R-ALPHA1 antitrypsin anion exchange chromatography

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.