Isolation of purified TGF- .beta.1 and TGF -.beta.2 from bone tissue 机翻标题: 暂无翻译,请尝试点击翻译按钮。

公开号/公开日
US2002119539 A1 2002-08-29 [US20020119539]US6492327 B2 2002-12-10 [US6492327] / 2002-08-292002-12-10
申请号/申请日
2000US-09742681 / 2000-12-19
发明人
JUNKER LOUIS;LEWIS MICHAEL;NELSON ROBERT;
申请人
ZIMMER ORTHOBIOLOGICS;
主分类号
IPC分类号
A61K-038/00A61P-017/02A61P-043/00C07K-001/14C07K-001/18C07K-001/20C07K-001/34C07K-014/495
摘要
(US6492327) A method for purifying bone-derived TGF-beta proteins including an anion exchange process, a cation exchange process, and a reverse phase HPLC process, and optionally, a filtration process, a Heparin-Sepharose process, and/or a reverse phase HPLC desalting process.  The filtration process preferably selects proteins having a nominal molecular weight between approximately 10 kilodaltons and approximately 100 kilodaltons.  Preferably, the anion exchange process uses a strongly cationic resin having quaternary amine functional groups.  Preferably, the cation exchange process uses a strongly anionic resin having sulfonic acid functional groups.  The TGF-beta proteins can be eluted from the reverse phase HPLC column with an acetonitrile solution in combination with aqueous trifluoracetic acid.  The purification processes yield highly enriched TGF-beta1 and TGF-beta2 proteins.
机翻摘要
暂无翻译结果,您可以尝试点击头部的翻译按钮。
地址
代理人
代理机构
;
优先权号
2000US-09742681 2000-12-19
主权利要求
(US6492327) What is claimed is: 1.  A process for purifying TGF- BETA  proteins from a first solution containing demineralized bone extract, comprising: subjecting said first solution to filtration to obtain a second solution containing proteins having a desired range of molecular weights; loading said second solution onto an anion exchange resin; collecting the void volume from loading said second solution onto said anion exchange resin; loading said void volume onto a cation exchange resin; eluting a protein fraction from said cation exchange resin with a gradient eluant to obtain a cation exchanged fraction eluate; loading said cation exchanged fraction eluate onto a reverse phase HPLC column;  and eluting a protein fraction from said HPLC column to obtain a HPLC fraction eluate enriched in a TGF- BETA  protein. 2. The process, as claimed in claim 1, further comprising the steps of loading said HPLC fraction eluate onto a Heparin-Sepharose column;  and eluting a protein fraction from said Heparin-Sepharose column to obtain an Heparin-Sepharose eluate enriched in a TGF- BETA  protein. 3. The process, as claimed in claim 2, further comprising the step of loading said Heparin-Sepharose eluate onto a reverse phase HPLC column. 4. The process, as claimed in claim 1, wherein said filtration comprises: (i) subjecting said first solution to filtration using a filtration membrane having a nominal molecular weight cut off of approximately 100 kD to produce a filtrate;  and (ii) subjecting said filtrate to filtration using a filtration membrane having a nominal molecular weight cut off of approximately 10 kD to produce a retentate as said second solution. 5. The process, as claimed in claim 1, wherein the conductivity of said second solution is adjusted to a conductivity of less than about 1,300 mhos (1.3 * 10-3 S) prior to loading onto said anion exchange resin. 6. The process, as claimed in claim 1, wherein the conductivity of said void is in a range from about 900 mhos (0.9 * 10-3 S) to about 1300 mhos (1.3 * 10-3 S). 7. The process, as claimed in claim 1, wherein said cation exchange eluant has a conductivity from about 1500 mhos (1.5 * 10-3 S) to about 40,000 mhos (4.0 * 10-2 S). 8. The process, as claimed in claim 1, further comprising the step of dialyzing said cation enchanged fraction eluate into deionized water using a 3.5 kD molecular weight cut off dialysis membranes. 9. The process, as claimed in claim 1, wherein said step of eluting a protein fraction from said HPLC column to obtain a HPLC fraction eluate comprises eluting a protein fraction from said HPLC column with a second eluant having a gradient of increasing acetonitrile concentration ranging from about 25 percent by volume to about 70 percent by volume to obtain a purified protein mixture. 10. The process, as claimed in claim 9, wherein said gradient of increasing acetonitrile concentration of said second eluant increases from about 29 percent by volume to about 55 percent by volume. 11. The process, as claimed in claim 9, wherein said step of eluting a protein fraction from said HPLC column comprises collecting aliquots eluting at approximately 35 to 37 percent by volume acetonitrile to produce a TGF- BETA 1 fraction. 12. The process, as claimed in claim 9, wherein said step of eluting a protein fraction from said HPLC column comprises collecting aliquots eluting at approximately 38 to 40 percent by volume acetonitrile to produce a TGF- BETA 2 fraction. 13. The process, as claimed in claim 9, wherein said HPLC column comprises either a plastic or hydrocarbon-modified silica packing. 14. The process, as claimed in claim 13, wherein said silica is modified by the addition of C4 through C18 hydrocarbons. 15. The process, as claimed in claim 9, wherein said HPLC column comprises a packing material selected from the group consisting of C4, C8 or C18 material. 16. The process, as claimed in claim 1, wherein said anion exchange resin comprises quaternary amine functional groups. 17. The process, as claimed in claim 1, wherein said cation exchange resin comprises sulfonic acid functional groups. 18. The process, as claimed in claim 1, wherein the pH of said second solution is from about pH 8 to about pH 9 prior to said step of loading said second solution onto said anion exchange resin. 19. The process, as claimed in claim 1, wherein the pH of said void volume is from about pH 8.0 to about pH 9.0 prior to said step of loading said void volume onto said cation exchange resin. 20. The process, as claimed in claim 1, wherein said second solution, said void volume and said cation exchanged fraction eluate comprise an effective amount of urea to maintain proteins therein in solution. 21. The process, as claimed in claim 1, wherein said cation exchange loading solution has a counterion concentration less than about 0.01 M NaCl. 22. The process, as claimed in claim 1, wherein said first eluant has a counterion concentration in a range from about 0.01 M NaCl to about 0.6 M NaCl. 23. The process, as claimed in claim 1, wherein said HPLC fraction eluate comprises from about 0.05 percent by volume to about 0.15 percent by volume trifluoracetic acid. 24. The process, as claimed in claim 1, wherein said first solution containing demineralized bone extract is prepared by extracting proteins from cleaned, crushed demineralized bone particles using a compound selected from the group consisting of guanidine and urea. 25. A process for purifying TGF- BETA  proteins from a first solution containing demineralized bone extract, comprising: i subjecting said first solution to filtration to obtain a second solution containing proteins having a nominal molecular weight cut off of between approximately 10 kD and approximately 100 kD; ii adjusting the conductivity of said second solution, if necessary, to a conductivity of less than about 1,300  MU mhos (1.3 * 10-3 S) prior to loading onto said anion exchange resin iii loading said second solution onto an anion exchange resin; iv collecting the void volume from loading said second solution onto said anion exchange resin; v wherein the conductivity of said void volume is in a range from about 900  MU mhos (0.9 * 10-3 S) to about 1300  MU mhos (1.3 * 10-3 S); vi loading said void volume onto a cation exchange resin; vii eluting a protein fraction from said cation exchange resin with a first eluant having a sodium chlorine concentration of about 10 mM to about 600 mM to obtain a cation exchanged fraction eluate, having a sodium chloride concentration of about 400 to about 550 mM NaCl; vi loading said cation exchanged fraction eluate onto a reverse phase HPLC column;  and vii eluting a protein fraction from said HPLC column with a second eluant having a gradient of increasing acetonitrile concentration ranging from about 25 percent by volume to about 70 percent by volume to obtain a purified protein mixture to obtain a HPLC fraction eluate enriched in a TGF- BETA  protein. 26. The process, as claimed in claim 25, wherein said step of eluting a protein fraction from said HPLC column comprises collecting aliquots eluting at approximately 35-37 percent by volume acetonitrile to produce a TGF- BETA 1 fraction. 27. The process, as claimed in claim 25, wherein said step of eluting a protein fraction from said HPLC column comprises collecting aliquots eluting at approximately 38-40 percent by volume acetonitrile to produce a TGF- BETA 2 fraction. 28. A process for purifying TGF- BETA  proteins from a first solution containing demineralized bone extract, comprising: i subjecting said first solution to filtration to obtain a second solution containing proteins having a nominal molecular weight cut off of between approximately 10 kD and approximately 100 kD; ii adjusting the conductivity of said second solution, if necessary, to a conductivity of less than about 1,300  MU mhos (1.3 * 10-3 S) prior to loading onto said anion exchange resin iii loading said second solution onto an anion exchange resin comprising quaternary amine functional groups; iv collecting the void volume from loading said second solution onto said anion exchange resin; v wherein the conductivity of said void volume is in a range from about 900  MU mhos (0.9 * 10-3 S) to about 1300  MU mhos (1.3 * 10-3 S); vi loading said void volume onto a cation exchange resin comprising sulfonic acid functional groups; vii eluting a protein fraction from said cation exchange resin with a first eluant having a sodium chlorine concentration of about 10 mM to about 600 mM to obtain a cation exchanged fraction eluate, having a sodium chloride concentration of about 400 to about 550 mM NaCl; viii loading said cation exchanged fraction eluate onto a reverse phase HPLC column comprising C4 through C18 hydrocarbon-modified silica packing; ix eluting a protein fraction from said HPLC column with a second eluant having a gradient of increasing acetonitrile concentration ranging from about 25 percent by volume to about 70 percent by volume to obtain a purified protein mixture; x collecting aliquots eluting from said HPLC column at approximately 35-37 percent by volume acetonitrile to produce a TGF- BETA 1 fraction;  and xi collecting aliquots eluting from said HPLC column at approximately 38-40 percent by volume acetonitrile to produce a TGF- BETA 2 fraction.
法律状态
(US6492327) LEGAL DETAILS FOR US2002119539  Actual or expected expiration date=2015-01-05    Legal state=DEAD    Status=LAPSED     Event publication date=2000-12-19  Event code=US/APP  Event indicator=Pos  Event type=Examination events  Application details  Application country=US US09742681  Application date=2000-12-19  Standardized application number=2000US-09742681     Event publication date=2000-12-19  Event code=US/EXMR  Event type=Administrative notifications  USPTO Examiner Name Primary Examiner: CHAKRABARTI, ARUN K    Event publication date=2000-12-19  Event code=US/ART  Event type=Administrative notifications  USPTO Art Group  ART=1634     Event publication date=2000-12-19  Event code=US/ENT  Event type=Administrative notifications  Entity Status Set to Undiscounted Business Entity Status: UNDISCOUNTED    Event publication date=2000-12-19  Event code=US/AIA  Event type=Administrative notifications  First Inventor File Indicated:  AIA=No     Event publication date=2000-12-19  Event code=US/DK  Event type=Examination events  Attorney Docket Number Docket Nbr: 3394.159US1    Event publication date=2000-12-19  Event code=US/CUST  Event type=Examination events  Attorney/Agent Customer Number Customer Nbr: 21186    Event publication date=2001-02-07  Event code=US/INCD  Event type=Examination events  Event type=OAO  Notice Mailed--Application Incomplete--Filing Date Assigned    Event publication date=2001-04-04  Event code=US/AS  Event type=Change of name or address  Event type=Reassignment  Assignment Owner: SULZER BIOLOGICS INC., TEXAS  Effective date of the event=2001-03-22  ASSIGNMENT OF ASSIGNORS INTEREST ASSIGNORS:JUNKER, LOUIS LEWIS, MICHAEL NELSON, ROBERT REEL/FRAME:011690/0477     Event publication date=2001-05-23  Event code=US/TRDD  Event type=APL  Decision by documentation    Event publication date=2001-06-18  Event code=US/DOCK  Event indicator=Pos  Event type=Examination events  Case Docketed to Examiner    Event publication date=2001-07-02  Event code=US/IDS  Event type=Examination events  Event type=OAI  Information Disclosure Statement Filed    Event publication date=2001-08-17  Event code=US/IDS  Event type=Examination events  Event type=OAI  Information Disclosure Statement Filed    Event publication date=2001-10-10  Event code=US/CTNF  Event type=Examination events  Event type=OA  Event type=OAO  Non-Final Rejection    Event publication date=2002-02-07  Event code=US/DOCK  Event indicator=Pos  Event type=Examination events  Case Docketed to Examiner    Event publication date=2002-02-26  Event code=US/CTNFR  Event type=Examination events  Event type=OAI  Response after Non-Final Action    Event publication date=2002-04-04  Event code=US/NOAM  Event indicator=Pos  Event type=Examination events  Event type=OAO  Mail Notice of Allowance    Event publication date=2002-07-26  Event code=US/APRDY  Event indicator=Pos  Event type=Examination events  Application Is Considered Ready for Issue    Event publication date=2002-08-29  Event code=US/A1  Event type=Examination events  Application published  Publication country=US  Publication number=US2002119539  Publication stage Code=A1  Publication date=2002-08-29  Standardized publication number=US20020119539     Event publication date=2002-11-21  Event code=US/ISSM  Event indicator=Pos  Event type=Examination events  Event type=OAO  Event type=Restitution or restoration  Issue Notification Mailed    Event publication date=2002-12-10  Event code=US/B2  Event indicator=Pos  Event type=Event indicating In Force  Granted patent as second publication  Publication country=US  Publication number=US6492327  Publication stage Code=B2  Publication date=2002-12-10  Standardized publication number=US6492327     Event publication date=2002-12-10  Event code=US/354  Event indicator=Pos  Event type=Extension of term of duration of protection  Patent term extended under  35 U.S.C 154(b) until/for Delays (A,B,C): 0  Overlap Delays: 0  Non Overlap Delays: 0   PTO Office Delays: 0  Applicant Delays: 115  Adjustment total:  Number of days of extension=0    Event publication date=2004-12-03  Event code=US/AS  Event type=Change of name or address  Event type=Reassignment  Assignment Owner: CENTERPULSE BIOLOGICS INC., TEXAS  Effective date of the event=2002-10-02  CHANGE OF NAME ASSIGNOR:SULZER BIOLOGICS INC. REEL/FRAME:015418/0585     Event publication date=2005-02-02  Event code=US/AS  Event type=Change of name or address  Event type=Reassignment  Assignment Owner: ZIMMER ORTHOBIOLOGICS, INC., TEXAS  Effective date of the event=2004-04-28  CHANGE OF NAME ASSIGNOR:CENTERPULSE BIOLOGICS INC. REEL/FRAME:015819/0707     Event publication date=2006-06-12  Event code=US/FPAY  Event indicator=Pos  Event type=Event indicating In Force  Event type=Payment or non-payment notifications  Fee payment recorded   Annual fees payment date=2006-06-12     Year of payment of annual fees=4     Event publication date=2009-04-09  Event code=US/POAC  Event type=Change of name or address  Change in Power of Attorney (May Include Associate POA)    Event publication date=2010-05-21  Event code=US/FPAY  Event indicator=Pos  Event type=Event indicating In Force  Event type=Payment or non-payment notifications  Fee payment recorded   Annual fees payment date=2010-05-21     Year of payment of annual fees=8     Event publication date=2011-06-13  Event code=US/POAC  Event type=Change of name or address  Change in Power of Attorney (May Include Associate POA)    Event publication date=2014-07-18  Event code=US/REMI  Event type=Administrative notifications  Maintenance fee reminder mailed    Event publication date=2014-12-10  Event code=US/LAPS  Event indicator=Neg  Event type=Event indicating Not In Force  Event type=Payment or non-payment notifications  Lapse for failure to pay maintenance fees    Event publication date=2014-12-10  Event code=US/FP  Event indicator=Neg  Event type=Event indicating Not In Force  Event type=Payment or non-payment notifications  Expired due to failure to pay maintenance fee  Effective date of the event=2014-12-10     Event publication date=2015-01-02  Event code=US/EXP  Event indicator=Neg  Event type=Event indicating Not In Force  Patent expired    Event publication date=2015-01-05  Event code=US/FP  Event indicator=Neg  Event type=Event indicating Not In Force  Event type=Payment or non-payment notifications  Expired due to failure to pay maintenance fee
专利类型码
A1B2
国别省市代码
若您需要申请原文,请登录。

最新评论

暂无评论。

登录后可以发表评论

意见反馈
返回顶部