TW546144B - Production process for intravenous immune serum globulin and resultant product - Google Patents

Abstract

A process for producing an infravenously-administrable gamma globulin solution substantially free of contaminating viruses by heat treating for viral inactivation and fractionating an impure gamma globulin solution and then treating the purified gamma globulin with a solvent-detergent for further viral inactivation.

Description

Printed by the Central Bureau of Standards of the Ministry of Economic Affairs and Consumer Cooperatives 546144 Α7 ------ --R7 V. Description of the Invention (1) Field The present invention relates to a complete, multi-step commercial preparation method for producing Immune plasma globulin for intravenous injection with IgG (y · globulin) as its main component. Background Various methods are known for obtaining a plant globulin solution for intravenous injection from a cohn fractionation starting material from human plasma. Some of the Cohen sections contain higher titers of gamma-globulin than others. The part which can be used as the starting material of the γ-globulin solution is the Kohen part '11 or Kohen part ΙΙ + ΙΙΙ. Although previous techniques used a variety of separation and aseptic techniques, the industry is still looking for preparation methods that can improve the purity, safety, and overall yield of the final product. The Wuye process does not use a solvent / detergent step to deactivate the virus That is, a heat treatment step is used to deactivate the virus. So far, no multi-step preparation method has been proposed that uses Kohen part II paste or Kohen part II + III paste as a starting material. This method includes using two different virus deactivation procedures as an effective, Part of the preparation method of high yield γ-globulin.

Kameyama et al., U.S. Patent Application No. 5, isi, is related to a method for preparing a deactivated viral protein composition, which is composed of a solvent / detergent step and a heat treatment step. Deactivating viruses without envelopes Page 4 This paper is suitable for Chinese elephants (~ ——-_ (Please read the precautions on the back before filling this page)

546144 A7 R7 V. Explanation of the invention (= teaching of the case, it is better to perform the solvent first in the presence of protease, * the detergent step, and then the heat treatment step. The heat treatment step is performed under the state of liquid ~ d), and Before the heat treatment, the protein is adsorbed from the solvent / detergent to the ion exchange column by adsorption method. The state heat treatment can be performed in the presence of sugar, sugar alcohol or amino acid line agent τ. Although listed in the patent case Many kinds of starting proteomic filaments have been produced, including immunoglobulins, and the ninth factor, thrombin, fibrinogen, and fibronectin are used in the preparation examples. It is not considered to remove the heat treatment by faceting. The denatured protein produced in the step. Some of the previous techniques are prepared for intravenous injection [globulin solution, "Wanfa," describes a liquid heat treatment process, which is based on the Kouheng part ΙΙ + ΙΙΙ paste It is a multi-step purification procedure as a starting material and is performed in the presence of a sorbitol thermal stabilizer. U.S. Patent Application No. 4,845,199 by Hirao et al. Is based on the use of sorbitol as an egg Before the liquid heat treatment step of the mass stabilizer, the Kouheng part Π + ΙΠ paste is fractionated by polyethylene glycol (hereinafter referred to as "PEG") (8% w / v PEG and then 12% w / v pEG), and The Department of Economics, Central Bureau of Standards, Employees' Cooperatives printed sub-exchange chromatography (DEAE-Sephadex) and removed human blood group antibodies. On the other hand, Hirao et al., US Patent Application No. 4,876, 〇88, described A method for preparing a γ-globulin solution for intravenous injection from a Kouheng part II + ΙΙΙ paste was prepared, wherein the paste was suspended in water, the pH was reduced to 5.5 and centrifuged at 33% w / v in the presence of sorbitol 'The supernatant obtained by centrifugation was heat-treated to remove viral activity, followed by fractional distillation with PEG (6% / 12%) to remove the denatured protein after heat treatment' followed by other purifications The steps include DEAE-Sephadex from page 5. This paper size applies the Chinese National Standard (CNS) Λ4 specification (210X 297 cm) 546144 V. Description of the invention (A7 Η 7 Printing of printed materials of the Consumers ’Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Layer chromatography One purpose is to provide one

Wt_atlon pr. Ii) Preparation of high-purity branch library procedures (C-commercially available preparation of protein solutions < complete, another object of the present invention is to provide non-toxic species-including all heat-sensitive diseases that do not contain or contain Membrane disease γ · globulin solution. The present invention provides two kinds of R for intravenous injection. The invention further provides a commercial globulin preparation method, which removes the denatured protein produced by heat before the virus activity is removed. Those skilled in the art of cloth armor should be able to understand the present invention- ^ ^ Right and other items, which contains γ-globulin, a partially purified alcoholic Kohen branch knife, which is in the presence of a heat stabilizer Heat treatment in a liquid state to remove = activity, and then 'repeated by PEG' followed by a solvent, preferably a solvated elixir mixture, to perform the second stage of virus deactivation to destroy the disease with the envelope 4. It is then separated from the solvent or solvent detergent mixture. In the preferred embodiment of the present invention, sorbitol is used as the thermal stabilizer and trialkyl phosphate is used as the solvent. In another preferred embodiment of the invention The pEG fractionation method is used to remove the denatured products produced during the heat treatment to remove the viral activity before the virus deactivation step in the second stage, in order to provide a very pure, heat treated page 6t paper which is suitable for China. Standard (CNS) Λ4 specification (210X297 public §) (Please read the notes on the back before filling out this page) 丨 Order 546144 A7 B7 Printed by the Consumers' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs Protein. In another preferred embodiment of the present invention, any impurities are removed before the solvent-detergent treatment step is performed. In another preferred embodiment of the present invention, an Gamma globulin for intravenous injection, heat sterilization and solvent-detergent sterilization. As detailed in the _ use of a facet containing immunoglobulin as a starting material. The remaining part is not particularly limited to the present Derived from human plasma and immunoglobulin-containing fraction A. Such immunoglobulin-containing fractionation fractions include fractions Π + ΙΠ and VII obtained from the Kouhenfa ethanol fractionation fraction, and are equivalent to immunoglobulin-containing fractions Fractionate the paste. Other useful starting materials are Parts I + III + III and II + IIIw. The starting materials may contain human blood group antibodies, proserotonin, plasmin, kallikrein Impurities such as prokinin release, radioactive enzyme activator, IgM, IgA, IgG polymer ("abbreviated as agglutinate" hereinafter), etc. The preferred starting materials are π and Π + ΠΙ in the Kouheng fractionation section. When it is used as the paste of part Π + ΙΙΙ in the Kouheng fractionation part, it is recommended that the cleaning step be performed first to generate part II + IIIw, and then this part is used to implement the invention. "Section II + IIIw Part of it is the precipitation of part II + III in the Kouheng fractionation section after washing with a phosphoric acid dibasic solution. Part II + IIIw was obtained by suspending part III + III in about 20 ml of water plus 1 kg of part II + III paste in suspension in cold water for injection. Add sodium phosphate solution so that the final sodium phosphate concentration is about 0 003M, page 7 This paper size is applicable to Chinese National Standard (CNS) Λ4 specification (210X 297 cm) (Please read the precautions on the back before filling this page)

546144 A7 --__ B7 V. Description of the invention () ~~ 1 ~ s ^ To dissolve oil, lipoprotein and albumin. Ice ethanol was added to bring the final concentration to about 20%. When adding ethanol, the temperature will slowly drop to _5t; rc, and maintain the pH value or adjust the value to 12 especially 0.1 with acetic acid buffer or diluted sodium hydroxide solution. Centrifugation and / or filtration at -5: L1 ° C to recover the H + IIIw part of the precipitate formed. Before performing the virus inactivation in the first stage of the present invention, various preliminary purification and / or reduction steps may be performed. For example, when used to protect the H + part paste containing about 20% ethanol and more than 70% IgG ^ It can be suspended at a temperature of about 0 to 5 ° C in a volume of 3 to 10, preferably疋 3 to 5 volumes of cold water, and adjust the pH to between 4.5 and 6.0 with acetic acid buffer or hydrochloric acid at pH 40, preferably between 50 and 5.5. The mixture is stirred for about 2 to 15 hours to allow all globulin to be dissolved in the solution. Thereafter, insoluble proteins such as albumin and aglobin are removed by centrifugation and / or filtration. When the starting material used is a different Cohen fractionation fraction, it is necessary to select an appropriate starting step so that a preliminary purification step capable of obtaining a high content of IgG can be performed. For example, 'When the Kouheng No. 11 fractionation part (containing more than 95% of IgG) has been separated from the Kouheng No. 11 fractionation part, the process just started can be maintained at Uemura et al. The acidic pH of ρΗ 3.2 to 50 as described in U.S. Patent No. 4,371,520, preferably between 3.8 and 4.2, in order to decompose the immunoglobulin aggregates into immunoglobulin monomers and dimers. Because these aggregates have anti-complement activity (ACA). Alternatively, if the starting material used is part II + III of Cohen's fractionation, it can be carried out before heat treatment to remove viral activity. Page 8 f Please read the > 1 notice on the back and fill in this page. } ,-mouth

The paper size printed by the Central Bureau of Standards and Boiler Consumer Cooperatives of China applies the Chinese National Standard (CNS) Λ4 specification (210 x 297 male f) 546144 Λ7 Η 7 V. Description of the invention (υ_: the lowest in the patent case et al. The pH treatment is followed by a preliminary purification step as described above. For the heat sterilization step, the immunoglobulin is dissolved in water, if it is in the form of a mixture of water such as the filtrate of the first part of the chitosan purified from the upper part. If present, it can be used directly and added with sugar, sugar alcohol and / or acid heat stabilizer. The heat stabilizer is preferably sucrose, maltose, sorbitol or mannitol, most preferably sorbitol. Sugar or sugar alcohol It is added to the immunoglobulin solution in powder form, or mixed with a small amount of water, and then added to make the final concentration from about 10i 50w / v% to a saturated concentration. At this time, the immunoglobulin aqueous solution contains enough Amount of water, so the total protein mass of this solution is about 1 to 6%. Generally the typical Π + ΙΠ starting material contains about 300 grams of protein per kilogram of paste. 1 After adding a heat stabilizer, Heat the mixture to about 5〇_〇〇〇, about 10-100 hours, preferably about 6 (rc, about 10-20 hours in order to remove heat-sensitive virus activity. The heat treatment step can not only inactivate the virus, but also by the effect of protein denaturation , Can reduce some undesired protein masses, such as kallikrein, plasmin, proserotonin, and IgA, which are usually combined with Cohen Section II + III. Shellfish Consumer Cooperative, Central Bureau of Standards, Ministry of Economic Affairs After the printing heat treatment step, add a sufficient amount of cold water to maintain the protein concentration between 0.3% and 2.0% in Europe. The solution is cooled to 0-2 ° C. Next, PEG fractionation is performed in the heat-treated solution. In immunoglobulin During purification, PEG fractionation is a well-known technique to separate the desired IgG monomers and dimers from IgG agglutinates and other impurities that are naturally present in the protein fraction of the starting plasma. However, in this process the 9th The paper size of this page applies the Chinese National Standard (CNS) A4 specification (210X297). 5. Printed by the Ministry of Economic Affairs, Central Standards and Construction Bureau, Consumer Cooperatives, 546144 A7 B7, and the description of the invention. It can achieve the purpose of separating the desired IgG monomers and dimers from the undesired denatured protein products generated by heat treatment. These denatured protein products are denatured kallikrein, plasmin, and plasminogen. , IgA, IgM, and agglutinates. Any PEG fractionation procedure previously mentioned in the art can be used. In general, the two-stage peg fractionation is used. In the first stage, a single IgG fraction is selected. The PEG concentration and pH remain in the solution, so that protein concentration such as agglutinate is not desired. After centrifugation and / or filtration, increase the PEG concentration and adjust the pH to make the desired IgG monomer and two Polymer precipitates. For example, when the starting material is the + IIIw paste, the first stage of ρE (} fractionation can be performed at a pH value of about 5.0 to 7.5, preferably 6. 5 to 7. 5; if When the starting material used is paste III + III, the pH is preferably between about 5.5 and 6.0; the concentration of PEG is between 4% and 8%, or when the starting material used is II + IIIw paste, preferably 4% to 6%, or when the starting material used is a III + III paste, it is preferably 6% to 5%. Keep the temperature at about 0-rc, the -Stage PEG separation takes about 1 to 8 hours, after which the precipitate is removed as described above. Adjust the pH of the filtrate to about 8.0 to 9.0, preferably 85 to 89, and add additional The final concentration of peg is about 10 to 15%, preferably about 12%. The formed sink, that is, purified white, is removed by centrifugation and / or centrifugation. PEG used to implement the present invention Detailed details of the sorting sequence, detailed fan # The above-mentioned US patent case 4 876 088 and US patent case filed by Hirao et al.

Page 10

546144 Printed by the Central Standards Bureau, Ministry of Economic Affairs, Consumer Cooperatives A7 Η 7 V. Description of Invention () 4,845,199. The last and most important step of the present invention is to carry out the second stage of the virus deactivation step with a solvent or a solvent-detergent mixture. As described below, further purification steps', especially those involving the use of ion exchange resins, may be performed before and / or after the detergent / detergent treatment. A particularly advantageous procedure is anion exchange treatment before solvent-detergent virus deactivation, followed by cation exchange treatment after solvent-detergent virus deactivation. After this procedure, certain undesired proteinaceous substances (such as kinin release enzyme activators, [gM, IgA, and albumin) can be removed from IgG by anion X exchange treatment, and so on Substances such as prokinin activators, IgM, IgA, albumin, and PEG, and solvents remaining in the solvent-detergent process can be removed by a cation exchange process. If these objectives cannot be achieved throughout the treatment process, then A must remove particulate materials including denatured protein in the solvent-detergent solution. Therefore, it is preferable to filter the solution with a filter paper of 1 micron or less after adding the solvent_detergent. This also reduces the chance that the virus will remain in the large particles without being exposed to the solvent-detergent. Today, the preferred solvent capable of deactivating enveloped viruses is trialkyl phosphate. The trialkyl phosphate suitable for use in the present invention is not limited in any way, but di (n-butyl) phosphate (hereinafter referred to as "TNBp") is preferred. Other suitable ^ trialkyl phosphates are tris (tertiary-butyl) phosphate, tris (n-eutyl), tris (2-ethylhexyl) phosphate, and others. Two or more different tris (3) can also be used. Mixtures of phosphoric acid esters. ___ page 11 This paper is scale 1 for p ------ (诮 Please read the precautions on the back before filling this page)

546144 A7

IP ------- ^ ----------------- V. Description of the invention () 1 of the trialkyl phosphate used is between 0.01 and 10 (w / v)%, preferably between 0.1 and 3 (w / v)%. Trialkyl phosphates can be used in the presence or absence of detergents or surfactants. The trialkyl phosphate is preferably used in combination with a detergent. The function of the detergent is to promote the opportunity for the virus in the immunoglobulin composition to contact the trialkyl phosphate. Examples of detergents include polyoxyethylene derivatives of a fatty acid, such as partial esters of anhydrous sorbitol such as polysorbate 80 (trade name: Tween 80 and others) and polysorbate 20 (trade name: Tween 20 and others). Compounds; and non-ionic oil bath soaking agents such as oxyethylated alkylphenols (trade name: Triton × X00 and others). Examples also include other surfactants and cleaners such as zwitterionic cleaners. When using a cleaning agent, it is not used by adding a certain amount; for example, it is used at a ratio between 0.001% and 10%, preferably between 0.001% and 3%. In the present invention, the composition containing trialkyl phosphate to treat the immunoglobulin-containing composition is consumed at a temperature of about 20 to 35 ° C, preferably about 25 to 35 It takes about 1 hour or more for the cooperative to print 30 C, which is about 5 to 8 hours, and more preferably about 6 to 7 hours. During the trialkyl phosphate treatment, the immunoglobulins are present in the form of an aqueous solution of about 3% to 8% protein. If it is not performed before the solvent-detergent treatment, an anion exchange treatment may be performed in the immunoglobulin solution after the solvent-detergent treatment. It is better to carry out at least-second positive ionization on the products treated with solvents and detergents. The paper size is applicable to China ~----546144 A7 ——-_______ Η 7 Fifth invention description () sub-exchange processing. Ion exchange treatment is performed in an aqueous immunoglobulin solution with a pH of about 5-8, and its lower ionic strength can lead to the maximum absorption of IgG. Protein concentration is generally between 1-15 w / v%, more preferably between 10 w / v /%. Intermediate and ion exchange resins are balanced by the solvent solution used, and can be carried out in batch or continuous systems. For example, the batch-type anion-exchange process is as follows. First, the pre-treated anion-exchange tree 9 (for example, 1 g of DEAE Sephadex A-50 resin in a 0.4% sodium chloride solution can swell to about 20 g wet weight) The immunoglobulin solution is mixed with the anion exchange resin at a ratio of about 10 to 100 ml of the immunoglobulin solution, and the mixture is stirred at about ο-rc for about 0.5 i for 5 hours, and then Centrifuge or centrifuge at 6,000 to 8,000 rpm for about 10 to 30 minutes, and recover the supernatant. In continuous processing, the immunoglobulin solution is passed through the anion exchange column at a rate of about 10 to 100 ml per ml of anion exchange resin, and the unadsorbed portion is recovered. Useful anion exchange resins include, for example, at least one anion exchange group bonded to a water-insoluble carrier. Anion exchange groups include diethylaminoethyl (DEAE) and quaternary aminoethyl (QAE) f. Water-insoluble carriers include agar, cellulose, polydextrose, polyethylenamine, and the like. The 1% ionic X-exchange resins printed by Shelley Consumer Cooperative of the Central Marking Bureau of the Ministry of Economic Affairs are carboxymethylated Sephadex CM-cellulose, SP · Sephadex, CM_ Sephadex Floss (CM-Sepharose) and S-Sepharose (s_Sepharose). 1 ml of pre-treated cation exchange resin (for example, 1 g of CM-Sephadex C-50 resin can swell to about 30-35 g wet weight in 0.4% sodium chloride solution) and 0.5 ml To 5 ml of immunoglobulin solution, and 〇-5 page 13 This paper scale applies Chinese national standards ([, 5) / \ 4 specifications (2 丨 0/297 Gong Jun) 44 Λ7 B7 Five faces- Printed by the Central Ministry of Agriculture and the Bureau of Shellfish, consumer cooperation, printing, invention description () ^ ~ ^^ — c. Stir the mixture for about 6 hours. Centrifuge or filter the suspension to

^ ^ pC and resin with I g G attached. This step can also be performed using a continuous program. In the above case, when used in combination with a cation exchange resin, I g G will be adsorbed. When washing the protein-adsorbed cation exchange resin, IgG can be washed out with a sodium chloride solution of about 1.4N. After performing the above steps, the IgG was purified, dialyzed, and concentrated to the desired temperature; It is also possible to add a stabilizer such as D-sorbitol and make final adjustments so that the composition solution contains about 100 mg / ml of IgG and about 50 mg / ¾L of D-sorbitol at a pH of 5.4. The solution is then sterilized by passing it through a bacteria-retaining filter, and the solution is stored in a small tube. The following examples are provided for the purpose of illustrating the invention, and the invention is not limited thereto. Other procedures for purifying immunoglobulins are also used in combination with the procedures described above as appropriate. For example, a beautiful clay purification step that is effective in reducing kallikrein and then kallikrein activators can be used. This is detailed in Example 1 below. Example 1: Heat-treated and solvent-detergent-treated γ-globulin A 685 g portion of the II + IIIw paste was suspended in about ili9 kg of cold water. Sodium acetate trihydrate solution was added to the suspension to a final concentration of 0.04M capable of selectively dissolving IgG. After 15 minutes of mixing, the pH was adjusted to 4.8 with a pH 4.0 acetic acid buffer. Ice ethanol (95%) was added to the suspension to a final concentration of 17%. During the addition of ethanol, the temperature of the suspension will drop to about ·. Pickled Celia purchased from Celite Corporation at 303 grams. Page 14 This paper applies Chinese National Standard (CNS) Λ4 regulations (210X 297 male f) 546144 Λ7 ___ _B7 V. Description of the invention () — ^ Celite 535 is used as a filter aid in suspension, and its final concentration is about 0.2%. After 1 hour of mixing, the Celite diatomaceous earth and the in part of the undesired protein such as plasmin, proserotonin, IgA and IgM were removed by filtration with a filter. The filtrate was further purified using 遽 45 micron and 0.2 micron paper. The pH of the III + IIIw purification solution was adjusted to 4.0 with 1 · N hydrochloric acid, and then concentrated to about 3.4 liters (about 1/5 of the original volume) by ultrafiltration. Add about the same amount of cold water as the amount removed in the first ultrafiltration method to the concentrate, and then concentrate it to / 5th of the original volume by ultrafiltration. The protein concentration of the 'solution at this time was about 2%. The solution was further concentrated to 4 ° /. Dialyze the cold water until the conductivity of the solution is less than 30 s / em, to help avoid protein aggregation and denaturation during heat treatment. The solution was further concentrated to a protein mass of 8.8%. D-sorbitol was added to the solution to a final concentration of 33%. After mixing for 30 minutes, the pH of the sorbitol-containing solution was adjusted to 5.5 with 0.5N sodium hydroxide. The solution was then heated at 60 t for 10 hours. After the heat treatment, cold water was added at 3 times the volume of the heat treatment solution and the diluted solution was cooled to 0-2 ° C. After printing by the Central Standard Bureau of the Ministry of Consumer Affairs, Shellfish Consumer Cooperative, the pH of the solution was adjusted to 6.9 with 0.25N sodium hydroxide, and 50% polyethylene glycol (PEG) 3350 was added to the solution so that the final concentration of PeG was 4〇 /. . Adjust the solution's chlorinated steel concentration to about 8Mm to help the impurities to come out of the temple at pH 6.9. The precipitate was removed by filtration ^ 1 · NH hydrochloric acid, the pH of the filtrate was adjusted to 4.8, and beautiful clay was added to a final concentration of 0.25%. The pH of the US clay suspension was adjusted to about 5 · 2 and then filtered to remove the US clay. The pH value of the filtrate was adjusted to 8.5 with 0.25v sodium hydroxide and 50% polyethylene glycol (PEG) 3350 was added to the solution. Page 15 This paper applies Chinese National Standard (CNS) Λ4 ^ (21〇χ 297々 > [Γ " One ~~ —

Printed by the Central Consumer Bureau of the Ministry of Economic Affairs, Consumer Cooperatives 346144 V. Description of the invention (In the final concentration of PEG is 12/0 to remove the precipitate (purified immunoglobulin) by filtration. Paste 175 g of the purified immunoglobulin prepared above The material was suspended in about 790 grams of a 0.3% sodium chloride solution. The spleen was adjusted to a pH of 5.5, and the suspension was allowed to mix for 2.5 hours. Too a ★ ▲, add 62 grams of The DEAE-Sephadex A50 tree moon dagger "M Γ ^ 1" (Ph 5.5, 0.3% sodium chloride solution) which has previously reached equilibrium, and the suspension is allowed to mix for 2 hours. Remove the resin by hardening. After adjusting the sodium chloride concentration to 0.4%, add a mixture of tri-n-butyl phosphate (TNBP) and polysorbate 80 to the base, and remove it. The final temperature is 0.3q / () TNBP and 1.0% polysorbate 80. After overnight training, adjust the pH of the falling liquid to 5. 8 and add about 860 CM Sephada c50 resin (pH 5.8, 0.4% sodium chloride solution) which has reached equilibrium in Cresen. After mixing for 2 hours, the poem solution is passed through. Wash the CM_Sephadex resin 3 times with 0.3% sodium chloride solution, and wash out the adsorbed IgG with 4N sodium chloride. The eluate is purified, dialyzed, and concentrated. D-sorbitol is added and the final adjustment is made to make the composition solution approximately Contains 100 mg / ml of IgG and about 50 mg / ml of D-sorbitol 'pH is 5.4. The solution is then sterilized by passing through a bacteria-retaining filter and the solution is stored in a small tube. The intermediate step of meclay used in the examples is very useful to further reduce the low-totenase enzymes such as kallikrein and prokallikrein activator. Page 16 This paper is applicable to the Chinese National Standard (CNS) Λ4 specification (210X 297 public welfare) ) (Please read the following notes before filling out this page)

546144 Printed by the Central Standards Bureau, Ministry of Economic Affairs, Consumer Cooperatives 11 A7 7 7 i. Description of the invention (Test results of the product of Example 1 Test parameters Anti-complement activity (CH5Q u / mg IgG) 0.34 IgG purity (%) 100 IgG content ( mg / ml) 112.7 Prokinin (% CBER Ref # 3) 21 Measles antibody (% CBER Ref # 176) 0.67 Volume distribution of IgG molecules on HPLC (%) Monomer 82.2 (%) Dimer 17.4 ( %) Fragment 0.10 (%) Agglutinate 0.3 Hepatitis A antibody (titer) 1: 200 Hepatitis B antibody (titer) 1: 1024 IgA (pg / ml) 22 IgM (pg / ml) 16 Prostaglandin ND Serotonin 16 pH 5.4 ND = Not measured. Example 2: Heat treated and solvent-cleaned γ-globulin treated 1 kg of part III + III paste was suspended in about 4 5 kg Cold water. After mixing for 1 hour, the pH of the suspension was adjusted to 5.0 with 1N hydrochloric acid. After mixing for 3 hours at pH 5.0, the precipitate was removed by centrifugation. D · sorbitol was added to the centrifuge solution to make the final solution. The concentration was 33% and mixed for 1 hour. The pH of the solution was adjusted to 5.5. Then the solution was heated at 60 ° C for 10 hours. Then, add 3 times the volume of cold water of the heat treatment solution and cool the diluted solution to 0-2. (: Add 50% polyethylene glycol (PEG) 3350 to the solution so that the final concentration of pEG is 6%. 5N sodium hydroxide adjusts the pH of the 6% pEG suspension to 5. 7 and mixes the suspension for 2 hours. Adds acid-washed Celite silicon page 17

Paper Size: Shicai Taojia County (CNS) (210x797iJ (Please read the notes on the back first and fill in this page)

546144 ΑΊ ΙΓ V. Description of the invention () ~~~ Swing the soil 5 3 5 to make its final concentration a ί 0 /, 廿 ,, Japanese, slavery is 1.5 / 0, and the suspension τ hours. Filter to remove Shen Dian, Selite Second, and algal soil. The pH value of the filtrate was adjusted to 8.8 with G 5N steel hydroxide and 50% polyethylene glycol (PEG) was added to the solution so that the final pEG concentration was 12%. The pH value of the 12% PEG suspension was adjusted to 8.8, the suspension was mixed for 1 hour and the purified immunoglobulin paste was collected by filtration. About 2 51 grams of purified immunoglobulin paste can be recovered. 251 g of the purified immunoglobulin paste was suspended in about 1.4 kg of a 0.3% sodium chloride solution. After i hours of mixing, the pH of the suspension was adjusted to 5.5 with 5% acetic acid. After the paste was completely dissolved, 104 g of DEAE-Sephadex A50 resin (Ph6.0, 0.3% sodium chloride solution) which had previously reached equilibrium was added to the solution, and the suspension was mixed for 2 hours. The suspension was filtered to remove resin. The filtrate was further passed through a 0.2 micron filter. The sodium chloride concentration in the filtrate was increased to 0.4% by adding sodium chloride. A mixture of tri-n-butyl phosphate (TNBP) and polysorbate 80 was added to the filtrate to a final concentration of 0.3% TNPB and 1.0% polysorbate 80. Incubate overnight in a cold box at 4 ° C. The next day, the pH of the solution was adjusted to 5.8 and about 1.8 kg of CM-Sephadex C50 resin (pH 5.8, 0.4% sodium chloride solution) that had previously reached equilibrium was added. After mixing for 2 hours, the resin was removed by filtration. The CM-Sephadex resin was washed 3 times with a 0.3% sodium chloride solution, and the adsorbed IgG was washed out with 1.4N chloride steel. The eluate was purified, dialyzed, and concentrated. D-sorbitol was added and finally adjusted so that the composition solution contained about 1000 mg / ml of IgG and about 50 mg / ml of D-sorbitol, and the pH was 5.4. The solution was then divided into two parts, part A and part B. The pH of Part A was adjusted to 5.4, and the pH of Part B was adjusted to 4.3. After that, I will apply the Chinese paper standard ([~ 5) / \ 4 size (210 '/ 297 cm) on page 18 of Part A individually. (Please read the precautions on the back before filling in this page.) Printed by the Standardization Bureau Shellfish Consumer Cooperative Co., Ltd. 546144 λ \ 7 B7 V. Description of the invention () The solution of part B and part B is sterilized through a filter membrane capable of retaining bacteria, and the solution is stored in a small tube. Example 2 Product test Results Test parameters Anti-complement activity (CH5G u / mg IgG) < 0.05 IgG purity (%) 100 IgG content (mg / ml) 104.2 Prokinin (% CBER Ref # 3) ND diphtheria antibody (antitoxin U / ml) 8.2 Volume distribution of IgG molecules on HPLC pH 5.4 pH 4.3 (%) monomer 88.8 97.5 (%) dimer 10.9 2.3 (%) fragment 0.3 ND (%) agglutinate < 0.3 < 0.3 Hepatitis A antibody (titer) 1: 100 IgA (, ug / ml) 78 IgM (pg / ml) 28 Kallikrein (a4 () 5) 0.09 Proserotonin (ng / ml) < 8.4 Plasma Element (ng / ml) < 8.4 ND = No person skilled in the art should know that the present invention can have many variations. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs Page 19 This paper size applies to China National Standards (CNS) Λ4 specifications (210X 297 cm)

Claims (1)

546144 ABCD No. Reverse Case X 'Yue Xiujian 6. Application for Patent Scope 1 · A method for preparing a γ · globulin solution for intravenous injection, the method includes at least the following steps: (a) Let the impure γ- The globulin solution is heat-treated at a time and temperature sufficient to deactivate the heat-sensitive virus therein; (b) The heat-treated γ-globulin solution is processed in the order of polyethylene glycol fractionation cake: 1. Purified γ-globulin solution, wherein the I ethylene glycol fractionation process is performed in at least two stages, and impurities are precipitated: the γ-globulin is removed in the first stage of the polyethylene glycol facet. It is collected in a sanitary manner during the second stage of polyethylene glycol fractionation; and (c) the purified γ-globulin solution is treated with an organic solvent so that no further polyethylene glycol fractionation treatment is required The enveloped virus is then deactivated. 2. The method according to item 1 of the scope of patent application, wherein step (a) the impure γ-globulin solution refers to part Ⅰ + ΙΙ + ΙΙΙ in the Cohen fractionation part, and part III + ΙΙΙ in the Cohen fractionation part , Part Il + niw in the Kouheng fractionation section, or part II in the Kouheng fractionation section. (Please read the notes on the back before writing this page) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 3 The method of item 1 in the scope of patent application, wherein the impure γ-globulin solution is subjected to at least one purification step before being subjected to the heat treatment step U). Use Chinese National Standard (CNS) A4 specification (210X 297 hall) on page 20 to declare the scope of patents 4 · If the method of applying for the scope of the patent application item 丨 contains an organic solvent which is more cleansing agent 5. If the scope of patents is applied Method (1), in which the first stage of polyethylene glycol dehydration can be followed by the -US clay purification step. 6. If the method of patent application_ item 1 is used, the heat treatment step is performed at about 50 to 7 (rc for about 10 to 100 hours. 7. If the method of the scope of patent application, the heat treatment step is Step ⑷ is performed at about 60 ° C for about 10 hours. 8. The method according to item 1 of the scope of patent application, wherein the organic solvent used in step 为 is an alkyl phosphate ester. Item, in which the alkyl phosphate is di-n-butyl phosphate g. C Please read the notes on the back before filling out this page) Line · 10 · If the method in the scope of patent application No. 8 of which Contains a cleaning agent. Organic solvent package 1 1 · The method according to item 1 of the patent application, wherein the γ-globulin solution after step (a) is treated with an anion exchange resin and a cation exchange resin. , Page 21 This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 546144 ABCD patent application scope 12. If the method of patent application scope item No. 11, the anion exchange resin treatment is in step (c) The cation exchange resin treatment is performed after step (c). (Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Page 22 This paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm)