Protein a chromatography

Abstract

A novel method for selectively removing leaked protein A from antibody purified by means of protein A affnitiy chromatography is disclosed.

1 . Method of purifying an antibody, preferably an IgG antibody, comprising the steps of: 
 firstly, purifying an antibody by means of protein A affinity chromatography wherein the protein A is a native protein A or a functional derivative thereof,    secondly, loading the purified antibody on an anion exchange material under conditions which allow for binding of the protein A or its derivative and    thirdly, collecting at least 70% of the amount of antibody loaded onto the anion exchange material in the flow-through of the ion exchanger whilst a contaminant protein A is bound to the ion exchange material    
   
   
       2 . Method according to  claim 1 , characterized in the method comprises, after the first anion exchanger step, the step of further purifying said antibody, preferably allowing of removal of aggregated antibody.  
   
   
       3 . Method according to  claim 2 , characterized in the step of further purifying said antibody by removal of aggregated antibody.  
   
   
       4 . Method according to  claim 1 , characterized in that the protein A is a recombinant protein A that is engineered such as to allow of single-point attachement to a column material.  
   
   
       5 . Method according to  claim 4 , characterized in that the recombinant protein A is attached by at least 50% via a thioether bond from a cysteine to the chromatographic support material of the protein A affinity chromatography.  
   
   
       6 . Method according to one of claims  1 - 5 , characterized in that the protein A or its functional derivative is reduced to a concentration of <1 ng/mg IgG in the flow-through of the ion-exchanger.  
   
   
       7 . Method according to one of claims  1 - 5 , characterized in that the antibody has a pI of at least 6.5 or above.  
   
   
       8 . Method according to one of claims  1 - 5 , characterized in that the antibody is a monoclonal antibody, preferably an IgG antibody wherein the IgG antibody may be chimeric or CDR-grafted IgG antibody.  
   
   
       9 . Method according to  claim 8 , characterized in that the IgG antibody is at least in those portions of the antibody that are relevant for binding to a protein A, preferably in its Fc portion that is relevant for binding to protein A, more preferably in the Cγ2-Cγ3 interface region of IgG comprising the binding sites for protein A, of such species origin and IgG subclass origin which origins allow for high affinity binding to protein A.  
   
   
       10 . Method according to  claim 9 , characterized in that the IgG antibody is selected from the group comprising human IgG1, IgG2 and IgG4 with regard to the Fc portion of the antibody.  
   
   
       11 . Method according to  claim 8 , characterized in that the antibody is harvested from a cell culture prior to purifying the antibody by means of protein A affinity chromatography.  
   
   
       12 . Method according to  claim 11 , characterized in that the antibody is harvested from a mammalian cell culture.  
   
   
       13 . Method according to  claim 11 , characterized in that the antibody that is to be purified by means of protein A affinity chromatography is not treated as to inactivate proteases, preferably is not in admixture with at least one protease inhibitor.  
   
   
       14 . Method according to  claim 12 , characterized in that the antibody that is to be purified by means of protein A affinity chromatography is not treated as to inactivate proteases, preferably is not in admixture with at least one protease inhibitor.  
   
   
       15 . Method of purifying an antibody comprising the steps of: 
 firstly, purifying an antibody by means of protein A affinity chromatography wherein the protein A is a native protein A or a functional derivative thereof,    secondly, loading the purified antibody on a first anion exchange material under conditions which allow for binding of the protein A or its derivative,    thirdly, collecting the antibody loaded onto the anion exchange material in the flow-through of the ion exchanger whilst a contaminant protein A is bound to the ion exchange material,    and further purifying the antibody by loading on, binding to and eluting it from a second ion exchanger.    
   
   
       16 . Method according to  claim 15 , characterized in that at least 70% of the amount of antibody loaded onto the first anion exchange material are recovered in the flow-through.  
   
   
       17 . Method according to one  claim 16 , characterized in that the antibody has a pI of at least 7.5 or above.  
   
   
       18 . Method according to  claim 16 , characterized in that the second ion exchanger is a cation exchanger.  
   
   
       19 . Method according to  claim 14 , characterized in that the purified antibody is monomeric antibody and that the second ion exchange step allows of removal of aggregated antibody.  
   
   
       20 . Method according to  claim 1  wherein said anion exchange material is a ceramic matrix anion exchange material.  
   
   
       21 . Method according to  claim 13  wherein said anion exchange material is a ceramic matrix anion exchange material.  
   
   
       22 . Method according to  claim 20  wherein said ceramic matrix comprises quaternary ammonium functional groups.  
   
   
       23 . Method according to  claim 21  wherein said ceramic matrix comprises quaternary ammonium functional groups.  
   
   
       24 . Method according to claims  6 , characterized in that the antibody has a pI of at least 6.5 or above.  
   
   
       25 . Method according to  claim 6 , characterized in that the antibody is a monoclonal antibody, preferably an IgG antibody wherein the IgG antibody may be chimeric or CDR-grafted IgG antibody.  
   
   
       26 . Method according to  claim 7 , characterized in that the antibody is a monoclonal antibody, preferably an IgG antibody wherein the IgG antibody may be chimeric or CDR-grafted IgG antibody.  
   
   
       27 . Method according to  claim 24 , characterized in that the antibody is a monoclonal antibody, preferably an IgG antibody wherein the IgG antibody may be chimeric or CDR-grafted IgG antibody.  
   
   
       28 . Method according to  claim 25 , characterized in that the IgG antibody is at least in those portions of the antibody that are relevant for binding to a protein A, preferably in its Fc portion that is relevant for binding to protein A, more preferably in the Cγ2-Cγ3 interface region of IgG comprising the binding sites for protein A, of such species origin and IgG subclass origin which origins allow for high affinity binding to protein A.  
   
   
       29 . Method according to  claim 26 , characterized in that the IgG antibody is at least in those portions of the antibody that are relevant for binding to a protein A, preferably in its Fc portion that is relevant for binding to protein A, more preferably in the Cγ2-Cγ3 interface region of IgG comprising the binding sites for protein A, of such species origin and IgG subclass origin which origins allow for high affinity binding to protein A.  
   
   
       30 . Method according to  claim 27 , characterized in that the IgG antibody is at least in those portions of the antibody that are relevant for binding to a protein A, preferably in its Fc portion that is relevant for binding to protein A, more preferably in the Cγ2-Cγ3 interface region of IgG comprising the binding sites for protein A, of such species origin and IgG subclass origin which origins allow for high affinity binding to protein A.  
   
   
       31 . Method according to  claim 28 , characterized in that the IgG antibody is selected from the group comprising human IgG1, IgG2 and IgG4.  
   
   
       32 . Method according to  claim 29 , characterized in that the IgG antibody is selected from the group comprising human IgG1, IgG2 and IgG4.  
   
   
       33 . Method according to  claim 30 , characterized in that the IgG antibody is selected from the group comprising human IgG1, IgG2 and IgG4 with regard to the Fc portion of the antibody.  
   
   
       34 . Method according to  claim 25 , characterized in that the antibody is harvested from a cell culture prior to purifying the antibody by means of protein A affinity chromatography.  
   
   
       35 . Method according to  claim 26 , characterized in that the antibody is harvested from a cell culture prior to purifying the antibody by means of protein A affinity chromatography.  
   
   
       36 . Method according to  claim 27 , characterized in that the antibody is harvested from a cell culture prior to purifying the antibody by means of protein A affinity chromatography.  
   
   
       37 . Method according to  claim 34 , characterized in that the antibody is harvested from a mammalian cell culture.  
   
   
       38 . Method according to  claim 35 , characterized in that the antibody is harvested from a mammalian cell culture.  
   
   
       39 . Method according to  claim 36 , characterized in that the antibody is harvested from a mammalian cell culture.  
   
   
       40 . Method according to  claim 34 , characterized in that the antibody that is to be purified by means of protein A affinity chromatography is not treated as to inactivate proteases, and optionally is not in admixture with at least one protease inhibitor.  
   
   
       41 . Method according to  claim 35 , characterized in that the antibody that is to be purified by means of protein A affinity chromatography is not treated with a chemical additive as to inactivate proteases, and optionally is not in admixture with at least one protease inhibitor.  
   
   
       42 . Method according to  claim 36 , characterized in that the antibody that is to be purified by means of protein A affinity chromatography is not treated with a chemical additive as to inactivate proteases, and optionally is not in admixture with at least one protease inhibitor.  
   
   
       43 . Method according to  claim 37 , characterized in that the antibody that is to be purified by means of protein A affinity chromatography is not treated with a chemical additive as to inactivate proteases, and optionally is not in admixture with at least one protease inhibitor.  
   
   
       44 . Method according to  claim 38 , characterized in that the antibody that is to be purified by means of protein A affinity chromatography is not treated with a chemical additive as to inactivate proteases, and optionally is not in admixture with at least one protease inhibitor.  
   
   
       45 . Method according to  claim 39 , characterized in that the antibody that is to be purified by means of protein A affinity chromatography is not treated with a chemical additive as to inactivate proteases, and optionally is not in admixture with at least one protease inhibitor.  
   
   
       46 . Method according to  claim 43 , characterized in that the purified antibody is monomeric antibody and that the second ion exchange step allows of removal of aggregated antibody.  
   
   
       47 . Method according to  claim 44 , characterized in that the purified antibody is monomeric antibody and that the second ion exchange step allows of removal of aggregated antibody.  
   
   
       48 . Method according to  claim 45 , characterized in that the purified antibody is monomeric antibody and that the second ion exchange step allows of removal of aggregated antibody.  
   
   
       49 . Method according to  claim 17 , characterized in that the purified antibody is monomeric antibody and that the second ion exchange step allows of removal of aggregated antibody.