Methods for purification of antibodies using aliphatic alcohols
This disclosure relates to methods for isolating antibodies from cell-free culture supernatant.
1. A method for isolating a protein from a cell culture supernatant, the method comprising:
a) combining a cell culture supernatant with a divalent cation salt under conditions suitable for the precipitation of impurities in the supernatant to produce a primary supernatant;
b) combining the primary supernatant with an aliphatic alcohol under conditions suitable for forming a protein-containing precipitate and isolating the protein-containing precipitate;
c) re-suspending the protein-containing precipitate in a buffer comprising a divalent cation salt under conditions suitable for the precipitation of impurities therefrom to produce a protein-containing solution; and,
d) combining the protein-containing solution with an aliphatic alcohol under conditions suitable for forming a purified protein-containing precipitate and isolating the purified protein-containing precipitate.
2. The method of claim 1 wherein the aliphatic alcohol is ethanol.
3. The method of claim 1 wherein the conditions of step a) comprise a phosphate concentration of less than or equal to about 6 mM.
4. The method of claim 1 wherein the conditions of step c) comprise a phosphate concentration of less than about 6 mM.
5. The method of claim 1 wherein the conditions in each of the steps comprise a pH of about 6.5 to about 8.5.
6. The method of claim 1 wherein the divalent cation salt is CaCl 2 .
7. The method of claim 1 wherein the conditions of steps a) and c) comprise a conductivity of about 8-80 mS/cm.
8. The method of claim 1 carried out at a temperature of from about −10° C. to about 4° C.
9. The method of claim 1 wherein the conditions of steps b) and d) are selected from the group consisting of a pH of about 6.5, a final concentration (v/v) of aliphatic alcohol of about 25%, and a temperature of about −10° C.
10. The method of claim 1 wherein the protein is an antibody.
11. The method of claim 10 , the method comprising:
a) combining a cell-free culture supernatant with CaCl 2 at about pH 8.5 and removing any resulting precipitate to produce a primary supernatant;
b) combining the primary supernatant with ethanol from a stock solution of less than about 50% over a time period of about 60 minutes or a feeding rate of about 30 μl/min or less at about pH 6.5 and isolating the antibody-containing precipitate;
c) re-suspending the antibody-containing precipitate in a buffer comprising CaCl 2 at about pH 8.5 and removing any resulting precipitate to produce an antibody-containing solution; and,
d) combining the antibody-containing solution with ethanol from a stock solution of less than about 50% over a time period of about 60 minutes or a feeding rate of about 30 μl/min or less at about pH 6.5 and isolating the purified antibody-containing precipitate.
12. The method of claim 1 wherein the cell culture supernatant is a cell-free culture supernatant.
13. A method for isolating a protein from a cell culture supernatant, the method comprising:
a) combining a cell culture supernatant comprising the protein with an aliphatic alcohol under conditions suitable for forming a protein-containing precipitate and isolating the protein-containing precipitate;
b) re-suspending the protein-containing precipitate in a buffer comprising a divalent cation salt under conditions suitable for the precipitation of impurities therefrom to produce a protein-containing solution; and,
c) combining the protein-containing solution with an aliphatic alcohol under conditions suitable for forming a purified protein-containing precipitate and isolating the purified protein-containing precipitate.
14. The method of claim 13 wherein the protein is an antibody.
15. The method of claim 14 , the method comprising:
a) combining a primary supernatant derived from a cell-free culture supernatant with ethanol from a stock solution of less than about 50% over a time period of about 60 minutes or a feeding rate of about 30 μl/min or less at about pH 6.5 and isolating the antibody-containing precipitate;
b) re-suspending the antibody-containing precipitate in a buffer comprising CaCl 2 at about pH 8.5 and removing any resulting precipitate to produce an antibody-containing solution; and,
c) combining the antibody-containing solution with ethanol from a stock solution of less than about 50% over a time period of about 60 minutes or a feeding rate of about 30 μl/min or less at about pH 6.5 and isolating the purified antibody-containing precipitate.
16. The method of claim 13 wherein the cell culture supernatant is a cell-free culture supernatant.
17. The method of claim 13 wherein the aliphatic alcohol is ethanol.
18. The method of claim 13 wherein the divalent cation salt is CaCl 2 .
19. The method of claim 13 wherein the conditions of step b) comprise a phosphate concentration of less than 6 mM.
20. The method of claim 13 wherein the conditions in each of the steps comprise a pH of about 6.5 to about 8.5.