# US 10,370,408 B2 · Method for purification of a DNase or RNase protein from a sample > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,370,408 B2 - Document ID: US10370408B2 - Status: Granted - Kind: B2 - Application number: 15/321,016 - Filed: 2015-06-25 - Published: 2017-07-13 - Granted: 2019-08-06 - Earliest claimed: 2014-06-25 - Assignees: JHL Biotech, Inc. - Applicants: Jhl Biotech, Inc.; Gregory Scott Blank - Inventors: Gregory Scott Blank; Jiun-Liang Lin - Web page: https://patentgrep.com/patent/US10370408B2 ## CPC - C07K 1/165: {mixed-mode chromatography} - B01D 15/361: {Ion-exchange} - B01D 15/3847: {Multimodal interactions} - B01D 15/426: {Specific type of solvent} - C07K 1/18: Ion-exchange chromatography - C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} - 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) - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present disclosure provides compositions and methods for purifying a protein such as DNase using a chromatographic process. The methods include a single chromatographic step and the use of high concentration salt buffers. ## Claims ### Claim 1 A method for purification of a DNase or RNase from a sample comprising: loading a sample comprising a DNase or RNase and a detergent onto a chromatography column, washing the column with a wash buffer comprising calcium chloride, wherein the wash buffer has a conductivity of 80 mS/cm or higher, and eluting the DNase or RNase from the column with an elution buffer comprising calcium chloride. ### Claim 2 (depends on 1) The method of claim 1, wherein the wash buffer comprises a salt selected from the group consisting of (NH4)2SO4, Na2SO4, and CH3COONa. ### Claim 3 (depends on 2) The method of claim 2, wherein the wash buffer comprises a concentration of about 0.8 M (NH4)2SO4. ### Claim 4 (depends on 1) The method of claim 1, wherein the elution buffer has a conductivity of 80 mS/cm or higher. ### Claim 5 (depends on 1) The method of claim 1, wherein the elution buffer comprises a salt selected from the group consisting of NaCl, KCl, and NaOAc. ### Claim 6 (depends on 5) The method of claim 5, wherein the elution buffer comprises a concentration of about 1 M NaCl. ### Claim 7 (depends on 1) The method of claim 1, wherein the method comprises at least two washing steps. ### Claim 8 (depends on 7) The method of claim 7, wherein a first washing step comprises washing the column with a buffer having a conductivity of 2 mS/cm or less. ### Claim 9 (depends on 8) The method of claim 8, further comprising a third washing step, wherein the third washing step comprises washing the column with a buffer having a conductivity of 2 mS/cm or less. ### Claim 10 (depends on 1) The method of claim 1, wherein the DNase is a recombinant DNase. ### Claim 11 (depends on 1) The method of claim 1, wherein the calcium chloride is 1 mM calcium chloride. ### Claim 12 (depends on 1) The method of claim 1, wherein the chromatography column is an ion exchange or a mixed mode chromatography column. ### Claim 13 (depends on 12) The method of claim 12, wherein the mixed mode column is a multimodal anion exchanger. ### Claim 14 (depends on 13) The method of claim 13, wherein the mixed mode column is a Capto Adhere column. ### Claim 15 (depends on 1) The method of claim 1, wherein the sample is a cell culture sample. ### Claim 16 (depends on 1) The method of claim 1, wherein the pH of the loaded sample is between 5.0 and 9.0. ### Claim 17 (depends on 16) The method of claim 16, wherein the pH of the loaded sample is between 6.0 and 8.0. ### Claim 18 (depends on 17) The method of claim 17, wherein the pH of the loaded sample is about 7.0. ### Claim 19 (depends on 1) The method of claim 1, wherein the pH of the loaded sample is the same or higher than the pI of the protein. ### Claim 20 (depends on 1) The method of claim 1, wherein the pH of the wash buffer is between about 5.0 and about 9.0. ### Claim 21 (depends on 20) The method of claim 20, wherein the pH of the wash buffer is about 7.0. ### Claim 22 (depends on 1) The method of claim 1, wherein the pH of the elution buffer is between about 5.0 and about 9.0. ### Claim 23 (depends on 22) The method of claim 22, wherein the pH of the elution buffer is about 7.0. ### Claim 24 (depends on 1) The method of claim 1, wherein the detergent is Triton X-100. ### Claim 25 (depends on 24) The method of claim 24, wherein the detergent is added to the sample at a final concentration of 0.5%.