CS206294B1

CS206294B1 - Method of isolation of the albumine from the waste fraction of the humen fraction

Info

Publication number: CS206294B1
Application number: CS516979A
Authority: CS
Inventors: Jozef Bulik
Original assignee: Jozef Bulik
Priority date: 1979-07-25
Filing date: 1979-07-25
Publication date: 1981-06-30

Description

Vynález sa týká spósobu izolácie albuminu z odpadnej frakcie ludskej plazmy, ktorý sa v roztoku stabilizuje a ostatně bielkoviny sa tepelne denaturujú a potom precipitujú etanolom.The invention relates to a process for the isolation of albumin from the human plasma waste fraction, which is stabilized in solution and, moreover, the proteins are thermally denatured and then precipitated with ethanol.

Podlá literárnych údajov najznámejšie izolačně postupy albuminu z ludskej plazmy sú: Etanolová frakcionácia [E. J. Cohn, L. E. Strong, W. L. Hughes, D. J. Mulford, J. Ashworth, M. Melin, H. L. Taylor: J. Amer. Chem. Soc., 68, 459, (1946)] a jej modifikácia (Nitschmann H., Kistler P., Lergie W.: Helv. Chim. Acta, 37, 866, [1954]).According to literature, the best known isolation procedures for human plasma albumin are: Ethanol fractionation [E. J. Cohn, L. E. Strong, W. L. Hughes, D. J. Mulford, J. Ashworth, M. Melin, H. L. Taylor: J. Amer. Chem. Soc., 68, 459, (1946)] and modifications thereof (Nitschmann, H., Kistler, P., Lergie, W., Helv. Chim. Acta, 37, 866, [1954]).

Rivanolová metoda· [Rejnek J., Bednářik T., Mašek J.: Cs. farm., 10, 407, (1961)].Rivanol method · [Rejnek J., Bednářik T., Mašek J .: Cs. farm., 10, 407 (1961)].

Izolácia pomocou iontomeničov [Rybák M., Rejnek J.: Cs. farm., 7, 80, (1958)].Ion-exchange isolation [Rybák M., Rejnek J .: Cs. farm., 7, 80, (1958)].

Izolácia etylenglykolom [Curling J. M., Borglóf J., Lindouist L. O., Briksson S.: Vox sang., 33, 97, (1977), Vilioen M.: Vox sang., 35, 268, (1978) (Garcia L. A., Ordotnez G. A.: Pat. USA No 4025500)].Ethylene glycol isolation [Curling JM, Borglof J., Lindouist LO, Briksson S .: Vox sang., 33, 97, (1977), Vilioen M .: Vox sang., 35, 268, (1978) (Garcia LA, Ordotnez GA : U.S. Pat. No. 4025500)].

Tepelno-etanolová metoda [Hoch H., Chanutin A.: Arch. of biochem. and biophysic, 51, 27, (1954), Lundblad J. I.: Vox sang., 5, 122, (1960), Gabr Y., Soliman H. R.: Acta biolog, et med. ger., 27, 341, (1971), Schneider W., Lefewre H., Friedler H., Mc Carty L. I.: Blut, 30j 121) (1875), Schneider W,, Wolter D., Mc Carty L. J.: Folia haematol., 104, 116, (1977)].Thermal-ethanol method [Hoch H., Chanutin A .: Arch. of biochem. and biophysic, 51, 27, (1954), Lundblad J.I .: Vox sang., 5, 122, (1960), Gabr Y., Soliman H. R., Acta biolog, et med. ger., 27, 341, (1971), Schneider W., Lefewre H., Friedler H., Mc Carty LI: Blut, 30j 121) (1875), Schneider W. ,, Wolter D., Mc Carty LJ: Folia haematol 104, 116 (1977)].

Pri izolácii albuminu etanolovou frakcionáciou krvnej plazmy vzniká bielkovinový odpad vo formě frakcie IV. Táto sa izoluje pri pH 5,8, iónovej sile 0,1, 40 objemových % etanolu a teplote —6 °C. Uvedená frakcia obsahuje niekolko druhov plazmatických bielkovín, včítane albuminu, kvantitativné získanie ktorého bežne používanými metodami sa nedaří realizovať.Isolation of albumin by ethanol fractionation of blood plasma produces protein waste in the form of Fraction IV. This was isolated at pH 5.8, an ionic strength of 0.1, 40 vol% ethanol and a temperature of - 6 ° C. Said fraction contains several kinds of plasma proteins, including albumin, a quantitative recovery of which the commonly used methods cannot be realized.

Jedným z možných spósobov kvantitativného získania albuminu z odpadnej frakcie IV je modifikácia tepelno-etanolovej metody, ktorá povodně ako východzí materiál využívá l’udskú plazmu, v ktorej zastúpenie jednotlivých bielkovín je podstatné iné ako v odpadnej frakcií IV. Nevýhodou týchto metod je, že sa pracuje pri nízkom percente bielkovín východzieho materiálu, čo zvyšuje nároky na pracovně objemy, nízkej koncentrácii stabilizátora (0,004 M Na kaprylát) a vysokom percente etanolu, čo pri hraničných teplotách (70 °C) móže dochádzať k inreverzibilným změnám v molekule albuminu. Podstatným problémom pri tepelno-etanolovej metóde přípravy albuminu je separácia denaturovaných balastných bielkovín, ktorá sa spravidla prevádza na špeciálnych filtroch, alebo sa vyzráža polyetylénglykolom.One possible way of quantitatively recovering albumin from waste fraction IV is to modify the heat-ethanol method, which uses floods as the starting material for human plasma, in which the proportion of individual proteins is substantially different than in waste fraction IV. The disadvantage of these methods is that they work at a low percentage of protein of the starting material, increasing the demands on working volumes, a low stabilizer concentration (0.004 M per caprylate) and a high percentage of ethanol, which can cause inversible changes at 70 ° C. in an albumin molecule. A major problem in the heat-ethanol method of preparing albumin is the separation of denatured ballast proteins, which is usually converted to special filters or precipitated by polyethylene glycol.

Originálnost navrhovanej izolácie albuminu z odpadnej frakcie IV je v tom, že pri de206294 náturách balastných bielkovín sa používá optimálna koncentrácia kombinovaných stabilizátorov za přítomnosti nízkého· percenta etanolu a vysokého percenta bielkovín, čo má priaznivý vplyv na kvalitu výrobku a výrobnú kapacitu.The originality of the proposed albumin isolation from waste fraction IV is that the de206294 ballast protein coatings use the optimum concentration of the combined stabilizers in the presence of low percent ethanol and high percent protein, which has a beneficial effect on product quality and production capacity.

Jeden z najnáročnějších technologických stupňov, a to izolácia denaturovaných bielkovín sa prevádza účinným, jednoduchým a zatial’ pri tomto spósobe izolácie albuminu neopísaným postupom, precipitáciou etanolom.One of the most demanding technological steps, namely the isolation of denatured proteins, is carried out by an efficient, simple and yet in this method of albumin isolation by an unwritten procedure, ethanol precipitation.

Podstata izolácie albuminu z odpadnej frakcie IV spočívá v tom, že albumin přítomný vo frakcii IV sa v roztoku stabilizuje kaprylátom sodným a acetyl-DL-tryptofanom za podmienok 4—5 obj. % etanolu, pH 6,5— 6,7 a 4,5—5,5 hmot. % bielkovín sa ostatně bielkoviny tepelne denaturujú pri teplote 68 až 69 °C po dobu 30 až 40 minút. Denaturované bielkoviny sa potom precipitujú etanolom za podmienok 12 obj. % etanolu, pHThe essence of isolating albumin from waste fraction IV is that the albumin present in fraction IV is stabilized in solution with sodium caprylate and acetyl-DL-tryptophan under conditions of 4-5 vol. % ethanol, pH 6.5-6.7 and 4.5-5.5 wt. In addition, the protein is thermally denatured at 68 to 69 ° C for 30 to 40 minutes. The denatured proteins are then precipitated with ethanol under conditions of 12 vol. % ethanol, pH

5,1 až 5,3, iónovej sile 0,09 a separujú centrifugáciou. Supernatant obsahujúci albumin sa lyofilizuje, resp. albumin zo supernatantu sa vyzráža etanolom, popřípadě koncentruje iným vhodným spósobom.5.1 to 5.3, an ionic strength of 0.09 and separated by centrifugation. The albumin-containing supernatant is lyophilized, respectively. the albumin from the supernatant is precipitated with ethanol, optionally concentrated by another suitable method.

Příklad prevedeniaExecution example

1000 g pasty frakcie IV sa suspenduje v 5000 ml apyrogénnej vody. K roztoku sa přidá 19 g kaprylátu sodného a 21 g acetylDL-tryptofanu a pri pH 6,5—6,7 a teplote 68 —69 °C sa roztok zohrieva po dobu 30—40 minút. Denaturované bielkoviny sa vyprecipitujú přidáním 650 ml 96 % etanolu pri pH1000 g of Fraction IV paste is suspended in 5000 ml of pyrogen-free water. 19 g of sodium caprylate and 21 g of acetyl DL-tryptophan are added to the solution and the solution is heated for 30-40 minutes at pH 6.5-6.7 and 68-69 ° C. Denatured proteins are precipitated by adding 650 ml of 96% ethanol at pH

5,1—5,3, iónovej sile 0,09, teplote —2°C po dobu 12 hodin. Vyprecipitované bielkoviny sa separujú na vysokootáčkových prietokových centrifugách rýchlosťou 25 1/hod. pri teplote —3 °C a supernatant, obsahujúci albumin sa číri cez azbestocelulózové vložky, filtrát sa rozplňuje do NTS fliaš, rotačně namrazuje pri —45 °C a lyofilizuje za nízkého vákua na špeciálnych sušiacich aparátoch KS 30, alebo LZ45.5.1-5.3, an ionic strength of 0.09, a temperature of -2 ° C for 12 hours. The precipitated proteins are separated on high speed flow centrifuges at a rate of 25 L / h. at -3 ° C and the albumin-containing supernatant is cleared through asbestos cellulose pads, the filtrate is filled into NTS bottles, rotary frozen at -45 ° C and lyophilized under low vacuum on special KS 30 or LZ45 drying apparatuses.

1000 g pasty frakcie IV sa suspenduje v 5000 ml apyrogénnej vody. K roztoku sa přidá 19 g kapry látu sodného a 21 g acetylDL-tryptofanu a pri pH 6,5—6,7 a teplote 68—69 °C sa roztok zohrieva po dobu 30—40 minút. Denaturované bielkoviny sa vyprecipitujú přidáním 650 ml 96 % etanolu pri pH1000 g of Fraction IV paste is suspended in 5000 ml of pyrogen-free water. To the solution were added 19 g of sodium carboxylate and 21 g of acetyl DL-tryptophan, and the solution was heated for 30-40 minutes at pH 6.5-6.7 and 68-69 ° C. Denatured proteins are precipitated by adding 650 ml of 96% ethanol at pH

5,1—5,3, iónovej sile 0,09, teplote —2 °C po dobu 12 hodin. Vyprecipitované bielkoviny sa separujú na vysokootáčkových prietokových centrifugách rýchlosťou 25 1/hod. pri teplote —3 °C a albumin obsiahnutý v supernatante sa vyzráža etanolom za podmienok pH 4,80, iónovej sile 0,1, 40 obj. % etanolu a teplote —8 °C.5.1-5.3, an ionic strength of 0.09, a temperature of -2 ° C for 12 hours. The precipitated proteins are separated on high speed flow centrifuges at a rate of 25 L / h. at -3 ° C and the albumin contained in the supernatant is precipitated with ethanol under conditions of pH 4.80, an ionic strength of 0.1, 40 vol. % ethanol and a temperature of -8 ° C.

5,1—5,3, iónovej sile 0,09, teplote —2 °C po dobu 12 hodin. Vyprecipitované bielkoviny sa separujú na vysokootáčkových prietokových centrifugách rýchlosťoiu 25 1/hod. pri teplote —3 °C a albumin přítomný v supernatante sa koncentruje na špeciálnom zariadení typu Centriterm, alebo DC 30.5.1-5.3, an ionic strength of 0.09, a temperature of -2 ° C for 12 hours. The precipitated proteins are separated on high speed flow centrifuges at 25 L / h. at - 3 ° C and the albumin present in the supernatant is concentrated on a special Centriterm or DC 30 device.

Výťažnosťyield

Výťažnosť albuminu sa pohybuje od 120 do 140 g lyofilizovaného produktu na 1 kg pasty odpadnej frakcie IV. Dosahovaná výťažnosť je závislá na obsahu albuminu v odpadnej frakcii.The recovery of albumin ranges from 120 to 140 g of lyophilized product per 1 kg of waste fraction IV. The yield obtained is dependent on the albumin content of the waste fraction.

Analytické hodnotenieAnalytical evaluation

V následuj úcej tabufke sú uvedené analytické hodnoty zistené u komerčného přípravku a přípravku vyrobeného podl’a vynálezu a tieto porovnané s požadovanými kritériami normy na albumin.The following table shows the analytical values found for the commercial preparation and the preparation made according to the invention and compared with the required criteria of the albumin standard.

Požadované sfcúáky requested sfcúáky Kritériá normy criteria standards Dosliahnuté výsledky Results achieved komerč. príp. commerce. respectively. ipríp. podlá vynálezu ipríp. according to the invention Sterilita sterility sterilně aseptically sterilně aseptically sterilně aseptically Neškodnost Sfcúška na Harmless Sfcúška na neškodné harmless vyhovuje suits vyhovuje suits pyr. látky Obsah PYR. substances Content apyrogénne non-pyrogenic vyhovuje suits vyhovuje suits bielkovín protein 4,5-5,5 g/ 100 ml 4.5-5.5 g / 100 ml 4^94 g/lOOml 4? 94 g / 100ml 4,72 g/100 ml 4.72 g / 100 ml Alkohol kvallrtaitíviny alcohol kvallrtaitíviny negativna negativna negativna negativna negativna negativna PH Tepelná PH heat 6,2-7,4 6.2 to 7.4 6,95 6.95 6,75 6.75 stabilita stability max. ©0 ine- max. © 0 ine- 16 nefel. 16 nefel. 16 nefel. jed. 16 nefel. poison. íel. jed. IEL. poison. jed. poison. Obsah Na Contents Na max, 87 mmol/1 max, 87 mmol / L 61 tnm'ol/1 61 nmol / L 46 ,mmol/1 46, mmol / L Elektrofo- retloká čistota Elektrofo- retloká purity min, 96 % min, 96% 96,0 % 96.0% 96,4 % 96.4%

Uvedené výsledky ukazujú, že kvalitativně kritériá přípravku vyrobeného podl’a vynálezu sa nelíšia od komerčného přípravku a vyhovujú normě na albumin.The results shown show that the qualitative criteria of the preparation produced according to the invention do not differ from the commercial preparation and comply with the albumin standard.

Claims

2 naturation of ballast proteins, the optimum concentration of combined stabilizers is used in the presence of low percentage of ethanol and a high percentage of protein, with a beneficial effect on product quality and production capacity. One of the most sophisticated technological steps, namely the isolation of denatured white wines, is carried out in an efficient, simple, and in this way isolation of albumin by the non-described process, ethanol precipitation. The principle of isolating albumin from waste fraction IV is that the albumin present in the fraction IV is stabilized in solution with sodium prylate and acetyl-DL-tryptophan under conditions of 4-5 vol% ethanol, pH 6.5-6.7 and 4.5-5.5 wt. % of the proteins, the other proteins are thermally denatured at 68-69 ° C for 30-40 minutes. The denatured proteins are then precipitated with ethanol under conditions of 12 vol% ethanol, pH 5.1 to 5.3, ionic strength 0.09 and separated by centrifugation. The albumin-containing supernatant is lyophilized, respectively. the albumin from the supernatant is precipitated with ethanol, optionally in a suitable manner. An exemplary embodiment of 1000 g of paste IV paste is suspended in 5000 ml of pyrogen-free water. 19 g of sodium caprylate and 21 g of acetyl-DL-tryptophan are added to the solution and the solution is heated for 30-40 minutes at pH 6.5-6.7 and 68-69 ° C. The denatured proteins are purified by adding 650 ml of 96% ethanol at pH 5.1-5.3, ionic strength 0.09, 2 ° C for 12 hours. The precipitated proteins are separated on high speed flow centrifuges at a rate of 25 l / h. at 33 ° C and the supernatant containing albumin is sieved through asbestocellulose inserts, the filtrate is filled into NTS bottles, rotatively frozen at 4545 ° C, and lyophilized under low vacuum in special drying apparatuses KS 30 or LZ45. 1000 g of paste IV paste are suspended in 5000 ml of pyrogen-free water. 19 g of sodium carboxylate and 21 g of acetyl-DL-tryptophan are added to the solution and the solution is heated for 30-40 minutes at pH 6.5-6.7 and at 68-69 ° C. The denatured proteins are purified by adding 650 ml of 96% ethanol at pH 5.1-5.3, ionic strength 0.09, 2 ° C for 12 hours. The precipitated proteins are separated on high speed flow centrifuges at a rate of 25 l / hr. at -3 ° C and the albumin contained in the supernatant is precipitated with ethanol under conditions of P 4,80, an ionic strength of 0.1, 40 vol% ethanol and a temperature of -8 ° C. 1000 g of paste IV paste are suspended in 5000 ml of pyrogen-free water. 19 g of sodium caprylate and 21 g of acetyl-DL-tryptophan are added to the solution and the solution is heated for 30-40 minutes at pH 6.5-6.7 and 68-69 ° C. The denatured proteins are purified by adding 650 ml of 96% ethanol at pH 5.1-5.3, ionic strength 0.09, 2 ° C for 12 hours. The precipitated proteins are separated on high speed flow centrifuges at a rate of 25 l / h. at-3 ° C and the albumin present in the supernatant is concentrated on a Centriterm or DC 30 special apparatus. Yield The albumin yield ranges from 120 to 140 g of lyophilized product per kg of paste fraction IV. The yield obtained is dependent on the albumin content of the waste fraction. Analytical Evaluation The following table shows the analytical values found in the commercial preparation and the preparation prepared according to the invention and compared with the desired criteria for albumin. Required Partitions Standard Criteria Achieved Commercial Results eventually. , or. sterility sterile sterile sterile harmlessTest for harmless satisfies pyr. substancesContent pyrogen-free satisfies protein 4,5-5,5 g / 100 ml 4 ^ 94 g / 100ml 4,72 g / 100 ml Alcohol quality negative negative negative PH Heat 6,2-7,4 6,95 6,75 stability max © 0 ine- 16 nephel. 16 nefel. poison. clays. poison. poison. Content Na max, 87mmol / 1 61mm'ol / 1 46mmol / l Electrophoretic purity min, 96% 96.0% 96.4% The results show that the quality criteria of the preparation made according to the invention do not differ from commercial product and comply with the albumin standard. SUBJECT OF THE INVENTION

What is claimed is: 1. A method for isolating albumin from a human plasma waste fraction comprising alpha globulins, beta metal-binding globulins, a large portion of lipoproteins, alpha lipoprotein, alpha2 mucoprotein, alpha2 glycoprotein aalbumin, wherein the paste fraction IV is suspended in water, the albumin is stabilized and, moreover, the proteinaceous present in fraction IV is thermally denatured at 68-69 ° C for 30-40 minutes, precipitated with ethanol, separated by centrifugation, and the supernatant containing the cesiumbumin is filtered, lyophilized, or concentrated. 3

2. A process as claimed in claim 1, wherein the albumin is stabilized with 0.02 sodium metaprylate and 0.02 M acetyl-DL-tryptophan and the proteins are denatured in the presence of 4-5 vol. up to 5.5% by weight of protein.

3. A process according to claim 1 or 2 wherein the denatured proteins are saprecipitated at 12 vol% ethanol and a pH of 5.1 to 5.3.