Purified vegetarian protein A and process for production thereof
Purification of vegetarian (non-animal derived) Protein A using a multidimensional purification process to remove undesirable non-animal derived component impurities in the Protein A from the non-animal fermentation media used to produce the Protein A so as to produce a vegetarian Protein A free of animal-origin components and essentially free of components derived from non-animal derived growth media.
1 . A non-animal derived Protein A purification process that comprises:
(a) providing a non-animal derived Protein A solution that has been produced by fermenting a secretor strain of Staphylococcus aureus in a media containing vegetarian amino acids or peptides, said media being free of animal products;
(b) loading the filtered Protein A solution onto a first column;
(c) washing the column with a buffer-salt mixture to rinse off the column undesirable color and contaminating proteins;
(d) eluting the Protein A from the first column to produce a Protein A solution pool;
(e) adjustment of the pH and conductivity of the Protein A solution pool;
(f) loading the Protein A from the Protein A solution pool onto a second column;
(g) washing the second column with a buffer-salt mixture to rinse off the column undesirable color and contaminating proteins; and
(h) eluting the Protein A from the second column to produce a purified Protein A solution;
wherein the first column is selected from (1) the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column, or (2) a cation exchange column; and the second column is a cation exchange column when the first column is a column selected from the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column, and the second column is a column selected from the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column when the first column is a cation exchange column.
2 . A non-animal derived Protein A purification process according to claim 1 wherein the first column is selected from the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column; and the second column is a cation exchange column.
3 . A purification process according to claim 1 comprising the additional step:
(i) monitoring the absorbance of each fraction of purified Protein A of out of the first column and the second column at 275 nm and at 340 nm to obtain a A275/A340 ratio in order to choose appropriate fractions for pooling.
4 . A purification process according to claim 2 , wherein the first column is an anion exchange column.
5 . A purification process according to claim 2 , wherein the first column is a HIC column.
6 . A purification process according to claim 2 , wherein the first column is a ceramic hydroxyapatite column.
7 . A purification process according to claim 1 comprising the further step:
(k) drying the purified Protein A solution to a dry product.
8 . A purification process according to claim 7 , wherein the drying occurs by lyophilization.
9 . A purification process according to claim 1 comprising the further step of filtering said Protein A solution to remove bacterial cells, particulates and low molecular weight molecules (<10,000 daltons) before loading the Protein A solution onto the first column.
10 . A non-animal derived Protein A purification process according to claim 1 , wherein the first column is a cation exchange column and the second column is a column selected from the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column.
11 . A purification process according to claim 10 comprising the additional step:
(i) monitoring the absorbance of each fraction of purified Protein A out of the first and the second column at 275 nm and at 340 nm to obtain a A275/A340 ratio in order to choose appropriate fractions for pooling.
12 . A purification process, according to claim 10 , wherein the second column is a anion exchange column.
13 . A purification process according to claim 10 , wherein the second column is a HIC column.
14 . A purification process according to claim 10 , wherein the second column is a ceramic hydroxyapatite column.
15 . A purification process according to claim 10 comprising the further step of:
(j) drying the purified Protein A solution to a dry product.
16 . A purification process according to claim 15 , wherein the drying occurs by lyophilization.
17 . A purification process according to claim 10 comprising the further step of filtering said Protein A solution to remove bacterial cells, particulates and low molecular weight molecules (<10,000 daltons) before loading the Protein A solution onto the cation exchange column.
18 . A purified vegetarian Protein A produced by the following process of production and then of claim 1 immobilized on a chromatographic resin to form a vegetarian Protein A-chromatographic resin, the process of production comprising:
(a) providing a non-animal derived Protein A solution that has been produced by fermenting a secretor strain of Staphylococcus aureus in a media containing vegetarian amino acids or peptides, said media being free of animal products;
(b) loading the filtered Protein A solution onto a first column;
(c) washing the column with a buffer-salt mixture to rinse off the column undesirable color and contaminating proteins;
(d) eluting the Protein A from the first column to produce a Protein A solution pool;
(e) adjustment of the pH and conductivity of the Protein A solution pool;
(f) loading the Protein A from the Protein A solution pool onto a second column;
(g) washing the second column with a buffer-salt mixture to rinse off the column undesirable color and contaminating proteins; and
(h) eluting the Protein A from the second column to produce a purified Protein A solution;
wherein the first column is selected from (1) the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column, or (2) a cation exchange column; and the second column is a cation exchange column when the first column is a column selected from the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column, and the second column is a column selected from the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column when the first column is a cation exchange column
19 . (canceled)
20 . A vegetarian protein A chromatographic resin according to claim 18 , wherein the chromatographic resin is a synthetic polymer selected from the group consisting of polymethacrylate or polygalactose.
21 . (canceled)
22 . (canceled)
23 . A vegetarian purified vegetarian Protein A immobilized on a chromatographic resin to form a Protein A-chromatographic resin according to claim 18 , wherein in the process of production the first column is selected from the group consisting of an anion exchange column, a HIC column and a ceramic hydroxyapatite column; and the second column is a cation exchange column.
24 . (canceled)
25 . A vegetarian protein A chromatographic resin according to claim 23 , wherein the chromatographic resin is a synthetic polymer selected from the group consisting of polymethacrylate or polygalactose.
26 . (canceled)
27 . (canceled)
28 . A method of binding antibodies or antibody fragments to a chromatographic resin comprising contacting the antibodies or antibody fragments with the vegetarian Protein A chromatographic resin of claim 23 .
29 . A method of binding antibodies or antibody fragments to a chromatographic resin comprising contacting the antibodies or antibody fragments with the vegetarian Protein A chromatographic resin of claim 18 .
30 . (canceled)
31 . (canceled)
32 . (canceled)
33 . (canceled)
34 . (canceled)
35 . (canceled)
36 . The antibody or antibody fragments bound to the vegetarian Protein A in accordance with the process of claim 29 .
37 . (canceled)
38 . (canceled)
39 . (canceled)
40 . (canceled)