Dual Affinity Polypeptides for Purification

Abstract

The present invention relates to a process for purification of a target biomolecule, comprising the steps: (a) contacting (i) a target biomolecule, (ii) a dual affinity polypeptide, and (iii) a solid support comprising a catching ligand, wherein the ratio between the equilibrium dissociation constants of the dual affinity polypeptide, [KD,t/KD,s], is at least 10° at standard conditions; and (b) recovering the target biomolecule by elution.

1 - 18 . (canceled) 
     
     
         19 . A process for purification of a target biomolecule, comprising the steps: (a) contacting (i) a target biomolecule, (ii) a dual affinity polypeptide, and (iii) a solid support comprising a catching ligand or dual affinity polypeptide binding site, wherein the ratio between the equilibrium dissociation constants of the dual affinity polypeptide, [K D,t /K D,s ], is at least 10 0  at standard conditions; and (b) recovering the target biomolecule by elution, wherein the target affinity polypeptide and the dual affinity polypeptide are contacted in solution before the mixture is contacting the solid support comprising a catching ligand or dual affinity polypeptide. 
     
     
         20 . The process according to  claim 19 , wherein the solid support is selected from the group comprising solid phase matrices and particles. 
     
     
         21 . The process according to  claim 19 , wherein the dual affinity polypeptide has an equilibrium dissociation constant, K D,t  towards the target biomolecule in the range from 10 −2  to 10 −13  M, more particularly from 10 −4  to 10 −13  M, preferably in the range from 10 −6  to 10 −13  M and an equilibrium dissociation constant, K D,s  towards the catching ligand in the range from 10 −9  to 10 −16  M, preferably in the range from 10 −11  to 10 −16  M. 
     
     
         22 . The process according to  claim 19 , wherein the ratio between the equilibrium dissociation constants of the dual affinity polypeptide, [K D,t /K D,s ], is at least 10 1 , more particularly at least 10 2 , more particularly 10 3 , and even more particularly at least 10 4 . 
     
     
         23 . The process according to  claim 19 , wherein elution of the target is accomplished by changing either of pH, ionic strength, or content of chaotropic ions in solution, or any combinations thereof. 
     
     
         24 . The process according to  claim 19 , wherein the dual affinity polypeptide is a fusion polypeptide, preferably selected from the group consisting of protein A, antibodies, antibody fragments, protein A fragments, protein A derived IgG binding domains, lipocalins, lectins. 
     
     
         25 . The process according to  claim 19 , wherein the ligand binding part of the dual affinity polypeptide is selected from the group consisting of avidin, streptavidin, neutravidin, steroid receptor, antibody, antibody fragment, lipocalins, lectins, amyloglucosidase, cellulose binding domains. 
     
     
         26 . The process according to  claim 24 , wherein the antibody is selected from the group consisting of llama and camel antibodies. 
     
     
         27 . The process according to  claim 24 , wherein the fusion polypeptide is made by fusion of at least one IgG binding domain of protein A or protein A derived IgG binding domain and at least one biotin binding domain of avidin, streptavidin, or neutravidin. 
     
     
         28 . The process according to  claim 19 , wherein the ligand is selected from the group consisting of biotin, acarbose, steroids, hapten, epitope-peptides, dyes, and enzyme inhibitors. 
     
     
         29 . The process according to  claim 27 , wherein the catching ligand attached to the solid support is biotin and the target biomolecule is IgG. 
     
     
         30 . The process according to  claim 19 , wherein the solid support is a solid phase matrix, preferably selected from the group consisting of agar-agar, agaroses, celluloses, cellulose ethers, carboxymethyl cellulose, polyamides, polyvinylalcohols, silicas, and controlled pore glasses. 
     
     
         31 . The process according to  claim 24 , wherein the fusion polypeptide is produced as a recombinant polypeptide in a recombinant host cell. 
     
     
         32 . The process according to  claim 31 , wherein the fusion polypeptide and the target biomolecule is expressed in the same type of host cell. 
     
     
         33 . The process according to  claim 31 , wherein the host cell is selected from the group consisting bacterial cells, fungal cells, mammalian cells, plant cells, and insect cells. 
     
     
         34 . The process according to  claim 19 , wherein the dual affinity polypeptide is chemically fused. 
     
     
         35 . A process for purification of a target biomolecule, comprising the steps: (a) contacting (i) a target polypeptide, (ii) a dual affinity polypeptide, and (iii) a solid support comprising a catching ligand, wherein the dual affinity polypeptide has an equilibrium dissociation constant, K D,t  towards the target biomolecule in the range from 10 −2  to 10 −13  M, preferably from 10 −4  to 10 −13  M, more preferred from 10 −6  to 10 −13  M at standard conditions, and wherein binding of the dual affinity polypeptide to the catching ligand on the solid support is provided by cleavage of a para-substituted benzyl guanine resulting in a thioether bond; and (b) recovering the target biomolecule by elution, where the target polypeptide and the dual affinity polypeptide are contacted in solution before the mixture is contacting the solid support comprising a catching ligand.