R-ALPHA1 antitrypsin anion exchange chromatography

1. A method for obtaining highly purified recombinant alpha 1-antitrypsin (rAAT) from a composition comprising the rAAT and at least one host cell protein impurity comprising hexokinase originating from cultivation of cells used for generating rAAT, the method comprising:

(a) purifying rAAT present in the composition away from the impurity by sample displacement chromatography, comprising the steps of:

(i) loading the composition comprising rAAT and at least one host cell protein impurity onto a column comprising an anion exchange material comprising an anion exchange ligand selected from the group consisting of quaternary ammonium and quaternary aminoethyl;

(ii) washing the anion exchange material using a buffer comprising 1 to about 80 mM phosphate ions and 0.1 to about 50 mM N acetylcysteine (NAC);

(iii) eluting the rAAT from the anion exchange material using a phosphate ion gradient starting with a first buffer composition comprising 1 to about 80 mM phosphate ions and 0.1 to about 50 mM NAC and ending with a second buffer composition comprising 50 to 1000 mM phosphate ions and 0.1 to 50 mM NAC, wherein the concentration of phosphate ions at the end of the gradient is greater than the concentration of phosphate ions at the beginning of the gradient, and further wherein the pH of the first and second buffer compositions is maintained between 7.3 to 7.8; thereby generating an eluent in fractions;

(iv) collecting fractions of the eluent containing rAAT that has been purified away from the impurity; and

(b) pooling the fractions containing purified rAAT to form an rAAT pool, wherein the rAAT pool contains at least 50% less mass of hexokinase impurity as compared to an rAAT pool generated under the same elution conditions except the elution gradient is sodium chloride in a Tris buffer.

2. The method according to claim 1 , wherein the at least one impurity is a yeast host cell protein.

3. The method according to claim 1 , wherein the anion exchange material is selected from the group consisting of Q Sepharose HP, Cellufine Q, SourceQ, and Toyopearl QAE.

4. The method according to claim 1 , wherein step (i) comprises loading about 2 to 50 mg of rAAT per ml anion exchange material onto the column.

5. The method according to claim 1 , wherein step (iii) comprises eluting the rAAT from the anion exchange material within about 30 column volumes (CV) of the gradient.

6. The method according to claim 1 , wherein the starting point for the pooling in step (b) is determined by UV absorption of the eluent at a wavelength in the range of 340 nm to 430 nm and the end point for the pooling is determined by UV absorption of the eluent at 280 nm.

7. The method according to claim 6 , wherein the rAAT fraction pooling starts when the UV absorption at 395 nm of the peak just preceding the rAAT main peak has declined to less than 10% of the maximum absorbance and wherein the rAAT fraction pooling is terminated when the UV absorption at 280 nm has declined to 40% of the UV absorption monitored at the beginning of the pooling.

8. The method of claim 1 , wherein the phosphate ion gradients ends with a buffer composition comprising 100 to 300 mM phosphate ions.

9. The method of claim 8 , wherein the phosphate ion gradients begins with a buffer composition comprising 20 to 50 mM phosphate ions.

10. The method of claim 1 , wherein the rAAT is eluted from the anion exchange material within 5 to 100 column volumes of the gradient.

11. The method according to claim 1 , wherein the rAAT pool contains at least 60% less mass of hexokinse.

12. The method according to claim 11 , wherein the rAAT pool contains at least 70% less mass of hexokinase.

13. The method according to claim 12 , wherein the rAAT pool contains at least 80% less mass of hexokinase.