Isolation and purification of anti-IL-13 antibodies using protein A affinity chromatography
View Patent ↗Disclosed herein are methods for the isolation and purification of anti-IL-13 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 (e.g., Protein A affinity chromatography), 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 antiIL-13 antibody, or antigen-biding fragment thereof, preparation from a sample mixture comprising an anti-IL-13 antibody, or antigen-binding fragment thereof, and at least one HCP, said method comprising:
(a) contacting said sample mixture to Protein A affinity chromatography resin, washing said affinity chromatomphy resin with a buffer comprising 0.5 M NaCl, 20 mM Na citrate, at pH 6, and a buffer comprising 25 mM Tris, 100 mM NaCl, pH 7.2 and collecting an affinity chromatography sample;
(b) subjecting said affinity chromatography sample to a reduction in pH thus forming a reduced pH sample, wherein said reduction in pH is from about 3 to about 4;
(c) adjusting said reduced pH sample to a pH of about 4.5 to about 6.5 and contacting said adjusted pH sample to an ion exchange resin and collecting an ion exchange sample;
(d) 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 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 sample is applied to a cation exchange resin and a cation exchange sample is collected prior to application to the hydrophobic interaction chromatography resin.
5. The method of claim 1 , wherein said ion exchange resin is a cation exchange resin.
6. The method of claim 5 , wherein said cation exchange comprises a substituted matrix wherein the substituents are selected from the group consisting of SO 3 − , carboxymethyl, sulfoethyl, sulfopropyl, phosphate and sulfonate.
7. The method of claim 6 , wherein said substituent is SO 3 − .
8. The method of claim 1 , wherein said ion exchange resin is an anion exchange resin.
9. The method of claim 8 , 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.
10. The method of claim 9 , wherein said substituent is a quaternary amine.
11. The method of claim 1 , wherein said HIC resin comprises a substituted matrix wherein the substituents consist of one or more hydrophobic groups.
12. The method of claim 11 , wherein said substituents are selected from the group consisting of alkyl-, aryl-groups, and a combination thereof.
13. The method of claim 12 , wherein said substituents are selected from the group consisting of: phenyl, 3-octoxypropane-1,2-diol and ether, propyl, methyl, or butyl groups.
14. The method of claim 13 , wherein said resin comprises an agarose matrix comprising phenyl substituents.
15. 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.
16. The method of claim 10 , wherein said anti-IL-13 antibody or antigen-binding portion thereof is a humanized antibody, a chimeric antibody, or a multivalent antibody.
17. The method of claim 16 , wherein said anti-IL-13 antibody or antigen-binding portion thereof is a humanized antibody.
18. The method of claim 1 , wherein said preparation is substantially free of HCPs.
19. The method of claim 1 further comprising a depth filtration step.