CN1214042C - Production process for intravenous immune serum globulin and resultant product - Google Patents
Production process for intravenous immune serum globulin and resultant product Download PDFInfo
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- CN1214042C CN1214042C CNB988043769A CN98804376A CN1214042C CN 1214042 C CN1214042 C CN 1214042C CN B988043769 A CNB988043769 A CN B988043769A CN 98804376 A CN98804376 A CN 98804376A CN 1214042 C CN1214042 C CN 1214042C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Abstract
A process for producing an intravenously-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
Background of the present invention
The present invention relates to a kind of be used to produce intravenous administration contain commercialization method set, that multistep rapid of IgG (gamma globulin) as the immune globulin of main component.
Known have the multiple parent material that produces from human plasma Cohn classification to obtain the method for the gamma globulin solution of intravenous administration.Specific Cohn fraction contains the gamma globulin than the higher titre of other fraction.The common parent material of gamma globulin is Cohn fraction II or Cohn fraction II+III.
Though prior art has been used multiple separation and sterilising technology, the improvement of people in the method for seeking arranged always, with the purity and the security of increase finished product, and total output.
Many commercialization methods utilize the solvent/detergent step to carry out inactivation of virus, or use heat treatment step to carry out inactivation of virus.Up to now, do not provide a kind of in the prior art and begin to comprise two kinds of different virus inactivation step as effective, the rapid method of multistep of a high yield gamma globulin manufacturing process part with Cohn fraction II or II+III paste (paste) shape thing.
Kameyama etc. are at United States Patent (USP) 5,151, point out a kind of method of making the protein composition of inactivation of viruses in 499, wherein use solvent/detergent treatment protein composition deactivation envelope virus, with thermal treatment protein composition deactivation nonenveloped virus.The instruction of ' 499 patents is preferably at first carried out the solvent/detergent step in the presence of proteinase inhibitor, follow by thermal treatment.Because thermal treatment is to carry out at solution state, before any thermal treatment, at first needs to reclaim protein by being adsorbed onto ion exchange column from solvent/detergent.Solution thermal treatment can be carried out in the presence of sugar, sugar alcohol or amino acid stabilizers.Though ' 644 patents have been listed the initiation protein composition that comprises immunoglobulin (Ig), its production example usage factor IX, zymoplasm, fibrinogen and fibronectin.Do not consider to remove the denatured protein that in heat treatment step, produces by classification.
But described in the method for the gamma globulin solution of some generation intravenous injection of prior art in the multistep rapid purifying flow process initial, to be included in the presence of the Sorbitol Powder thermo-stabilizer and carried out solution thermal treatment with Cohn fraction II+III mashed prod.United States Patent (USP) 4 at Hirao etc., 845, in 199, Cohn fraction II+III is used for polyoxyethylene glycol (after this being called for short " PEG ") classification (8%W/V PEG, 12%W/V PEG then) classification was carried out ion exchange chromatography (DEAE-Sephadex) and was removed human blood type antibody before Sorbitol Powder carries out solution thermal treatment in the presence of as stablizer then.On the other hand, United States Patent (USP) 4 at Hirao etc., 876, but described among 088 the embodiment 1 from the gamma globulin solution of Cohn fraction II+III settling preparation intravenous injection, wherein mashed prod has been suspended in water, the pH value has been adjusted to 5.5 and centrifugal, then in the presence of the 33%W/V Sorbitol Powder with supernatant thermal treatment inactivation of viruses, then being to remove heat denatured protein matter by PEG classification (6%/12%), is other purification step then, comprises the DEAE-Sephadex ion exchange chromatography.
The present invention sums up
An object of the present invention is to provide method a kind of integration, that can be used for commercialization production to produce highly purified gamma globulin solution from the Cohn classification process.
Another object of the present invention provides very pure, but does not contain the gamma globulin solution of the intravenously medication of the coating that comprises all thermo-responsive viruses and nonenveloped virus.
A further object of the present invention provides a kind of commercialization gamma globulin technology, makes to remove any denatured protein that produces in the heat sterilization before the subordinate phase inactivation of virus.
The invention provides above-mentioned is conspicuous purposes with other to those of skill in the art, wherein in water-bearing media in the presence of the thermo-stabilizer may for the partially purified but pure Cohn fraction thermal treatment of being rich in gamma globulin with inactivation of viruses, use earlier the PEG classification subsequently, then at the solvent of broken enveloped virus, preferably there is down inactivation of viruses for the second time in solvent-detergent, separates from solution or solution-stain remover mixture at last.
In a preferred embodiment of the invention, Sorbitol Powder is a thermo-stabilizer, and trialkylphosphate is a solvent.
In another preferred embodiment of the present invention, the denatured products when utilizing the PEG classification to remove the thermal treatment inactivation of viruses before the inactivation of viruses second time is to provide extremely pure heat treated gamma globulin.
In another preferred embodiment of the present invention, before handling, removes solvent-detergent the particle of any existence.
In yet another embodiment of the present invention, provide a kind of gamma globulin that is suitable for the heat-killed and solvent-detergent sterilization of intravenously medication.
Detailed description of the present invention
The fraction that contains immunoglobulin (Ig) is as parent material.As long as the not specific qualification of this fraction is from human serum and contain immunoglobulin fraction.This specific examples that contains immunoglobulin fraction comprises fraction II+III and the fraction II by the alcohol grading acquisition of Cohn, and the mashed prod that contains immunoglobulin fraction of equal value therewith.Other parent material is fraction I+II+III and fraction II+IIIw.Parent material may contain impurity, such as human blood type antibody, and plasminogen, fibrinolysin, kallikrein, PKA, IgM, IgA, IgG polymer (back is called " aggregate "), etc.
Preferred parent material is Cohn fraction II or Cohn fraction II+III.When using Cohn fraction II+III mashed prod, recommend to carry out washing step earlier and form fraction II+IIIw, use it for flow process of the present invention then." fraction II+IIIw " is the Cohn fraction II+III throw out with the Di-Sodium Phosphate solution washing.
Can obtain fraction II+IIIw by fraction II+III mashed prod being suspended in injection cold water, ratio approximately is 1 kilogram of II+III mashed prod of per 20 volume water.Add sodium phosphate to the about 0.003M of final concentration, be used to dissolve fat, lipoprotein and white protein.Cold ethanol is added to whole determining alcohol about 20%.When adding ethanol, temperature is progressively reduced to-5 ± 1 ℃ also for example by using acetate buffer or dilute hydrogen sodium oxide that pH is remained on 7.2 ± 0.1.Maintain the temperature at-5 ± 1 ℃, by centrifugal and/or filtered and recycled fraction II+IIIw mashed prod.
Before the present invention's inactivation of viruses step first time, can carry out various purifying in advance and/or reduce the aggregation step.For example, when using fraction II+IIIw mashed prod, typically contain about 20% ethanol and surpass 70%IgG, can it be suspended in 3 to 10 volumes in about 0 to 5 ℃ of temperature, preferably in 3 to the 5 volume cold water and use pH and acetate buffer or hydrochloric acid with pH regulator between 4.5 to 6.0, preferably between 5.0 to 5.5.Mixed solution is vibrated about 2 to 15 hours, so that all gamma globulins enter solution.After this, can be by centrifugal and/or remove by filter undissolved protein, such as white protein and alpha-globulin.
When using different initial Cohn level timesharing, can suitably select to carry out required initial step or the process flow steps of purifying in advance, to obtain to be used for the further high IgG content fraction of processing.For example, with fraction II (contain surpass 95% IgG) when Cohn fraction III separates, fraction II is used for further processing, initial process can be as Uemura etc. at United States Patent (USP) 4, described in 371,520 at 3.2 to 5.0 acid pH, preferably 3.8 to 4.2, thereby the immunoglobulin (Ig) aggregation is degraded into immunoglobulin monomer and disome, because known aggregation has anticomplementary activity (ACA).On the other hand, be parent material with Cohn fraction II+III, Uemura etc. apply for a patent the step of hanging down the pH processing adding behind the aforesaid initialization step and before making virally inactivated heat treatment step.
For hot degerming step, immunoglobulin (Ig) is water-soluble or if such as the mixed liquid of the filtrate of collecting from above-mentioned partially purified fragment II+III, can use like this, and to wherein adding sugar, sugar alcohol and/or amino acid thermo-stabilizer.Thermo-stabilizer is sucrose preferably, maltose, and Sorbitol Powder or mannitol are most preferably Sorbitol Powder.Sugar or sugar alcohol are added to immunoglobulin solution or earlier itself and mixes with small amount of water are added again with Powdered, so that about final concentration of 10 to 5W/V% to be provided, until saturated.At this moment, the immunoglobulin (Ig) aqueous solution contains enough water, thereby this solution contains about total protein of 1 to 6%, and the per kilogram mashed prod contains the proteinic typical fraction II+III parent material of about 300 grams.
After adding thermo-stabilizer, mixed solution was heated to about 50-70 ℃ 10-100 hour, preferably at about 60 ℃ 10 to 20 hours, with the thermo-responsive virus of deactivation.Thermal treatment is inactivation of viruses not only, and can produce the protein denaturation effect, can preferably reduce some common and Cohn fraction II+III links unwanted Protein content, such as prekallikrein, and fibrinolysin, plasminogen and IgA.
After thermal treatment, the cold water that adds aequum makes protein concn remain on about 0.3 to 2.0%.Solution is chilled to 0-2 ℃.
After this, heat treated solution is carried out the PEG fractional separation.The PEG fractional separation is the method that present technique field purifying immunoglobulin (Ig) is known, and is for required IgG monomer is separated with naturally occurring other impurity in IgG polymkeric substance and the initial plasma proteins fraction with disome.Yet in the method, the PEG classification also can separate the unwanted metaprotein product that produces in required IgG monomer and disome and the thermal treatment.The protein product of these sex change is prekallikreins of sex change, plasminogen, fibrinolysin, IgA, IgM and polymkeric substance thereof.
Can use any PEG fractionation method described in the prior art.Usually carry out two stage PEG fractional separation.In the PEG fractionated fs, select PEG concentration and pH, required IgG monomer and dimer are remained in the solution and will go out solution such as the unwanted albumen precipitation of aggregation.After centrifugal and/or filtration, increase PEG concentration and regulated pH, make required IgG monomer and dimer precipitation.
For example, when using fraction II+IIIw mashed prod as parent material, the PEG classification of fs can be at pH about 5.0 to 7.5, preferably pH value about 6.5 to 7.5 is carried out, when using fraction II+III mashed prod as parent material preferably in pH value about 5.5 to 6.0, the PEG concentration range is about 4 to 8%, when using fraction II+IIIw mashed prod as parent material preferably 4 to 6%, or when using fraction II+III mashed prod 6 to 8% as parent material.Keep about 0-2 ℃ of low temperature, can carry out about 1 to 8 hour of the PEG classification of fs, after this remove mashed prod as mentioned above.Then with the filtered liquid pH regulator to about 8.0 to 9.0, preferably about 8.5 to 8.9, and PEG added to final concentration about 10 to 15%, preferably about 12%.By filtering and/or the centrifugal mashed prod of removing formation, the i.e. immunoglobulin (Ig) of purifying.
In the United States Patent (USP) 4,845,199 of the United States Patent (USP) 4,876,088 of above-mentioned Hirao etc. and Hirao etc., can find other details of actual in the present invention available PEG fractionation method.
Final key step of the present invention is to use solvent or solvent-detergent mixed solution to carry out the inactivation of virus step second time.As described below, further purification step, particularly those comprise and make the spent ion exchange resin step, can and/or carry out afterwards before solvent-detergent is handled.A kind of special effective means is to handle and to carry out cationic exchange then handle behind solvent stain remover inactivation of virus carrying out anionresin before the solvent stain remover inactivation of virus.By this program, some the unwanted proteinaceous substances that uses anionresin to remove from IgG to exist in the human plasma is (such as PKA, IgA, IgM and white protein) further remove this class material (PKA by cationic exchange then, IgA, IgM, white protein and PEG) and the residual reagent that uses in handling of solvent-detergent.
If do not finish in addition in whole flow process, the solution that the need processing is used for solvent-detergent is to remove all particulates matter, and it can comprise denatured protein.Thereby, preferably before adding solvent-detergent with 1 micron or second filter filtering solution more.Exist the possibility of virus may avoid being exposed to solvent-detergent in the macrobead thereby so also can be reduced in.
Now, the preferred solvent of deactivation envelope virus is a trialkylphosphate.The trialkylphosphate that uses among the present invention does not have specific limited, but preferably use three (just-and butyl) phosphoric acid ester (after this being called " TNBP ").Other available trialkylphosphate is three (tributyl) phosphoric acid ester, three (n-hexyl) phosphoric acid ester, three (ethylhexyl) phosphoric acid ester or the like.Can use 2 kinds or how different trialkylphosphate mixtures.
The amount of the trialkylphosphate that uses is at 0.01 to 10 (W/V) %, preferably about 0.1 to 3 (W/V) %.
Can use trialkylphosphate having or do not have in the presence of stain remover or the tensio-active agent.Preferably trialkylphosphate and stain remover are used in combination.The effect of stain remover is the contacting of virus and trialkylphosphate in the enhancing immunity globulin composite.
Stain remover comprises the polyoxyethylene deriv of lipid acid for example, part ester such as the Polysorbate 80 of anhydrous Sorbitol Powder (trade(brand)name: tween 80, etc.) and Polysorbate 20 (trade(brand)name: polysorbas20, etc.); And nonionic oil bath clean-out system such as the oxygen alkylphenol (trade(brand)name: Triton X100, etc.) that ethylizes.Embodiment comprises that also other tensio-active agent and stain remover are such as zwitterionic detergent or the like.
When using stain remover, do not have strict add-on; For example, can use about 0.001% to about 10% ratio, preferably 0.01% to 3%.
Among the present invention, the trialkylphosphate processing that contains immune globulin composite is carried out at about 20 to 35 ℃, and preferably 25 to 30 ℃, the time surpasses 1 hour, and preferably about 5 to 8 hours, more preferably about 6 to 7 hours.
In trialkylphosphate was handled, immunoglobulin (Ig) was about protein solution of 3 to 8% in water solvent.
If do not carry out before solvent-detergent is handled, the immunoglobulin (Ig) of crossing with the solvent detergent-treatment is handled in then available anionresin.Preferably, at least solvent-detergent is handled product and carry out the cationic exchange processing.In ion exchange treatment, immunoglobulin (Ig) is dissolved in the aqueous solution, the common about 5-8 of pH value, and have required low ionic strength and make the IgG maximum adsorption.Protein concn is usually in the scope of 1-15W/V%, more preferably about 3 to 10W/V%.With employed identical aqueous equilibrium ion-exchange, can use system in batches or continuously.For example, can be by with about 10 to 100 milliliters of every milliliter of pretreated anionic exchange mediums (for example with immunoglobulin solution and anionic exchange medium, 1 gram DEAE Sephadex A-50 resin expand into about 20 gram weight in wet bases in 0.4% sodium chloride solution) amount mix, mixture was vibrated about 0.5 to 5 hours at about 0.5 ℃, filter then or 6,000 to 8,000rpm reclaimed supernatant liquor in centrifugal 10 to 30 minutes and carries out the anionresin batch treatment.Continuously processing can be undertaken by immunoglobulin solution is passed anion-exchange column with the ratio of about 10 to 100 milliliters of every milliliter of ion-exchangers, and reclaims non-adsorbable fraction.
The anionite that uses comprises the anion exchange groups that links to each other with soluble carrier for example.Anion exchange groups comprises diethyl amido ethyl (DEAE), a kind of tetravalence amino-ethyl (QAE) group etc., and soluble carrier comprises agarose, Mierocrystalline cellulose, dextran, polyacrylamide etc.
Spendable cationite is CM-sephadex (CM-Sephadex), CM-Mierocrystalline cellulose, SP-Sephadex, CM-sepharose (CM-Sepharose) and S-Sepharose.With 1 milliliter of pretreated cationite (for example 1 gram CM-Sephadex C-50 resin is expanded to about 30-35 gram weight in wet base in 0.4% sodium chloride solution) and mixed 0-5 ℃ of vibration 1-6 hour that be incorporated in of 0.5 milliliter to 5 milliliters immunoglobulin solution.Centrifugal or filtered and recycled has absorbed the resin of IgG with suspension.Also can use continous treatment process.
When above-mentioned condition is used for cationite, IgG will be adsorbed, and after this wash by for example about 1.4N sodium chloride solution and adsorbed proteic Zeo-karb, but wash-out go out IgG.
Follow above-mentioned steps, clarification, diafiltration IgG and be concentrated into required degree.If desired, can add such as the D-Sorbitol Powder and the most at last solution component be adjusted to and contain about 100 mg/ml IgG and 50 mg/ml D-Sorbitol Powders, the pH value is 5.4.Then solution being kept filter by aseptic bacterium carries out sterile filtration and charges in the tubule.
Statement the following example illustrates the present invention but is not restriction.
If desired, other immunoglobulin purification method suitably can be combined with flow process described herein.For example, can use the wilkinite clarification steps that can reduce kallikrein and PKA.In the following examples 1 this is illustrated.
Embodiment 1: the gamma globulin that thermal treatment and solvent-detergent are handled
685 gram fraction II+IIIw mashed prod are suspended in about 11.9 kilograms of cold water.Sodium acetate trihydrate is added in the suspension to the about 0.04M of final concentration, selective dissolution IgG.After mixing about 15 minutes, the pH value of suspension is adjusted to 4.8 with the acetate buffer of pH4.0.Cold ethanol (95%) is added in the suspension to final concentration 17%.When adding ethanol, the temperature of suspension is reduced to approximately-6 ℃ gradually.The Celite 535 that in suspension, adds the Celite company of 303 gram pickling, final concentration about 2.0%.Mix after one hour, remove Celite and contain unwanted protein such as fibrinolysin, plasminogen, the fraction III of IgA and IgM by the strainer filtering under pressure.By 0.45 micron and 0.2 micron further clear filtrate of filter.
With 1.0N hydrochloric acid with the pH value of fraction II+IIIw settled solution be adjusted to 4.0 then by ultrafiltration and concentration to about 3.4 liters (original volumes 1/5).To be added to concentrated solution with the cold water of ultrafiltration first time removal amount equivalent and arrive about 1/5 of original volume by ultrafiltration and concentration again.In this step, protein concentration about 2% in the solution.Solution further is concentrated into about 4% and the cold water diafiltration is lower than 300 μ s/cm until the electric conductivity of solution, to prevent protein aggregation and the sex change in the thermal treatment.Solution further is concentrated to about 8.8% protein.The D-Sorbitol Powder is added in the solution to final concentration about 33%.Mix after 30 minutes, the pH that will contain the solution of Sorbitol Powder is adjusted to 5.5 with 0.5N sodium hydroxide.Then solution was heated 10 hours at 60 ℃.After the thermal treatment, add the cold water be equivalent to 3 times of volumes of solution after the thermal treatment and diluting soln is cooled to 0-2 ℃.
With 0.25N sodium hydroxide pH value of solution is adjusted to 6.9 and add 50% polyoxyethylene glycol (PEG) 3350 in solution to make the PEG final concentration be 4%.Sodium chloride concentration in the solution is adjusted to about 8mM, to help the precipitation of impurity and aggregation under pH6.9.By removing by filter the mashed prod of formation.With 1.0N hydrochloric acid with the pH regulator to 4.8 of filtrate and add wilkinite to final concentration about 0.25%.With the pH of bentonite suspension readjust to about 5.2 then filtering suspension liquid remove wilkinite.With 0.25N sodium hydroxide with filtrate pH regulator to 8.5 and to add 50% PEG 3350 solution to PEG final concentration be 12%.By the centrifugal precipitation (immunoglobulin (Ig) of purifying) of removing formation.
The purifying immunoglobulin (Ig) mashed prod that about 175 grams are as above obtained is suspended in about 790 grams, 0.3% sodium chloride solution.PH of suspension is adjusted to 5.5 also mixes them 2.5 hours subsequently.The DEAE-Sephadex A-50 resin of 62 gram pre-balances (is contained 0.3% sodium-chlor, pH5.5) adds solution and suspension was mixed 2 hours.Filtering suspension liquid is removed resin then.After sodium chloride concentration is adjusted to 0.4%, three-just-butyl phosphoric acid ester (TNBP) and Polysorbate 80 mixtures are added in the filtrate, make TNBP final concentration 0.3% in the solution, Polysorbate 80 final concentrations 1.0%.After being incubated overnight, with pH value of solution be adjusted to 5.8 and add about 860 the gram pre-balances CM-Sephadex C-50 (contain 0.4% sodium-chlor, pH5.8).Mix after 2 hours, with suspension filtered.After washing CM-Sephadex resin 3 times with 0.3% sodium-chlor, with the IgG of 1.4N sodium-chlor wash-out absorption.With the elutriant clarification, diafiltration also concentrates.Add the D-Sorbitol Powder and regulate to make at last and about 100 mg/ml IgG are arranged, 50 mg/ml D-Sorbitol Powders, pH5.4 in the solution composition.Keep strainer with solution sterile filtration and charge in the tubule with aseptic bacterium then.
Intermediary wilkinite step is useful among this embodiment, can further reduce the existence of ypotension enzyme such as kallikrein and PKA.
The detected result of product among the embodiment 1
| Detect parameters | |
| Anticomplementary activity (CH 50Unit/milligram IgG) | 0.34 |
| IgG purity (%) | 100 |
| IgG content (mg/ml) | 112.7 |
| Prekallikrein (%CBER is with reference to No. 3) | 21 |
| Measles antibody (%CBER reference number 176) | 0.67 |
| HPLC distribution (%) monomer (%) dimer (%) fragment (%) aggregate of IgG molecular size | 82.2 17.4 0.10 0.3 |
| Hepatitis A antibody (titre) | 1∶200 |
| Hbv antibody (titre) | 1∶1024 |
| IgA (mcg/ml) | 22 |
| IgM (mcg/ml) | 16 |
| Plasminogen (nanograms/milliliter) | ND |
| Fibrinolysin (nanograms/milliliter) | 16 |
| pH | 5.4 |
ND=does not measure
Embodiment 2: the heat treated gamma globulin of handling with solvent-detergent
1 kilogram of fraction II+III mashed prod is suspended in 4.5 kilograms 0-2 ℃ the cold water.Mix after 1 hour, pH of suspension is adjusted to 5.0 with 1N hydrochloric acid.After pH5.0 mixes 3 hours, the centrifugal precipitation of removing.Be added in the centrifugal thing D-Sorbitol Powder to final concentration 33% and mixed 1 hour.PH of suspension is adjusted to 5.5 also subsequently 60 ℃ of heating 10 hours.After the thermal treatment, add the cold water be equivalent to 3 times of volumes of solution after the thermal treatment and diluting soln is cooled to 0-2 ℃.Add 50% polyoxyethylene glycol (PEG), 3350 solution to final concentration 6%PEG.With 0.5N sodium hydroxide the 6%PEG pH of suspension is adjusted to 5.7 and suspension mixed 2 hours.Adding pickling Celite 535 mixed 1 hour to final concentration 1.5% and with suspension.Remove by filter precipitation and Celite., and add 50%PEG solution and make PEG concentration adjustment to 12% filtrate pH regulator to 8.8 with 0.5N sodium hydroxide.With the pH regulator to 8.8 of 12%PEG suspension, suspension was mixed 1 hour and filtered the immunoglobulin (Ig) mashed prod of collection purifying.Be recovered to the immunoglobulin (Ig) mashed prod of 251 gram purifying.
The 251 immunoglobulin (Ig) mashed prod that restrain purifying that as above obtain are suspended in about 1.4 kilogram of 0.3% sodium chloride solution.Mix after 1 hour, with the pH regulator to 6.0 of 5% acetate suspension.After mashed prod dissolves fully, be added to the 104 gram DEAE-Sephadex A-50 resins that use 0.3% sodium-chlor in the pH6.0 pre-balance in the solution and mixed 2 hours.Remove by filter resin.By the further clear filtrate of 0.2 micron filter.By adding sodium-chlor the concentration of sodium-chlor in the settled solution is increased to 0.4%.Three-just-butyl phosphoric acid salt (TNBP) and Polysorbate 80 mixtures are added to make its final concentration in the solution be 0.3%TNBP and 1.0%Polysorbate 80.Then with solution 27 ℃ of incubations 1 hour and place cold box to spend the night for 4 ℃.Second day, pH value of solution is adjusted to 5.8 and add 1.8 kilograms use 0.4% sodium-chlor equilibrated CM-Sephadex C-50 resin in advance under pH5.8.Mix after 2 hours, by the filtering separation resin.Wash CM-Sephadex resin 3 times with 0.3% sodium-chlor after, with the IgG of 1.4N sodium-chlor wash-out absorption.With the elutriant clarification, diafiltration also concentrates.Add the solution that D-Sorbitol Powder and last adjusting generation have about 100 mg/ml IgG and about 50 mg/ml D-Sorbitol Powder components.Solution is divided into 2 parts, A part and B part.A part pH value is adjusted to 5.4 and B part is adjusted to pH4.3.Respectively A part and B part solution are carried out sterile filtration and are filled to tubule with aseptic bacterium reservation filter then.
The measurement result of embodiment 2 products
| Detect parameters | ||
| Anticomplementary activity (CH50 unit/milligram IgG | ＜0.05 | |
| IgG purity (%) | 100 | |
| IgG content (mg/ml) | 104.2 | |
| Prekallikrein (%CBER is with reference to No. 3) | ND | |
| Diphtheria antibody (antitoxic unit/milliliter) | 8.2 | |
| IgG molecule HPLC size distribution | pH5.4 88.8 10.9 0.3 ＜0.3 | pH4.3 97.5 2.3 ND ＜0.3 |
| (%) monomer | ||
| (%) dimer | ||
| (%) fragment | ||
| (%) aggregate | ||
| Hepatitis A antibody (titre) | 1∶100 | |
| IgA (mg/ml) | 78 | |
| IgM (mg/ml) | 28 | |
| Kallikrein (A405) | 0.09 | |
| Plasminogen (nanograms/milliliter) | ＜8.4 | |
| Fibrinolysin (nanograms/milliliter) | ＜8.4 | |
ND=does not measure
For those of skill in the art's variation of the present invention will be conspicuous.
Claims (11)
1. method for preparing intravenous injection gamma globulin solution comprises:
(a) the impure gamma globulin solution of thermal treatment under time of the enough thermo-responsive virus of deactivation and temperature condition, wherein impure gamma Globulin solution is Cohn fraction I+II+III, Cohn fraction II+III, Cohn fraction II+IIIw, or Cohn fraction II;
(b) heat treated gamma globulin solution only being carried out scalping with polyoxyethylene glycol separates to obtain the gamma globulin solution of purifying, polyoxyethylene glycol fractional separation was wherein carried out through at least two stages, in the fs of polyoxyethylene glycol fractional separation, impurity is removed with coprecipitation mode, in the subordinate phase of polyoxyethylene glycol fractional separation, gamma globulin is removed; And
(c) do not carry out further polyoxyethylene glycol classification, the gamma globulin solution of handling purifying with organic solvent is with the deactivation envelope virus, and wherein said organic solvent is an alkyl phosphate.
2. the method in the claim 1 is wherein carried out purification step at least one time to impure gamma Globulin solution in that heat treatment step (a) is preceding.
3. the method in the claim 1, heat treatment step wherein (a) carried out 10 to 100 hours at 50 to 70 ℃.
4. the method in the claim 3, wherein heat treatment step (a) carried out 10 hours at 60 ℃.
5. the method in the claim 1, alkyl phosphate wherein is three-just-butyl phosphoric acid ester.
6. the method in the claim 1, organic solvent wherein contains stain remover.
7. the method in the claim 1 is handled gamma Globulin solution after the step wherein (a) with anionite-exchange resin and Zeo-karb.
8. the method in the claim 7, anion exchange process wherein step (c) before and Zeo-karb handle afterwards in step (c).
9. the method in the claim 1 is wherein carried out the wilkinite clarification steps after fs of isolating polyoxyethylene glycol fractional separation.
10. the intravenous injection gamma Globulin solution of making by the method in the claim 1.
11. the intravenous injection gamma Globulin solution of making by the method in the claim 8.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US99795297A | 1997-12-24 | 1997-12-24 | |
| US08/997,952 | 1997-12-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1252729A CN1252729A (en) | 2000-05-10 |
| CN1214042C true CN1214042C (en) | 2005-08-10 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB988043769A Expired - Fee Related CN1214042C (en) | 1997-12-24 | 1998-12-07 | Production process for intravenous immune serum globulin and resultant product |
Country Status (15)
| Country | Link |
|---|---|
| EP (1) | EP0971727A4 (en) |
| JP (1) | JP2001514672A (en) |
| KR (1) | KR20000075562A (en) |
| CN (1) | CN1214042C (en) |
| AR (1) | AR018260A1 (en) |
| AU (1) | AU747893B2 (en) |
| BR (1) | BR9807598A (en) |
| CA (1) | CA2281938A1 (en) |
| CO (1) | CO4970799A1 (en) |
| IL (1) | IL131471A0 (en) |
| MY (1) | MY117328A (en) |
| RU (1) | RU2198668C2 (en) |
| TW (1) | TW546144B (en) |
| UA (1) | UA64742C2 (en) |
| WO (1) | WO1999033484A1 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2527915T3 (en) | 1998-06-09 | 2015-02-02 | Csl Behring Ag | Liquid immunoglobulin G (IgG) product |
| EP1185290A4 (en) * | 1999-06-15 | 2005-08-31 | Alpha Therapeutic Corp | Manufacturing method for intravenous immune globulin and resultant product |
| JP2004525077A (en) * | 2000-09-28 | 2004-08-19 | リサーチ コーポレイション テクノロジーズ，インコーポレイテッド | Treatment of immune-mediated diseases by oral administration of immunoglobulin G enriched plasma fraction |
| ES2184594B1 (en) * | 2001-01-17 | 2004-01-01 | Probitas Pharma Sa | PROCEDURE FOR THE PRODUCTION OF GAMMAGLOBULINA G HUMANA INACTIVADA OF VIRUS. |
| US6893639B2 (en) | 2001-10-19 | 2005-05-17 | Hemacare Corporation | Method for high yield purification of immune globulins from blood plasma and blood plasma intermediates |
| ES2558303T3 (en) | 2002-09-11 | 2016-02-03 | Chugai Seiyaku Kabushiki Kaisha | Protein Purification Method |
| TWI320788B (en) | 2005-05-25 | 2010-02-21 | Hoffmann La Roche | Method for the purification of antibodies |
| KR100791502B1 (en) * | 2006-09-29 | 2008-01-03 | 한스바이오메드 주식회사 | Production methods of virus inactivated and cell-free body implant |
| EP2350271B1 (en) * | 2008-11-20 | 2016-01-27 | Biogen MA Inc. | Arginine inactivation of enveloped viruses |
| IL212911A0 (en) * | 2011-05-16 | 2011-07-31 | Omrix Biopharmaceuticals Ltd | Immunoglobulin reduced in thrombogenic contaminants and preparation thereof |
| ES2381828B1 (en) * | 2012-03-20 | 2012-11-16 | Grifols, S.A. | PROCEDURE TO OBTAIN A COMPOSITION OF IgG THROUGH THERMAL TREATMENT |
| ES2785375T3 (en) | 2014-03-11 | 2020-10-06 | Green Cross Holdings Corp | Immunoglobulin purification procedure |
| CN107880116B (en) * | 2016-09-30 | 2023-02-03 | 盖立复集团公司 | Method for producing immunoglobulins |
Family Cites Families (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2604759C2 (en) * | 1976-02-07 | 1983-06-01 | SCHURA Blutderivate GmbH & Co KG, 4150 Krefeld | Method of Obtaining IV Compatible Gamma Globulins |
| US4820805A (en) * | 1983-07-14 | 1989-04-11 | New York Blood Center, Inc. | Undenatured virus-free trialkyl phosphate treated biologically active protein derivatives |
| US4835257A (en) * | 1984-07-07 | 1989-05-30 | Armour Pharma Gmbh | Process for preparing gamma globulin suitable for intravenous administration using peg and a citrate buffer |
| FR2582515B1 (en) * | 1985-05-30 | 1988-11-04 | Merieux Inst | PROCESS FOR THE PREPARATION OF GAMMA-GOBULINS ADMINISTRABLE BY THE INTRAVENOUS ROUTE AND GAMMA-GLOBULINS OBTAINED |
| JPH0662436B2 (en) * | 1986-05-19 | 1994-08-17 | 株式会社ミドリ十字 | Method for producing intravenous immunoglobulin preparation |
| US4841023A (en) * | 1986-06-25 | 1989-06-20 | New York Blood Center, Inc. | Inactivation of viruses in labile protein-containing compositions using fatty acids |
| DE3825429C2 (en) * | 1988-07-27 | 1994-02-10 | Biotest Pharma Gmbh | Method for producing an intravenously administrable polyclonal immunoglobulin preparation with a high IgM content |
| JP2965069B2 (en) * | 1989-01-13 | 1999-10-18 | 吉富製薬株式会社 | Method for producing protein-containing composition |
| EP0378208B2 (en) * | 1989-01-13 | 2003-01-15 | Mitsubishi Pharma Corporation | Production method for protein-containing composition |
| CA2034489C (en) * | 1989-06-15 | 1996-07-16 | Michael E. Hrinda | Methods for the inactivation of viruses in viral-contaminated pharmaceutical compositions |
| US5177194A (en) * | 1990-02-01 | 1993-01-05 | Baxter International, Inc. | Process for purifying immune serum globulins |
| JP3145696B2 (en) * | 1990-10-05 | 2001-03-12 | 日本ケミカルリサーチ株式会社 | Method for producing secretory immunoglobulin A preparation |
| US5110910A (en) * | 1991-03-25 | 1992-05-05 | Miles Inc. | Virucidal euglobulin precipitation |
| DE4431833C1 (en) * | 1994-09-07 | 1995-05-18 | Blutspendedienst Der Drk Lande | Prepn. of an anti-haemophilic factor from a cryo-precipitate |
| JP4003235B2 (en) * | 1994-09-30 | 2007-11-07 | 三菱ウェルファーマ株式会社 | Method for producing intravenous immunoglobulin preparation |
-
1998
- 1998-07-12 UA UA99084781A patent/UA64742C2/en unknown
- 1998-12-07 AU AU17038/99A patent/AU747893B2/en not_active Ceased
- 1998-12-07 CN CNB988043769A patent/CN1214042C/en not_active Expired - Fee Related
- 1998-12-07 IL IL13147198A patent/IL131471A0/en unknown
- 1998-12-07 EP EP98961803A patent/EP0971727A4/en not_active Withdrawn
- 1998-12-07 BR BR9807598-5A patent/BR9807598A/en not_active Application Discontinuation
- 1998-12-07 KR KR1019997007622A patent/KR20000075562A/en not_active Application Discontinuation
- 1998-12-07 CA CA002281938A patent/CA2281938A1/en not_active Abandoned
- 1998-12-07 JP JP53498599A patent/JP2001514672A/en not_active Ceased
- 1998-12-07 RU RU99120190/14A patent/RU2198668C2/en not_active IP Right Cessation
- 1998-12-07 WO PCT/US1998/025208 patent/WO1999033484A1/en not_active Application Discontinuation
- 1998-12-22 AR ARP980106616A patent/AR018260A1/en unknown
- 1998-12-23 MY MYPI98005848A patent/MY117328A/en unknown
- 1998-12-23 CO CO98076460A patent/CO4970799A1/en unknown
- 1998-12-23 TW TW087121571A patent/TW546144B/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1999033484A1 (en) | 1999-07-08 |
| CN1252729A (en) | 2000-05-10 |
| KR20000075562A (en) | 2000-12-15 |
| EP0971727A1 (en) | 2000-01-19 |
| IL131471A0 (en) | 2001-01-28 |
| MY117328A (en) | 2004-06-30 |
| TW546144B (en) | 2003-08-11 |
| CA2281938A1 (en) | 1999-07-08 |
| JP2001514672A (en) | 2001-09-11 |
| AU1703899A (en) | 1999-07-19 |
| UA64742C2 (en) | 2004-03-15 |
| RU2198668C2 (en) | 2003-02-20 |
| AR018260A1 (en) | 2001-11-14 |
| AU747893B2 (en) | 2002-05-30 |
| BR9807598A (en) | 2000-02-22 |
| CO4970799A1 (en) | 2000-11-07 |
| EP0971727A4 (en) | 2005-01-19 |
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