Method of Cleaning and/or Sanitizing a Separation Matrix

Abstract

The present invention concerns a method of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support. The method comprises the steps of: a) optionally purifying a mixture comprising a first immunoglobulin using the separation matrix; b) providing a cleaning liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; and c) cleaning and/or sanitizing the separation matrix by contacting the cleaning liquid with the separation matrix for a predetermined contact time. The alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of Staphylococcus Protein A (SpA), as defined by SEQ ID NO 51 or SEQ ID NO 52, wherein the amino acid residues at positions 13 and 44 of SEQ ID NO 51 or 52 are asparagines and wherein at least the asparagine residue at position 3 of SEQ ID NO 51 or 52 has been mutated to an amino acid selected from the group consisting of glutamic acid, lysine, tyrosine, threonine, phenylalanine, leucine, isoleucine, tryptophan, methionine, valine, alanine, histidine and arginine.

1 . A method of cleaning and/or sanitizing a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the method comprises the steps of:
 a) optionally purifying a mixture comprising a first immunoglobulin using the separation matrix;   b) providing a cleaning liquid comprising at least 50% by volume of an aqueous alkali metal hydroxide solution; and   c) cleaning and/or sanitizing the separation matrix by contacting the cleaning liquid with the separation matrix for a predetermined contact time;   wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of  Staphylococcus  Protein A (SpA), wherein the parental Fc-binding domain of SpA is defined by SEQ ID NO: 53   
       
         
           
                 
                 
               
                     
                   X 1 Q X 2 AFYEILX 3 LP NLTEEQRX 4 X 5 F IX 6 X 7 LKDX 8 PSX 9   
                 
                     
                     
                 
                     
                   SX 10 X 11 X 12 LAEAKX 13  X 14 NX 15 AQ 
                 
             
                
                
                
               
            
           
         
         wherein individually of each other: 
         X 1 =A, Q or is deleted 
         X 2 =E,K,Y,T,F,L,W,I,M,V,A,H or R 
         X 3 =H or K 
         X 4 =A or N 
         X 5 =A, G, S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, such as S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K 
         X 6 =Q or E 
         X 7 =S or K 
         X 8 =E or D 
         X 9 =Q, V or is deleted 
         X 10 =K, R, A or is deleted 
         X 11 =A, E, N or is deleted 
         X 12 =I or L 
         X 13 =K or R 
         X 14 =L or Y 
         X 15 =D, F,Y,W,K or R. 
       
     
     
         2 . The method according to  claim 1 , wherein individually of each other:
 X 1 =A or is deleted   X 2 =E   X 3 =H   X 4 =N   X 6 =Q   X 7 =S   X 8 =D   X 9 =V or is deleted   X 10 =K or is deleted   X 11 =A or is deleted   X 12 =I   X 13 =K   X 14 =L.   
     
     
         3 . The method according to  claim 1 , wherein individually of each other:
 X 1 =A, X 2 =E, X 3 =H, X 4 =N, X 6 =Q, X 7 =S, X 8 =D, X 9 =V, X 10 =K, X 11 =A, X 12 =I, X 13 =K, X 14 =L.   
     
     
         4 . The method according to  claim 1 , wherein individually of each other:
 X 2 =E, X 3 =H, X 4 =N, X 5 =A, X 6 =Q, X 7 =S, X 8 =D, X 12 =I, X 13 =K, X 14 =L and X 15 =D and one or more of X 1 , X 9 , X 10  and X 11  is deleted.   
     
     
         5 . The method according to  claim 1 , wherein individually of each other:
 X 1 =A, X 2 =E, X 3 =H, X 4 =N, X 8 =S,Y,Q,T,N,F,L,W,I,M,V,D,E,H,R or K, X 6 =Q, X 7 =S, X 8 =D, X 9 =V, X 10 =K, X 11 =A, X 12 =I, X 13 =K, X 14 =L and X 15 =D, or alternatively X 1 =A, X 2 =E, X 3 =H, X 4 =N, X 8 =A, X 6 =Q, X 7 =S, X 8 =D, X 9 =V, X 10 =K, X 11 =A, X 12 =I, X 13 =K, X 14 =L and X 15 =F,Y,W,K or R.   
     
     
         6 . The method according to  claim 1 , wherein individually of each other:
 a) X 1 =A or is deleted, X 2 =E, X 3 =H, X 4 =N, X 6 =Q, X 7 =S, X 8 =D, X 9 =V or is deleted, X 10 =K or is deleted, X 11 =A or is deleted, X 12 =1, X 13 =K, X 14 =L;   b) X 1 =A, X 2 =E, X 3 =H, X 4 =N, X 8 =A, X 6 =Q, X 7 =S, X 8 =D, X 9 =V, X 10 =K, X 11 =A, X 12 =I, X 13 =K, X 14 =L and X 15 =D;   c) X 1  is A, X 2 =E, X 3 =H, X 4 =N, X 6 =Q, X 7 =S, X 8 =D, X 9 =V, X 10 =K, X 11 =A, X 12 =I, X 13 =K, X 14 =Land X 15 =D; or   d) X 1  is A, X 3 =H, X 4 =N, X 5 =A, X 6 =Q, X 7 =S, X 8 =D, X 9 =V, X 10 =K, X 11 =A, X 12 =I, X 13 =K, X 14 =L and X 15 =D.   
     
     
         7 . The method according to  claim 1 , wherein the parental Fc-binding domain of SpA comprises a polypeptide having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50. 
     
     
         8 . The method according to  claim 1 , wherein the parental Fc-binding domain of SpA comprises a polypeptide having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. 
     
     
         9 . The method according to  claim 1 , wherein the parental Fc-binding domain of SpA comprises a polypeptide having at least 90%, 95% or 98% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48. 
     
     
         10 . The method according to  claim 1 , wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains are homomultimers selected from the group consisting of dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers and nonamers. 
     
     
         11 . The method according to  claim 1 , wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains each comprise a C-terminal cysteine residue for covalent coupling to the porous support. 
     
     
         12 . The method according to  claim 1 , wherein the multimers of immunoglobulin-binding alkali-stabilized Protein A domains are coupled to the porous support via thioether links. 
     
     
         13 . The method according to  claim 1 , wherein the separation matrix comprises at least 11 mg/ml of the multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to the porous support. 
     
     
         14 . The method according to  claim 1 , wherein the porous support is highly cross-linked agarose beads. 
     
     
         15 . The method according to  claim 1 , wherein the aqueous alkali metal hydroxide solution is sodium hydroxide solution, potassium hydroxide solution or a mixture thereof. 
     
     
         16 . The method according to  claim 1 , wherein the aqueous alkali metal hydroxide solution has a molarity of from 500 mM to 5 M. 
     
     
         17 . The method according to  claim 1 , wherein the cleaning liquid further comprises a C2-C7 alcohol. 
     
     
         18 . The method according to  claim 1 , wherein the cleaning liquid comprises at least 70% by volume aqueous alkali metal hydroxide solution. 
     
     
         19 . The method according to  claim 1 , wherein the predetermined contact time is a time sufficient to provide a 6-log 10 reduction in an endotoxin concentration and/or a microorganism concentration in the separation matrix. 
     
     
         20 . The method according to  claim 1 , wherein the predetermined contact time is from 10 minutes to 50 hours. 
     
     
         21 . The method according to  claim 1 , wherein the steps a)-c) are repeated at least 10 times. 
     
     
         22 . A method of preventing carryover in the purification of immunoglobulins with a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the alkali-stabilized Protein A domains comprise mutants of a parental Fc-binding domain of  Staphylococcus  Protein A (SpA), wherein the parental Fc-binding domain of SpA is defined by  claim 1 ; and wherein the method comprises:
 purifying a first immunoglobulin and cleaning and sanitizing the separation matrix by performing the steps a)-c) of the method of cleaning and/or sanitizing a separation matrix according to  claim 1 ; and further comprises the step of   d) purifying a mixture comprising a second immunoglobulin using the separation matrix, wherein the second immunoglobulin is different from the first immunoglobulin.   
     
     
         23 . The method according to  claim 22 , wherein the separation matrix retains at least 80% of its original dynamic binding capacity after step c). 
     
     
         24 . A method of using a cleaning liquid comprising at least 70% by volume of an aqueous alkali metal hydroxide solution for the sanitization of a separation matrix comprising multimers of immunoglobulin-binding alkali-stabilized Protein A domains covalently coupled to a porous support, wherein the method comprises cleaning and/or sanitizing the separation matrix by containing the cleaning liquid with the separation matrix for a predetermined contact time; and
 wherein the Protein A domains comprise mutants of a parental Fc-binding domain of  Staphylococcus  Protein A (SpA) as defined by  claim 1 .