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Method of Preparing Highly Purified Alpha-1-Proteinase Inhibitor

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Method of Preparing Highly Purified Alpha-1-Proteinase Inhibitor ~" ' MM II II II II I II II II I Ml II II I II J European Patent Office © Publication number: 0 288 841 B1 Office europeen* des.. brevets , © EUROPEAN PATENT SPECIFICATION © Date of publication of patent specification: 30.12.92 © Int. CI.5: C07K 3/02, A61 K 37/64 © Application number: 88106017.2 @ Date of filing: 15.04.88 © Method of preparing highly purified alpha-1-proteinase inhibitor. ® Priority: 27.04.87 US 42925 © Proprietor: MILES INC. 1127 Myrtle Street @ Date of publication of application: Elkhart Indiana 4651 4(US) 02.11.88 Bulletin 88/44 @ Inventor: Shearer, Michael A. © Publication of the grant of the patent: 4927 Silverado Drive 30.12.92 Bulletin 92/53 Suisun California 94585(US) © Designated Contracting States: AT BE CH DE ES FR GB GR IT LI NL SE © Representative: Adrian, Albert, Dr. et al c/o BAYER AG Konzernverwaltung RP Paten- © References cited: tabteilung EP-A- 0 067 293 W-5090 Leverkusen 1, Bayerwerk(DE) EP-A- 0 097 274 ANALYTICAL BIOCHEMISTRY, vol. 124, 1982, pages 364-371, Academic Press, Inc.; C.B. GLASER et al.: "The isolation of alpha- 1 -protease inhibitor by a unique procedure designed for industrial application" 00 00 00 00 CM O Note: Within nine months from the publication of the mention of the grant of the European patent, any person ^ may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition qj shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid (Art. 99(1) European patent convention). Rank Xerox (UK) Business Services 1 EP 0 288 841 B1 2 Description agents which can be utilized are dithiothreitol, dithioerythritol, 2-mercaptoethanol, mercaptoacetic This invention relates to and has among its acid, sodium borohydride and cysteine. The reduc- objects a method of preparing a high purity alpha- ing agents are present in a concentration of about 1 -proteinase inhibitor (PI) concentrate from blood 5 2mM to 8mM. The concentration of reducing agent plasma, blood plasma fractions and concentrates. will vary depending on the type of solution contain- Further objects of the invention will be evident from ing PI and the choice of reducing agent. The the following description wherein parts and per- amount of reducing agent should be sufficient to centages are by weight unless otherwise specified. reduce the disulfide bonds without denaturing or Alpha-1 -proteinase inhibitor is a glycoprotein io damaging PI or any other protein to be recovered. having molecular weight of 54,000. The protein The pH of the solution containing PI and the reduc- consists of a single polypeptide chain to which ing agent can be adjusted to optimize the rate of several oligosaccharide units are covalently bound. reaction and also to stop the reaction. Heat de- Human PI has a role in controlling tissue destruc- naturation of the reduced protein can be accom- tion by endogenous serine proteinases. A genetic 75 plished by raising the temperature to about 35 °C deficiency of PI, which accounts for 90 % of the or higher. trypsin inhibitory capacity in blood plasma, has Suitable pH ranges are of about 7.9 to about been shown to be associated with premature devel- 8.5 whereas the rates of reaction are about 5 to opment of pulmonary emphysema. The degrada- about 95 minutes. tion of elastin associated with emphysema prob- 20 Heat denaturation can also be accomplished in ably results from a local imbalance of elastolytic combination with a heat treating viral inactivation enzymes and the naturally occurring tissue and step. In U.S. Patent 4,470,968, there is disclosed a plasma proteinase inhibitors. PI rapidly inhibits hu- method of stabilizing coagulation Factors II, VIII, IX, man pancreatic and leukocyte elastases and X against heat during pasteurization or heating (Biochem. Biophys. Res. Comm., Vol. 72, No. 1, 25 at a temperature of about 60°-100°C by mixing pages 33-39, 1976; ibid., Vol. 88, No. 2, pages 346- with heat-stabilizing or pasteurization-stabilizing, 350, 1979). amounts of a polyol and a source of citrate ions. A number of methods employed to isolate PI Pasteurized compositions of these coagulation Fac- from blood plasma are set forth in U.S. Patents tors were obtained by heating a mixture of un- 4,439,358, 4,379,087, and 4,656,254 and are incor- 30 pasteurized protein composition, a polyol, and a porated herein by reference. source of citrate ions suspended or solubilized Pannell et al., Biochemistry, Vol. 13, pages usually in an aqueous medium at a temperature 5439-5445, (1974), teaches a method of depleting and for a time sufficient to pasteurize the protein albumin from plasma using Sepharose-Blue Dex- composition. Following pasteurization or heat treat- tran adsorption are then pooled and fractionated 35 ment, the polyol and citrate ions are removed to- with solid ammonium sulfate. tally, or in part, as desired, from the protein com- Glaser et al., EP-A- 0.067.293 , teach the use position by conventional techniques, and the pas- of reducing agents, such as dithiothreitol to break teurized protein composition is processed accord- disulfide bonds and precipitation by salting out with ing to conventional procedures for its ultimate 50% (Nr-U)2504, to remove unwanted proteins. 40 therapeutic use. By way of illustration and not The invention described herein is a method for limitation, one may employ the method of U.S. preparing a highly purified PI concentrate by de- Patent No. 4,470,968 (herein incorporated by refer- pleting the albumin from a solution containing PI by ence) or any of the methods of the prior art de- adding a mild reducing agent sufficient to break the scribed therein for heat inactivation. disulfide bonds in the albumin and heating the 45 One advantage of the invention is the availabil- solution containing the PI and mild reducing agent ity of an essentially pure PI preparation that does to prevent the destabilized proteins from recom- not require the addition of ammonium sulfate to bining. remove unwanted protein. In preferred embodiments, PI is separated from Another advantage of the invention is the incor- blood plasma or blood plasma fractions according 50 poration of a step to remove unwanted protein with to the Coan methods, U.S. Patents 4,379,087 and a heat inactivation step. The mild reducing agent 4,439,358 and U.S. Serial No. 793,807. As well as can be added to the aqueous solution any time the improvements set forth in U.S. Patent prior to the heat inactivation step and unwanted 4,656,254, Improved Methods of Preparing alpha-1 - proteins removed when the pasteurized protein proteinase inhibitor. The reducing agent can be 55 composition is processed according to convention- added to a solution of separated PI to break any al procedures for ultimate therapeutic use. protein disulfide bonds without reducing the PI, The reducing agent and denatured proteins can since PI does not have disulfide bonds. Reducing be removed by ultrafiltration and anion exchange 2 3 EP 0 288 841 B1 4 methods without the addition of a salting out step. proteinase inhibitor can be accomplished in modi- Current methods for obtaining PI from blood fied form by the steps comprising plasma use as a starting material: Cohn Fraction IV (a) adding a mild reducing agent to a mixture of and IV-1 and includes reworks of these fractions plasma proteins containing alpha-1 -proteinase wherein other proteins are first removed, Cohn 5 inhibitor, said reducing agent being in a con- Effluent II + III and Cohn Effluent I and cryosuper- centration sufficient to reduce disulfide bonds natant solution. Blood plasma is fractionated ac- without reducing alpha-1 -proteinase inhibitor; cording to the Cohn ethanol fractionation technique (b) mixing the composition of Step (a) with a or its modifications [see for example, Cohn et al., polyol selected from sucrose or reduced sugars J.Am. Chem. Soc, 68,459, (1946); Oncley eTal., io and a source of citrate ions in an aqueous ibid., 71,541 (1949); U.S. Patent 2,390,074; "'The medium, said polyol and citrate ions being Plasma Proteins", second edition, Vol. Ill, pages present in an amount sufficient to stabilize the 548-550, Academic Press, New York, N.Y.] to give protein during pasteurization; a Cohn fraction containing PI. This method for (c) and heating the mixture at a temperature and removing proteins containing disulfide bonds could is for a time sufficient to pasteurize the composi- have application in genetically engineered PI, in tion. those situations where the solution containing PI, Generally the amount of polyol and citrate ions such a tissue culture medium or other eluates, also in the mixture of Step (b) is about 20% to satura- contained disulfide proteins, such as albumin. tion of polyol. On a weight to volume basis and PI was produced following the method outlined 20 about 0.1-1.0 moles of citrate ion per liter of solu- by Coan et al., Vox Sang. 48:333-342, 1985; U.S. tion. Patents 4,379,087" and 4,439,358, and improve- Heat denaturation can be accomplished inde- ments set forth by Shearer et al., U.S. Patent pendently of a viral inactivation step by raising the 4,656,254. temperature of the solution containing PI and the Dithiothreitol was added to Coan's purified col- 25 mild reducing agent sufficient to reduce the disul- umn eluate at a concentration of 5mM, pH was fide bonds to a level above 35 °C. The reducing adjusted to 8.0-8.2.
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