TWI226333B - Method for the recovery of fibrinogen, prothrombin complex, immunoglobulin, albumin and antithrombin III from frozen plasma - Google Patents

Abstract

In this patent application we demonstrate our success in the establishment of a method that could efficiently recovers fibrinogen, prothrombin complex, immunoglobulin, antithrombin III and albumin from frozen plasma. The protein fractions are recovered sequentially in separate steps: fibrinogen is recovered as a precipitate; prothrombin complex and immunoglobulin are recovered by adding 20% ethanol while aqueous supernate remains antithrombin III and albumin. This fraction is further purified into separated proteins by using 40% to 42% ethanol. Each fraction could be purified further, such as antithrombin III is recovered by adsorption to aluminum salt. The combined method described here is highly efficient in retrieving proteins from frozen plasma and those purified proteins are suitable for further method into different biological preparations.

Description

1226333 发明 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a method for producing fibrinogen, fibrinogen complex, third antithrombin, immunoglobulin, and albumin from plasma, particularly The invention relates to a method for producing fibrinogen, fibrinogen complex, third antithrombin, immunoglobulin and albumin from frozen plasma. [Prior art] 1. Traditional purification method of plasma protein

The blood components are complex and difficult to store. Especially, the storage conditions of plasma and blood cells obtained by using blood donations are very different, so there is a defect in the specificity and convenience of blood transfusion therapy; since the early 1940s, plasma components have been used in cold alcohol. The different principles of solubility in Shen Dian (1'2), respectively, in order to purify a variety of proteins convenient for medical use. In the early days, this "cold alcohol precipitation method" was used to simultaneously prepare four basic blood paddle preparations, such as the eighth coagulation factor, the ninth coagulation factor, immunoglobulin, and albumin, so it must be rapidly frozen (freezing within 4 hours. -30 ° C) fresh frozen plasma as raw materials, the above four proteins are separated by continuous changes in alcohol concentration; however, in recent years, the eighth and ninth coagulation factors have been manufactured by genetic engineering. (3_6), the demand for plasma protein purification products is relatively reduced, so the "cold alcohol precipitation" steps for plasma protein purification must also be relatively developed and changed to achieve the purpose of making good use of precious blood resources.

2. Difficulties of the plasma protein purification step Because it is not easy to simultaneously purify multiple proteins in plasma without affecting the yield of each other, the design of the production method is very important. In addition, a large amount of plasma must be used to purify and isolate the final product, but fresh frozen plasma is costly and limited; if frozen plasma at -18 ° C is used as the raw material, the amount of effective coagulation factor is not rapidly frozen before storage. Very few; coupled with the higher temperature during storage and slightly more impurities, there are not many production methods that have been successfully separated by the traditional standard "low-temperature alcohol precipitation method"; or some separation methods use medical treatments that have not been accepted Chemicals that make the final product not immediately available for clinical use. The content of this patent application is to overcome the above-mentioned difficulties and establish a production method using various frozen plasma as raw materials to recover fibrinogen, profibrin complex, third antithrombin, immunoglobulin and albumin. [Summary of the Invention] 6 1226333 The purpose of the present invention is to establish a production method to effectively use frozen plasma to separate various proteins. The frozen plasma used in the step of the present invention may be first subjected to a solvent-detergent. (7) Isolate after treatment, so that the separated fibrinogen, fibrinogen complex, the third antithrombin, immunoglobulin and albumin are first deactivated by the virus step (8) to improve Product safety. In addition, foreign technology often uses fresh frozen plasma as a raw material, but fresh frozen plasma is more expensive. In order to effectively use blood resources, it is urgent to develop a method using frozen plasma as a raw material. In order to solve the two major problems of safety and raw materials, we have established a new production method (such as the first picture), which has three characteristics

1. In terms of safety: Since 1985, more than 25 plasma separation plants in many countries have used tri (n-butyl) phosphate [TNBP], an organic solvent such as Tween 80 and other cleaning agents. Agent for treating plasma-separated preparations (9); because of its unique virus inactivation effect on enveloped viruses, it greatly improves the safety of the preparations. Moreover, the present invention is processed first in the raw material stage, so that each product goes through the virus inactivation step, so it is safe and worry-free.

2. In terms of raw materials: Frozen plasma is obtained by centrifugation of whole blood. After centrifugation, whole blood can obtain platelets and red blood cells in addition to frozen plasma. The valid periods of the three are five years, five days, and five days after blood collection. 42 days; countries around the world suffer from oversupply of frozen plasma in bags. Using it as a raw material, not only does not worry about scarcity, it is more than 1/3 cheaper than fresh frozen plasma. In addition, most platelets and red blood cells of the same source have been transfused. If there is an adverse reaction Frozen plasma is eliminated, thus ensuring safety. 3. In terms of principle: The principle of the "Cohn's Low-Temperature Alcohol Precipitation Method" used in this production method is simple. It only needs to adjust three variables such as temperature, alcohol concentration, and pH 値, which can stratify plasma proteins, which is the majority in the world. Used in the plasma separation factory, this method adjusts the conditions according to its precipitation principle, not only achieves the function of virus inactivation, but also confirms that this reaction can also be successfully applied to frozen plasma raw materials; the biological agent industry has already developed mature, The process widely accepted and used for many years and modern requirements can be balanced. Based on this production process, further application of various biochemical separation technologies can refine different proteins separately, and then cooperate with other necessary processing processes to prepare plasma separation preparations. [Embodiment] 7 1226333 The method provided by the present invention generally includes: treating frozen plasma with a solvent-detergent for virus deactivation, low-temperature alcohol precipitation for preliminary purification, and different methods based on the plasma protein produced. Subsequent purification and other steps are described in detail below: Thaw frozen plasma at low temperature (0 ~ 2 ° C), mix with TNBP solvent and Tween 80 detergent to make the final concentration of 0.5% ~ 5% TNBP Solvent and 0.5% ~ 5% Tween 80 cleaner (Calbiochem, La Jolla, CA), mixed with a shaker and placed in a water bath and increased the reaction temperature to 30 ° C. After shaking for six hours, the The container was placed on an ice bath and cooled to 0 ~ 2 ° C (first picture), and the virus was inactivated.

Into. The "centrifugation sinking steps" described in this section are all achieved by centrifugation (Beckman J2MC with JA-21 roter) at each stage at a reaction temperature of 10,000 xg for 30 minutes; the alcohol concentration is achieved by 50% (v / v) Alcohol adjustment, please refer to the first picture. The detailed description of the first figure is as follows: Phase 1 (1) The plasma that has undergone the solvent-detergent treatment step described above or that has not been treated in this step is subjected to a centrifugal precipitation step to be separated into the first Colloid (11) and first supernatant (12). The second stage (2)-add the alcohol and acetic acid buffer to the first supernatant (I2) of the first stage, so that it finally contains 8% ~ 10% alcohol and 0.1M acetic acid Buffer,

pH7.3. If the plasma is not deactivated by the solvent-detergent virus, the final alcohol concentration described above is 8%. After the foregoing solutions are thoroughly mixed, the mixture is rapidly cooled to 0 ° C ~ 2 ° C (Stuart Scientific, Surrey, UK), and continuously stirred at 0 ° C ~ 21: for 24 minutes, and then subjected to a centrifugal precipitation step, Layer to obtain a second colloid (21) and a second supernatant (22), wherein fibrinogen (23) can be obtained from the second colloid (21) and the first colloid (11). The third stage (3)-add the second supernatant (2¾) of the second stage (2¾) to alcohol and acetic acid buffer so that it finally contains 20% alcohol and 0.1M acetic acid buffer, pH 7.2 ~ 7.6. After thorough mixing, the reaction solution is maintained at 0 ~ 4 QC and continuously stirred for 20 minutes, and the third colloid (31) and the third supernatant (32) are separated through the centrifugal precipitation step. 8 1226333 Fourth Stage (4)-Dissolve the third colloid (31) in the third stage (3) in alcohol and acetic acid buffer so that it finally contains 17% alcohol and 0.3 M acetic acid buffer, ρΗ5 · 2 ~ 5 · 4. After thorough mixing, the reaction solution is rapidly cooled to -5 ° C and continuously stirred for 19 minutes. After the centrifugal precipitation step, the fourth colloid (41) and the fourth supernatant (42) are separated into layers. Fifth stage (5) —Add alcohol and sodium bicarbonate buffer to the third supernatant (32) in the third stage (3), so that it finally contains 40% ~ 42% alcohol and 0.2M sodium carbonate buffer , PH 5.75. After thorough mixing, the reaction solution is maintained at _5 ° C and continuously stirred for 14 minutes, and the fifth colloid (51) and the fifth are obtained through the centrifugal precipitation step. Supernatant (52). In the sixth stage, the fifth supernatant (52) in the fifth stage (5) is added with alcohol and acetic acid, so that it finally contains 40% to 42% alcohol and 0.8M. After the acetic acid buffer solution, ρΗ 4.80, was thoroughly mixed, the reaction solution was maintained at -5 ° C and continuously stirred for 40 minutes. After the centrifugal precipitation step, the sixth colloid (61) and the sixth supernatant (62) were separated by layers. . Purification of fibrinolytic enzymes with yin-named facial fat. The fourth colloid (41) precipitated in the fourth stage (4) is further purified by anion exchange resin purification (7). The detailed process is shown in the second figure. As shown in the figure, the fourth colloid (41) precipitated in the fourth stage (4) was completely dissolved in the citrate buffer (ρΗ7.44), and the solution containing PEG 6000 was added to make it final. The concentration of PEG 6000 was 1%, and it was stirred for 45 minutes. After centrifugation, the supernatant was purified with an anion exchange resin (DEAE Sepharose Fast Flow (FF)) (Amersham-Pharmacia Biotech, Uppsala, Sweden), and 2M sodium chloride. -Citrate buffer (NaCl-citrate buffer) wash out the fibroin enzyme complex adsorbed on the resin, Then to cut (cut-off molecular weight of Fen 3〇K of JumbosepTM (Pall Gilman Sciences, Ann Arbor, MI) ultra-filtration and removal of citrate, to obtain a raw material complex enzyme plasmin (71). . I »ei * (gel filtmtiom) and ion-cross-linked lipid-purified immunoglobulin The fourth supernatant (42) of the fourth stage (4) was further purified by colloid filtration and ion exchange resin purification (8) The detailed process is shown in the third figure. Transfer the fourth supernatant (42) of the fourth stage to 1226333

Sephadex G25C resin (Amersham-Pharmacia Biotech) was gelled, and the buffer was replaced with ρΗ6.5 and a conductivity (1.40mS) solution, and then anion exchange resin (Q Sepharose FF) (Amersham-Pharmacia Biotech) was used. ) Was purified, and the immunoglobulin adsorbed on the resin was washed with 0.02 M sodium acetate buffer solution (ρΗ6.5), and then purified by cation exchange resin (CM Sepharose FF) (Pharmada Biotech) to adsorb impurities. Finally, ultrafiltration with a cut-off molecular weight of 30K in Jumbosep ™ (Pall Gilman Sciences) and removal of excess salts yields immunoglobulin (81). Purification of the third antithrombin by aluminum salt gel adsorption and heat treatment The fifth colloid (51) precipitated in the fifth stage (5) is further purified by aluminum salt gel adsorption and heat treatment purification (9). The detailed process is shown in Figure 4. The fifth colloid (51) precipitated in the fifth stage is completely dissolved in a mixed solution of sodium citrate and sodium chloride (the volume ratio of 0.1M sodium citrate to 0.15M sodium chloride is 1: 4, ρΗ7 · 3) Then, it was heat-treated at 56 ° C for three hours, and then reacted with aluminum hydroxide gel (Sigma, St. Louis, MO) to adsorb proteins. Finally, it was treated with 1M sodium phosphate buffer (sodium phosphate, ρΗ7 ·). 0) (1 ()) Wash the voice containing the third antithrombin (91) [5 points. The sixth colloid (61) precipitated in the sixth stage (6) is purified by diafiltration and cation exchange resin purification, and further purification is performed by diafiltration and cation exchange resin purification (10). The detailed process is as follows: The fifth figure is shown. After completely dissolving the sixth colloid (61) precipitated in the sixth stage with pure water, diafiltration was performed with Jmnbosq) ™ (Pall Gilman Sciences) with a molecular weight cut-off of 30K, and the concentration of the buffer solution was adjusted to 0.02M. Purification was performed with cation exchange resin (CM Sepharose FF) (Amersham-Pharmacia Biotech), and then the albumin adsorbed on the resin was washed out with 0.1 M sodium acetate (NaAc, pH 5.5) to obtain white Protein (101). Test of agglutination time of fibrinase (11) Add bovine thrombin (Sigma) to 5 NIH units (in 100 μΐ) to 0.2 m 啲 diluted fresh plasma (1: 1 in normal saline), And record the time required to form a transparent fibrin film on the petri dish (approximately 10 minutes); in the experiment, replace the diluted fresh plasma with fibrinogen solution or add a third anticoagulant to the cow's fibrinase After the enzyme, the time required to form a transparent blood fibrous film 1226333 was measured and compared with diluted fresh plasma. Enzyme immunoassay The sandwich enzyme immunoassay [Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)] was compared with a standard solution of known concentration to measure the production of immunoglobulin and albumin: 50 mM carbonate buffer, pH 9.6 Solution (carbonate buffer) 4,000-fold diluted rabbit anti-human immunoglobulin and rabbit anti-human albumin (Dakopatts, Copenhagen, Denmark) at 37 ° C 96-well ELIS A dish for one hour After washing, add 50 μΐ of test solution to each well. After 37T: reaction, rinse at room temperature, add 3,000-fold diluted rabbit anti-human

IgG (peroxidase-labeled rabbit anti-human IgG and anti-human albumin; Dakopatts) »After color reaction, measure its absorbance at 490 nm; standard solutions of immunoglobulin and albumin were purchased from Dakopatts and Sigma, respectively. The amount of immunoglobulin and albumin was measured by enzyme immunoassay and recorded. The results are shown in Table 1.

11 1226333 Schedule 1: Representative production of fibrinogen, fibrinogen complex, third antithrombin, immunoglobulin and albumin from frozen plasma as raw material Original # 70.0 Original blood fiber enzyme complex # 30.2 Immunoglobulin 48.0 Third anticoagulant enzyme # 7.6 Albumin 76.8 * Yield = final product amount obtained / content of raw materials in frozen plasma # According to testing the same volume of raw material frozen plasma And the change in the "fibrinase agglutination time" of the final product. 12 1226333 References 1. Cohn EJ? Strong LE, Hughes WL Jr, et al Preparation and properties of serum and plasma proteins: IV. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J Am Chem Soc 1946; 68: 459. 2. Richter P. Present state and perspectives of the farctionation of human plasma, serum and placenta. Ann Immunol Hung 1973; 17:85. 3. Moll S, White GC 2nd. Treatment of the hemophilias Curr Opin Hematol 1995; 2: 386. 4. Green C, Akehurst R. For debate. New therapeutic products and difficult decisions. The case of recombinant factor VIII in the management of haemophilia A · / Public Health Med 1998; 20: 137 5. Aygoren-Pursun E, Scharrer I. A multicenter pharmaco-surveillance study for the evaluation of the efficacy and safety of recombinant factor VIII in the treatment of patients with hemophilia A. German Kogenate Study Group. Thromb Haemost 1997; 78: 1352 6. White Q Shapiro A, Ragni M, Garzone P, Goodfellow J, Tubr idy K, Courier S. Clinical evaluation of recombinant factor IX. Semin Hematol 1998; 35 (2 Suppl 2): 33. 7. Horowitz B. Investigations into the application of tri (nbutyl) phosphate / detergent mixtures to blood derivatives. Curr Studies Hematol Blood Transf \ 9S9 \ 56:83. 8. Peerlinck K, Amout J, Tamise M, Vanherle A, Fondu P, Vermylen J. In vitro and in vivo evaluation of two factor VIII concentrates virally inactivated by solvent-detergent or by pasteurization . Acta

Clin 5 ^ / g 1991; 46: 298. 9. Horowitz MS, Rooks C, Horowitz B, Hilgartner MW. Vims safety of solvent / detergent-treated antihaemophilic factor concentrate. Lancet 1988; 23: 186. 13 1226333 10. Seegers WH Antithrombin III: a backward glance o'er travel'd roads. MYing / Ying 1975; 52: 195. 11. Wohllebe W, Taube C, Fiedler H. Behavior of thrombin time, activities of antithrombin II and 3 following administration of cobalt (II) compounds in rabbits. Z Gesamte Inn Med 1968; 23: 537.

Rosenthal RL? Sloan E. Assay of clotting factors in outdated blood bank plasma and its potenial use for therapy in hemophilia and other hemorrhagic dyscrasias. Transfusion 1966; 6: 289.

14 1226333 [Schematic description] The first picture: using frozen plasma as the material to purify and recover fibrinogen, prothrombin complex, and immunogloblo by immunoprecipitation and stratification. -schematic diagram of bulin), third antithrombin III and albumin. Figure 2: Flow chart for further purification of fibrinase complex by anion exchange resin. Figure 3: Flow chart for further purification of immunoglobulins by gel filtration and ion exchange resin. Figure 4: Flow chart of the third antithrombin enzyme obtained by further purification by aluminum salt gel adsorption and heat treatment. Figure 5: Flow chart of obtaining albumin by diafiltration and further purification of cation exchange resin. The first colloid 12 the first supernatant the second colloid 22 the second supernatant third stage 31 the third colloid fourth stage 41 the fourth colloid fifth stage 51 the fifth colloid sixth stage 61 the sixth colloid [element supplement number Explanation] 1 The first stage 11 2 The second stage 21 23 Fibrinogen 3 32 The third supernatant 4 42 The fourth supernatant 5 52 The fifth supernatant 6 62 The sixth supernatant 71 The original fibrinase complex Body 81 Immunoglobulin 91 Third antithrombin 101 Albumin 7 Purification of anion exchange resin 8 Purification of colloidal filtration and ion exchange resin 9 Purification of aluminum salt gel and heat treatment purification 10 Diafiltration and purification of cation exchange resin 15

Claims (1)

1226333 colloid and fifth supernatant; (i) the fifth colloid in (h) is purified by aluminum salt gel adsorption and heat treatment to recover the third antithrombin; ① the fifth in (h) Add the alcohol and buffer solution to the final solution to make it into a mixed solution containing 40-42% alcohol and pH 4.8. After that, maintain the level of -5 ° C and continue stirring for a period of time, and then perform centrifugation. Then, the colloid is separated from the supernatant to obtain a sixth colloid and a sixth supernatant; and (k) the sixth colloid in G) is purified by diafiltration and cation exchange resin, and albumin is recovered. 2. The method for producing fibrinogen, fibrinogen complex, immunoglobulin, third antithrombin and albumin from frozen plasma as described in item 1 of the scope of patent application, wherein (f) Purification of anion exchange resin includes: i. Adding the fourth colloid in mash to PEG 6000 to make it finally a mixed solution containing 1% PEG 6000, stirring continuously for a period of time, then centrifuging to precipitate, and then colloid and supernatant Liquid separation; ii. The supernatant obtained by i was purified by an anion exchange resin DEAE Sepharose, and the fibrinogen enzyme complex extracted with 2M sodium chloride-citrate buffer was recovered; iii. The fibrinogen enzyme complex is obtained by dialysis and desalting. 3. The method for producing fibrinogen, fibrinogen complex, immunoglobulin, third antithrombin and albumin from frozen plasma as described in item 1 of the scope of the patent application, wherein (g) Gel filtration and ion exchange resin purification include: A. The fourth supernatant in (e) is passed through the gel filtration resin Sephadex G25C to recover the solution containing the immunoglobulin; B. The solution containing the immunoglobulin in A The buffer solution was replaced with a buffer solution with a conductivity of 1.40ms, pH 6.5; C. The B was replaced with a buffer solution, and the solution containing the immunoglobulin was purified by anion exchange resin Q 17 1226333 Sephamse, and the solution containing the immunoglobulin was recovered. D. Purifying the purified recovery solution of C by cation exchange resin CM Sepharose to recover the solution containing the immunoglobulin; and E. Desalting the salt by dialysis to obtain the immunoglobulin. 4. The method for producing fibrinogen, fibrinogen complex, immunoglobulin, third antithrombin, and albumin from frozen plasma as described in item 1 of the scope of the patent application, wherein the aluminum salt in the tincture Gel adsorption purification and heat treatment include: a) dissolving the fifth colloid in (h) in sodium chloride-citrate buffer solution (pH7.3); b) heating the mixed solution of a) to 56QC, heat treatment 3 hours; and c) purification of the solution containing the third antithrombin by b) heat treatment with an aluminum hydroxide gel, and extraction of the solution containing the third antithrombin with 1M sodium phosphate buffer, pH 7.0. 5. The method for producing fibrinogen, fibrinogen complex, immunoglobulin, third antithrombin and albumin from frozen plasma as described in item 1 of the scope of the patent application, wherein (k) is dialysis Filtration and cation exchange resin purification include: I dissolving the sixth colloid in (D) in pure water; diafiltration of the mixed solution of II and adjusting the buffer concentration to 0.02M; and III of the mixed solution obtained from II Purified by cation exchange resin CM Sepharose with 0.11M vinegar Sodium buffer was washed out and albumin was recovered. 6. A method for producing fibrinogen, fibrinogen complex, immunoglobulin, third antithrombin and albumin using frozen blood paddle as described in any one of the scope of application patents 1-5, The frozen plasma is frozen plasma stored at -18 ° C. 18