Isolation and purification of antibodies using protein A affinity chromatography
View Patent ↗Disclosed herein are methods for the isolation and purification of antibodies wherein the use of an affinity chromatographic step results in an antibody composition sufficiently pure for pharmaceutical uses. The methods described herein comprise pH viral reduction/inactivation, ultrafiltration/diafiltration, affinity chromatography, preferably Protein A affinity, ion exchange chromatography, and hydrophobic chromatography. Further, the present invention is directed toward pharmaceutical compositions comprising one or more antibodies of the present invention.
1. A method for producing a host cell-protein (HCP) reduced anti-IL-12 antibody, or antigen binding portion thereof, preparation from a sample mixture comprising an anti-IL-12 antibody, or antigen binding portion thereof, and at least one HCP, said method comprising:
(a) subjecting said sample mixture to a reduction in pH thus forming a reduced pH primary recovery sample, wherein said reduction in pH is from about 3 to about 4;
(b) adjusting said reduced pH primary recovery sample to a pH of about 6.0 to about 8 thus forming an adjusted pH primary recovery sample followed by;
(c) contacting said adjusted pH primary recovery sample to a Protein A affinity chromatography resin, washing said affinity chromatography resin with a buffer comprising 0.5 M NaCl, 20 mM citric acid/Na citrate, at pH 6, and collecting an affinity chromatography sample;
(d) contacting said affinity chromatography sample to an ion exchange resin and collecting an ion exchange sample;
(e) contacting said ion exchange sample to a hydrophobic interactive chromatography (HIC) resin and collecting an HIC sample, wherein said HIC sample comprises said HCP-reduced anti-IL-12 antibody, or antigen binding portion thereof, preparation.
2. The method of claim 1 , wherein said reduction in pH is accomplished by admixing a suitable acid with said sample mixture, and wherein said suitable acid is selected from the group consisting of citric acid, acetic acid, and caprylic acid.
3. The method of claim 1 , wherein said Protein A resin comprises Protein A coupled to crosslinked agarose beads.
4. The method of claim 1 , wherein said ion exchange resin is a cation exchange resin.
5. The method of claim 4 , wherein said cation exchange resin comprises a substituted matrix wherein the substituents are selected from the group consisting of SO 3 − , carboxymethyl, sulfoethyl, sulfopropyl, phosphate and sulfonate.
6. The method of claim 5 , wherein said substituent is SO 3 − .
7. The method of claim 1 , wherein said ion exchange resin is an anion exchange resin.
8. The method of claim 7 , wherein said anion exchange resin comprises a substituted matrix wherein the substituents are selected from the group consisting of diethylaminoethyl, quaternary aminoethyl, and quaternary amine groups.
9. The method of claim 8 , wherein said substituent is a quaternary amine.
10. The method of claim 1 , wherein said HIC resin comprises a substituted matrix wherein the substituents consist of one or more hydrophobic groups.
11. The method of claim 10 , wherein said substituents are selected from the group consisting of alkyl-, aryl-groups, and a combination thereof.
12. The method of claim 10 , wherein said substituents are selected from the group consisting of phenyl, 3-octoxypropane-1,2-diol, ether, propyl, methyl, and butyl groups.
13. The method of claim 12 , wherein said resin comprises an agarose matrix comprising phenyl substituents.
14. The method of claim 1 , further comprising a filtration step, wherein said HIC sample is subjected to filtration to remove viral particles and to facilitate buffer exchange.
15. The method of claim 1 , wherein said anti-IL-12 antibody or antigen-binding portion thereof is a humanized antibody, a chimeric antibody, or a multivalent antibody.
16. The method of claim 15 , wherein said anti-IL-12 antibody or antigen-binding portion thereof is a humanized antibody.
17. The method of claim 1 , wherein said preparation is substantially free of HCPs.
18. The method of claim 1 , further comprising depth filtration.