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. A method of purifying one or more immunoglobulins from a sample, the method comprising the steps of:
a. providing a sample comprising one or more immunoglobulins;
b. contacting the sample with a matrix under conditions such that the one or more immunoglobulins bind to the matrix, wherein the matrix comprises an affinity chromatography ligand attached to a solid support and 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 3 or 4 consecutive amino acids from the N-terminus starting at position 1 or 2 corresponding to wild-type B, Z or C domain positions and further having a mutation to reduce Fab binding; and
c. recovering the one or more bound immunoglobulins by elution.
2. The method of claim 1 , wherein the sample is selected from the group consisting of a cell culture liquid, a cell culture supernatant and a fermentation broth.
3. The method of claim 1 , wherein the mutation to reduce Fab binding comprises the replacement of glycine amino acid residue at position 29 with a lysine amino acid residue in case of the B domain and the C domain and the replacement of the an alanine amino acid residue at position 29 with a lysine amino acid residue in case of the Z domain.
4. The method of claim 1 , wherein the ligand exhibits reduced fragmentation compared to its wild-type counterpart following exposure to 0.5M NaOH for at least 5 hours.
5. The method of claim 1 , wherein the matrix retains at least 95% of its initial binding capacity after 5 hours incubation in 0.5M NaOH.
6. The method of claim 1 , wherein the matrix retains at least 95% of its initial binding capacity after 25 hours incubation in 0.1M NaOH.
7. A method of separating an immunoglobulin from one or more of host cell proteins (HCPs), DNA, viruses, endotoxins, nutrients, one or more components of a cell culture medium and product related impurities, the method comprising the steps of:
a. providing a sample comprising an immunoglobulin and one or more of host cell proteins (HCPs), DNA viruses, endotoxins, nutrients, one or more components of a cell culture medium and product related impurities;
b. contacting the sample with a matrix under conditions such that the immunoglobulin binds to the matrix, wherein the matrix comprises an affinity chromatography ligand attached to a solid support and 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 3 or 4 consecutive amino acids from the N-terminus starting at position 1 or 2 corresponding to wild-type B, Z or C domain positions and further having a mutation to reduce Fab binding; and
c. recovering the bound immunoglobulin by elution,
thereby to separate the immunoglobulin from one or more of host cell proteins (HCPs), DNA, viruses, endotoxins, nutrients, one or more components of a cell culture medium and product related impurities.
8. The method of claim 7 , wherein the sample is a fermentation broth.
9. The method of claim 7 , wherein the mutation to reduce Fab binding comprises the replacement of glycine amino acid residue at position 29 with a lysine amino acid residue in case of the B domain and the C domain and the replacement of the an alanine amino acid residue at position 29 with a lysine amino acid residue in case of the Z domain.
10. The method of claim 7 , wherein one or more components of a cell culture medium are selected from the group consisting of antifoam agents and antibiotics.
11. The method of claim 7 , wherein the product related impurities comprise misfolded species and aggregates.
12. The method of claim 1 , wherein the sample is a clarified cell culture broth.
13. The method of claim 7 , wherein the sample is a clarified cell culture broth.
14. The method of claim 7 , wherein the matrix retains at least 95% of its initial binding capacity after 5 hours incubation in 0.5M NaOH.
15. The method of claim 7 , wherein the matrix retains at least 95% of its initial binding capacity after 25 hours incubation in 0.1 M NaOH.
16. The method of claim 7 , wherein the ligand exhibits reduced fragmentation compared to its wild-type counterpart following exposure to 0.5M NaOH for at least 5 hours.