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CN101972472A - Method for purifying and preparing allergen vaccine - Google Patents

Method for purifying and preparing allergen vaccine
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Publication number
CN101972472A
CN101972472ACN 201010527006CN201010527006ACN101972472ACN 101972472 ACN101972472 ACN 101972472ACN 201010527006CN201010527006CN 201010527006CN 201010527006 ACN201010527006 ACN 201010527006ACN 101972472 ACN101972472 ACN 101972472A
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allergen
ultrafiltration
extracting solution
preparation
buffer
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白彩明
裴潇竹
姜敏
任雅丽
汤承祁
何立峰
牛占坡
乔秉善
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Beijing Xinhualian Xiehe Pharmaceutical Co Ltd
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Beijing Xinhualian Xiehe Pharmaceutical Co Ltd
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Abstract

The invention discloses a method for removing impurity macromolecular and micromolecular substances irrelevant to allergen from activated protein extracting solution containing allergen. The method removes free micromolecular pigments and pigment molecules combined with protein through static electricity and hydrophobicity from the protein extracting solution which is prepared by the traditional technique. The discoloring extracting solution preparation method does not damage the activity and immunologic specificity of allergen. The invention can be used for preparing a high-activity protein extracting solution without macromolecular and micromolecular substances, and the preparation made from the extracting solution has the advantages of high safety, high diagnosis accuracy and high effectiveness for immunotherapy. From another point of view, the protein purification method improves the quality of the raw materials in a chemical essential way, widens the application range of the raw materials, and prepares the attenuated vaccine for immunotherapy.

Description

The method of a kind of allergen vaccine purification and preparation thereof
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method of purification allergen vaccine and prepare the method for allergen vaccine.
Background technology
The allergen vaccine of the purifying process preparation of existing allergen vaccine, contain allergenic activity composition and no allergenic activity and with the incoherent composition of allergen.
" allergen vaccine " is meant the allergen preparation by appropriate solvent is extracted, purification obtains from the active component of animal or plant." allergenic prod " comprises the biological product of the various allergenic extracts that are used for allergic disease diagnosis, treatment, prevention.In European Pharmacopoeia, allergenic prod belongs to pharmaceutical preparation, refers to contain the particular matter that nature can cause and/or excite anaphylactic disease.
At the beginning of last century, the water formulation of grass, weeds, tree and other plant pollen is widely used in the hay hot body and external diagnosis, and this Susceptible population also is called hereditary anaphylaxis patient.1911, Noon and Freman proposed this notion of pollinosis first, and utilized pollen allergen to treat the success that this pollenogenic allergic rhinitis is got first.Since then, people utilize and to cause that material extracting solution hypersensitive treats this disease, by desensitize, subtract quick, immunization therapy to patient injection.
Allergen vaccine/extracting solution is a complex, and its composition is not single, comprise protein, carbohydrate, enzyme, pigment in the composition, and the allergen composition is a small part in constituent.Studies show that, cause allergic main allergic protein molecular weight at 20~70KD, and molecular weight be sure of there is not allergenicity less than the molecule of 10KD.The document record, the micromolecule pigment in the allergen vaccine not only can bring the risk of local and systemic side effects in immunization therapy, and the release of pigment micromolecule inhibition histamine, has a strong impact on the result of allergen medical diagnosis on disease.
Along with the change in development of human society and bad border, anaphylactic disease patient's number is also increasing year by year, and is especially obvious with Europe.Anaphylactic disease the patient increase, and development characteristic is with multiple class, and the characteristics of multizone are spreading, and some rare allergens become conventional allergen, and some allergens that are confined to certain areas can spread to other area.20 years in the past, short artemisiifolia allergy was confined to north America region, present Continental Europe, and short artemisiifolia autopath rises year by year.So, improve the accuracy of allergen medical diagnosis on disease and the safety and the effectiveness of treatment is necessary with the quality that improves the allergen vaccine product.
Now, in allergen is used for diagnosing production technology with the immunization therapy vaccine, with molecular cut off is that the film of 5~10KD is dialysed or the ultrafiltration purpose is in order to remove molecular weight at 5~10KD and the incoherent molecule of allergen, the molecule that has kept 10~100KD has like this improved the quality that is used for clinical allergen extracting solution.But also have considerable part impurity to exist in the allergen vaccine, so the purification process of allergen vaccine is most important for the diagnosis and the immunization therapy of allergen disease.Spectrum shows, is used to diagnose and treats product after the traditional handicraft dialysis, firm being combined on the protein molecular of a large amount of pigment (flavonoid).In the allergen extracting solution, the micromolecule pigment not only exists with free state, and to exist with protein bound mode.So in traditional handicraft, dialysis can only be removed a part of pigment impurity, also have quite a few pigment impurity to exist.
The object of the invention is to overcome the many defectives of the impure composition of traditional allergen vaccine technology, thereby obtains a kind of safer, effective standardization allergen.
Summary of the invention
One of purpose of the present invention provides a kind of purification process of allergen vaccine.
Another object of the present invention provides a kind of preparation method of allergen vaccine.
The invention provides a kind of purification process of allergen vaccine, the allergen extracting solution is prepared into allergen vaccine then through ultrafiltration purification, ultrafiltration decolouring, acid treatment, dialysis treatment.
Ultrafiltration purification can adopt 100KD~300KD membrane ultrafiltration, preferred 100KD film.
The ultrafiltration decolouring can be adopted 1KD~5KD membrane ultrafiltration decolouring, preferred 3KD film.In the process of ultrafiltration, constantly add buffer,, with conductivity meter test filter liquor electrical conductivity and buffering liquid phase simultaneously, be the ultrafiltration terminal point when filter liquor when being colourless.The pigment molecular that exists in free mode in the allergen extracting solution is constantly being removed in the ultra-filtration process, when filter liquor is colourless when consistent with buffer with electrical conductivity, illustrates that the pigment that dissociates in the allergen extracting solution removes substantially.
Acid treatment is a ultrafiltration decolouring back extracting solution, dropwise adds strong acid, and the pH value of regulating stock solution is 1.5~2.5, and the pigment that is attached on the protein is dissociated.Preferred 6~8M the HCl of strong acid, the pH value of preferred stock solution is adjusted to 2.
Dialysis is that the extracting solution after the acid treatment is dialysed with 1KD~5KD bag filter, preferred 3KD bag filter.On magnetic stirring apparatus, stir 10-30min at ambient temperature, preferred 30min; 12~the 36h that in the water for injection that 50-100 times of volume PH is 6-8.5, dialyses under 4 ℃ of conditions, preferred 24h.In dialysis procedure, container is put on the magnetic stirring apparatus, allow the bag filter freedom slowly rotate.
Allergen extracting solution after purified can adopt the common method of this area to be prepared into allergen vaccine.
Allergen extracting solution before the purification, allergen that can be by being selected from tree pollen, showy flowers of herbaceous plants powder, fungus, animal cuticle and hair, insecticide makes through defat, extraction, centrifugal and clarification filtration.
Defat: in the allergen raw material, added acetone or ether in 1: 2~1: 10 with volume ratio, room temperature defat under magnetic agitation, repeatable operation is colourless up to supernatant, in the ventilation drying.
Extract: the raw material after the defat with 1: 10~1: 50 (w/v) ratio with buffer, buffer can be phosphate buffer, sodium bicarbonate buffer liquid and ammonium bicarbonate buffers, concentration is 50~200mM, PH is 6~8.5, stir ormechanical agitation extraction 2~24h preferred 24h at 2~8 ℃ of condition lower magnetic forces.Preferred 1: 25 of ingredient proportion.
Centrifugal: extract mixture at 4000~6000rpm, centrifugal 15min under 4 ℃ of conditions gets supernatant then at 6000~8000rpm, centrifugal 30min under 4 ℃ of conditions.Collect supernatant, discard precipitation or precipitate and extract again with same condition.
Clarification filtration: supernatant adopts a step or a plurality of step to filter clarification by filter paper, cardboard or other aperture 10-0.22um film.Preferred cardboard filter.
The purification process of allergen vaccine of the present invention has overcome the deficiency of traditional handicraft, the purification process of this allergen vaccine not only can be removed impurity macromole and free pigment micromolecule, and can remove the pigment molecular that is attached on the albumen by destroying static and hydrophobic interaction.Pigment molecular in the allergen extracting solution so will remove the pigment molecular that is attached on the albumen, at first will destroy pigment molecular and protein-bonded hydrophobic and electrostatic interaction with hydrophobic and electrostatic interaction and protein binding.Destroying pigment and protein-bonded hydrophobic and mode electrostatic interaction has add strong acid in extracting solution, highly basic, neutral salt and in electrolytic mode.The present invention makes pigment and conjugated protein dissociating by add strong acid in the allergen extracting solution, and dissociated pigment molecular is removed in dialysis or ultrafiltration again, can remove the pigment micromolecule more completely.Purifying process provided by the invention can remove the unnecessary material that has nothing to do with allergen in the allergen extracting solution, prepares a kind of allergenic activity composition and low allergen vaccine of impurity content of containing.This purifying process can prepare a kind of allergen vaccine of high-purity high-activity, and reduce traditional handicraft and prepare the side effect that allergen vaccine brings, be a kind of attenuated vaccine of allergen immunotherapy.
Allergen can be tree pollen, showy flowers of herbaceous plants powder, fungus, animal cuticle and allergens such as hair, insecticide among the present invention.
Tree pollen comprises: Hainan sago cycas (Cycas hainanensis); Semen Ginkgo (Ginkgo biloba); PiceameyeriRehd. Et Wils. (Picea meyeri); Larix principis-rupprechtii (Larix principis-ruppredhtii); Cedrus deoclar (Roxb.) G. Don (Cedrus deodara); Pinus tabuliformis (Pinus tabulaeformis); Lacebark pine (Pinus bungeana); Pinus armandi Franch-P. Komavovii Lavl. (Pinus armandii); Lignum seu Ramulus Cunninghamiae Lanceolatae (Cunninghamia lanceolata); Cortex Cryptomeriae Fortunei Radicis (Cryptomeria foutunei); Japanese cedar (Crytomeria japonica); Pond shirt (Taxodium ascendens); Cacumen Platycladi (Platycladus orientalis); Chinese juniper (Sabina chinensis); Fokienia hodginsii (Dunn) Henry et Thomas. (Fokienia hodginsis); Cupressus funebris Endl. (Cupressus funebriis); Podocarpus nagi (Podocarpus nagi); Short Ramulus et folium taxi cuspidatae (Taxus cuspidata); Caephalotaxus sinensis (Cephalotaxus sinensis); Casuarina equisetifolia L. (Casuarina equisetifolia); Cathay poplar (Populus cathayana); Add poplar (Populus Canadensis); Dry land willow (Salix matsudana); Salix babylonica L. (Salix babylonica); Purple osier (Salix purpurea); Cotton Gossypii willow (Salix linearistipularis); Salix wallichiana (Salix wallichinan); Pterocarya Stenoptera (Pterocarya stenoptera); Semen Juglandis (Juglans regia); Juglans mandshurica (Juglans mandshurica); Betula platyphylla Suk. (Betula platyphylla); Black birch (Betula dahurica); Red China (Betula albo-sinensis); Hard birch (Betula chinensis); Indian birch (Betula utilis); Alnus nepalensis (Alnus nepalensis); Hazel (Corylus heterophylla); Corylus mandshurica (Coypus mandshurica); Birch hazel (Corylus chinensis); Ostryopsis davidiana (Ostryopsis davidiana); Semen Castaneae (Castanea mollissima); Cork oak (Quercus variabilis); Quercus liaotungensis (Qaercus liaotungensis); Quercus mongolica (Quercus mongolica); Oriental white oak (Quercus aliena); Toothed oak (Quercus aliena); Hainan evergreen chinquapin (Castanopsis hainanensis); Family elm (Ulmus pumila); Wingceltis (Pteroceltis tatarinowii); Family Mulberry (Morus alba); Chicken Mulberry (Morus australis); Broussonetia papyrifera (Broussonetia papyrifera); Buxus sinica (Rehd.et Wils.) (Buxus sinica); The Cortex Eucommiae (Eucommia uimoides); Oriental plane tree (Platanus acerifolia); Button ball (Platanus occidentalis); Fraxinus Pennsylvanica Marsh var. lanceolata (Borkh.) Sarg (Fraxinus pennsylvanica); Chinese ash (Fraxinus chinensis); Fraxinus rhynchophylla (Fraxinus rhyohophylla); Syringa reticulata var mandshurica (Syringa reticulate); Peking lilac (Syringa pekinensis); Ligustrum quihoui (Ligustrum quihoui); Ligustrum X vicaryi (Ligustrum ovalifolium Var.aureo-marginatum); Ailanthus altissima (mill.) swingle (Ailanthus altissima); Tonnae Sinensis (Toona sinensis); Chinaberry (Melia azedarach); Torch tree (Rhus typhina); Acer truncatum (Acer truncatum); Cacumen Tamaricis (Tamarix chinensis); Beijing mockorange (Philadelphus pekinensis); Uanchurian currant Fischer (Ribes mandshuricum); Yin Chai (Aporosa chinensis); White Chinese catalpa (Mallotus paniculatus); False indigo (Amorpha fruticosa); Herba Albiziae (Albizia julibrissin); Acacia farnesiana Willd. (Acacia farnesiana); Acacia confusa (Acacia confusa); America Herba Albiziae (Calliandra haematocephala); Cassia surattensis (Cassia surattensis); Bauhinia purpurea (Bauhinia variegata); Winged euonymus (Euonymus alatus); Euonymus japonicus (Euonymus japonicus); Folium Eucalypti Robustae (Eucalyptus robusta); Gray gum (Eucalyptus tereticornis); Callistemon rigidus (Callistemon rigidus); Cortex Melaleucae leucadendrae (Melaleuca leucadendra); Jambul (Syzygium jambos); Lobule Hibiscus syriacus (Tilia mongolica); Shoulder pole wood (Grewia biloba); Hibiscus rosa-sinensis (Hibiscus rosea-sinensis); Cortex Sorbariae Arboreae (Sorbaria Ririlowii); Cotoneaster multiflorus Bge (Cotoneaster multiflorus); Fructus Crataegi (Crataegus pinnatifida); White pear (Pyrus bretschneideri); Fructus Mali pumilae (Malus pumila); Mountain peach (Prunus davidiana); Flowering peach (Prunus duplex); Prunus mume (sieb.) sieb.et zucc. (Prunus mume); Luan Shu (Koelreuteria paniculata); Wood brocade (Gossampinus malabarica); Punica granatum L. (Punica granatum); Fructus Caricae (Carica papaya); Vitex chinensis Mill. (Vitex negundo); Paulownia (Paulownia tomentosa); Ramulus Sambuci Williamsii (Sambucus williamsii); Fructus Corni (Macrocarpium officinalis) etc.
The showy flowers of herbaceous plants powder comprises: Fructus Cannabis (Cannabis sativa); Herba humuli scandentis (Humulus scandens); Semen Ricini (Ricinus communis); Brassica campestris L (Brassica campestris); Herba Rorippae Islandicae (Rorippa globosa); Descurainia sophia (l.) webb ex prantl (Descurainia Sophia); Herba Spinaciae (Spinacia oleracea); Fructus Kochiae (KochiaScoparia); Chenopodium album (Chenopodium album); Chenopodium glaucum linn (Chenopodium glaucum); Tip leaf Herba chenopodii (Chenopodium acuminatum); Little Herba chenopodii (Chenopodium sertinum); Amaranthusspinosus L. (Amaranthus spinosus); Amaranthus caudatus (Amaranthus caudatus); Amaranthus retroflexus (Amaranthus retroflexus); Recessed Herba Amaranthi tricoloris (Amaranthus lividus); Wild Herba Amaranthi tricoloris (Amaranthus viridis); Tampala (Amaranthus tricolor); Fructus Gleditsia (GleditsiaSinensis); Flos Robiniae Pseudoacaciae (Robinia pseudoacia); Herba Mimosae Pudicae (Mimosa pudica); T.repens (Trifolium repens); Folium Cannabis Herba Urticae Cannabinae (Urtica cannabina); Semen Fagopyri Esculenti (Fagopyrum esculentum); Smartweed (Polygonum orientale); Pale persicaria (Polygonum lapathifolium); Rumex patientia Linn. (Rumex patientia); Curled dock (Rumex crispus); Mount Lushan Radix Berberidis Amurensis (Berberis virgetorum); Althaea rosea (L.) Cavan. (Althaea rosea); Herba Lythri Salicariae (Lythrum salicaria); Radix seu Herba Tetrastigmatis Hypoglauci (Ipomoea cairica); The wolf decorative pattern at the end of a poem, article, etc. where there is a small blank space (Lysimachia barystachys); American lotus (Lysimachia davurica); Ramulus Sambuci Williamsii (Sambucus williamsii); Herba Leonuri (Leonurus japonicus); Arbitrarily careless (Physostegia virginisna); Herba Apocyni veneti (Apocynum venetum); Before the dolly (Plantago depressa); Big Semen Plantaginis (Plantago major); Fructus Cucurbitae moschatae (Cucurbita moscbata); Fructus Luffae (Luffa cylindrica); Fructus Benincasae (Benincasa hispida); Parthenocissus quinquefolia (Parthenocissus quinquefolia); (Campsis grandiflora) reaches the clouds; Firecracker flower (Pyrostegia venusta); Celery (Apium graveolens); Hemlock (Cicuto virosa); Fructus Foeniculi (Foneiculum vulgare); Artemisia sieversiana Willd. (Artemisia sieversiana); Herba Artemisiae annuae (Artemisia annua); Tarragon (Artemisia capillaries); Radix Artemisia ordosicae (Artemisia argyi); Wild Radix Artemisia ordosicae (Artemisia lavandulaefolia); Artemisiifolia (Ambrosia artemisiifolia); Ambrosia trifida (Ambrosia trifida); Helianthi (Helianthus annuus); Jerusalem artichoke (Helianthus thberosus); Herba Xanthii (Xanthium sibiricum); Little fluffy grass (Conyza canadensis); Herba Agerati Conyzoidis (Ageratum conyzoides); Flos Inulae (Inula japonica); Herba Cirsii (Cirsium segetum); Herba Hemisteptae Lyratae (Hemistepta lyrata); Chrysanthemum carinatum Schousb. (Chrysanthemum carinatum); Herba Typhae (Typha angustifolia); Typha minima (Typha minima); Flat bar Herba Scirpi triqueteris (Scirpus planiculmis); Scirpus scirpus (Scirpus tabernaemontani); Taihang Mountain Herba Scirpi triqueteris (Scirpus schansiensis); Pin Lin (Eleocharis valleculosa); Middle type pin Lin (Eleocharis intersita); Different fringe sedge (Carex heterostachya); Hemerocallis fulva L. (Hemerocallis fulva); Herba Alii fistulosi (Allium fistulosum); Folium Allii tuberosi (Allium tuberosum); Chinese small iris (Iris Lactea): annual bluegrass (Poa annue); Herba rutae (Melica scabrosa); Cilium goose hat grass (Roegneria ciliaris); Semen Tritici aestivi (Triticum aestivum); Rye grass (Lolium perenne); Itanlian rye (Lolium multiflorum); Buffalograss (Buchloe dactyloides); Beckmannia syzigachne (Steud.) Fernald [B.erucaeformis (L.)Host var. uniflora Sckibn. Ex A.Grag (Bechmannia syzigachne); Amur foxtail (Alopecurus aequalis); Herba Setariae Viridis (Setaria viridis); Herba penniseti (Pennisetum alopecuroides); Sorghum vulgare Pers. (Sorghum vulgare); Corn (Zea mays); Semen livistonae chinensis (Livistona chinensis); Petiolus Trachycarpi (Trachycarpus fortunei); Short fringe fishtail palm (Caryota mitis); Phoenix rebelenii (Phoenix roebelenii); Elaeis guineensis Jacq. (Elaeis guineensis); Cortex cocois radicis (Cocos mucifera); Yagua (Roystonea regia); Archontophoenix alexandrae Wendl et Orude (Archontophoenix dlexandrae); Chrysaliclocarpus lutescens H. Wendl. (Chrysalidocarpus lutescens) etc.
Fungus comprises: alternaric bacteria (Alternaria alternata), De Shi mould (Helminthosporium sorokinanum), branch spore bud branch bacterium (Cladosporium cladosporioides), big spore cladosporium (Cladosporium macrocarpum), bunch spore handle mould (Stemphylium botryosum) of crawling, Curvularia lunata (Curvularia lunata), rhizopus stolonifer (Rhizopus stolonifer), Syncephalastrum racemosum (Syncephalastrum racemosum), Mucor racemosus Fres (Mucor racemosum), Penicllium chrysogenum (Penicillium chrysogenum), aspergillus niger (Aspregillus niger), absidia rasmosa (Absidia ramosa), paecilomyces varioti (Paecilomyces varioti), clump stalk spore mould (Monilia sitophlia), scopulariopsis brevicaulis (Scopulariopsis brevicaulis), Fusarium graminearum (Fusarium graminearum), sturdy top cover mould (Acrothecium robustum), ball Tuber Melanosporum (Nigrospora sphaerica), special red verticillium sp (Verticillium lateritium), cephalosporium acremonium (Cephalosporium chrysogenum), spherical abundant spore bacterium (Papularia sphaerospermanum), the wart spore bacterium (Heterosporium allii) that wriggles, spherical single head spore mould (Monotospora sphaerocephala), Ustilago maydis (D C.) Corola. (Ustilago maydis), wheat loose smut (Ustilago nuda), bakery yeast (Saccharomyces cerevisiae), Botrytis cinerea bacterium (Botrytis einerea), yellow ball tumor spore bacterium (Sepedonium chrysospermum), black attached ball mould (Epicoccum nigrum) etc.
Animal cuticle and hair comprise: the camel hair; Scurf (Camel Hair ﹠amp; Dander); Ox hair Fa ﹠amp; Scurf (Cattle Hair ﹠amp; Dander); Deer Mao Fa ﹠amp; Scurf (Deer Hair ﹠amp; Dander); The goat hair; Scurf (Goat Hair ﹠amp; Dander); Pilus Sus domestica Fa ﹠amp; Scurf (Dog Hair ﹠amp; Dander); Horsehair Fa ﹠amp; Scurf (House Hair ﹠amp; Dander); Pilus Canitis Fa ﹠amp; Scurf (Dog Hair ﹠amp; Dander); Cat hair Fa ﹠amp; Scurf (Cat Hair ﹠amp; Dander); Chicken feather (Chicken Feathers); Drake feather (Duck Feathers); Pluma Anseris domestica (Goose Feathers); Pluma Columba livia (Pigeon Feathers); People's dandruff (Human Dander); Monkey Mao Fa ﹠amp; Epithelium (Monkey Hair ﹠amp; Epithelium); Mus Mao Fa ﹠amp; Epithelium (Mouse Hair ﹠amp; Epithelium); Rabbit hair Fa ﹠amp; Epithelium (Monkey Hair ﹠amp; Epithelium) etc.
The insecticide allergen comprises: dust demodicid mite (Dermatophagoides farina); Dermatophagoides pteronyssinus (Dermatophagoides pteronyssinus); Tyrophagus putrescentiae (Tyrophagus putrescentiae); Groton bug (Blatella germanica); Periplaneta americana (Peripalanete Americana); Peroplaneta fluligginosa (Periplaneta fuliginosa); Housefly (Musca domestica); Mayfly tadpole (Ephemeridae); Four joint Fu sections (Baetidae); Anopheles minius (Anophelesnminimus); Culex pipiens pallens (Culex pipiens pappens); Aedes albopictus (Aedes albopictus); Monomorium pharaonis (Monomorium pharaonis); Major part ant (Pheidole spp); Rice weevil (SitophilusOryzae); Callosobruchus chinensis (Callosobruchuschinensis); Broad bean weevil (Laria rufimanus); Mythimna separata (Leucania separate); Pyrausta nubilalis (Hubern). (Pyrausta nubilalis) etc.
The present invention also provides a kind of preparation method of allergen vaccine, may further comprise the steps:
(1) defat: in the allergen raw material, added acetone or ether in 1: 2~1: 10 with volume ratio, room temperature defat under magnetic agitation, repeatable operation is colourless up to supernatant, in the ventilation drying.The allergen raw material is the allergen that is selected from tree pollen, showy flowers of herbaceous plants powder, fungus, animal cuticle and hair, insecticide.
(2) extract: the raw material after the defat with 1: 10~1: 50 (w/v) ratio with buffer, buffer can be phosphate buffer, sodium bicarbonate buffer liquid and ammonium bicarbonate buffers, concentration is 50~200mM, PH is 6~8.5, stir ormechanical agitation extraction 2~24h preferred 24h at 2~8 ℃ of condition lower magnetic forces.Preferred 1: 25 of ingredient proportion.
(3) centrifugal: extract mixture at 4000~6000rpm, centrifugal 15min under 4 ℃ of conditions gets supernatant then at 6000~8000rpm, centrifugal 30min under 4 ℃ of conditions.Collect supernatant, discard precipitation or precipitate and extract again with same condition.
(4) clarification filtration: supernatant is by filter paper, and cardboard or other aperture 10-0.22um film adopt a step or a plurality of step to filter clarification.Preferred cardboard filter.
(5) ultrafiltration purification: the filtrate after the clarification is again through 100KD~300KD membrane ultrafiltration, and preferred 100KD film is collected filter liquor, removes macromolecule impurity.
(6) ultrafiltration decolouring: the extracting solution after macromole impurity is removed in ultrafiltration is through 1KD~5KD membrane ultrafiltration decolouring, preferred 3KD film.In the process of ultrafiltration, constantly add buffer,, with conductivity meter test filter liquor electrical conductivity and buffering liquid phase simultaneously, be the ultrafiltration terminal point when filter liquor when being colourless.The pigment molecular that exists in free mode in the extracting solution in allergen is constantly being removed in the ultra-filtration process, when filter liquor is colourless when consistent with buffer with electrical conductivity, illustrates that the pigment that dissociates in the allergen extracting solution removes substantially.
(7) acid treatment: extracting solution behind 1KD~5KD membrane ultrafiltration, dropwise add 6~8M HCl, the pH value of regulating stock solution is 1.5~2.5, preferred pH value is 2, and the pigment that is attached on the protein is dissociated.
(8) dialysis: dialysis is that the extracting solution after the acid treatment is dialysed with 1KD~5KD bag filter, preferred 3KD bag filter.Acid treatment extracting solution is later being stirred 10-30min at ambient temperature on magnetic stirring apparatus, preferred 30min.The 24-36h that in the water for injection that 50-100 times of volume PH is 6-8.5, dialyses under 4 ℃ of conditions, preferred 24h.In dialysis procedure, container is put on the magnetic stirring apparatus, allow the bag filter freedom slowly rotate.
(9) pH value is regulated: dropwise drip 1M NaOH in the extracting solution after the purification dialysis and regulate PH to 6.5~7.5.
(10) add protective agent: add 1%~3% mannitol and 0.1%~1% human albumin in the extracting solution after regulating pH value, preferred 2% mannitol and 0.1% human albumin.
(11) aseptic filtration: under hundred grades of conditions with 0.22 μ m poly (ether sulfone) film aseptic filtration.
(12) lyophilizing: lyophilization obtains the allergen vaccine lyophilized powder.
A further object of the present invention has provided new allergen preparation.New allergen preparation of the present invention is the allergen preparation according to allergen vaccine preparation method preparation of the present invention.
Allergen preparation of the present invention can be any dosage form that gives the patient treatment anaphylactic disease, comprises injection, pricking method liquid, tablet, capsule, sublingual administration agent, oral liquid, aerosol, nasal cavity agent, patch, drop pill or spraying etc.
The preparation of allergen injection: with above-mentioned lyophilized injectable powder is diluted with the isopyknic normal saline of lixiviating solution,, be diluted to 1/100 to 1/1,000 according to desired concn, the preparation of a plurality of concentration in 000 scope, injection must desensitize.
Dissolve lyophilizing dry powder in proportion with buffer, add 0.4% phenol and 0.5%NaCl and be mixed with desensitization stock solution or add 50% glycerol again and be mixed with pricking method liquid.Allergen vaccine stock solution according to method for preparing can be as subcutaneous injection desensitization, Sublingual immunity desensitization and subcutaneous injection pricking method diagnosis.
The allergen vaccine stock solution of preparation can be added Al (OH)3, tyrosine, MPL adjuvant is mixed with and contains adjuvant allergen vaccinate.
The present invention compares with traditional allergen vaccine technology, macromole, micromolecule and the pigment molecular of free mode in the allergen extracting solution have not only been got rid of, and removed with the allergen extracting solution in the pigment molecular that combines of albumen, improve the effectiveness and the safety of allergenic prod, really accomplished the attenuated vaccine of allergen vaccine, the allergen vaccine behind the purification can be realized standardized production simultaneously.
Description of drawings
Fig. 1 is the process chart of a kind of purification and preparation allergen vaccine
Fig. 2 is that Herba Artemisiae annuae pollen allergen extracting solution is at purification different phase protein component sds polyacrylamide gel electrophoresis figure.Wherein M is a reference substance.
Fig. 3 is that dermatophagoides pteronyssinus allergen extracting solution is at purification different phase protein component sds polyacrylamide gel electrophoresis figure.Wherein M is a reference substance.
The specific embodiment
The preparation ofembodiment 1 Herba Artemisiae annuae pollen allergen vaccine
The Herba Artemisiae annuae pollen of learning from else's experience and being up to the standards adds acetone with 1: 2 (volume ratio) and is interrupted defat repeatedly, is colourless up to the supernatant solvent that leaves standstill, and outwells solvent, and residue is in the fume hood drying, and the pollen after the defat can not residual degreasing solvent.Take by weighing defat pollen, with 1: 25 with buffer (PH:8.0200mmolNa2HPO4NaH2PO450mmol NaCl 0.4% phenol), 4 ℃ are stirred 24h on magnetic stirring apparatus, mixture is forwarded in the centrifuge bottle of 500ml.4 ℃ of 6000rpm, centrifugal 15min gets supernatant and abandons residue, and 4 ℃ of centrifugal 30min of 8000rpm of supernatant get supernatant and abandon residue, and supernatant is clarified with common filter paper filtering.Extracting solution after the clarification is through the 100KD membrane ultrafiltration, collects filter liquor, and filter liquor is constantly added buffer again through the 5KD membrane ultrafiltration in ultra-filtration process, is colourless and electrical conductivity and buffering liquid phase finish ultrafiltration simultaneously up to filter liquor, probably needs 4~5 volume buffer.Collect backflow, backflow is the extracting solution that has concentrated, and dropwise drips 6M HCl in said extracted liquid, and adjusting stock solution PH is 2, stir 30min in room temperature on magnetic stirring apparatus.Then stock solution is transferred in the bag filter, added buffer (the PH7.550mmol NH of 50~100 times of volumes4HCO3) 4 ℃ of dialysis 24h, in the dialysis procedure, on magnetic stirring apparatus, keep bag filter to rotate slowly.Collect the extracting solution in the bag filter, dropwise add 1M NaOH, under magnetic stir bar stirs, regulate pH value to 7, be the allergen stock solution behind the purification, add 2% mannitol and 0.1% human albumin in stock solution, with the aseptic filtration of 0.22um film, lyophilizing then.
The preparation ofembodiment 2 dermatophagoides pteronyssinus allergen vaccines
The pure demodicid mite body of dermatophagoides pteronyssinus through being up to the standards adds acetone with 1: 2 ratio and is interrupted and stirs defat, till the supernatant solvent is colourless, outwells degreasing solvent, and residue is dry in ventilating kitchen, keeps in the defat raw material not residual acetone.Take by weighing the raw material after the defat, add 50mmol NH with 1: 25 ratio4HCO3, 4 ℃ are stirred 24h on magnetic stirring apparatus, mixture is forwarded in the centrifuge bottle of 500ml.4 ℃ of 6000rpm, centrifugal 15min gets supernatant and abandons residue, and supernatant is clarified with common filter paper filtering.Extracting solution 100KD membrane ultrafiltration after the clarification is collected filter liquor, and filter liquor is constantly added buffer again through the 5KD membrane ultrafiltration in ultra-filtration process, be colourless up to filter liquor, probably needs 4~5 volume buffer.Collect backflow, backflow is the extracting solution that has concentrated, and dropwise drips 6M HCl, and adjusting stock solution PH is 2, stir 30min in room temperature on magnetic stirring apparatus.Then it is transferred in the bag filter, to buffer (the PH7.550mmol NH of 50~100 times of volumes4HCO3) 4 ℃ of dialysis 24h.In the dialysis procedure, on magnetic stirring apparatus, keep bag filter to rotate slowly.Collect the extracting solution in the bag filter, dropwise add 1M NaOH, under magnetic stir bar stirs, regulate pH value to 7.5, be the allergen stock solution behind the purification, add 2% mannitol and 0.1% human albumin in stock solution, with the aseptic filtration of 0.22um film, lyophilizing then.
The preparation ofembodiment 3 PiceameyeriRehd. Et Wils. pollen allergen vaccines
PiceameyeriRehd. Et Wils. pollen through being up to the standards with the defat repeatedly that adds diethyl ether of 1: 2 ratio, is colourless up to the supernatant solvent that leaves standstill, and outwells solvent, and residue is in the fume hood drying, the not residual degreasing solvent of the pollen after the defat.Take by weighing defat pollen, with 1: 25 with buffer (PH:8.0200mmolNa2HPO4NaH2PO450mmol NaCl 0.4% phenol), 4 ℃ are stirred 24h on magnetic stirring apparatus, mixture is forwarded in the centrifuge bottle of 500ml.4 ℃ of 6000rpm, centrifugal 15min gets supernatant and abandons residue, 4 ℃ of centrifugal 30min of 8000rpm of supernatant, get supernatant and abandon residue, supernatant is clarified through common filter paper filtering, and then filters through the filter of 0.45um film, stock solution 100kKD membrane ultrafiltration after the clarification, collect filter liquor, filter liquor is constantly added buffer again through the 5KD membrane ultrafiltration in ultra-filtration process, up to filter liquor is that colourless and electrical conductivity and buffering liquid phase finish ultrafiltration simultaneously, probably needs 4~5 volume buffer.Collect backflow, backflow is the extracting solution that has concentrated, and dropwise adds 6M HCl in said extracted liquid, and adjusting PH is 2, stir 30min in room temperature on magnetic stirring apparatus.Then extracting solution is transferred in the bag filter, to buffer (the PH7.550mmol NH of 50~100 times of volumes4HCO3) 4 ℃ of dialysis 24h, in the dialysis procedure, on magnetic stirring apparatus, keep bag filter to rotate slowly.Collect the extracting solution in the bag filter, dropwise add 1M NaOH, under magnetic stir bar stirs, regulate pH value to 7.5, be the allergen stock solution behind the purification, add 2% mannitol and 0.1% human albumin in stock solution, with the aseptic filtration of 0.22um film, lyophilizing then.
Allergen vaccine purity analysis behindembodiment 4 purification
In the process of Herba Artemisiae annuae pollen allergen vaccine and dermatophagoides pteronyssinus allergen vaccine purification and preparation, behind filtration clarification back, the 100KD membrane ultrafiltration, after the decolouring of 5KD membrane ultrafiltration, collect the sample 100ml of three phases, by concentrating the adjusting protein concentration all is 300ug/ml, calculates the concentration of three phases stock solution 50% suppression ratio by the method for ELISA competition inhibition.This method is carried out as follows: the internal reference product stock solution bag of using Herba Artemisiae annuae allergen vaccine and dermatophagoides pteronyssinus allergen vaccine is by 96 orifice plates (4 ℃ of overnight incubation), after washing plate, with 1%BSA or the sealing of 10% calf serum, wash plate after the sealing, bag add in by the hole with 4 times extraordinarily dilution to form dilution factor be 1: 2,1: 8,1: 32,1: 128, the corresponding autopath's of 1: 512 sample and equivalent serum, hatch 1h for 37 ℃, flush away is bound substances not, adding the link coupled sheep anti human IgE two of horseradish peroxidase resists, hatch 1h for 37 ℃, wash and add ABTS substrate Color Appearance System behind the plate.Hatch 30min for 37 ℃,, under the 405nm wavelength, measure absorption value with 2N sulphuric acid cessation reaction.
Calculate each dilution percent inhibition according to the percent inhibition formula:
Percent inhibition (%)=[F (0)-FNSB-F (x)]/[F (0)-FNSB] * 100%;
F (x): the detection fluorescent value under the allergen of different dilution factors (x) suppresses;
F (0): the patients serum does not have in the pond detection fluorescent value that allergen suppresses, blank;
FNSB: the normal human serum pond does not have the detection fluorescent value that allergen suppresses, background;
Logarithm value withdilution 1/2 is an abscissa, and corresponding percent inhibition is the vertical coordinate mapping, the dilution factor when drawing 50% suppression ratio (seeing thefollowing form 1 and 2);
The sample of four purification phase separates through 14%SDS-PAGE glue, analyzes the variation (see accompanying drawing 2 and 3) of purification front and back at 10KD~100KD protein ingredient.
The concentration of table 1 Herba Artemisiae annuae pollen purification different phase sample 50% suppression ratio
Figure BSA00000326817600131
The concentration of table 2 dermatophagoides pteronyssinus purification different phase sample 50% suppression ratio
Figure BSA00000326817600141

Claims (10)

1. the purification process of an allergen vaccine, the allergen extracting solution is prepared into allergen vaccine after ultrafiltration purification, ultrafiltration decolouring, acid treatment, dialysis purification process.
2. purification process according to claim 1, wherein: ultrafiltration purification adopts 100KD~300KD membrane ultrafiltration; 1KD~5KD membrane ultrafiltration decolouring is adopted in the ultrafiltration decolouring; Acid treatment is a ultrafiltration decolouring back extracting solution, dropwise adds 6~8M HCl, and the pH value of regulating stock solution is 1.5~2.5; Dialysis is that the extracting solution after the acid treatment is dialysed with 1KD~5KD bag filter.
3. purification process according to claim 2, wherein: ultrafiltration purification adopts the 100KD membrane ultrafiltration; The decolouring of 3KD membrane ultrafiltration is adopted in the ultrafiltration decolouring; The pH value that stock solution is regulated in acid treatment is 2; Dialysis is that the extracting solution after the acid treatment is dialysed with the 3KD bag filter.
4. according to the arbitrary described purification process of claim 1-3, wherein the allergen extracting solution prepares as follows:
1) defat: in the allergen raw material, added acetone or ether in 1: 2~1: 10 with volume ratio, room temperature defat under magnetic agitation, repeatable operation is colourless up to supernatant, in the ventilation drying;
2) extract: the raw material after the defat with 1: 10~1: 50 (w/v) ratio with buffer, buffer can be phosphate buffer, sodium bicarbonate buffer liquid and ammonium bicarbonate buffers, concentration is 50~200mM, PH is 6~8.5, stir or mechanical agitation extraction 2~24h preferred 1: 25 of ingredient proportion at 2~8 ℃ of condition lower magnetic forces;
3) centrifugal: extract mixture at 4000~6000rpm, centrifugal 15min under 4 ℃ of conditions gets supernatant then at 6000~8000rpm, and centrifugal 30min under 4 ℃ of conditions collects supernatant, discards precipitation or precipitates and extract with same condition again;
4) clarification filtration: supernatant is by filter paper, and cardboard or other aperture 10-0.22um film adopt a step or a plurality of step to filter clarification.
5. the preparation method of an allergen vaccine, the allergen that is selected from tree pollen, showy flowers of herbaceous plants powder, fungus, animal cuticle and hair, insecticide makes allergen vaccine through defat, extraction, centrifugal, clarification filtration, ultrafiltration purification, ultrafiltration decolouring, acid treatment, dialysis, aseptic filtration, dilution packing, lyophilizing.
6. according to the preparation method of claim 5, wherein:
Defat: be selected from the allergen raw material of tree pollen, showy flowers of herbaceous plants powder, fungus, animal cuticle and hair, insecticide and added acetone or ether in 1: 2~1: 10 with volume ratio, room temperature defat under magnetic agitation, repeatable operation is colourless up to supernatant, in the ventilation drying;
Extract: the raw material after the defat with 1: 10~1: 50 (w/v) ratio with buffer, buffer can be phosphate buffer, sodium bicarbonate buffer liquid and ammonium bicarbonate buffers, concentration is 50~200mM, PH is 6~8.5, stir or mechanical agitation extraction 2~24h ingredient proportion 1: 25 at 2~8 ℃ of condition lower magnetic forces;
Centrifugal: extract mixture at 4000~6000rpm, centrifugal 15min under 4 ℃ of conditions gets supernatant then at 6000~8000rpm, and centrifugal 30min under 4 ℃ of conditions collects supernatant, discards precipitation or precipitates and extract with same condition again;
Clarification filtration: supernatant is by filter paper, and cardboard or other aperture 10-0.22um film adopt a step or a plurality of step to filter clarification;
Ultrafiltration purification: the filtrate after the clarification is again through 100KD~300KD membrane ultrafiltration, and preferred 100KD film is collected filter liquor, removes macromolecule impurity;
The ultrafiltration decolouring: the extracting solution after macromole impurity is removed in ultrafiltration is through 1KD~5KD membrane ultrafiltration decolouring, in the process of ultrafiltration, constantly add buffer, when filter liquor when being colourless, with conductivity meter test filter liquor electrical conductivity and buffering liquid phase while, be the ultrafiltration terminal point, the pigment molecular that exists in free mode in the extracting solution in allergen is constantly being removed in the ultra-filtration process, when filter liquor is colourless when consistent with buffer with electrical conductivity, illustrates that the pigment that dissociates in the allergen extracting solution removes substantially; Acid treatment: extracting solution behind 1KD~5KD membrane ultrafiltration, dropwise add 6~8M HCl, the pH value of regulating stock solution is 2~3, and the pigment that is attached on the protein is dissociated;
Dialysis: acid treatment extracting solution is later being stirred 10-30min at ambient temperature on magnetic stirring apparatus, 24-36h dialyses under 4 ℃ of conditions in the water for injection that 50-100 times of volume PH is 6-8.5, in dialysis procedure, container is put on the magnetic stirring apparatus, allows the bag filter freedom slowly rotate;
Regulate PH: dropwise drip 1M NaOH in the extracting solution after the purification dialysis and regulate PH to 6.5~7.5;
Add 1%~3% mannitol and 0.1%~1% human albumin in the extracting solution after regulating pH value;
Lyophilization obtains the allergen vaccine lyophilized powder.
7. according to the preparation method of claim 6, wherein: clarification filtration uses cardboard filter; Ultrafiltration purification adopts the 100KD film; The 3KD film is adopted in the ultrafiltration decolouring; The pH value of regulating stock solution in the acid treatment step is 2; Stirred 30 minutes in the dialysis step, dialysed 24 hours.
8. an allergen preparation is characterized in that the preparation method preparation according to claim 6 or 7.
9. the original preparation of allergic effect according to claim 8, dissolve preparation lyophilizing dry powder in proportion with buffer according to claim 6 or 7, add 0.4% phenol and 0.5%NaCl and be mixed with desensitization stock solution, the allergen vaccine stock solution of preparation can be as subcutaneous injection desensitization, Sublingual immunity desensitization and subcutaneous injection pricking method diagnosis.
10. the original preparation of allergic effect according to claim 9 is characterized in that: add 50% glycerol and be mixed with pricking method liquid, or add Al (OH) 3, tyrosine, MPL adjuvant and be mixed with and contain adjuvant allergen vaccinate.
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CN110078803A (en)*2018-03-232019-08-02中国医学科学院北京协和医院A kind of ocean Chinese wax pollen allergen extract, its immersion liquid and preparation method thereof
CN109939227A (en)*2018-03-232019-06-28中国医学科学院北京协和医院 A kind of ragweed pollen allergen extract, its infusion and preparation method thereof
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CN110201188A (en)*2019-05-242019-09-06中国医学科学院北京协和医院A kind of summer cypress pollen allergen extract, its leaching liquor and preparation method thereof
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