Chromatography matrices including novel
View Patent ↗The present invention relates to chromatography matrices including ligands based on one or more domains of immunoglobulin-binding proteins such as, Staphylococcus aureus Protein A (SpA), as well as methods of using the same.
1. An affinity chromatography ligand attached to a solid support, wherein the ligand is based on two or more B domains or two or more Z domains or two or more C domains of Staphylococcus aureus Protein A, each domain having a deletion of at least 4 consecutive amino acids from the N-terminus starting at position 1 corresponding to wild-type B, Z or C domain position and further having a mutation to reduce Fab binding, wherein the ligand exhibits reduced fragmentation, relative to its wild-type counterpart, following exposure to 0.5M NaOH for at least 5 hours.
2. The affinity chromatography ligand according to claim 1 , wherein the mutation to reduce Fab binding is at position 29 corresponding to the wild-type domain position.
3. The affinity chromatography ligand according to claim 2 , wherein the mutation comprises replacing a glycine amino acid residue at position 29 with a lysine amino acid residue when the domain is a B domain or a C domain and replacing an alanine amino acid residue at position 29 with a lysine amino acid residue when the domain is a Z domain.
4. The affinity chromatography ligand according to claim 1 , wherein the ligand is attached to the solid support via multiple point attachment.
5. The affinity chromatography ligand according to claim 1 , comprising at least five C domains, wherein each C domain comprises a deletion of at least 4 consecutive amino acids from the N-terminus starting at position 1 corresponding to wild-type C domain position and further comprises a mutation to reduce Fab binding, wherein the ligand exhibits reduced fragmentation, relative to its wild-type counterpart, following exposure to 0.5M NaOH for at least 5 hours.
6. An affinity chromatography matrix comprising a ligand according to claim 1 or 5 .
7. The affinity chromatography matrix according to claim 6 , wherein the matrix retains at least 95% of its initial binding capacity after 5 hours incubation in 0.5M NaOH.
8. The affinity chromatography matrix according to claim 6 , wherein the matrix retains at least 95% of its initial binding capacity after 25 hours incubation in 0.1M NaOH.
9. The affinity chromatography ligand according to claim 1 , wherein the solid support is selected from the group consisting of controlled pore glass, silica, zirconium oxide, titanium oxide, agarose, polymethacrylate, polyacrylate, polyacrylamide, polyvinylether, polyvinyl alcohol and polystyrene and derivatives thereof.
10. A method of affinity purifying one or more target molecules from a sample, the method comprising the steps of:
a. providing a sample comprising one or more target molecules;
b. contacting the sample with a matrix of claim 6 under conditions such that the one or more target molecules bind to the matrix; and
c. recovering the one or more bound target molecules by elution.
11. The method of claim 10 , further comprising after step (c), a step of cleaning the matrix with sodium hydroxide.
12. The method of claim 11 , wherein steps (a)-(c) of claim 10 are repeated after the cleaning step.