# US 2018/0022608 A1 · Apatite pretreatment > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2018/0022608 A1 - Document ID: US20180022608A1 - Status: Published application - Kind: A1 - Application number: 15/719,958 - Filed: 2017-09-29 - Published: 2018-01-25 - Earliest claimed: 2014-06-23 - Assignees: Bio-Rad Laboratories, Inc. - Applicants: Bio-Rad Laboratories, Inc. - Inventors: Larry Cummings - Web page: https://patentgrep.com/patent/US20180022608A1 ## CPC - C01B 25/32: Phosphates of magnesium, calcium, strontium, or barium - B01D 15/203: {Equilibration or regeneration} - B01D 15/3847: {Multimodal interactions} - B01J 20/048: {containing phosphorus, e.g. phosphates, apatites, hydroxyapatites} - B01J 20/281: Sorbents specially adapted for preparative, analytical or investigative chromatography - C01B: NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; {METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C} - 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) - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS ## Abstract Apatite pretreatment methods are provided. The method is applied to the apatite solid surface prior to first chromatographic use. In one embodiment, the method may be achieved by contacting an apatite solid surface with a phosphate buffered solution at a pH of at least about 6.5 and contacting the apatite solid surface with a solution having a hydroxide. ## Claims ### Claim 1 A method of treating a ceramic hydroxyapatite solid surface prior to first use in a chromatographic procedure, the method comprising: (a) providing a ceramic hydroxyapatite solid surface, wherein the ceramic hydroxyapatite surface has not been used in any chromatographic procedure and has not previously been contacted with any sample; (b) contacting the ceramic hydroxyapatite solid surface with a phosphate buffered solution at a pH of at least about 6.5; and then (c) contacting the ceramic hydroxyapatite solid surface obtained in step (b) with a solution having a hydroxide; and then (d) purifying protein from a sample with the ceramic hydroxyapatite solid surface obtained in step (c). ### Claim 2 (depends on 1) The method of claim 1, wherein the phosphate buffered solution is a solution having from about 0.1 M to about 1.0 M phosphate at a pH of from about 6.5 to about 10.0. ### Claim 3 (depends on 2) The method of claim 2, wherein the phosphate buffered solution is 400 mM phosphate at a pH of 8.0. ### Claim 4 (depends on 1) The method of claim 1, wherein the hydroxide is an alkaline metal hydroxide. ### Claim 5 (depends on 4) The method of claim 4, wherein the alkaline metal hydroxide is sodium or potassium hydroxide. ### Claim 6 (depends on 1) The method of claim 1, wherein the purifying of step (d) comprises contacting the ceramic hydroxyapatite solid surface with the sample, thereby separating the protein from one or more biological compounds; then (e) collecting the protein from the ceramic hydroxyapatite solid surface; and then (f) regenerating the ceramic hydroxyapatite solid surface, the regenerating comprising, (i) contacting the ceramic hydroxyapatite solid surface with a buffered calcium solution having a calcium ion at a concentration of at least about 5 mM and a zwitterionic buffer, wherein the ratio of zwitterionic buffer concentration to calcium ion concentration is at least about 2, and the pH of the solution is at least about 6.5; (ii) contacting the ceramic hydroxyapatite solid surface with a phosphate buffered solution at a pH of at least about 6.5; and (iii) contacting the ceramic hydroxyapatite solid surface with a solution having an hydroxide. ### Claim 7 (depends on 6) The method of claim 6, wherein (d) comprises binding the protein to the ceramic hydroxyapatite solid surface, and (e) comprises eluting the protein from the ceramic hydroxyapatite solid surface. ### Claim 8 (depends on 6) The method of claim 6, wherein (d) comprises contacting the ceramic hydroxyapatite solid surface to the protein, thereby flowing the protein through the ceramic hydroxyapatite solid surface, and (e) comprises collecting the protein in the flow through. ### Claim 9 (depends on 6) The method of claim 6, wherein the zwitterionic buffer is a sulfonic acid containing buffer. ### Claim 10 (depends on 9) The method of claim 9, wherein the sulfonic acid containing buffer is MES, PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, TAPS, TAPSO, POPSO, or HEPPSO, EPPS, CAPS, CAPSO, or CHES. ### Claim 11 (depends on 9) The method of claim 9, wherein the sulfonic acid containing buffer is MES. ### Claim 12 (depends on 6) The method of claim 6, wherein the calcium ion is at least about 25 mM or at least about 50 mM. ### Claim 13 (depends on 6) The method of claim 6, wherein the ratio of zwitterionic buffer concentration to calcium ion concentration is at least about 2.5. ### Claim 14 (depends on 6) The method of claim 6, wherein the buffered calcium solution comprises calcium chloride or calcium nitrate. ### Claim 15 (depends on 6) The method of claim 6, wherein the phosphate buffered solution comprises a solution containing from about 0.1 M to about 1.0 M phosphate at a pH of from about 6.5 to about 8. ### Claim 16 (depends on 15) The method of claim 15, wherein the phosphate buffered solution comprises 400 mM phosphate at a pH of 7.0. ### Claim 17 (depends on 6) The method of claim 6, wherein the regenerating step reverses or eliminates degradation of a column that occurs during protein purification or column cleaning steps. ### Claim 18 (depends on 6) The method of claim 6, wherein the regenerating step is performed before a phosphate cleaning/stripping step. ### Claim 19 (depends on 7) The method of claim 7, wherein the regenerating step is performed after elution of the protein. ### Claim 20 (depends on 6) The method of claim 6, wherein step (f) (ii) further comprises: contacting the ceramic hydroxyapatite solid surface with a solution comprising phosphate at a concentration of 10 mM, or less than about 10 mM, at a pH of at least about 6.5; and then contacting the ceramic hydroxyapatite solid surface with a solution comprising phosphate at a concentration of at least about 100 mM at a pH of at least about 6.5. ### Claim 21 (depends on 6) The method of claim 6, wherein the regenerating step consists of (i), a wash, (ii), and (iii). ### Claim 22 (depends on 1) The method of claim 1, wherein the protein is an antibody.