Antibody purification

Abstract

This invention relates to the application of hydrophobic interaction chromatography combination chromatography to the purification of antibody molecule proteins.

1. A method for purifying monomeric IgG antibody from a mixture comprising said monomeric antibody and at least one of immunoglobulin aggregates, mis-folded species, host cell protein or protein A comprising contacting said mixture with a hydrophobic interaction chromatographic support and selectively eluting the monomer from the support. 
     
     
       2. The method according to  claim 1  wherein the IgG is selected from the group consisting of anti-RSHZ-19 and CH-CD4. 
     
     
       3. The method according to  claim 1  wherein the HIC support is selected from the group consisting of alkyl C2-C8  agarose, aryl-agarose, alkyl-silica, aryl-silica alkyl organic polymer resin and aryl organic polymer resin. 
     
     
       4. The method according to  claim 3  wherein the support is selected from the group consisting of butyl-, phenyl- and octyl-agarose and butyl-, phenyl- and ether- organic polymer resin. 
     
     
       5. The method according to  claim 4  wherein the support is phenyl-organic polymer resin. 
     
     
       6. The method according to  claim 4  wherein the support is butyl-organic polymer resin. 
     
     
       7. The method according to  claim 1  wherein the antibody is selectively eluted with a low salt buffer. 
     
     
       8. The method according to  claim 7  wherein the antibody is selectively eluted with a gradient decreasing in salt to 50 mM phosphate, pH7.0. 
     
     
       9. A method for the purification of an IgG antibody from conditioned cell culture medium containing same comprising sequentially subjecting the medium to (a) Protein A affinity chromatography, (b) ion exchange chromatography, and (c) hydrophobic interaction chromatography. 
     
     
       10. The method according to  claim 9   A method for the purification of an IgG antibody from conditioned cell culture medium containing same comprising sequentially subjecting the medium to ( a )  Protein A affinity chromatography,  ( b )  ion exchange chromatography, and  ( c )  hydrophobic interaction chromatography  wherein the ion exchange chromatography employs a support selected from the group consisting of CM-23-, CM-32-, CM-52- cellulose; CM  carboxymethyl- and, SP  sulfopropyl-cross-linked dextrans, CM  carboxymethyl- and S-argose  sulfonate- agarose ; CM  carboxymethyl-organic polymer resin; DEAE-QAE-Q  diethylaminoethyl- quaternary aminoethyl - quaternary amine -cross-linked dextians  dextrans; DEAE-,QAE-,Q  diethylaminoethyl- , quaternary aminoethyl - , quaternary amine -linked agarose; and DEAE  diethylaminoethyl-organic polymer resins and is by a  buffered by a salt solution. 
     
     
       11. The method according to  claim 10  wherein the support is CM  carboxymethyl-agarose Fast Flow  and the salt is NaCl. 
     
     
       12. The method according to  claim 10  wherein the buffered salt solution is 40 mM citrate containing, 100 mM, NaCl, pH 6.0. 
     
     
       13. The method according to  claim 9  wherein the hydrophobic interaction chromatographic employs a support selected from the group consisting of alkyl C2-C8 -agarose, aryl-agarose, alkyl-silica, aryl-silica, alkyl-organic polymer resin and aryl-organic polymer resin. 
     
     
       14. The method according to  claim 13  wherein the support is selected from the group consisting of butyl-, phenyl- and octyl-agarose and butyl-, phenyl- and ether-organic polymer resin. 
     
     
       15. The method according to  claim 14  wherein the support is phenyl-organic polymer resin or butyl-organic polymer resin. 
     
     
       16. The method according to  claim 9  wherein the support is phenyl- or butyl-organic polymer resin and the antibody is selectively eluted with a low salt buffer. 
     
     
       17. The method according to  claim 16  wherein the antibody is selectively eluted with a gradient decreasing to 50 mM sodium phosphate buffer, pH 7.0. 
     
     
       18. The method according to  claim 9  wherein the Protein A chromatography employs as a support Protein A linked to controlled pore glass and elution is by a low pH buffer. 
     
     
       19. The method according to  claim 18  wherein said buffer is 25 mM citrate, pH 3.5. 
     
     
       20. A method for purifying antibody from a conditioned cell medium comprising:
 (a) adsorbing the antibody onto a Protein A chromatographic support;    (b) washing the adsorbed antibody with at least one buffer;    (c) eluting the antibody from step (b);    (d) adsorbing the antibody from step (c) onto an ion exchange chromatographic support;    (e) washing the adsorbed antibody with at least one buffer;    (f) selectively eluting the antibody from step (e);    (g) adsorbing the eluate of step (f) onto a hydrophobic interaction chromatographic support;    (h) washing the adsorbed antibody with at least one buffer;    (i) eluting the adsorbed antibody; and    (j) recovering the antibody.    
     
     
       21. The method according to  claim 20  which includes one or more optional steps of inactivating viruses if present. 
     
     
       22. The method according to  claim 21  wherein a viral inactivation step is performed after step (f) and before step (g). 
     
     
       23. The method according to  claim 22  wherein said viral inactivation step comprises treatment of the eluate with guanidine hydrochloride for a period of time sufficient to inactivate virus followed by the addition of an ammonium sulfate solution. 
     
     
       24. The method according to  claim 23  wherein the guanidine hydrochloride is present at 2.0M and following treatment eluate from step (f) is adjusted to 1.3M ammonium sulfate. 
     
     
       25. The method according to  claim 22  wherein an additional viral inactivation step is performed after step (c) and before step (d). 
     
     
       26. The method according to  claim 25  wherein said additional viral inactivation step comprises treatment of the eluate of step (c) with acid. 
     
     
       27. The method according to  claim 26  wherein the pH of the eluate is adjusted to pH 3.5 and maintained at that pH for a period of time sufficient to inactivate virus, and terminating the treatment by adjusting the pH to 5.5. 
     
     
       28. The method according to  claim 20  wherein the ion exchange support of step (d) is selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate(P), diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quarternary (Q), substituted cellulosic resins, cross linked dextrans, agarose and organic polymer resins. 
     
     
       29. The method according to  claim 28  wherein the cationic support is CM-agarose. 
     
     
       30. The method according to  claim 20  wherein the hydrophobic interaction chromatographic support is selected from the group consisting of alkyl  C2-C8 -agarose, aryl-agarose, alkyl-silica, aryl-silica, alkyl-organic polymer resin and aryl-organic polymer resins. 
     
     
       31. The method according to  claim 30  wherein the support is selected from the group consisting of butyl-, phenyl- and octyl-agarose and phenyl-, ether- and butyl-organic polymer resins. 
     
     
       32. The method according to  claim 31  wherein the support is phenyl- or butyl-organic polymer resins. 
     
     
       33. The method according to  claim 20  wherein said protein is recovered by pooling and concentrating the protein containing fractions from chromatography step (i) by ultrafiltration. 
     
     
       34. The method according to  claim 20  wherein the chromatographic support of step (a) is Protein A linked to controlled pore glass. 
     
     
       35. The method according to  claim 20  wherein the absorbed antibody of step (h) is washed with two buffers, a first equibration buffer and a second low pH wash buffer. 
     
     
       36. The method according to  claim 35  wherein the pH of the second buffer is less than 4.0. 
     
     
       37. The method according to  claim 36  wherein the second buffer is 1M ammonium sulfate, 50 mM sodium citrate, pH 3.5. 
     
     
       38. A method of removing Protein A from a mixture comprising Protein A and IgG antibodies comprising contacting the mixture with a hydrophobic interaction chromatography support and selectively eluting the antibody from the support. 
     
     
       39. The method according to  claim 38  which includes washing the support prior to elution with a buffer having a pH less than 7.0. 
     
     
       40. The method according to  claim 39  wherein the pH of the wash buffer is less than 4.0. 
     
     
       41. The method according to  claim 40  wherein the buffer is (1M ammonium sulfate, 50 mM sodium citrate, pH 3.5). 
     
     
       42. A method for purifying monomeric IgG antibody from a mixture comprising said monomeric IgG antibody and at least one of immunoglobulin aggregates, misfolded species, and protein A, wherein said method comprises the steps of ( i )  contacting said mixture with a hydrophobic interaction chromatographic support and  ( ii )  selectively eluting the monomeric IgG antibody from the support wherein the monomeric IgG antibody is selectively eluted with a low salt buffer gradient decreasing in salt to  50  mM phosphate, pH  7 . 0 .