen Phenyl; ToyoScreen Butyl; ToyoScreen Hexyl; GE HiScreen Butyl FF; and HiScreen Octyl FF.
17 . The method of claim 1 , wherein said hydrophobic interaction chromatography sample is subjected to a filtration step.
18 . The method of claim 17 , further comprising a depth filtration step, nanofiltration step, ultrafiltration step or absolute filtration step or combination thereof.
19 . A pharmaceutical composition comprising a process-related impurity-reduced and/or product-related substance-reduced composition comprising a protein of interest and a pharmaceutically acceptable carrier
20 . The pharmaceutical composition of claim 19 , wherein said composition is substantially free of product-related aggregates, product-related fragments, and HCPs.