# US 8,148,502 B2 · R-ALPHA1 antitrypsin anion exchange chromatography > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,148,502 B2 - Document ID: US8148502B2 - Status: Granted - Kind: B2 - Application number: 11/693,609 - Filed: 2007-03-29 - Published: 2007-11-08 - Granted: 2012-04-03 - Earliest claimed: 2006-03-30 - Assignees: Baxter International Inc.; Baxter Healthcare S.A. - Inventors: Artur Mitterer; Meinhard Hasslacher; Christian Fiedler - Web page: https://patentgrep.com/patent/US8148502B2 ## CPC - C07K 14/8125: {Alpha-1-antitrypsin} - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract The present invention relates to a method for obtaining highly purified recombinant alpha1-antitrypsin (rAAT) using anion exchange chromatography, wherein a composition comprising rAAT and at least one impurity originating from cultivation of cells used for generating of rAAT is loaded onto a column containing anion exchange material and the anion exchange chromatography is carried out with buffers containing phosphate ions and N-acetylcysteine. ## Claims ### Claim 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. ### Claim 2 (depends on 1) The method according to claim 1, wherein the at least one impurity is a yeast host cell protein. ### Claim 3 (depends on 1) 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. ### Claim 4 (depends on 1) 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. ### Claim 5 (depends on 1) 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. ### Claim 6 (depends on 1) 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. ### Claim 7 (depends on 6) 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. ### Claim 8 (depends on 1) The method of claim 1, wherein the phosphate ion gradients ends with a buffer composition comprising 100 to 300 mM phosphate ions. ### Claim 9 (depends on 8) The method of claim 8, wherein the phosphate ion gradients begins with a buffer composition comprising 20 to 50 mM phosphate ions. ### Claim 10 (depends on 1) The method of claim 1, wherein the rAAT is eluted from the anion exchange material within 5 to 100 column volumes of the gradient. ### Claim 11 (depends on 1) The method according to claim 1, wherein the rAAT pool contains at least 60% less mass of hexokinse. ### Claim 12 (depends on 11) The method according to claim 11, wherein the rAAT pool contains at least 70% less mass of hexokinase. ### Claim 13 (depends on 12) The method according to claim 12, wherein the rAAT pool contains at least 80% less mass of hexokinase.