CN1225020A - Methods of terminal sterilization of biological products - Google Patents
Methods of terminal sterilization of biological products Download PDFInfo
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- CN1225020A CN1225020A CN 97196248 CN97196248A CN1225020A CN 1225020 A CN1225020 A CN 1225020A CN 97196248 CN97196248 CN 97196248 CN 97196248 A CN97196248 A CN 97196248A CN 1225020 A CN1225020 A CN 1225020A
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Abstract
The invention relates to methods of sterilizing biologically active products, particularly therapeutic or prophylactic products and the compositions obtained thereby. The methods include obtaining a dried sample containing an amount of trehalose sufficient to render heat stability to the product and exposing the dried sample to heating conditions at a temperature and for a duration sufficient to substantially inactivate viruses, especially non-lipid encapsulated viruses. The drying methods include both ambient drying conditions and lyophilization. The heating conditions include any known in the art and cover a wide range of temperatures and heating times. The compositions obtained contain stable products and do not contain measurable infectious virus, particularly parvovirus.
Description
Technical field
The present invention relates to the sterilization of product of autoblood and other biogenetic derivation.The present invention relates in the presence of the trehalose and be enough to kill virus heating biologic activity product a period of time under the condition of parvovirus particularly.
Background technology
Removing virus removal and other pollutant from the biologic activity product fully is essential to the production and the use of miscellaneous treatment and prevention product.Use certain methods at present.These methods mainly are dry heat treatment, chromatography, solvent-detergent (SD) processing and pasteurization.These methods all have shortcoming and neither one eliminate all known viral aspect success.Also has the virus that these methods can not deactivation of passing through that is not characterized as yet.About summarizing referring to (1991) Blood Separation and Plasma Fractionation such as Cuthbertson Wiley-Liss, Inc. 385-435 page or leaf; Mozen (1993), J.Clin.Apheresis 8:126-130; Ingerslev (1994) Haemostasis 24:311-323; Dorner etc. (1993) VirologicalSafety Aspects of Plasma Derivatives, Brown edits, Dev.Biol.Stand., Basel, Karger, the 81st volume, 137-143 page or leaf; Mannucci (1993) Vox Sang.64:197-203; With (1994) Vox Sang.67:72-77 such as Hamman.
Miscellaneous product with treatment effectiveness is the biogenetic derivation that derives from such as blood plasma and cell line.The blood plasma that is used for fractionated at U.S.'s great majority is to obtain by plasmapheresis at the collection center that spreads all over whole nation distribution.This center provides commercial fractionated product at US and European.Collect about 9,000,000 liters of blood plasma every year from about 13,000,000 donations.The Red Cross increases about 800,000 and is raised in this number.
Product from human plasma can be divided into several groups: albumin product, immunoglobulin, AHG, coagulation prod and protease inhibitor.Described albumin product is also referred to as the V portioned product, is mainly used to recover colloid osmotic pressure in the shock disease such as burn or hemorrhagic shock, and in these diseases, loss of fluid is a basic problem.
Described immunoglobulin or " gamma Globulin " are isolating from the II part, and comprise the mixture of the antibody of representing the blood plasma storehouse source.The many hyperimmune globulins that are used for passive immunity are from the donor separating plasma with high-level protection antibody.Described AHG comprises the fibrinogen and the von WillebrandShi factor.
Described coagulation prod comprises the antihemophilic factor VIII and the factor IX complex of the replacement therapy that is respectively applied for hemophilia A and B.Prepared activated form be called counter inhibitor condense complex factor IX complex and be used for the treatment of patient with factor VIII inhibitor.Described protease inhibitor comprises α l protease inhibitor, is also referred to as α l antitrypsin, and it is used for the treatment of birth defect.Antithrombin also is the inhibitor that causes the birth defect of thrombosis complication.
Other body fluid is the source of treatment product.For example, in the past from aplastic amenia people's blood or urine purification of erythropoietin generate plain.United States Patent (USP) the 4th, 677, No. 195.Highly purified albumin also derives from people's Placenta Hominis.Grandgeorge and Veron (1993) Virological SafetyAspects of Plasma Derivatives, Brown edits, Dev.Biol.Stand., Basel, Karger, the 81st volume, 237-244 page or leaf.Producing recombiant protein now in the milk of transgenic sheep is the fact of a commerce.
A large amount of treatment products derives from the cell culture of express recombinant protein now.The generally growth in the presence of animal or human's serum of described cell culture.Described product derives from these cells or cell culture supernatant, therefore comprises the virus from culture medium or cell self.The product of these acquisitions includes but not limited to colony stimulating factor, monoclonal antibody and its derivant, somatomedin, such as erythropoietin, interleukin.Somatomedin is represented the industry of millions of dollar separately.About summarizing referring to Erikson (1991) Sci.Am., Feb.1991 126-127 page or leaf.
Though it is more much smaller than the risk relevant with blood plasma product to derive from the proteic viral pollution risk of cell culture, the risk of this viral pollution is always arranged when relating to cell.For this reason, will be for inactivation of viruses such as the product of monoclonal antibody through heat-treated.And adding human serum albumin (HSA) is common way with the preparation of stablizing recombiant protein.
The virus of the main blood delivery of clinical concern is hepatitis B and hepatitis C virus and HIV and HTLV retrovirus.As for blood derivatives, as if the HTL VI is relevant with cell with II and cytomegalovirus (CMB), and therefore not dangerous in acellular product.
New when viral when finding, change ablation method to adapt to them.For example, the discovery that human immunodeficiency virus (HIV) holds out against the standard processing of factor VIII need change the method that requirement adds HSA, to stablize this product under new, stricter condition.Mozen(1993)。
The method of deactivation HIV and hepatitis virus is known in the blood plasma part.As described above, in the presence of HSA, heat 10 hours deactivation HIV at 60 ℃.In the preparation of factor VIII and factor IX by under lyophilised state, finding that non-first, non-hepatitis B (NANBH) was inactivated in 72 hours 80 ℃ of heating.The research group at Britain hemophilia center promptly monitors director (1988) the Lancet Oct.8 that transmits virus by concentrate, the 814-816 page or leaf.
In recent years, identified the disease that several blood transfusions are propagated, though these diseases are uncommon from the publilc health viewpoint, the use of blood plasma and blood plasma derivant and cell products is had real and potential impact.These comprise the propagation of parvovirus (B19).As if these etiology factors resist the method that is used for inactivation of virus at present.Sherwood (1993) Brown edits Virological Safety Aspects of Plasma Derivatives, and Brown edits, Dev.Biol.Stand., Basel, Karger, the 81st volume, 25-33 page or leaf.
At present the virus inactivating method that uses also may cause acquisition biology product the change of biologic activity.The immunogenicity of described product especially stretches with sterilization treatment possibility induced protein and/or assembles relevant.For example, found that factor shows the active evidence of F VIII, one-step method is than the polypeptide of the effectiveness of two step method higher, faster generation FVa and increase lower molecular weight.The change of virus inactivating method also may be induced non--F VIII composition, and these changes may partly cause some immunosuppressive activity of these concentrate.Barrowcliffe (1993) Virological Safety Aspects of Plasma Derivatives, Brown edits, Dev.Biol.Stand., Basel, Karger, the 81st volume, 125-135 page or leaf.
In the patient who frequently is exposed to the biological origin product in vivo and exsomatize and all to have found noticeable variation aspect function of immune system.In the patient of HIV feminine gender, when by they estimate, change the number and the function reduction that comprise immunologically competent cell to replying of stimulating with according to their mark of cell turnover (turnover).When chronic viral diseases exists, these change generation probably.And the degeneration of factor concentrate albumen impurity of the same race also may cause immunosuppressant with other pollutant.Summarize referring to Ingerslev (1994).
The human parvovirus is the factor of recent findings, and the coding that gives is called B19.Cossart etc. (1975) Lancet 1:72-73.It is the single-stranded DNA viruses of very little (24nm), has a very simple protein coat, but does not have lipid envelope.It causes the of short duration viremia in 1-2 week, but can obtain very high every ml at least 10
12Virion circulating virus titre.Though cause the relative minor illness that does not often have clinical manifestation under the parvovirus normal condition, cause the slight rubella sample rash that is called erythema infectiosum or erythema infectiosum, it also can cause more serious reaction.The parvovirus infections bone marrow stem cell, and this can cause serious, life-threatening disease in the patient of that pre-exist, potential anemia is arranged.Aplastic crisis as the result of acute hemopoietic blocking-up may take place in the patient that congenital hemolytic anemia and immune deficiency state are arranged.Parvovirus also causes fetus edema the pregnant woman.Therefore parvovirus is accepted the patient of the therapeutic agent in blood plasma source to those, particularly at those danger that representative is infected among patient of disorder of hemostasis is arranged.Although it should be noted that and use the method for effectively killing the virus and chromatography to remove, should virus still propagate by some concentrate, the parvovirus transmission danger is not only arranged, and owing to may exist other to have the pathogenic virus of same feature.
There is of child of haemolysis disease to discover that parvovirus is still infecting in the nonheat-treated blood plasma derivant fast.In a research, a child of group (N=9) who uses the heat treatment factor is not by parvovirus infections.Williams etc. (1990) Vox Sang.58:177-181.Yet other people has found that heat treated product propagates parvovirus really.Corsi etc. (1988), Med.Virol.25:151-153.Do not resemble hepatitis and HIV, parvovirus in individual blood plasma donations, detect less than, and therefore be present in stock's the blood plasma.
As mentioned above, various processing all are the deactivations that is intended for use in or is used for virus in the treatment product of biological origin.About summarizing referring to Soumela (1993) Trans.Med.Rev. VII: 42-57.Allocation step by all being used for reducing viral load and inactivation step in conjunction with obtaining final product.
Normally used heat treatment has several.What the dialogue protein product was commonly used is to heat in solution.In other words this method is called pasteurization.In the presence of few stable agent such as caprylate or tryptophan salt, by with fluid sample at least 10 hours inactivation of viruses of 60 ℃ of heating.Yet this method is not suitable for other products, because the degeneration under these conditions of most of albumen.But shown the miscellaneous virus of pasteurization deactivation, comprised HIV, HBV, HCV, HAV, HSV, poliovirus CMV, mumps virus, Measles virus and rubella virus.Nowak etc. (1993) Virological Safety Aspects of PlasmaDerivatives, Brown edits, Dev.Biol.Stand., Basel, Karger, the 81st volume, 169-176 page or leaf; And Soumela (1993).In these researchs, do not detect parvovirus.And, relevant with the use of pasteurization coagulation factor with the formation of neoantigen.Ingerslev(1994)。
When the high temperature to 68 ℃ of lyophilizing, labile protein tolerance, carry out the heating of drying product.The morning method that is included in 60 ℃ of heating is used for the deactivation hepatitis virus.As referring to United States Patent (USP) the 4th, 456, No. 590.Yet these conditions are not enough to deactivation HIV, propagate as the coagulation factor of virus by purification to confirm.Also find at 68 ℃ of products of handling 72 hours dangerous.Soumela(1993)。Recently, with higher temperature and longer time as 80 ℃ of deactivations that guaranteed hepatitis virus and HIV in 72 hours.As referring to (1994) Vox Sang.66:89-95 such as Knevelman.Yet the activity of most of unsettled biologic activity, particularly bio-pharmaceutical can not stand to be exposed under this extreme temperature/time conditions.Another shortcoming of this method is the frequent unexpected result about the deactivation of parvovirus.Santagostino etc. (1994) Lancet 343:798; With (1995) Lancet345:794 such as Yeed.
Solvent/detergent (SD) deactivation of virus depends on the breaking of film of lipid envelope virus.By the destruction of structure breaking or cell receptor recognition site, making this virus is non-infectious virus.Though most of human pathogenic virus have lipid envelope, parvovirus and HAV do not have, and can not be inactivated by this method.About summarizing referring to (1993) Ann.Hematol.67:259-266 such as Wieding.This SD method is used in many countries.Horowitz etc. (1993) Virological Safety Aspects of Plasma Derivatives, Brown edits, Dev.Biol.Stand., Basel, Karger, the 81st volume, 147-161 page or leaf.In a relevant method, lipid is used for the virus that deactivation has lipid envelope.Isaacs etc. (1994) Ann.NY Acad, Sci.724:457-464.
Some other methods have been developed or have developed.For example, by plasma exposure is carried out the cold sterilization of blood plasma in the 9-propanoic acid lactone and the ultraviolet of combination.Yet this method reduces the activity of labile protein.The various chemical treatments that proposed comprise the use of psoralen and UVA, BPD-MA and light, though these methods may be limited to the deactivation that lipid envelope virus is arranged.Found that also caprylate and sodium chlorite can kill the virus.Fixed various physical separation method comprises chromatography, the separation of cold alcohol grading, pore film and perfluocarbon emulsion.Lawrence (1993) Virological Safety Aspects of Plasma Derivatives, Brown edits, Dev.Biol.Stand., Basel, Karger, the 81st volume, 191-197 page or leaf; Burnouf (1993) is the same, the 199-209 page or leaf; Teh (1993) Vox Sang 65:251-257; Lebing etc. (1994) VoxSang.67:117-124; DiScipio (1994) Prot.Exp.Purif 5:178-186; Morgenthaler and Omar (1993) Virological Safety Aspects of plasmaDerivatives, Brown edits, Dev.Biol.Stand., Basel, Karger, the 81st volume, 185-190 page or leaf; Erickson (1992) Sci.Am.9 month 163-164 page or leaf; With (1993) J.Chromatog.629:201-213 such as McCreath.
Determine that in the production of plasma protein successful inactivation of virus need possess three prerequisite.At first, production routine must as far as possible fully reduce.The second, must select relevant detection virus to adding indicator (spiking) experiment.The 3rd, must correctly analyze the sample of generation with regard to infectious virus.The detailed description of this detection method is for example edited referring to (1993) Brown such as Hifenhaus, Virological Safety Aspects of Plasma Derivatives, Dev.Biol.Stand., Basel, Karger, the 81st volume, 117-123 page or leaf.These guides have been followed this paper.
Trehalose (α-D-glucopyranose-α-D-glycopyranoside) be a kind of natural generation, non-reducible disaccharide, it originally be found with some can be dry and do not damage and can the plant and animal of taskwork when the aquation again in the protection of dehydration damage relevant.Trehalose is commercially available with the form of dihydrate.It is useful to have shown that trehalose prevents in dehydration aspect the degeneration of albumen, virus and food.Referring to United States Patent (USP) the 4.891st, 319; 5,149,653; 5,026, No. 566; Blakeley etc. (1990) Lancet 336:854-855; Roser (1993) BioPharm.4:47-53; Colaco etc. (1992) Bio/Tech.10:1007-1011; With (in July, 1991) Trends in Food Sci.and Tech.166-169 such as Roser; Colaco etc. (1992) Biotechnol.Internat 345-350; Roser etc. (in May, 1993) New Scientist, the 25-28 page or leaf.The trehalose dihydrate compound uses with the crystallization preparation of excellent production process (GMP) level.The method of the trehalose of preparation dehydration, anhydrous form is described in No. the 600730th, European patent publication.This method relates in the presence of crystal seed heating trehalose syrup and reclaims anhydrous trehalose.
In such as the diverse animal and plant kind of antibacterial, yeast, fungus, insecticide and invertebrates, extensively find trehalose.In many insecticides, it is main blood sugar.For the only main source of the mankind is such as the mushroom or the food of yeast product.Madsarovova-Nohejlova(1973)Gastroenterol.65:130-133。
At United States Patent (USP) the 4th, 879, trehalose is described for PDS in No. 280, wherein puts down in writing it utilizes the prior art system of glucose as replacement several disaccharide a kind of.The record trehalose is used for dialysis system as the disaccharide that is not easy it is decomposed into glucose and therefore avoids increasing blood glucose levels.Trehalose also is described to be applicable to parenteral formulation at first, because it can be sterilized by autoclaving, and does not follow traditional parenteral formulation palm fibre to become.Japan Patent 6-70718 number.
Trehalose is the common constituent of human diet, can obtain its metabolic data.After the per os digestion, trehalose remains untouched and is not absorbed by gastrointestinal tract, because only monosaccharide can pass through enteric epithelium.Ravich and Bayless (1983) Clin.Gast.12:335-356.By trehalase is the glucose of two molecules with Trehalose Metabolism.Sacktor(1968)Proc.Natl.Acad,Sci.USA?60:1007-1041。Trehalose is the normal components of most of mammals bodies (comprising the mankind), and identifies in human serum, lymphocyte regulating liver-QI, but is located substantially on the brush border and the kidney proximal tubule of intestinal.Belfiore etc. (1973) Clin.Chem.19:447-452; Eze (1989) Biochem.Genet.27:487-495; Yoshida etc. (1993) Clin.Chim.Acta215:123-124; With Kramers and Catovsky (1978) Brit.J.Haematol.38:4453-461.Trehalose is a kind of embrane-associated protein of humans and animals intestinal.Bergoz etc. (1981) Digestion 22:108-112; Riby and Galand (1985) Comp.Biochem.Physiol.82B:821-827; With (1987) Biochem.J.247:715-724 such as Chen.
All lists of references that this paper quotes all are attached to herein by reference.
Description of the present invention
The present invention relates to the sterilizing methods that product particularly derives from the treatment product of biogenetic derivation, and the compositions that obtains by described method.Described method is included in down dry this product of existence of trehalose that is enough to this product is provided the amount of heat stability, and will this exsiccant sample the persistent period of inactivation of viruses is exposed in the heating condition with being enough to substantially with certain temperature.Preferably, this heating condition is enough to the non-lipid envelope virus of deactivation.Described drying means is any method well known in the art, comprises environmental drying condition (comprising spray drying and vacuum drying) and lyophilizing.This heating condition has covered the temperature of wide region and the combination of heat time heating time.
The present invention also comprises the compositions that obtains by described method.These compositionss comprise stable product biology, and do not comprise detectable infectious virus, particularly parvovirus.
Preferred forms of the present invention
The end last sterilizing methods that the biological product in a large amount of blood sources are arranged as described in detail herein.These methods are being known in the art, the list of references of quoting as this paper, and do not need to describe in detail.Though the purification process such as the strictness of antibody affinity chromatography may produce virus-free biological product, the method for still not finding any viable commercial can provide does not consistently have infectious virus, the product of lipid envelope virus nothing but particularly.In addition, the stringency that increases sterilising conditions for the product that does not have infectious virus is provided has that to lower product active and/or increase the immunogenic shortcoming of product.
Product except the blood source also has a large amount of biologic activity products that can benefit from sterilizing methods provided herein.These include but not limited to recombinate albumen, natural isolating albumen, antibody, enzyme, cytokine and somatomedin of producing and such as the pharmaceutically active molecule of analgesics, anesthetis, anti-emetic, antibiotic, chemotherapeutics, hormone vitamin and steroid.
Claimed method also is applicable to any aseptic individual material that imports.These include but not limited to the diagnostic reagent of medicine, antibiotic, photographic developer.Importantly, when product that will be to be given is unstable, for example cephalosporin, treatment antibody and erythropoietin, but the invention provides stable, exsiccant, the aseptic composite of heavy waterization before use.That trehalose is particularly suitable for is injectable, molten etc. medicine, because it fragments into 2 glucose molecules by the trehalase that is exposed in the blood flow.This glucose can cause that blood sugar level is little, instantaneous increase, but this does not almost have clinical impact.
The present invention includes needs the aseptic end last sterilizing methods that gives individual product.The step of this method comprises that acquisition contains this product and the drying sample that is enough to the α-D-glycopyranosyl-α-D-glycopyranoside (trehalose) to the heat-staple substantially amount of this product; And be enough to temperature and this exsiccant sample of persistent period (preferably under the heating condition of the non-lipid envelope virus of deactivation) heating of inactivation of viruses basically.This exsiccant sample also can comprise suitable buffer, adjuvant etc.Produce the amount of suitable concn during preferably with rehydration.
This product can derive from multiple material.Preferably, this product derives from any known biogenetic derivation, includes but not limited to blood, blood plasma, serum, Placenta Hominis, milk, urine, cell culture and cell culture supernatant.In addition, this product can be synthetic or utilize recombinant DNA technology synthetic deutero-by chemosynthesis or enzymatic.These preparation methods and described product separation method are well known in the art.
Usually, separate or the product that obtains includes but not limited to albumin product, immunoglobulin, coagulation prod and protease inhibitor from blood, blood plasma and serum.The albumin product includes but not limited to HSA, AHG and fibrinogen.Immunoglobulin includes but not limited to tetanus, pertussis, hepatitis B, Rho (D), varicella zoster and rabic antibody.Coagulation prod comprises that load is not limited to antihemophilic factor VIII, factor IX complex and activated factor IX complex.Protease inhibitor includes but not limited to α-1 protease inhibitor, α-1 antitrypsin and antithrombin.Also can obtain these products in other source, for example, albumin can derive from the Placenta Hominis source.
When this biogenetic derivation was cell culture or cell culture supernatant, described product included but not limited to colony stimulating factor, monoclonal antibody and its derivant and somatomedin.Typical somatomedin includes but not limited to natural acquisition and erythropoietin, cytokine and interleukin reorganization.
When this product is that described product includes but not limited to analgesics, anesthetis, chemotherapeutics, hormone and vaccine when needing aseptic administered agents.Analgesics includes but not limited to morphine, benzocaine, Pethidine and dolantin, and anesthetis includes but not limited to bupivacaine, atracurium and Vecuronium.
Chemotherapeutics includes but not limited to radiosiotope, such as vinblastine, the vinca of vincristine and vindesine sulfate (vinca alkaloids), amycin, the sulphuric acid bleomycin of fighting, carboplatin, cisplatin, cyclophosphamide, cytosine arabinoside, dacarbazine, D actinomycin D, daunomycin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, mustine hydrochlcride, chloramphenalan, purinethol, methotrexate, mitomycin, mitotane, penta system rhzomorph, pipobroman, procarbazine hydrochloride (procarbaze), streptozotocin, paclitaxel, thioguanine and uracil mustard.
The hormone that is fit to includes but not limited to estrogen, testosterone, progesterone and synthetic analog thereof.Typical vaccine comprises the former subunit vaccine of monoclonal antibody and the former subunit vaccine of multi-resistance and the antibacterial that kills and virus formulation and cancer antigen.Typical antibiotics includes but not limited to cephalosporin and aminoglycoside.
This method comprises the first step that obtains drying sample.Can use the several different methods drying sample.That these methods include but not limited to is air-dry, vacuum drying, spray drying and lyophilizing.These methods have detailed illustrating in the embodiment that this paper proposes, and are being known in the art.Referring to United States Patent (USP) the 4th, 891,319; 5,026,566 and 5,149, No. 653.
Described sample is usually with solution or suspension preparation, and comprises this product, is enough to provide trehalose and any other general additive of the amount of this product heat stability, as suitable buffer, adjuvant etc.Usually, yet the amount of trehalose is the 1-50% of solution weight, and this trehalose can be rarer or denseer.If not too rare, then may play inhibition drying time, and if denseer, this solution thickness that may become then.The accurate initial concentration of product, trehalose and any additives will need to determine by rule of thumb, but known embodiment provided herein, then this is fully in those skilled in the art's technical scope.The concentration of preferred this product and trehalose makes the active loss of the product that produces after drying less than about 30%.More preferably it is less than 15% loss of activity, and most preferably it less than 10% loss.
In case obtained exsiccant sample, then it is stood heating condition, its temperature and persistent period are enough to inactivation of viruses.Usually, this exsiccant sample had lipid envelope virus with deactivation in 72 hours in 80 heating, and in 72 hours non-lipid envelope virus of further deactivation of 90 ℃ of heating.The best of breed of the heating and the duration of heat will rule of thumb be determined.Known embodiment provided herein then thisly determines in those skilled in the art's technical scope.Usually, by or with the virus for the treatment of deactivation or add indicator with virus to specimen and determine described optimum condition with homologue Li Tezheng.Because 4log
10Titre to descend be forfeiture, be administrative organization to inactivation of virus/remove the requirement of method, so 4log
10The titre forfeiture that adds indicator virus be considered to " deactivation " usually.
This method causes the thermally-stabilised of this product substantially.Preferred this method causes this product activity loss less than about 30%.More preferably this method causes this product activity loss less than 15%.Most preferably this method causes this product activity loss less than 10%.
This method provides the drying sample that stability is high and energy is long time stored.This bin stability relates to the residual moisture of sterilising prods.Preferred this method produces residual water capacity less than 4% drying sample.More preferably this residual water capacity is less than 2%.Most preferably this residual water capacity is approximately 0.8-1%.Residual water capacity can be measured by several different methods, includes but not limited to various differential thermal analyses, thermal gravimetric analysis and Fischer coulometric titration.
The range of heating-up temperature and time is very wide, and the knowledge in this field and embodiment provided herein then can rule of thumb determine the Best Times of specific sample.The result that this paper proposes shows, about 72 hours is effectively in about 80 ℃ of heating, and is effective too 90 ℃ of heating 20 hours.Can use longer time cycle and/or higher temperature, be accompanied by the active forfeiture of product simultaneously potentially.
Preferably, this method causes 4log
10The infective reduction of Virus Pollution.To the certain methods of carrying out this detection is known.Wherein manyly quote hereinbefore, and some other is being known in the art.Owing to have lipid envelope virus that heat treated resistance is not so good as non-lipid envelope virus, show that all lipid envelope virus that has all is inactivated so determine the forfeiture of non-lipid envelope viral infection.This non-lipid envelope virus includes but not limited to hepatitis A virus (HAV) and parvovirus.Preferably, this non-lipid envelope virus is parvovirus.
The present invention also comprises the compositions that obtains by the claimed method of the present invention.Described compositions does not have detectable virus and stores highly stable.In addition, the minimizing of the degeneration of treatment product and chemical degradation causes this product receiver's immunoreactive incidence rate is descended in the course of processing.
Said composition is preferably single dose form, particularly for such as analgesics and chemotherapeutics.Single dose form can by with original solution or suspension five equilibrium in suitable containers and process this container separately and produce.Preferably, this five equilibrium and processing are automatizatioies.Perhaps, this material can be processing more than the branch of a dosage in batches, and with exsiccant production sharing single dose.Therefore the present invention comprises the single dose form of claimed compositions.
To the product such as HSA, batch mode is best.Can process in enormous quantities and use with a large amount of providers.Therefore the present invention also comprises the claimed compositions of form in enormous quantities.
It is in order to illustrate that following examples are provided, but not the present invention of requirement for restriction protection.
Embodiment 1
Different sugar is to parvovirus infections and alkaline phosphatase activities
Influence relatively
To be formulated in the 25mM HEPES buffer that contains 50mM Ammonium bicarbonate food grade and 2%HSA at the storage of the 1mg/ml alkali phosphatase in 50% aqueous trehalose liquid, this storage liquid is with 10
6.5TCID 50/ml Canine Parvovirus adds indicator.This storage liquid is with 10
6.5TCID 50/ml Canine Parvovirus adds indicator.Adopt FTS exsiccator or Labconco lyophil apparatus, this of 250 μ l equal portions added vacuum drying or the lyophilizing in the medicinal glass tube vial of 3ml Wheaton glass of indicator preparation.
The against vacuum drying temperature of shelf is arranged on 30 ℃ at first, and when shelf temperature increased to 60 ℃, vacuum was reduced to 30mTorr with segmented mode, and drying was carried out 12-16 hour again.For lyophilizing, with described sample by reducing shelf temperature to-40 ℃ of lyophilizing with 5 ℃ of per minutes, and glass tube vial was frozen 1 hour fully and with sample-40 ℃ of dryings 40 hours.Then with this shelf temperature with 0.05 ℃ of per minute be increased to+20 ℃, and with sample+20 ℃ of dryings 3 hours, at last with this shelf temperature with 0.05 ℃ of per minute be increased to 40 ℃ and with sample+40 ℃ dry 2 hours again.Glass tube vial built under vacuum and sample is remained on 4 ℃ as dry contrast.For end sterilization eventually, glass tube vial was handled 72 hours or handled 20 hours in 90 ℃ in 80 ℃ in the Heraeus drying baker.Estimate Log
10Active minimizing of parvovirus and alkaline phosphatase activities reduce %.
Analyze parvovirus by the titration cell culture, determine terminal point by the agglutination of pig erythrocyte.Speak briefly, must keep in the culture medium (EMEM) 10 times of dilution series of preparation with sterile distilled water with in the Eagles limit, with exsiccant sample with its initial volume reconstruction.Cell by trypsinization fusion bottle prepares the A72 cell suspension among the EMEM that contains 5% hyclone, and will contain 2-5 * 10
3The 1ml cell suspension of cell/ml is distributed in the hole of 24 porocyte culture plates.Each dilution glass tube vial sample of 100 μ l is inoculated in 4 parallel assay holes of Tissue Culture Plate, and with described plate at 37 ℃ of 5%CO
2Environment in cultivated 14 days.
After 14 days, test hemagglutinin activity from each hole collecting cell culture medium and with 1% pig erythrocyte.With the quantitative Karber formula of terminal point in viral infection is analyzed is calculated with log
10The virus titer that TCID 50/ml represents, promptly the Karber formula=-log dilution factor interval (dilution interval)-(positive test summation)-0.5.
With commercial reagent Sigma fast
TMAlkaline phosphatase substrate is analyzed, by the colorimetric analysis alkali phosphatase.Speak briefly, sample and standard alkaline type phosphatase formulations (1 mg/ml) are prepared double dilution series, add 100 T1 substrates in the 100 T1 samples in the EIA plate, and hatched 30 minutes at the dark place.Read colour developing with reading the titertek multi-channel scanning instrument (multiscan) that plate instrument software kit connects at 405 nm with 6 soft boards, and from calculate the activity of described sample corresponding to the absorption value of standard curve linear segment.Exsiccant contrast is defined as 100% activity, at percentage ratio from the active minimizing of these value computing samples.
For all virus analysis, by log from the drying contrast
10Deduct log in the TCID 50/ml value
10The virus that TCID 50/ml reclaims, the log of computing sample
10The minimizing of titre.The results are shown in table 1.In table 1, this virus of 〉=expression is below the detection level of this analysis, and RIT represents " titre reduction ", the HSA represent human serum albumin,
*Be illustrated in the log of the parvovirus in the dry contrast
10TCID 50/ml=6.5,
*Be illustrated in dry contrast neutral and alkali phosphatase activity=100%, GPS represents the glucopyranosyl sorbitol, and the n.d. representative is not done.All the embodiment part is all identical with the abbreviation in the table.
Table 1
| Sample | Log after 90 ℃ sterilized 20 hours in the end eventually 10Parvovirus titre reduction/alkaline phosphatase activities reduces % | |||
| Lyophilizing | Vacuum drying | |||
| Log 10The RIT parvovirus * | Alkali phosphatase reduces % ** | Log 10The RIT parvovirus * | Alkali phosphatase reduces % ** | |
| Trehalose | ????≥4.4 | ????7.0 | ???≥4.0 | ????0 |
| Trehalose+HSA | ????≥4.4 | ????9.5 | ???≥4.0 | ????0 |
| Sucrose | ????≥4.4 | ????65 | ???≥4.0 | ????48 |
| Sucrose+HSA | ????≥4.4 | ????68 | ???≥4.0 | ????56 |
| Lactose (lactitol)+HSA | ????≥5.6 | ????≥99 | ???n.d. | ????n.d. |
| Lactose+HSA | ????≥5.0 | ????44 | ???n.d. | ????n.d. |
| Sorbitol | ????≥4.0 | ????≥99 | ???n.d. | ????n.d. |
| Sorbitol+HSA | ????≥4.0 | ????≥99 | ???n.d. | ????n.d. |
| GPS | ????≥4.0 | ????≥99 | ???n.d. | ????n.d. |
| GPS+HSA | ????≥4.0 | ????≥99 | ???n.d. | ????n.d. |
Embodiment 2 does not lose the sterilization of end eventually of biologic activity to eliminate parvovirus infections by vacuum drying in trehalose; The influence of temperature and time
To be formulated in the 25mM HEPES buffer that contains 50mM Ammonium bicarbonate food grade and 2%HSA at the storage of the 1mg/ml alkali phosphatase in 50% aqueous trehalose liquid, this storage liquid is with 10
6.5TCID 50/ml Canine Parvovirus adds indicator.With the manual program of the final operating parameter that reaches 30 mTorr vacuum and 60 ℃ of shelf temperatures, in the FTS exsiccator that the preparation of 250 μ l equal portions is dry in the 30ml glass tube vial.Glass tube vial built under vacuum and sample is remained on 4 ℃ as dry contrast.
For eventually end sterilization, glass tube vial was handled 72 hours or handled as many as 144 hours in 90 ℃ in 80 ℃ in the Heraeus drying baker.In the parvovirus activity, estimate Log
10Reduce, reduce and in alkaline phosphatase activities, estimate %.Describe by the titration cell culture as embodiment 1, then carry out the haemagglutination of pig erythrocyte and analyze parvovirus.With the Karber formula of describing among the embodiment 1 with log
10TCID 50/ml calculates virus titer.Describe with commercial reagent Sigma fast as embodiment 1
TMAlkaline phosphatase substrate is analyzed, by the colorimetrically analysing alkali phosphatase.
The result's who obtains summary is shown in table 2, and table 2 is described in the log of 144 hours sterilization back, end eventually parvovirus titres of 80 ℃ of 72 hours or 90 ℃
10Reduce and alkaline phosphatase activities reduction %.In table 2,
*Representative is log in the drying contrast
10TCID50/ml parvovirus=6.5.
*Representative contrasts neutral and alkali phosphatase activity=100% in drying, and 〉=represent below the detection limit of this analysis.
Table 2
| Handle | The log of parvovirus titre 10Reduce * | Alkaline phosphatase activities reduces % ** |
| 80 ℃/72 hours | ????3.0 | ????0 |
| 90 ℃/20 hours | ????3.0 | ????0 |
| 90 ℃/24 hours | ????4.4 | ????0 |
| 90 ℃/48 hours | ??≥5.0 | ????0.8 |
| 90 ℃/72 hours | ??≥5.0 | ????0 |
| 90 ℃/96 hours | ??≥5.0 | ????3.3 |
| 90 ℃/120 hours | ??≥5.0 | ????0 |
| 90 ℃/144 hours | ??≥5.0 | ????3.4 |
Embodiment 3 eliminates the sterilization of end eventually peplos and non-infection of coated virus are arranged and do not lose biologic activity by vacuum drying in trehalose
In this experiment, the preparation identical with embodiment 1 added indicator with 3 kinds of different virus formulations: poliovirus; Parvovirus (the non-enveloped virus that contains RNA and DNA respectively); And Measles virus (peplos RNA viruses).Estimate the log of virus titer as previous or following description
10Reduce and alkaline phosphatase activities reduction %.With cytopathy assay poliovirus.Speak briefly,, exsiccant sample with its initial volume reconstruction, is inoculated into 100 μ l diluents in 5 parallel assay holes of the 96 porocyte culture plates that contain Vero cell fusion monolayer with sterile distilled water and 10 times of dilution series in EMEM, preparing.Described plate was cultivated 7 days and the cytopathy effect by writing down virus induction with the described hole of observation by light microscope.Use again then the quantitative Karber formula of terminal point in the viral infection analysis with log
10The virus that TCID 50/ml reclaims is calculated virus titer.For all virus analysis, by log from the drying contrast
10Deduct log in the TCID 50/ml value
10The virus that TCID 50/ml reclaims, the log of computing sample titre
10Reduce.
Infectivity with plaque analyzing and testing Measles virus.Speak briefly, with sterile distilled water and 10 times of dilution series in EMEM, preparing, with exsiccant sample with its initial volume reconstruction.Every kind of diluent of 200 μ l is inoculated in the double parallel assay hole of the 6 porocyte culture plates that contain Vero cell fusion monolayer.Absorb cell after 1 hour in 37 ℃ of viruses, each hole is added 2ml upper strata culture medium (EMEM that contains 1% carboxymethyl cellulose and 5% hyclone).Then with described plate at 37 ℃ of 5%CO
2Cultivated 7 days in the environment.Form inductive virus by Gentian Violet dyeing perusal plaque in cell monolayer.
Counting plaque, and by calculating viral quantitative that every ml plaque forming unit will reclaim, every ml plaque forming unit is number * coefficient of dilution * 5 of PFU/ml=plaque.For all virus analysis, by log from the drying contrast
10Deduct log in the PFU/ml value
10The virus that PFU/ml reclaims, the log of computing sample titre
10Reduce.The result who obtains is illustrated in table 3, after table 3 is presented at 80 ℃ of 72 hours or 90 ℃ of sterilizations of end eventually of 20 hours, and the log of poliovirus, parvovirus and Measles virus titre
10Reduce and reduce % at alkaline phosphatase activities.In table 3,
*Representative is log in the drying contrast
10TCID 50/ml poliovirus=4.5;
*Representative is log in the drying contrast
10PFU/ml Measles virus=5.10;
* *Representative is log in the drying contrast
10TCID 50/ml parvovirus=6.5;
* * *Representative is at drying contrast neutral and alkali phosphatase activity=100%; And 〉=represent below the detectable limit of this analysis.
Table 3
| Handle | The log of virus titer 10Reduce | Alkaline phosphatase activities reduces (%) **** | ||
| Poliovirus * | Measles virus ** | Parvovirus *** | ||
| 80 ℃/72 hours | ???≥3.0 | ???≥4.1 | ????≥4.0 | ????0-3 |
| 90 ℃/20 hours | ???≥3.0 | ???≥4.1 | ????≥4.0 | ????0-5 |
Embodiment 4 eliminates the sterilization of end eventually of not losing biologic activity from the parvovirus of blood products by vacuum drying in trehalose
With fibrinogen (component 1, the 1-S type, Sigma Chemical company) is dissolved in and contains in the solution 10% sodium citrate and 15% sodium chloride or 10% trehalose or 25% sucrose, and with described solution centrifugal to remove any insoluble material, it is 5mg/ml that protein concentration is adjusted to the fibrinogen final concentration.Fibrinogen is store liquid with 10
6.5TCID 50/ml Canine Parvovirus adds indicator, and the fibrinogen solution branch of 12ml equal portions is installed in the 5mlWheaton pharmaceutical glass glass tube vial, with described sample vacuum drying or lyophilizing in the Labconco freeze dryer in the FTS exsiccator.The against vacuum drying sample is chilled to 10 ℃ and vacuum reduced to 30,000 mTorr in advance with the exsiccator frame.Reduce to 30,000 mTorr 2 minutes, 20,000m mTorr 2 minutes and 10,000 mTorr 20 minutes with frame temperature rise to 40 ℃ and with vacuum then.Then this vacuum is risen to 30,000 mTorr 5 minutes, and then reduce to 30mTorr, and described sample is 60 ℃ of frame temperature dried overnight.For the lyophilizing sample, described sample is refrigerated to-40 ℃ with 5 ℃/min, kept 16 hours at-40 ℃, then with this temperature rise to-35 ℃ and with sample under the 10mTorr vacuum dry 80 hours.With this temperature rise to 25 ℃ and with sample under the 10mTorr vacuum dry 5 hours again.All glass tube vials are at the vacuum lower seal, by will be somebody's turn to do the sample heat sterilization of end sterilization eventually in 90 ℃ of heating 20 hours or 48 hours samples in the Heraeus stove.
Glass tube vial is rebuild, measured total soluble protein and condensable albumen.Coagulation assay to fibrinogen is the modification of state-run biologic criteria institute to the thrombin coagulation assay.Speak briefly,, fibrin raw sample and standard are condensed, and quantitatively absorb, measure proteic concentration in the grumeleuse by dissolving grumeleuse in 7M carbamide and at 280nm by adding thrombin to fibrinogen solution.
When adding 3ml water, except being exposed to fibrinogen/sucrose preparation of 90 ℃/20 hours, all preparations are all rebuild easily.Lyophilized fibrinogen as fibrinogen/the sucrose preparation that has been exposed to 90 ℃/20 hours shows tangible brown.Analysis of protein shows that most albumen dissolves (～90%) during reconstruction, and except in 90 ℃/20 hours situation of fibrinogen/sucrose, glass tube vial shows soluble protein seldom.This coagulation assay shows that for soluble albumen, the albumen that adds thrombin time～95% condenses.The result who obtains sums up and is shown in table 4, and table 4 shows the log of coagulable fibrinogen and parvovirus titre
10Reduce.Table 4
| 80 ℃/72 hours | 90 ℃/20 hours | |||
| % condenses | Log. reduce | % condenses | Log. reduce | |
| Trehalose lyophilizing vacuum drying sucrose lyophilizing vacuum drying | ????87 ????94 ????0 ????68 | ????≥4 ????≥3.75 ????≥5 ??≥4.75 | ????83 ????89 ????44 ????43 | ????4.0 ????3.75 ????≥5.0 ????≥4.75 |
Though the purpose for explanation and understanding has described aforementioned invention in detail to a certain extent by description and embodiment, those skilled in the art can obviously find out can implement some variation and modification.Therefore, not will be understood that described description and embodiment have limited scope of the present invention, scope of the present invention is described by appended claim.
Claims (58)
1. be used for the end last sterilizing methods of the aseptic biologic activity product that gives, comprise following steps:
(a) obtain to contain the drying sample of this product and a certain amount of α-D-glucopyranose-α-
The dry products of D-glycopyranoside (trehalose), the amount of described trehalose is enough to this product
Product provide elementary heat stability; And
(b) heat this drying sample, heating-up temperature and persistent period are enough to deactivation infectious virus basically.
2. according to the process of claim 1 wherein that described product gets autoblood and is selected from albumin product, immunoglobulin, coagulation prod and protease inhibitor.
3. according to the method for claim 2, wherein said albumin product is selected from HSA, cold solvable globulin and fibrinogen.
4. according to the method for claim 2, wherein said immunoglobulin is selected from tetanus, pertussis, hepatitis, herpes, varicella zoster and rabic antibody.
5. according to the method for claim 2, wherein said coagulation prod is selected from antihemophilic factor VIII, factor IX complex and activated factor IX complex.
6. according to the method for claim 2, wherein said protease inhibitor is selected from α-1 protease inhibitor and antithrombin.
7. according to the process of claim 1 wherein that described biologic activity product derives from the biogenetic derivation that is selected from blood, blood plasma, serum, Placenta Hominis, milk, urine, cell culture and cell culture supernatant.
8. according to the method for claim 7, wherein said biogenetic derivation is cell culture or cell culture supernatant, and this product is selected from colony stimulating factor, monoclonal antibody and its derivant and somatomedin.
9. according to the method for claim 7, wherein said biogenetic derivation is cell culture or cell culture supernatant, and this product is a recombinant.
10. according to the method for claim 8, wherein said somatomedin is selected from erythropoietin, cytokine and interleukin.
11. according to the process of claim 1 wherein that described biologic activity product is an analgesics.
12. according to the method for claim 11, wherein said analgesics is selected from morphine, benzocaine, Pethidine and dolantin.
13. according to the process of claim 1 wherein that described biologic activity product is an anesthetis.
14. according to the method for claim 13, wherein said anesthetis is selected from bupivacaine, atracurium and Vecuronium.
15. according to the process of claim 1 wherein that described biologic activity product is a chemotherapeutics.
16. according to the method for claim 15, wherein said chemotherapeutics is selected from radiosiotope, vinca (vinca alkaloids), amycin, the sulphuric acid bleomycin of fighting, carboplatin, cisplatin, cyclophosphamide, cytosine arabinoside, dacarbazine, actinomycin D, daunomycin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, mustine hydrochlcride, chloramphenalan, purinethol, methotrexate, mitomycin, mitotane, penta system rhzomorph, pipobroman, procarbazine hydrochloride (procarbaze), streptozotocin, paclitaxel, thioguanine and uracil mustard.
17. according to the process of claim 1 wherein that described biologic activity product is a hormone.
18. according to the method for claim 17, wherein said hormone is selected from estrogen, testosterone, progesterone and synthetic analog thereof.
19. according to the process of claim 1 wherein that described biologic activity product is a vaccine.
20. according to the method for claim 19, wherein said vaccine is selected from former subunit vaccine of monoclonal antibody and the former subunit vaccine of multi-resistance, and the antibacterial that kills and virus formulation and cancer antigen.
21. according to the process of claim 1 wherein that described drying sample is air-dry by being selected from, vacuum drying, spray drying and freeze dried method obtain.
22. according to the process of claim 1 wherein that described elementary heat stability causes this product activity forfeiture less than about 30%.
23. according to the method for claim 22, wherein said stability causes this product activity forfeiture less than about 15%.
24. according to the method for claim 23, wherein said stability causes this product activity forfeiture less than about 10%.
25. according to the process of claim 1 wherein that the residual water capacity of described drying sample is less than about 4%.
26. according to the method for claim 25, wherein said residual water capacity is less than about 2%.
27. according to the method for claim 26, wherein said residual water capacity is less than about 1%.
28. according to the process of claim 1 wherein that described heating-up temperature is about 80 ℃, and the persistent period of heating be approximately at least 72 hours.
29. according to the process of claim 1 wherein that described heating-up temperature is about 90 ℃, and the persistent period of heating be approximately at least 20 hours.
30. according to the process of claim 1 wherein that the basic deactivation of infectious virus causes this viral infection to reduce about 10
4Doubly.
31. according to the process of claim 1 wherein that described deactivation causes non-lipid envelope viral infection to reduce about 10
4Doubly.
32. according to the process of claim 1 wherein that described non-lipid envelope virus is selected from hepatitis A virus (HAV) and parvovirus.
33. according to the method for claim 32, wherein said non-lipid envelope virus is parvovirus.
34. according to the obtainable compositions of the method for claim 1.
35. according to the compositions of claim 34, wherein said product gets autoblood and is selected from and is selected from albumin product, immunoglobulin, coagulation prod and protease inhibitor.
36. according to the compositions of claim 35, wherein said albumin product is selected from HSA, cold solvable globulin and fibrinogen.
37. according to the compositions of claim 35, wherein said immunoglobulin is selected from tetanus, pertussis, hepatitis B, Rho (D), varicella zoster and rabic antibody.
38. according to the compositions of claim 35, wherein said coagulation prod is selected from antihemophilic factor VIII, factor IX complex and activated factor IX complex.
39. according to the compositions of claim 35, wherein said protease inhibitor is selected from α-1 protease inhibitor and antithrombin.
40. according to the compositions of claim 33, wherein said biologic activity product derives from the biogenetic derivation that is selected from blood, blood plasma, serum, Placenta Hominis, milk, urine, cell culture and cell culture supernatant.
41. according to the compositions of claim 40, wherein said biogenetic derivation is cell culture or cell culture supernatant, and this product is selected from colony stimulating factor, monoclonal antibody and its derivant and somatomedin.
42. according to the compositions of claim 41, wherein said biogenetic derivation is cell culture or cell culture supernatant, and this product is a recombinant.
43. according to the compositions of claim 41, wherein said somatomedin is selected from erythropoietin, cytokine and interleukin.
44. according to the compositions of claim 34, wherein said biologic activity product is an analgesics.
45. according to the compositions of claim 44, wherein said analgesics is selected from morphine, benzocaine, Pethidine and dolantin.
46. according to the compositions of claim 34, wherein said biologic activity product is an anesthetis.
47. according to the compositions of claim 46, wherein said anesthetis is selected from bupivacaine, atracurium and Vecuronium.
48. according to the compositions of claim 34, wherein said biologic activity product is a chemotherapeutics.
49. according to the compositions of claim 48, wherein said chemotherapeutics is selected from radiosiotope, vinca, amycin, sulphuric acid fight bleomycin, carboplatin, cisplatin, cyclophosphamide, cytosine arabinoside, dacarbazine, actinomycin D, daunomycin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, mustine hydrochlcride, chloramphenalan, purinethol, methotrexate, mitomycin, mitotane, penta system rhzomorph, pipobroman, procarbazine hydrochloride, streptozotocin, paclitaxel, thioguanine and uracil mustard.
50. according to the compositions of claim 34, wherein said biologic activity product is a hormone.
51. according to the compositions of claim 50, wherein said hormone is selected from estrogen, testosterone, progesterone and synthetic analog thereof.
52. according to the compositions of claim 34, wherein said biologic activity product is a vaccine.
53. according to the compositions of claim 52, wherein said vaccine is selected from the former subunit vaccine of monoclonal antibody and the former subunit vaccine of multi-resistance, the antibacterial that kills and virus formulation and cancer antigen.
54. according to the compositions of claim 34, the residual water capacity of wherein said drying sample is less than about 4%.
55. according to the compositions of claim 54, wherein said residual water capacity is less than about 2%.
56. according to the compositions of claim 55, wherein said residual water capacity is less than about 1%.
57. according to the compositions of claim 34, wherein said non-lipid envelope virus is selected from hepatitis A virus (HAV) and parvovirus.
58. according to the compositions of claim 57, wherein said non-lipid envelope virus is parvovirus.
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|---|---|---|---|
| CN 97196248 CN1225020A (en) | 1996-05-14 | 1997-05-14 | Methods of terminal sterilization of biological products |
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| Application Number | Priority Date | Filing Date | Title |
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| US08/647,515 | 1996-05-14 | ||
| CN 97196248 CN1225020A (en) | 1996-05-14 | 1997-05-14 | Methods of terminal sterilization of biological products |
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| CN110038149A (en) * | 2019-04-22 | 2019-07-23 | 石河子大学 | A kind of inactivation of biology laboratory waste cell and Cytology Lab improve sterilization method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110038149A (en) * | 2019-04-22 | 2019-07-23 | 石河子大学 | A kind of inactivation of biology laboratory waste cell and Cytology Lab improve sterilization method |
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