NOVEL PURIFICATION OF HUMAN, HUMANIZED, OR CHIMERIC ANTIBODIES USING PROTEIN A AFFINITY CHROMATOGRAPHY
Disclosed herein are compositions and methods for the isolation and purification of antibodies from a sample matrix. In particular, the present invention relates to compositions and methods for isolating and purifying antibodies exhibiting low or high binding capacity for Protein A resin. In certain embodiments, the methods herein employ a kosmotropic salt solution, an affinity chromatographic step, and may include one or more additional chromatography and/or filtration steps to achieve the desired degree of purification. The present invention is also directed toward pharmaceutical compositions comprising one or more antibodies purified by a method described herein.
1 . A method for producing a host cell-protein (HCP)-reduced antibody preparation from a sample mixture comprising an antibody and at least one HCP, said method comprising:
(a) subjecting said sample matrix to a kosmotropic salt solution thus forming a primary recovery sample;
(b) contacting said primary recovery sample to a Protein A affinity chromatography resin and obtaining a Protein A affinity chromatography eluate sample,
wherein said Protein A affinity chromatography sample comprises an HCP-reduced antibody preparation.
2 . The method of claim 1 , wherein said antibody has weak binding strength and low binding capacity for the affinity chromatography resin.
3 . The method of claim 1 , wherein said antibody is a human antibody, humanized antibody, a chimeric antibody, a multivalent antibody, a dual-variable domain antibody, or an antigen-binding portion thereof.
4 . The method of claim 1 , wherein said antibody in said primary recovery sample that is contacted to said affinity chromatography resin is concentrated such that it has a concentration of from about 1 g/L to about 10 g/L.
5 . The method of claim 1 , wherein the kosmotropic salt in said kosmotropic salt solution is selected from the group consisting of ammonium sulfate, sodium sulfate, sodium citrate, potassium sulfate, potassium phosphate, sodium phosphate, and a combination thereof.
6 . The method of claim 5 , wherein said kosmotropic salt is present in said kosmotropic salt solution at a concentration of from about 0.3 M to about 1.1 M.
7 . The method of claim 1 , wherein said Protein A affinity chromatography resin is selected from any commercial Protein A resins including MabSelect SuRe™, MabSelect, MabSelect SuRe LX, MabSelect Xtra, rProtein A Sepharose Fast Flow, Poros® MabCapture A, Amsphere™ Protein A JWT203, ProSep HC, ProSep Ultra, and ProSep Ultra Plus.
8 . The method of claim 1 , comprising contacting said affinity chromatography eluate sample to:
(a) an ion exchange media and obtaining an ion exchange eluate sample, wherein said ion exchange eluate sample comprises an HCP-reduced antibody preparation;
(b) a hydrophobic interaction chromatography (HIC) media and obtaining a HIC eluate sample, wherein said HIC eluate sample comprises an HCP-reduced antibody preparation; or
(c) a depth filter and obtaining a filtrated sample.
9 . A method for producing a host cell-protein (HCP)-reduced antibody preparation from a sample mixture comprising an antibody and at least one HCP, said method comprising:
(a) concentrating the said sample matrix to obtain a conditioned sample matrix;
(b) contacting said conditioned sample matrix to a Protein A affinity chromatography resin and obtaining a Protein A affinity chromatography eluate sample, wherein said antibody in said conditioned sample matrix that is contacted to said Protein A affinity chromatography resin has a concentration of from about 1 g/L to about 10 g/L; and
wherein said Protein A affinity chromatography eluate sample comprises an HCP-reduced antibody preparation.
10 . The method of claim 9 , wherein said antibody has weak binding strength and low binding capacity for the affinity chromatography resin.
11 . The method of claim 9 , wherein said antibody is a human antibody, humanized antibody, a chimeric antibody, a multivalent antibody, a dual-variable domain antibody, or an antigen-binding portion thereof.
12 . The method of claim 9 , wherein said Protein A affinity chromatography resin is selected from any commercial Protein A resins including MabSelect SuRe™, MabSelect, MabSelect SuRe LX, MabSelect Xtra, rProtein A Sepharose Fast Flow, Poros® MabCapture A, Amsphere™ Protein A JWT203, ProSep HC, ProSep Ultra, and ProSep Ultra Plus.
13 . The method of claim 9 , comprising contacting said affinity chromatography eluate sample to:
(a) an ion exchange media and obtaining an ion exchange eluate sample, wherein said ion exchange eluate sample comprises an HCP-reduced antibody preparation;
(b) a hydrophobic interaction chromatography (HIC) media and obtaining a HIC eluate sample, wherein said HIC eluate sample comprises an HCP-reduced antibody preparation; or
(c) a depth filter and obtaining a filtrated sample.
14 . A pharmaceutical composition comprising an HCP-reduced antibody preparation produced by the method of claim 1 , and a pharmaceutically acceptable carrier.
15 . A method for improving HCP clearance during Protein A capture purification of an antibody from a mixture comprising the antibody of interest and at least one HCP, said method comprising:
(a) subjecting said sample matrix to a kosmotropic salt solution thus forming a primary recovery sample;
(b) contacting said primary recovery sample to a Protein A affinity chromatography resin and obtaining a Protein A affinity chromatography eluate sample,
wherein said Protein A affinity chromatography eluate sample comprises significantly reduced HCP level as compared to that obtained from the typical Protein A capture process in which a kosmotropic salt solution is not used.
16 . The method of claim 15 , wherein said antibody is a human antibody, humanized antibody, a chimeric antibody, a multivalent antibody, a dual-variable domain antibody, or an antigen-binding portion thereof.
17 . The method of claim 15 , wherein the kosmotropic salt in said kosmotropic salt solution is selected from the group consisting of ammonium sulfate, sodium sulfate, sodium citrate, potassium sulfate, potassium phosphate, sodium phosphate, and a combination thereof.
18 . The method of claim 17 , wherein said kosmotropic salt is present in said kosmotropic salt solution at a concentration of from about 0.3 M to about 1.1 M.
19 . The method of claim 15 , wherein said Protein A affinity chromatography resin is selected from any commercial Protein A resins including MabSelect SuRe™, MabSelect, MabSelect SuRe LX, MabSelect Xtra, rProtein A Sepharose Fast Flow, Poros® MabCapture A, Amsphere™ Protein A JWT203, ProSep HC, ProSep Ultra, and ProSep Ultra Plus.
20 . The method of claim 15 comprising contacting said affinity chromatography eluate sample to:
(a) an ion exchange media and obtaining an ion exchange eluate sample, wherein said ion exchange eluate sample comprises an HCP-reduced antibody preparation;
(b) a hydrophobic interaction chromatography (HIC) media and obtaining a HIC eluate sample, wherein said HIC eluate sample comprises an HCP-reduced antibody preparation; or
(c) a depth filter and obtaining a filtrated sample.