CN116479088A - Reagent combination, kit, method and application for freeze-drying preservation of biological reagent - Google Patents
Reagent combination, kit, method and application for freeze-drying preservation of biological reagent Download PDFInfo
- Publication number
- CN116479088A CN116479088A CN202211437899.4A CN202211437899A CN116479088A CN 116479088 A CN116479088 A CN 116479088A CN 202211437899 A CN202211437899 A CN 202211437899A CN 116479088 A CN116479088 A CN 116479088A
- Authority
- CN
- China
- Prior art keywords
- freeze
- reagent
- drying
- biological
- surfactant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 88
- 238000004108 freeze drying Methods 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000004321 preservation Methods 0.000 title claims abstract description 12
- 150000001720 carbohydrates Chemical class 0.000 claims abstract description 30
- 150000001413 amino acids Chemical class 0.000 claims abstract description 25
- 239000004094 surface-active agent Substances 0.000 claims abstract description 21
- 229920000642 polymer Polymers 0.000 claims abstract description 20
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 claims abstract description 17
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 claims abstract description 17
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 claims abstract description 17
- 239000004475 Arginine Substances 0.000 claims abstract description 13
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims abstract description 13
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims abstract description 12
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 8
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 8
- 229960003080 taurine Drugs 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 48
- 239000008188 pellet Substances 0.000 claims description 48
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 36
- 229910052757 nitrogen Inorganic materials 0.000 claims description 18
- 239000003124 biologic agent Substances 0.000 claims description 14
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims description 12
- 229940050526 hydroxyethylstarch Drugs 0.000 claims description 12
- 238000000859 sublimation Methods 0.000 claims description 9
- 230000008022 sublimation Effects 0.000 claims description 9
- 229940119744 dextran 40 Drugs 0.000 claims description 8
- 238000007710 freezing Methods 0.000 claims description 8
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 claims description 8
- 230000008014 freezing Effects 0.000 claims description 7
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 7
- 229920000053 polysorbate 80 Polymers 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 125000000647 trehalose group Chemical group 0.000 claims description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 abstract description 4
- 230000008569 process Effects 0.000 description 20
- 238000011156 evaluation Methods 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 15
- 238000012360 testing method Methods 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 239000003223 protective agent Substances 0.000 description 10
- 229930182555 Penicillin Natural products 0.000 description 8
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 8
- 230000032683 aging Effects 0.000 description 8
- 239000007798 antifreeze agent Substances 0.000 description 8
- 229940049954 penicillin Drugs 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 238000004806 packaging method and process Methods 0.000 description 7
- 239000003755 preservative agent Substances 0.000 description 7
- 238000010521 absorption reaction Methods 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 238000011056 performance test Methods 0.000 description 6
- 241001678559 COVID-19 virus Species 0.000 description 5
- 238000004090 dissolution Methods 0.000 description 5
- 239000000945 filler Substances 0.000 description 5
- 230000002335 preservative effect Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000004660 morphological change Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000009477 glass transition Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 239000012807 PCR reagent Substances 0.000 description 2
- 239000004353 Polyethylene glycol 8000 Substances 0.000 description 2
- 230000002528 anti-freeze Effects 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229940085678 polyethylene glycol 8000 Drugs 0.000 description 2
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- UNXHWFMMPAWVPI-QWWZWVQMSA-N D-threitol Chemical compound OC[C@@H](O)[C@H](O)CO UNXHWFMMPAWVPI-QWWZWVQMSA-N 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001112090 Pseudovirus Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000012858 packaging process Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000001012 protector Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a reagent combination, a kit, a method and application for freeze-drying preservation of biological reagents, and belongs to the technical field of biology. The reagent combination comprises saccharides and amino acids, wherein the saccharides are trehalose and/or raffinose; the amino acid is taurine and/or arginine. Further, the reagent combination further comprises a polymer and a surfactant. The biological reagent is freeze-dried by using the reagent and reagent combination or the kit, the whole freeze-drying curve can be finished within 6 hours, the freeze-drying efficiency is obviously improved, and the method has great application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a reagent combination, a reagent kit, a method and application of freeze-drying preservation of biological reagents.
Background
The nucleic acid detection technology is a simple, rapid, high-sensitivity and specific gene detection method, wherein the activities of primers, dNTPs and enzymes used in the PCR technology are key factors for detecting success or failure. However, the above components have a short shelf life in aqueous solutions and need to be stored in a low temperature environment below-20 ℃, and repeated freeze thawing in practical operation can greatly reduce their activity and even inactivate, which can directly affect the detection result. On the other hand, in order to ensure reliability, accuracy, consistency and simplicity, large-scale commercial detection kits are also required to add and split each reactant at a time as much as possible, and to ensure that the biological activity thereof is not affected during normal storage and transportation.
The products currently used for nucleic acid detection are increasingly being presented in solid form, so that the storage and transport of reagents is no longer limited by low temperatures, with the most widely used dehydrated form being a freeze-drying process. Freeze-drying is an efficient drying technique that causes frozen solid ice in a mixture to sublimate directly into a gaseous state without melting into liquid water, eventually removing water and retaining other effective components. The stability of the bioactive raw materials in the solid dry powder state is far higher than that of the bioactive raw materials in the liquid state, so that the long-term storage and stable transportation of each component in the nucleic acid detection reagent at room temperature can be realized through freeze-drying and dehydration treatment.
Lyophilization technology has been rapidly developed in the eighth nineties of the last century, and is thereafter widely used in the preservation of biological tissues, pharmaceuticals and foods. The freeze-dried nucleic acid detection reagent is also gradually appeared on the market, and can be stably stored for 12-24 months under the room temperature condition without cold chain transportation. At present, physical parameters of vacuum freeze drying, influence factors, process parameters, process mechanism, process optimization control and the like are well known in the art, so that the freeze-drying technology is programmed gradually, and a certain reference basis is provided for key control factors in the freeze-drying process of the nucleic acid detection reagent and stability analysis of the freeze-dried product.
The conventional freeze-drying process mainly comprises 3 stages of pre-freezing, sublimation and analysis, and the total time for producing a batch of freeze-dried reagent is at least 15 hours and is difficult to finish in one working day, wherein the packaging time is longer than the liquid preparation split charging before freeze-drying and after freeze-drying. In addition, the existing single-person single-part freeze-dried reagent container is usually an 8-joint tube, a 96-hole plate and the like, the container occupies most of the freeze-dried plate layers, the contact area between the reagent and the plate layers is small, the production efficiency is low, and the increasing yield requirement is difficult to meet.
Disclosure of Invention
The invention aims to provide a quick freeze-drying solution, which can simplify the freeze-drying process and realize the whole process from the preparation of biological reagent to the packaging of freeze-dried finished products in one working day; and the utilization rate of the freeze dryer plate layer is improved, the energy consumption is reduced, and the yield is increased. In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a reagent combination for freeze-drying preservation of biological reagents, characterized in that the reagent combination comprises saccharides and amino acids,
wherein the saccharide is trehalose and/or raffinose;
the amino acid is taurine and/or arginine.
In the invention, the saccharides are used as antifreeze agents, protein protectants and fillers to protect the biological agents from changing performance during the freeze-drying process; the amino acids may protect the protein by adjusting the pH.
In some embodiments of the invention, the saccharide and amino acid may be stored separately or mixed together at all times. When stored separately, the composition was used as a 1: 1-2: mixing the materials according to the proportion of 1, and adding the materials into biological reagents or mixing the materials according to the proportion of 1: 1-2: 1 ratio is added to the biological agent. In some preferred embodiments of the invention, the saccharide is raffinose, the amino acid is arginine, and further, the ratio of raffinose to amino acid is 2:1.
in some embodiments of the invention, the reagent combination further comprises a polymer that increases the glass transition temperature of the solution, increases sublimation efficiency, and stabilizes the morphology of the lyophilized product. Preferably, the polymer is selected from at least one of hydroxyethyl starch and dextran 40. Further, the ratio of the saccharide to the polymer is 3:1 to 4:1.
in some embodiments of the invention, the reagent combination further comprises a surfactant that, upon reconstitution, acts as a wetting agent to enhance the reconstitution of the lyophilized reagent. Preferably, the surfactant is selected from at least one of Tween-80 and Thebit, wherein Thebit is also a potent protein and enzyme stabilizing protector. Further, the ratio of the saccharide to the surfactant is 6:1 to 8:1.
in a second aspect, the invention provides a kit for freeze-drying preservation of biological agents, comprising a combination of agents according to the first aspect of the invention, i.e. comprising a saccharide and an amino acid,
wherein the saccharide is trehalose and/or raffinose;
the amino acid is taurine and/or arginine.
In some embodiments of the invention, the kit has a mass ratio of saccharide to amino acid of 1: 1-2: 1.
in some embodiments of the invention, the kit further comprises a polymer selected from at least one of hydroxyethyl starch and dextran 40. Further, the ratio of the saccharide to the polymer is 3:1 to 4:1.
in some embodiments of the invention, the kit further comprises a surfactant selected from at least one of Tween-80 and Thebit. Further, the ratio of the saccharide to the surfactant is 6:1 to 8:1.
a third aspect of the present invention provides a method for freeze-drying and preserving biological reagents using the reagent combination according to the first aspect of the present invention, comprising the steps of:
s1, adding saccharides and amino acids into biological reagents to ensure that the final concentration of the saccharides and the amino acids is 3 to 5 percent and 1.5 to 3 percent respectively,
s2, dripping the biological reagent into a container filled with liquid nitrogen to freeze into freeze-dried pellets, wherein the liquid nitrogen pre-frozen reagent is spherical, so that the sublimation surface area is greatly increased, and the subsequent sublimation analysis stage can be shortened;
s3, freeze-drying the freeze-dried pellets in a freeze dryer, wherein the freeze-drying procedure is as follows:
| step (a) | Ply temperature | Time to rise/fall | Hold time | Pressure of |
| Sublimation | -30℃ | 0min | 150min | 20pa |
| Parse | 30℃ | 60min | 150min | 10pa |
In order to ensure that the whole process from liquid preparation to packaging is completed in one working day, the freeze-drying curve needs to be shortened to be within 6 hours, and according to the common temperature in the sublimation analysis stage, the biological reagent can be freeze-dried by utilizing the freeze-drying curve by utilizing the reagent combination in the first aspect of the invention,
s4, after the freeze-drying is finished, the container is closed under the condition that the vacuum degree is controlled to be minus 0.04 MPa. The freeze-dried pellets are not contacted with air in the whole process, so that the possibility of moisture absorption is reduced to the greatest extent, the requirements on the temperature and humidity of a freeze-drying workshop are lower, strict temperature and humidity control is not needed, and the energy consumption is reduced.
In some embodiments of the invention, in step S1, a step of adding a polymer to the biological agent is further included such that the final concentration of the polymer is 0.5% to 2%, the polymer being selected from at least one of hydroxyethyl starch and dextran 40.
In some embodiments of the invention, in step S1, a step of adding a surfactant to the biological agent is further included such that the final concentration of the surfactant is 0.3% to 0.8%, the surfactant being selected from at least one of Tween-80 and Thesit.
In some embodiments of the invention, the container is an octant, 96-well plate, or penicillin bottle. Preferably, the container is a penicillin bottle, and the penicillin bottle is used for replacing an eight-connection pipe and a 96-hole plate as the container, so that the use ratio of the plate layer of the freeze dryer can be increased, the freeze dryer can be directly capped after freeze drying is finished, and freeze-dried pellets are not contacted with air in the whole process, so that the risk of moisture absorption is reduced to the greatest extent.
According to a fourth aspect of the present invention, there is provided a biological reagent capable of freeze-drying preservation, the biological reagent comprising 3% -5% of saccharides and 1.5% -3% of amino acids, wherein the saccharides are trehalose and/or raffinose; the amino acid is taurine and/or arginine.
The biological reagent can complete freeze-drying by using a freeze-drying curve within 6 hours due to the addition of saccharides and amino acids.
In some embodiments of the invention, the biological agent further comprises 0.5% to 2% of a polymer and/or 0.3% to 0.8% of a surfactant, wherein the polymer is selected from at least one of hydroxyethyl starch and dextran 40; the surfactant is at least one selected from Tween-80 and Thesit.
In the present invention, the biological agent refers to a temperature-sensitive agent, and when the temperature is higher than 10 ℃, the biological function is reduced or even lost or the biological agent itself is degraded so as not to exert the biological function, and generally refers to an agent having biological activity or having a specific biological structure, for example, an agent having biological enzyme catalytic activity, or a biological agent such as nucleic acid, enzyme, protein fragment, etc.
The beneficial effects of the invention are that
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a reagent combination suitable for a quick freeze-drying curve, which is used as a freeze-drying preservative and preferably comprises 4 parts of raffinose, 2 parts of arginine, 1 part of hydroxyethyl starch and 0.5 part of Thesit in parts by weight; wherein raffinose is used as an antifreeze agent, a filler and a protein protectant at the same time; arginine as a supplemental protein protectant increases the shelf life of the lyophilized reagent; hydroxyethyl starch increases the overall glass transition temperature of the lyophilized reagent, reducing the final moisture content; the threit can obviously improve the re-dissolution rate of the freeze-dried reagent, and can further prolong the validity period of the freeze-dried reagent as a protein protecting agent.
2. The method for freeze-drying is utilized, 3 steps of pre-freezing, reagent sub-packaging and reagent out-of-box are simplified, the whole freeze-drying curve can be finished within 6 hours, the liquid preparation sub-packaging and packaging processes can be completed within one working day, the freeze-drying efficiency is obviously improved, and the method has great application value.
3. By utilizing the freeze-drying method disclosed by the invention to freeze-dry the RT-PCR reagent, the obtained freeze-dried pellets are round in appearance, good in uniformity, have certain rigidity, are not easy to absorb moisture, and can be re-dissolved within 5-10 sec. The quick freeze-drying scheme is applied to the novel Guanyin flow RT-PCR reagent, experimental data prove that the reagent has consistent performance before and after freeze-drying, the biological activity of the reagent is not affected by liquid nitrogen quick freezing under the action of a freeze-drying protective agent, single person can be realized by matching with clamping tools such as tweezers in the actual use process, and the freeze-dried pellet form can be stably kept for one month in the room temperature environment after bottle opening. The freeze-dried pellets produced by the freeze-drying process can be stably stored for 2 months at 45 ℃ and 80% humidity, namely 8 months at 25 ℃ and 32 months at 4 ℃ and the storage life of the liquid reagent is far longer.
4. The freeze-drying method can be carried out in a conventional workshop without strict temperature control and humidity control, and reduces energy consumption compared with the traditional freeze-drying process, and the freeze-drying method disclosed by the invention introduces liquid nitrogen pre-freezing and penicillin bottle split-packaging freeze-dried pellets, and is carried out simultaneously, so that the cost is low, the whole operation steps are simple, and the large-scale production of freeze-drying reagents is easy.
Drawings
FIG. 1 shows the effect on PCR amplification curves after cryopreservation of different lyoprotectants in example 1 of the present invention. A:2%, 4% and 6% of sucrose are respectively used as freeze-drying preservative; b:2%, 4% and 6% of trehalose are respectively used as freeze-drying preservative; c:2%, 4% and 6% raffinose are respectively used as freeze-drying preserving agents; d:2%, 4% and 6% mannitol are respectively used as freeze-drying preservative; e:2%, 4% and 6% sorbitol are respectively used as freeze-drying preservative; f:2%, 4% and 6% polyethylene glycol 8000 were used as freeze-drying preserving agents, respectively.
Detailed Description
Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this application is incorporated by reference in its entirety, and the equivalent patents to those cited are incorporated by reference, particularly as they relate to the definitions of terms in the art. If the definition of a particular term disclosed in the prior art does not conform to any definition provided in this application, the definition of that term provided in this application controls.
Numerical ranges in this application are approximations, so that it may include the numerical values outside of the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.
The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or procedure, and are not related to whether or not such other component, step or procedure is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising," "including," or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant, or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.
In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.
Examples
The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
EXAMPLE 1 screening of anti-Freeze Agents
1. Preparation of new Guanyujin liquid reagent
| Component (A) | Single person use amount/. Mu.L | 10 person consumption/. Mu.L |
| 5 Xdetection buffer | 4 | 40 |
| New crown nail and B flow primer probe (50 mu M) | 1 | 10 |
| dNTPs(25mM) | 0.5 | 5 |
| Taq enzyme (5U/. Mu.L) | 0.4 | 4 |
| Rt enzyme (200U/. Mu.L) | 0.1 | 1 |
| Freeze-drying protective agent | 0～9 | 0～90 |
| DEPC water | 0～9 | 0～90 |
| Total volume of | 15 | 150 |
2. Performance test of antifreeze Agents
2.1 preparation of antifreeze Agents
3 saccharide protectants (sucrose, trehalose and raffinose) and 3 polyhydroxy compounds (mannitol, sorbitol and polyethylene glycol 8000) with the mass-volume ratio of 15% are respectively prepared, and the effect of the antifreeze is verified.
2.2 preparation of PCR mixture
Groups 1 to 18 were added as antifreeze to the new crown gall liquid reagent in the amounts shown in the following table, and each system was made up to 15. Mu.L or 150. Mu.L with DEPC water.
2.3 liquid nitrogen treatment: and respectively dripping the 18 groups of PCR mixed solutions into liquid nitrogen by using a liquid transfer gun, maintaining for 2min after molding, and respectively split charging into eight-joint tubes.
2.4PCR test
After the 18 groups of PCR mixed liquids treated by liquid nitrogen in 2.3 are frozen at normal temperature, the performance of the antifreeze agent is tested by taking the 18 groups of PCR mixed liquids which are not treated by liquid nitrogen as a control and 2019-nCoV pseudovirus particles as templates, and an amplification curve is shown in figure 1.
As can be seen from FIG. 1, A, B, C, 3 saccharides at a concentration of between 2 and 6% can be used as cryoprotectants, with sucrose being the best cryoprotectant (A). After the ultralow temperature freezing treatment of liquid nitrogen, the 3 polyhydroxy compounds can not perform the freezing protection function on active substances in the PCR mixed solution when being singly acted (D, E, F).
From this, it can be seen that the saccharide can be used as a protein protectant, an anti-freeze agent and a bulking agent at the same time, and according to the results of example 1, a one-component protectant can be formulated to verify the protective effect of the saccharide.
EXAMPLE 2 screening of fillers
1. Preparation of new Guanyujin liquid reagent
| Component (A) | Single person use amount/. Mu.L | 1000 person consumption/. Mu.L |
| 5 Xdetection buffer | 4 | 4000 |
| New crown nail and B flow primer probe (50 mu M) | 1 | 1000 |
| dNTPs(25mM) | 0.5 | 500 |
| Taq enzyme (5U/. Mu.L) | 0.4 | 400 |
| Rt enzyme (200U/. Mu.L) | 0.1 | 100 |
| Freeze-drying protective agent | 0～9 | 0～9000 |
| DEPC water | 0～9 | 0～9000 |
| Total volume of | 15 | 15000 |
2. Performance testing of fillers
2.1 preparation of PCR mixture
Groups 1-9 of example 1 were added to the new crown gall liquid reagent as antifreeze agent and filler simultaneously in amounts as shown in the following table, with each system being supplemented to 15 μl or 15mL with DEPC water.
2.2 liquid separation and liquid nitrogen Pre-freezing
(1) Soaking the cold plate in liquid nitrogen for 30min, and placing a penicillin bottle on the cold plate to keep the temperature low;
(2) And (3) subpackaging liquid nitrogen into each penicillin bottle, then using a gun to quickly drop the prepared new Guanyin full premix RT-PCR amplification reagent into the penicillin bottles (the volume of the pellets is 15 mu L), instantly forming the liquid pellets after the liquid pellets are dropped into the liquid nitrogen, waiting for 5sec, and dropping the liquid pellets again after the pellets are settled to ensure that the pellets are not adhered to each other, wherein 50 pellets are added into each bottle, and the total amount of 20 bottles.
(3) After the split charging is finished, half-pressing the penicillin bottle rubber plug, and after the split charging is finished, immediately transferring all cold plates into a freeze dryer with the temperature reduced in advance for freeze drying after liquid nitrogen volatilizes.
2.3 lyophilization
| Step (a) | Ply temperature | Time to rise/fall | Hold time | Pressure of |
| Sublimation | -30℃ | 0min | 150min | 20pa |
| Parse | 30℃ | 60min | 150min | 10pa |
2.4 gland discharging
After freeze-drying, controlling the vacuum degree to be under the condition of-0.04 MPa, capping, discharging air from the box, and directly packaging after capping.
3. Evaluation of Performance
3.1 appearance evaluation
3.2 evaluation of Performance
Further performance evaluations were performed on visually acceptable groups 5, 6, 8, 9.
(1) Resolubility of
Randomly extracting 4 bottles, clamping 2 freeze-dried pellets from each bottle by using tweezers, placing the pellets into a PCR eight-joint tube, and adding 15 mu L DEPC water into each hole for re-dissolution.
(2) Moisture absorption resistance test in use process of freeze-dried pellets
Selecting groups 5, 6 and 8, randomly extracting 4 bottles of freeze-dried pellets, opening once every 5 days, taking 4 freeze-dried pellets, placing the 4 freeze-dried pellets in an eight-joint tube, timely covering a rubber plug after opening, opening 6 times, placing the pellets at room temperature for 30 days, observing morphological changes and testing amplification performance, and obtaining the results shown in the following table:
(3) Accelerated aging stability test
The freeze-dried reagents of the group 5 and the group 8 are placed into a constant temperature and constant humidity incubator for an accelerated aging experiment, and the accelerated condition is that the temperature is 45 ℃ and the humidity is 80%. And (3) respectively placing for 3, 7 and 15 days, and taking freshly prepared liquid reagent as a control to perform a comparison test with the lowest detection limit. The performance of the lyophilized reagents was tested using 8 pseudovirions as templates (H1N 1, H1N12009, H3N2, H5N1, H7N9, BV, BY, 2019-nCoV) and the results are shown in the following table:
according to the results of example 2, the lyophilized reagent with saccharide as one-component protectant can only be stored stably for 7 days at 45℃and 80% RH; there is a need to add additional protein protectants to extend shelf life.
EXAMPLE 3 screening of protein protectants
1. Preparation of new Guanyujin liquid reagent
| Component (A) | Single person use amount/. Mu.L | 1000 person consumption/. Mu.L |
| 5 Xdetection buffer | 4 | 4000 |
| New crown nail and B flow primer probe (50 mu M) | 1 | 1000 |
| dNTPs(25mM) | 0.5 | 500 |
| Taq enzyme (5U/. Mu.L) | 0.4 | 400 |
| Rt enzyme (200U/. Mu.L) | 0.1 | 100 |
| Freeze-drying protective agent | 0～9 | 0～9000 |
| DEPC water | 0～9 | 0～9000 |
| Total volume of | 15 | 15000 |
2. Performance test of protein protectants
2.1 preparation of PCR mixture
According to the results of example 2, amino acid protein protectant was added based on the final concentration of 4% raffinose, multicomponent protectant was formulated, groups 8.1 to 8.9 were added to the fresh crown gall liquid reagent, respectively, in amounts as shown in the following table, and each system was made up to 15 μl or 15mL with DEPC water.
2.2 liquid separation, liquid nitrogen prefreezing, freeze drying and capping
The same as in example 2.
3. Evaluation of Performance
3.1 appearance evaluation
3.2 evaluation of Performance
Further performance evaluations were performed on the appearance-acceptable groups 8.4, 8.5, 8.7, 8.8 and 8.9
(1) Resolubility of
Randomly extracting 4 bottles, clamping 2 freeze-dried pellets from each bottle by using tweezers, placing the pellets into a PCR eight-joint tube, and adding 15 mu L DEPC water into each hole for re-dissolution. The results are shown in the following table:
(2) Moisture absorption resistance test in use process of freeze-dried pellets
Randomly extracting 4 bottles of freeze-dried pellets, opening once every 5 days, taking 4 freeze-dried pellets, placing the freeze-dried pellets in an eight-joint tube, timely covering a rubber plug after opening, opening 6 times, placing the freeze-dried pellets at room temperature for 30 days, and observing morphological change and amplification performance test. The results are shown in the following table:
(3) Accelerated aging stability test
The freeze-dried reagents of groups 8.4, 8.5, 8.7 and 8.8 are placed into a constant temperature and humidity incubator for accelerated aging test, wherein the accelerated conditions are that the temperature is 45 ℃ and the humidity is 80%. And (3) respectively placing for 7, 12 and 20 days, and taking freshly prepared liquid reagent as a control to perform a comparison test with the lowest detection limit. The performance of the lyophilized reagents was tested using 8 pseudovirions as templates (H1N 1, H1N12009, H3N2, H5N1, H7N9, BV, BY, 2019-nCoV). The results are shown in the following table:
according to the results of example 3, the lyophilized reagent of the saccharide and amino acid combination protectant can be stably stored for 20 days at 45 ℃ under 80% rh; according to the results of examples 2 and 3, the amplification performance was not satisfactory and the morphology was degraded, so that it was necessary to increase the protective agent for maintaining the morphology stable on the basis of the group 8.8 (4% raffinose, 2% arginine), that is, to increase the glass transition temperature of the lyophilized reagent, to increase the sublimation efficiency and to decrease the final water content.
Example 4 screening of Polymer
1. Preparation of new Guanyujin liquid reagent
| Component (A) | Single person use amount/. Mu.L | 1000 person consumption/. Mu.L |
| 5 Xdetection buffer | 4 | 4000 |
| New crown nail and B flow primer probe (50 mu M) | 1 | 1000 |
| dNTPs(25mM) | 0.5 | 500 |
| Taq enzyme (5U/. Mu.L) | 0.4 | 400 |
| Rt enzyme (200U/. Mu.L) | 0.1 | 100 |
| Freeze-drying protective agent | 0～9 | 0～9000 |
| DEPC water | 0～9 | 0～9000 |
| Total volume of | 15 | 15000 |
2. Performance testing of polymers
2.1 preparation of PCR mixture
According to the results of example 3, a polymer (hydroxyethyl starch, dextran 40 or polyvinylpyrrolidone) was added on the basis of group 8.8 (4% raffinose, 2% arginine) to prepare a multicomponent protectant, and groups 8.8.1 to 8.8.9 were added to the fresh crown gall liquid reagent, respectively, in amounts as shown in the following table, with each system being made up to 15 μl or 15mL with DEPC water.
2.2 liquid separation, liquid nitrogen prefreezing, freeze drying and capping
The same as in example 2.
3. Evaluation of Performance
3.1 appearance evaluation
3.2 evaluation of Performance
Further performance evaluations were performed on groups 8.8.1-8.8.9.
(1) Resolubility of
Randomly extracting 4 bottles, clamping 2 freeze-dried pellets from each bottle by using tweezers, placing the pellets into a PCR eight-joint tube, and adding 15 mu L DEPC water into each hole for re-dissolution. The results are shown in the following table:
(2) Moisture absorption resistance test in use process of freeze-dried pellets
Randomly extracting 4 bottles of freeze-dried pellets, opening once every 5 days, taking 4 freeze-dried pellets, placing the freeze-dried pellets in an eight-joint tube, timely covering a rubber plug after opening, opening 6 times, placing the freeze-dried pellets at room temperature for 30 days, and observing morphological change and amplification performance test. The results are shown in the following table:
(3) Accelerated aging stability test
The freeze-dried reagents of the groups 8.8.1, 8.8.2, 8.8.7 and 8.8.8 are placed into a constant temperature and humidity incubator for accelerated aging test, wherein the accelerated conditions are that the temperature is 45 ℃ and the humidity is 80%. And (3) respectively placing for 20, 40 and 60 days, and taking freshly prepared liquid reagent as a control to perform a comparison test with the lowest detection limit. The performance of the lyophilized reagents was tested using 8 pseudovirions as templates (H1N 1, H1N12009, H3N2, H5N1, H7N9, BV, BY, 2019-nCoV). The results are shown in the following table:
/>
according to the result of the example 4, the freeze-dried reagent added with the group 8.8.2 freeze-dried protective agent can be stably stored for 40 days under the condition of 45 ℃ and 80% RH, and can meet the normal-temperature transportation at home and abroad; but the freeze-dried pellets obtained by the protective agent have a reconstitution time of 20-30 sec; further surfactant addition is required to increase the rate of reconstitution.
Example 5 screening of surfactants
1. Preparation of new Guanyujin liquid reagent
/>
2. Performance test of surfactants
2.1 preparation of PCR mixture
According to the results of example 4, a multicomponent protectant was formulated by adding surfactant based on group 8.8.2 (4% raffinose, 2% arginine, 1% hydroxyethyl starch), groups 8.8.2.1-8.8.2.9 were added to fresh crown gall liquid reagent, respectively, in the amounts shown in the following table, and each system was made up to 15 μl or 15mL with DEPC water.
2.2 liquid separation, liquid nitrogen prefreezing, freeze drying and capping
The same as in example 2.
3. Evaluation of Performance
3.1 appearance evaluation
3.2 evaluation of Performance
Further performance evaluations were performed on groups 8.8.2.1-8.8.2.9.
(1) Resolubility of
Randomly extracting 4 bottles, clamping 2 freeze-dried pellets from each bottle by using tweezers, placing the pellets into a PCR eight-joint tube, and adding 15 mu L DEPC water into each hole for re-dissolution. The results are shown in the following table:
(2) Moisture absorption resistance test in use process of freeze-dried pellets
And 5, randomly extracting 4 bottles of freeze-dried pellets from the group 8.8.2.1-8.8.2.6, opening the freeze-dried pellets once every 5 days, placing the freeze-dried pellets in an eight-joint tube, timely covering a rubber plug after opening the freeze-dried pellets, opening the freeze-dried pellets 6 times in total, placing the freeze-dried pellets at room temperature for 30 days, and observing morphological change and amplification performance test. The results are shown in the following table:
(3) Accelerated aging stability test
The freeze-dried reagents of the groups 8.8.2.1, 8.8.2.4 and 8.8.2.5 are placed into a constant temperature and humidity incubator for accelerated aging experiments, wherein the accelerated conditions are that the temperature is 45 ℃ and the humidity is 80%. And (3) respectively placing for 20, 40 and 60 days, and taking freshly prepared liquid reagent as a control to perform a comparison test with the lowest detection limit. The performance of the lyophilized reagents was tested using 8 pseudovirions as templates (H1N 1, H1N12009, H3N2, H5N1, H7N9, BV, BY, 2019-nCoV). The results are shown in the following table:
/>
according to the result of example 5, the freeze-dried reagent added with the group 8.8.2.5 freeze-dried protective agent not only has obvious improvement on the re-solubility, but also can be completely re-dissolved within 5-10 sec; and the stability of the freeze-drying reagent is prolonged, and the freeze-drying reagent can be stably stored for 60 days under the condition of 45 ℃ and 80% RH. 8.8.2.5 (4% raffinose, 2% arginine, 1% hydroxyethyl starch, 0.5% thesit) was therefore chosen as the final lyoprotectant combination.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (10)
1. A reagent combination for freeze-drying preservation of biological reagents is characterized by comprising saccharides and amino acids,
wherein the saccharide is trehalose and/or raffinose;
the amino acid is taurine and/or arginine.
2. The reagent combination of claim 1, further comprising a polymer selected from at least one of hydroxyethyl starch and dextran 40.
3. A reagent combination according to claim 1 or 2, further comprising a surfactant selected from at least one of Tween-80 and Thesit.
4. A kit for the lyophilized preservation of biological agents comprising the combination of agents of claim 1.
5. The kit according to claim 4, wherein the mass ratio of saccharide to amino acid in the kit is 1: 1-2: 1.
6. a method of freeze-drying preservation of biological agents using the combination of agents of claim 1, comprising the steps of:
s1, adding saccharides and amino acids into biological reagents to ensure that the final concentration of the saccharides and the amino acids is 3 to 5 percent and 1.5 to 3 percent respectively,
s2, dripping the biological reagent into a container filled with liquid nitrogen, and freezing into freeze-dried pellets;
s3, freeze-drying the freeze-dried pellets in a freeze dryer, wherein the freeze-drying procedure is as follows:
S4, after the freeze-drying is finished, the container is closed under the condition that the vacuum degree is controlled to be minus 0.04 MPa.
7. The method of claim 6, further comprising the step of adding a polymer to the biological agent such that the final concentration of the polymer is 0.5% to 2%, the polymer being at least one selected from the group consisting of hydroxyethyl starch and dextran 40, in step S1.
8. The method according to claim 6 or 7, further comprising the step of adding a surfactant to the biological agent in step S1 such that the final concentration of the surfactant is 0.3% to 0.8%, the surfactant being at least one selected from Tween-80 and Thesit.
9. A biological reagent capable of freeze-drying preservation, which is characterized by comprising 3-5% of sugar and 1.5-3% of amino acid, wherein the sugar is trehalose and/or raffinose; the amino acid is taurine and/or arginine.
10. The biological agent according to claim 9, further comprising 0.5-2% of a polymer and/or 0.3-0.8% of a surfactant, wherein the polymer is at least one selected from hydroxyethyl starch and dextran 40; the surfactant is at least one selected from Tween-80 and Thesit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211437899.4A CN116479088A (en) | 2022-11-15 | 2022-11-15 | Reagent combination, kit, method and application for freeze-drying preservation of biological reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211437899.4A CN116479088A (en) | 2022-11-15 | 2022-11-15 | Reagent combination, kit, method and application for freeze-drying preservation of biological reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116479088A true CN116479088A (en) | 2023-07-25 |
Family
ID=87220133
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211437899.4A Pending CN116479088A (en) | 2022-11-15 | 2022-11-15 | Reagent combination, kit, method and application for freeze-drying preservation of biological reagent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116479088A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117368454A (en) * | 2023-12-08 | 2024-01-09 | 普迈德（北京）科技有限公司 | Thromboelastography rapid activated blood coagulation reagent card, preparation method and application |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003087327A2 (en) * | 2002-04-11 | 2003-10-23 | Medimmune Vaccines, Inc. | Preservation of bioactive materials by freeze dried foam |
| WO2005065709A2 (en) * | 2003-12-24 | 2005-07-21 | Boehringer Ingelheim International Gmbh | Lyophilized antibody conjugate formulation |
| JP2009203223A (en) * | 2008-01-29 | 2009-09-10 | Sanyo Chem Ind Ltd | Protectant for freeze drying and production method of physiologically active substance |
| US20120114646A1 (en) * | 2009-06-18 | 2012-05-10 | Wyeth Llc | Lyophilized formulations for small modular immunopharmaceuticals |
| CN102793927A (en) * | 2012-08-29 | 2012-11-28 | 郑州后羿制药有限公司 | Swine fever vaccine heat-resistant freeze-drying protective agent, and preparation method and application thereof |
| CN106063933A (en) * | 2015-12-31 | 2016-11-02 | 武汉博沃生物科技有限公司 | General vaccines freeze drying protectant and application thereof |
| CN107049964A (en) * | 2008-09-03 | 2017-08-18 | 奥克塔法马股份有限公司 | The novel protected composition for the Factor IX being prepared by recombinant |
| US20200345000A1 (en) * | 2019-04-30 | 2020-11-05 | Regents Of The University Of Minnesota | Compositions and methods for storing liquid biospecimens |
| CN113652468A (en) * | 2021-07-19 | 2021-11-16 | 江苏硕世生物科技股份有限公司 | Freeze-drying protective agent suitable for freeze-drying of nucleic acid detection kit and freeze-drying method thereof |
-
2022
- 2022-11-15 CN CN202211437899.4A patent/CN116479088A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003087327A2 (en) * | 2002-04-11 | 2003-10-23 | Medimmune Vaccines, Inc. | Preservation of bioactive materials by freeze dried foam |
| US20030219475A1 (en) * | 2002-04-11 | 2003-11-27 | Medlmmune Vaccines, Inc. | Preservation of bioactive materials by freeze dried foam |
| WO2005065709A2 (en) * | 2003-12-24 | 2005-07-21 | Boehringer Ingelheim International Gmbh | Lyophilized antibody conjugate formulation |
| JP2009203223A (en) * | 2008-01-29 | 2009-09-10 | Sanyo Chem Ind Ltd | Protectant for freeze drying and production method of physiologically active substance |
| CN107049964A (en) * | 2008-09-03 | 2017-08-18 | 奥克塔法马股份有限公司 | The novel protected composition for the Factor IX being prepared by recombinant |
| US20120114646A1 (en) * | 2009-06-18 | 2012-05-10 | Wyeth Llc | Lyophilized formulations for small modular immunopharmaceuticals |
| CN102793927A (en) * | 2012-08-29 | 2012-11-28 | 郑州后羿制药有限公司 | Swine fever vaccine heat-resistant freeze-drying protective agent, and preparation method and application thereof |
| CN106063933A (en) * | 2015-12-31 | 2016-11-02 | 武汉博沃生物科技有限公司 | General vaccines freeze drying protectant and application thereof |
| US20200345000A1 (en) * | 2019-04-30 | 2020-11-05 | Regents Of The University Of Minnesota | Compositions and methods for storing liquid biospecimens |
| CN113652468A (en) * | 2021-07-19 | 2021-11-16 | 江苏硕世生物科技股份有限公司 | Freeze-drying protective agent suitable for freeze-drying of nucleic acid detection kit and freeze-drying method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 薛菲 等: "蛋白质的冻干保护剂及其保护机制研究进展", 中国药学杂志, no. 10, 22 May 2018 (2018-05-22), pages 765 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117368454A (en) * | 2023-12-08 | 2024-01-09 | 普迈德（北京）科技有限公司 | Thromboelastography rapid activated blood coagulation reagent card, preparation method and application |
| CN117368454B (en) * | 2023-12-08 | 2024-04-23 | 普迈德（北京）科技有限公司 | Thromboelastography rapid activated blood coagulation reagent card, preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110452956B (en) | Freeze-dried microsphere of PCR reaction reagent and preparation method thereof | |
| US6653062B1 (en) | Preservation and storage medium for biological materials | |
| EP2021508B1 (en) | Ambient temperature stable kits for molecular diagnostics | |
| Melin et al. | Optimisation and comparison of liquid and dry formulations of the biocontrol yeast Pichia anomala J121 | |
| EP2294222B1 (en) | Freeze-dried compositions for pcr and rt-pcr | |
| CN101971796B (en) | Nonprogrammed cell frozen stock solution free of proteins | |
| CN105274192A (en) | Nucleic acid amplification reaction mixture particle and application thereof | |
| AU2007203958A1 (en) | Cryoprotective compositions and methods of using same | |
| AU2001268057A1 (en) | Preservation and storage medium for biological materials | |
| CN116479088A (en) | Reagent combination, kit, method and application for freeze-drying preservation of biological reagent | |
| CN111662902A (en) | Freeze-drying protective agent and application thereof in nucleic acid amplification reagent | |
| CN110093403A (en) | The freeze drying protectant of fluorescent PCR reagent and the preparation method that chip is lyophilized | |
| WO2006029467A1 (en) | Rapid freeze drying process | |
| CA2458794C (en) | Plasticized hydrophilic glasses for improved stabilization of biological agents | |
| MX2011011616A (en) | A dried and stabilized ready-to-use composition containing nucleic acid polymerization enzymes for molecular biology applications. | |
| CN114752703A (en) | Novel freeze-drying detection reagent for coronavirus nucleic acid and preparation method thereof | |
| CN110551785B (en) | Cell-free lyophilized preparation for in vitro protein synthesis, preparation method and application thereof | |
| CN105535987A (en) | Heat-resisting protective agent, swine fever heat-resisting protective agent live vaccine and preparation method of swine fever heat-resisting protective agent live vaccine | |
| EP1196541A2 (en) | Storage of microorganisms, cells and tissue | |
| CN112485451B (en) | Interleukin 6 freeze-drying calibrator, preparation method thereof and freeze-drying protective solution | |
| KR20140053829A (en) | Inoculum and method of preparation | |
| CN114015758B (en) | Freeze-drying protective agent, fluorescent PCR detection kit and freeze-drying process | |
| KR102058338B1 (en) | Composition for cryopreservation of Canidae sperm comprising mesenchymal stem cell as effective component | |
| CN113025757A (en) | Freeze-drying protective agent for 2019-nCov multiplex amplification reaction reagent and application thereof | |
| CN116121339A (en) | Freeze-drying protective agent and application thereof in preparation of PCR freeze-drying reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |