Viral inactivation during purification of antibodies cross reference to related applications
View Patent ↗Described herein are methods for isolating and purifying antibodies from a sample matrix. One aspect of the present disclosure is directed to viral reduction/inactivation of samples generated in the various steps of antibody purification. In a particular aspect, methods herein employ an acidification step followed by one or more chromatography steps. The chromatography steps can include one or more of the following chromatographic procedures: ion exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography.
1. A method for producing a host cell-protein (HCP) reduced antibody, or antigen binding portion thereof, preparation from a sample mixture comprising an antibody, or antigen binding portion thereof, an HCP and a viral particle, wherein said sample mixture has not been exposed to Protein A, and wherein the preparation comprises a decreased number of viral particles or decreased viral activity in comparison to the sample mixture, said method comprising:
(a) subjecting said sample mixture to a reduction in pH thus forming a primary recovery sample, wherein said reduction in pH is to a pH of 3.8 and said reduction is achieved by the addition of citric or phosphoric acid;
(b) adjusting said primary recovery sample to a pH of about 5.0 followed by;
(c) applying said primary recovery sample to an ion exchange resin and collecting an ion exchange sample;
(d) applying 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 ion exchange resin is a cation exchange resin.
3. The method of claim 2 , wherein said cation exchange resin comprises a substituted matrix wherein the substituents are selected from the group consisting of carboxymethyl, sulfoethyl, sulfopropyl, SO 3 − , phosphate and sulfonate.
4. The method of claim 3 , wherein said substituent is SO 3 −.
5. The method of claim 1 , wherein said ion exchange resin is an anion exchange resin.
6. The method of claim 5 , 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.
7. The method of claim 6 , wherein said substituent is a quaternary amine.
8. The method of claim 1 , wherein said ion exchange sample is applied to a second ion exchange resin and a second ion exchange sample is collected prior to application to the hydrophobic interaction chromatography resin.
9. The method of claim 8 , wherein said primary recovery sample is applied to a cation exchange resin and said ion exchange sample is applied to an anion exchange resin.
10. The method of claim 8 , further comprising an intermediate step, wherein said intermediate step is a filtration step occurring after said ion exchange sample is collected and before said ion exchange sample is applied to said second ion exchange resin.
11. The method of claim 10 , wherein said filtration step is accomplished by capture ultrafiltration/diafiltration.
12. The method of claim 1 , wherein said HIC resin comprises a substituted matrix wherein the substituents consist of one or more hydrophobic groups.
13. The method of claim 12 , wherein said substituents are selected from the group consisting of alkyl-groups, aryl-groups, and combinations thereof.
14. The method of claim 12 , wherein said substituents are selected from the group consisting of: phenyl, 3-octoxypropane-1,2-diol, ether, propyl, methyl, and butyl groups.
15. The method of claim 14 , wherein said resin comprises an agarose matrix comprising phenyl substituents.
16. 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.
17. The method of claim 1 , wherein said HCP-reduced antibody, or antigen binding portion thereof, is adalimumab.
18. The method of claim 1 , wherein said HCP-reduced antibody, or antigen binding portion thereof, preparation comprises an anti-IL-12 antibody or an anti-IL-18 antibody or antigen-binding portions thereof.
19. The method of claim 18 , wherein said anti-IL-18 antibody or antigen-binding portion thereof neutralizes IL-18 both in vivo and in vitro.
20. The method of claim 1 , wherein said preparation is substantially free of HCPs.
21. The method of claim 18 , wherein said anti-IL-18 antibody or antigen-binding portion thereof is a humanized antibody, a chimeric antibody, or a multivalent antibody.
22. The method of claim 21 , wherein said anti-IL-18 antibody or antigen-binding portion thereof is a humanized antibody.
23. A method for producing a host cell-protein (HCP) reduced antibody, or antigen binding portion thereof, preparation from a sample mixture comprising an antibody, or antigen binding portion thereof, and at least one HCP, and wherein said sample mixture has not been exposed to Protein A, said method comprising:
(a) subjecting said sample mixture to a reduction in pH thus forming a primary recovery sample, wherein said reduction in pH is to 3.8 and said reduction is achieved by the addition of citric or phosphoric acid;
(b) adjusting said primary recovery sample to a pH of about 5.0 followed by;
(c) applying said primary recovery sample to a cation exchange resin and collecting a cation exchange sample;
(d) applying said cation exchange sample to an anion exchange resin and collecting a anion exchange sample; and
(e) applying said anion 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.
24. The method of claim 23 , wherein said HCP-reduced antibody, or antigen binding portion thereof, is adalimumab.
25. A method for producing a host cell-protein (HCP) reduced antibody, or antigen binding portion thereof, preparation from a sample mixture comprising an antibody, or antigen binding portion thereof, and at least one HCP, and wherein said sample mixture has not been exposed to Protein A, said method comprising:
(a) subjecting said sample mixture to a reduction in pH thus forming a primary recovery sample, wherein said reduction in pH is to 3.8 and said reduction is achieved by the addition of citric or phosphoric acid;
(b) adjusting said primary recovery sample to a pH of about 5.0 followed by;
(c) applying said primary recovery sample to a cation exchange resin and collecting a cation exchange sample;
(d) subjecting said cation exchange sample to filtration and collecting a filtrate;
(e) applying said filtrate from (d) to an anion exchange resin and collecting an anion exchange sample; and
(f) applying said anion 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.
26. The method of claim 25 , wherein said HCP-reduced antibody, or antigen binding portion thereof, is adalimumab.