# US 8,772,461 B2 · Two-stage ultrafiltration/diafiltration > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,772,461 B2 - Document ID: US8772461B2 - Status: Granted - Kind: B2 - Application number: 12/937,454 - Filed: 2009-04-14 - Published: 2011-09-01 - Granted: 2014-07-08 - Earliest claimed: 2008-04-15 - Assignees: Grifols Therapeutics Inc. - Applicants: Martin Gonzalez; Woody D. Wood; Fred H. Earp - Inventors: Martin Gonzalez; Woody D. Wood; Fred H. Earp - Web page: https://patentgrep.com/patent/US8772461B2 ## CPC - C07K 16/065: {Purification, fragmentation} - C07K 1/30: by precipitation - C07K 1/34: by filtration, ultrafiltration or reverse osmosis - C07K 1/36: by a combination of two or more processes of different types - 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 provides a method for concentrating a protein, in particular a method for concentrating a plasma product, in particular IgG, using glycine in a (two-stage ultrafiltration/diafiltration approach. ## Claims ### Claim 1 A method for concentrating a plasma protein of a solution comprising the protein, the method comprising: a) ultrafiltering the solution using a first membrane to form a first retentate solution comprising the protein at a first concentration, wherein the first membrane has a molecular weight cutoff sufficient to retain at least a portion of the protein present in the solution; b) diafiltering the first retentate solution with an aqueous solution using the first membrane to form a second retentate solution comprising the protein at about the first concentration; c) formulating the second retentate solution comprising the diafiltered protein with glycine and adjusting the pH to form a formulated second retentate solution; and d) ultrafiltering the formulated second retentate solution of step c using a second membrane to form a final retentate solution comprising the protein at a second concentration, wherein the second membrane has a molecular weight cutoff of about twice the molecular weight cutoff of the first membrane, wherein the second concentration is greater than the first concentration. ### Claim 2 (depends on 1) The method of claim 1, wherein the protein is IgG. ### Claim 3 (depends on 1) The method of claim 1, wherein the first membrane is a polyethersulfone C-screen membrane having a molecular weight cutoff of 50 kDa. ### Claim 4 (depends on 1) The method of claim 1, wherein the first concentration is about 5% protein (w/v). ### Claim 5 (depends on 1) The method of claim 1, wherein the aqueous solution is a water. ### Claim 6 (depends on 1) The method of claim 1, wherein the second membrane is a hollow fiber membrane. ### Claim 7 (depends on 1) The method of claim 1, wherein the second membrane has a molecular weight cutoff of 100 kDa. ### Claim 8 (depends on 1) The method of claim 1, wherein the second concentration is at least about 19% protein (w/v). ### Claim 9 (depends on 1) The method of claim 1, wherein the first membrane has a molecular weight cutoff sufficient to retain at least 90% of the protein present in the solution. ### Claim 10 (depends on 4) The method of claim 4, wherein the first concentration is about 5%, wherein, in step c), the about 5% diafiltered protein is formulated to about 0.16 to about 0.30 M glycine and the pH is adjusted to about 4.3. ### Claim 11 A method for concentrating a protein of a solution comprising the protein, the method comprising: a) ultrafiltering the solution using a first membrane to form a first retentate solution comprising the protein at a first concentration of about 5%, wherein the first membrane has a molecular weight cutoff sufficient to retain at least about 90% of the protein; b) diafiltering the first retentate solution using the first membrane with water to form a second retentate solution comprising the protein at about the first concentration; c) formulating the about 5% diafiltered protein of step b) to about 0.16 to about 0.30 M glycine, wherein the formulating further comprises adjusting pH to about 4.3; and d) ultrafiltering the second retentate solution using a second membrane to form a final retentate solution comprising the protein at a second concentration of about 19 to about 21%, wherein the second membrane has a molecular weight cutoff of about twice the molecular weight cutoff of the first membrane, wherein the second concentration is greater than the first concentration. ### Claim 12 (depends on 11) The method of claim 11, wherein the protein is IgG. ### Claim 13 (depends on 12) The method of claim 12, wherein the first membrane has a molecular weight cutoff of about 25 kDa to about 75 kDa. ### Claim 14 (depends on 13) The method of claim 13, wherein the first membrane has a molecular weight cutoff of about 50 kDa, wherein the second membrane has a molecular weight cutoff of about 100 kDa.