Cryoprotectant and use thereof
By using a cryoprotectant containing sugar protectants, pH buffers, Mg2+ and zwitterion donors, the problem of decreased viral activity during repeated freeze-thaw cycles was solved, achieving stability and effectiveness of the viral preparation and reducing the damage to the virus caused by freeze-thaw cycles.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- SUZHOU ROCROCK NO 1 BIOTECHNOLOGY CO LTD
- Filing Date
- 2024-11-06
- Publication Date
- 2026-05-15
AI Technical Summary
Existing methods for preserving viruses cannot maintain viral activity during repeated freeze-thaw cycles, leading to viral inactivation and failing to guarantee the stability and effectiveness of viral preparations.
A cryoprotectant containing 1wt%-10wt% sugar protectant, pH buffer, 0.5mM-3mM Mg2+, 1wt%-10wt% zwitterion donor and aqueous solvent is used to increase the preservation effect and stability of the virus through osmotic pressure and viscosity, ensuring that the virus remains active after repeated freeze-thaw cycles.
After three freeze-thaw cycles, the virus loss was less than 70%, and the infection efficiency was not affected. This reduced the damage to the virus caused by freeze-thaw cycles and ensured the stability and effectiveness of the viral preparation.
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Figure PCTCN2024130128-FTAPPB-I100001
Abstract
Description
Cryoprotectants and their uses Technical Field
[0001] This application belongs to the field of biological agents, specifically relating to a cryoprotectant and its uses. Background Technology
[0002] Viral vectors are receiving increasing attention as a powerful tool for introducing exogenous genes into target cells due to their high efficiency and modifiability.
[0003] Furthermore, with the continuous development of technology, the types of vaccines have become increasingly diverse, but inactivated vaccines remain the most widely used. A key factor affecting vaccine efficacy lies in the selection of the vaccine strain. On the one hand, it is crucial to ensure that the vaccine strain matches the circulating strain's antigen as closely as possible; on the other hand, it is essential to ensure the effective content of the antigen in the vaccine. In actual production applications, screening a strain that is both highly matched to the circulating strain and also high-yielding and stable is extremely difficult. Therefore, the preservation of selected superior vaccine strains is of paramount importance. According to relevant standards and regulations, seed virus with a potency below the standard line cannot be used for production.
[0004] Both viruses and viral vectors require extremely stringent preservation conditions, often necessitating storage at low temperatures, such as -80°C. In practice, achieving such transportation or storage conditions demands extremely expensive equipment, resulting in high costs. Furthermore, temperature instability can easily lead to viral inactivation. The optimal solution to address this issue at its source is to develop an effective viral protectant to extend virus preservation time as much as possible, ensure the consistency of production strains, reduce batch-to-batch variations, and improve virus quality.
[0005] Therefore, virus preservation or freezing reagents that can keep viruses active at higher temperatures play a crucial role in reducing logistics costs, maintaining viral activity, and ensuring the effectiveness of viral vaccines and gene therapy products.
[0006] Current viral preparation formulations typically consist of sucrose, Tris, and MgCl2, which cannot guarantee the survival quantity of the virus (viral preparation quality) after repeated freeze-thaw cycles, meaning they cannot maintain the stability of the viral preparation under repeated freeze-thaw conditions.
[0007] Therefore, there is an urgent need for a formulation that can protect the virus from repeated freeze-thaw cycles and maintain its activity.
[0008] Summary of the Invention
[0009] Therefore, it is necessary to provide at least one cryoprotectant and its uses.
[0010] In one aspect of this application, a cryoprotectant is provided, the cryoprotectant comprising 1 wt%-10 wt% of a sugar protectant, a pH buffer, and 0.5 mM-3 mM of Mg. 2+ 1 wt%-10 wt% zwitterionic donor and aqueous solvent; wherein the mass percentage is the mass percentage of the cryoprotectant, and the concentration of the pH buffer in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0011] In some embodiments, the sugar protectant is selected from the group consisting of monosaccharides and disaccharides.
[0012] In some embodiments, the monosaccharide includes one or more of glucose and galactose.
[0013] In some embodiments, the disaccharide includes one or more of trehalose and sucrose.
[0014] In some embodiments, the pH buffer is selected from the group consisting of tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, citrate, phosphate, acetate and borate.
[0015] In some embodiments, the zwitterionic donor includes betaine.
[0016] In some embodiments, the concentration of the zwitterionic donor in the cryoprotectant is 5 wt%-10 wt%.
[0017] In some embodiments, the cryoprotectant further comprises a polyol.
[0018] In some embodiments, the polyol is selected from the group consisting of glycerol, mannitol, sorbitol, xylitol, inositol, and calendula alcohol.
[0019] In some embodiments, the concentration of the polyol in the cryoprotectant is 1 g / L to 1.5 g / L.
[0020] In some embodiments, the cryoprotectant also includes a surfactant.
[0021] In some embodiments, the surfactant is selected from the group consisting of Triton 100, Tween 20, Tween 80 and S9.
[0022] In some embodiments, the surfactant accounts for 0.01 wt% to 0.05 wt% of the cryoprotectant by mass.
[0023] In some embodiments, the cryoprotectant also contains NaCl.
[0024] In some embodiments, the concentration of NaCl in the cryoprotectant is 10 mM-100 mM.
[0025] In some embodiments, the Mg 2+ It is derived from one or more of MgCl2 and MgSO4.
[0026] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0027] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, and 0.01 wt%-0.05 wt% Tween and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0028] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, 1 g / L-1.5 g / L glycerol, and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0029] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, 0.01 wt%-0.05 wt% Tween, 1 g / L-1.5 g / L glycerol, and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0030] In some embodiments, the pH value of the cryoprotectant is 6 to 8.
[0031] In a second aspect of this application, a viral preparation is provided, comprising a cryoprotectant as described in the first aspect, and a virus.
[0032] In some embodiments, the concentration of the virus in the viral preparation is 1e10 IFU / mL to 2e12 IFU / mL.
[0033] In a third aspect of this application, a method for freezing a virus is provided, the method comprising:
[0034] The virus to be frozen is mixed with a cryoprotectant as described in the first aspect to obtain a viral preparation;
[0035] The viral preparation is frozen.
[0036] In some embodiments, the concentration of the virus in the viral preparation is 1e10 IFU / mL to 2e12 IFU / mL.
[0037] In some embodiments, the freezing process is performed at least once. In some embodiments, the freezing process is performed at least twice. In some embodiments, the freezing process is performed at least three times.
[0038] The inventors of this application, through exploring the components of cryoprotectants, unexpectedly discovered that the osmotic pressure and viscosity of the cryoprotectant in this application can increase the preservation effect and stability of viruses.
[0039] The cryoprotectant provided in this application solves the problems of existing cryoprotectants, such as inability to undergo repeated freeze-thaw cycles and poor stability. Using the cryoprotectant disclosed in this application to preserve the virus, after three repeated freeze-thaw cycles, a comparison of the virus titer before and after the freeze-thaw cycles shows that the virus loss after freeze-thaw is low (e.g., less than 70%); and it can ensure that the damage to the virus from freeze-thaw cycles is reduced without affecting infection efficiency or adding operational steps. Detailed Implementation
[0040] Unless otherwise defined, 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0041] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.
[0042] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.
[0043] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.
[0044] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.
[0045] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."
[0046] In this application, "selected from...group" means that it may include one or more of them.
[0047] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.
[0048] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.
[0049] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.
[0050] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.
[0051] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.
[0052] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.
[0053] In one aspect of this application, a cryoprotectant is provided, the cryoprotectant comprising 1 wt%-10 wt% of a sugar protectant, a pH buffer, and 0.5 mM-3 mM of Mg. 2+ 1 wt%-10 wt% zwitterionic donor and aqueous solvent; wherein the mass percentage is the mass percentage of the cryoprotectant, and the concentration of the pH buffer in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0054] In some embodiments, the glycoprotectant is selected from the group consisting of monosaccharides and disaccharides. In some embodiments, a monosaccharide is used as the glycoprotectant in the cryoprotectant. In some embodiments, a disaccharide is used as the glycoprotectant in the cryoprotectant. In some embodiments, the cryoprotectant contains both monosaccharides and disaccharides as glycoprotectants.
[0055] Unless otherwise specified, the term "aqueous solvent" in this application refers to a liquid solvent that does not affect the physicochemical properties and functions of the cryoprotectant and its components, and may be a commonly used solvent in the art, such as water.
[0056] Unless otherwise specified, the term "monosaccharide" in this application refers to a molecule that cannot be further hydrolyzed into smaller sugar molecules. Common monosaccharides include glucose, galactose, fructose, and erythrose.
[0057] In some embodiments, the monosaccharide includes one or more of glucose and galactose. In some embodiments, the sugar protectant includes glucose. In some embodiments, the sugar protectant includes galactose. In some embodiments, the sugar protectant includes both glucose and galactose.
[0058] Unless otherwise specified, the term "disaccharide" in this application refers to a disaccharide molecule composed of two monosaccharide molecules linked by a glycosidic bond. Common disaccharides include sucrose, lactose, and maltose.
[0059] In some embodiments, the disaccharide includes one or more of trehalose and sucrose. In some embodiments, the sugar protectant includes trehalose. In some embodiments, the sugar protectant includes sucrose. In some embodiments, the sugar protectant includes both trehalose and sucrose.
[0060] In some embodiments, the pH buffer is selected from the group consisting of tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, citrate, phosphate, acetate, and borate. In some embodiments, the cryoprotectant includes at least one pH buffer from the group, such as tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, citrate, phosphate, acetate, or borate. In some embodiments, the cryoprotectant includes at least two pH buffers from the group, for example, tris(hydroxymethyl)aminomethane and borate, tris(hydroxymethyl)aminomethane and citrate, citrate and phosphate, etc.
[0061] In some embodiments, the concentration of the pH buffer is below 100 mM. In some embodiments, the concentration of the pH buffer is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, or a range or value between any two values.
[0062] Unless otherwise specified, the term "zwitterion donor" in this application refers to an ion or substance that can react with both hydrogen ions and hydroxide ions in solution.
[0063] In some embodiments, the zwitterionic donor includes betaine. In some embodiments, the zwitterionic donor in the cryoprotectant contains at least betaine.
[0064] In some embodiments, the zwitterionic donor is betaine; that is, the cryoprotectant contains only betaine as a zwitterionic donor.
[0065] The term "betaine" used in this application refers to an alkaloid, chemically named N,N,N-trimethylglycine, which has a chemical structure similar to amino acids and belongs to the quaternary ammonium base class, with the molecular formula C5H. 11 NO2.
[0066] In some embodiments, the concentration of the zwitterionic donor in the cryoprotectant is 5 wt%-10 wt%. In some embodiments, the concentration of the zwitterionic donor in the cryoprotectant is 5 wt%, 5.1 wt%, 5.2 wt%, 5.3 wt%, 5.4 wt%, 5.5 wt%, 5.6 wt%, 5.7 wt%, 5.8 wt%, 5.9 wt%, 6 wt%, 6.1 wt%, 6.2 wt%, 6.3 wt%, 6.4 wt%, 6.5 wt%, 6.6 wt%, 6.7 wt%, 6.8 wt%, 6.9 wt%, 7 wt%, 7.1 wt%, 7.2 wt%, 7.3 wt%, or 7.4 wt%. 7.5wt%, 7.6wt%, 7.7wt%, 7.8wt%, 7.9wt%, 8wt%, 8.1wt%, 8.2wt%, 8.3wt%, 8.4wt%, 8.5wt%, 8.6wt%, 8.7wt%, 8.8wt%, 8.9wt%, 9wt%, 9.1wt%, 9.2wt%, 9.3wt%, 9.4wt%, 9.5wt%, 9.6wt%, 9.7wt%, 9.8wt%, 9.9wt%, 10wt%, or any range or value between any two values.
[0067] In some embodiments, the cryoprotectant further comprises a polyol.
[0068] Unless otherwise specified, the term "polyol" in this application refers to a large class of alcohols containing two or more hydroxyl groups in their molecules. In some embodiments, the molecular weight of the polyol is less than 300 Da. In some embodiments, the molecular weight of the polyol is less than 200 Da. In some embodiments, the molecular weight of the polyol is less than 100 Da.
[0069] In some embodiments, the polyol is selected from the group consisting of glycerol, mannitol, sorbitol, xylitol, inositol, and calendula alcohol.
[0070] In some embodiments, the cryoprotectant contains only one polyol, such as glycerol, mannitol, sorbitol, xylitol, inositol, or calendula alcohol. In some embodiments, the cryoprotectant contains at least two polyols, such as glycerol and mannitol, glycerol and sorbitol, xylitol and inositol, etc. When the cryoprotectant contains at least two polyols, the relative amounts of the at least two polyols are not limited; taking two polyols as an example, their relative amounts (such as molar ratio or volume ratio) can be 1:99 to 99:1.
[0071] In some embodiments, the concentration of the polyol in the cryoprotectant is 1 g / L to 1.5 g / L. In some embodiments, the concentration of the polyol in the cryoprotectant is 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, or any range or value between any two values.
[0072] In some embodiments, the cryoprotectant also includes a surfactant.
[0073] Unless otherwise specified, the term "surfactant" in this application, also known as an interfacial surfactant, is a compound that can significantly reduce the surface tension or interfacial tension between two liquids, between a liquid and a gas, or between a liquid and a solid.
[0074] In some embodiments, the surfactant is selected from the group consisting of Triton 100, Tween 20, Tween 80, and S9 (Tetronic 1307). In some embodiments, the cryoprotectant contains only one surfactant, such as Triton 100, Tween 20, Tween 80, or S9. In some embodiments, the cryoprotectant contains at least two surfactants, such as Triton 100 and Tween 20, Tween 20 and Tween 80, or Tween 80 and S9. When the cryoprotectant contains at least two surfactants, the relative amounts of the at least two surfactants are not limited; for example, the relative amounts (e.g., molar ratio or volume ratio) of two surfactants can be 1:99 to 99:1.
[0075] In some embodiments, the surfactant comprises 0.01 wt% to 0.05 wt% of the cryoprotectant by mass. In some embodiments, the surfactant comprises 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt% of the cryoprotectant by mass, or any range or value between two such values.
[0076] In some embodiments, the cryoprotectant further comprises Na. + .
[0077] In some embodiments, the Na + It is derived from one or more of NaCl and Na2SO4.
[0078] In some embodiments, the Na + The concentration of the cryoprotectant is 10 mM-100 mM. In some embodiments, the Na... +The concentration of the cryoprotectant is 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, or any range or value between two such values.
[0079] In some embodiments, the Mg 2+ Derived from magnesium salts. Exemplary magnesium salts may include, for example, magnesium chloride (MgCl2), magnesium sulfate (MgSO4), magnesium citrate, magnesium glycinate, and magnesium malate. In some embodiments, the cryoprotectant comprises MgCl2. In some embodiments, the cryoprotectant comprises MgSO4. In some embodiments, the cryoprotectant comprises both MgCl2 and MgSO4.
[0080] In some embodiments, the Mg 2+ The concentration is 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, or any range or value between two values.
[0081] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 1-3 mM MgCl2, 5 wt%-10 wt% betaine, and an aqueous solvent; wherein the mass percentage is the mass percentage of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0082] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 1-3 mM MgCl2, 5 wt%-10 wt% betaine, and 0.01 wt%-0.05 wt% Tween; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0083] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 1-3 mM MgCl2, 5 wt%-10 wt% betaine, 1 g / L-1.5 g / L glycerol, and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0084] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 1-3 mM MgCl2, 5 wt%-10 wt% betaine, 0.01 wt%-0.05 wt% Tween, 1 g / L-1.5 g / L glycerol, and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0085] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, and 0.01 wt%-0.05 wt% Tween and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0086] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, 1 g / L-1.5 g / L glycerol, and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0087] In some embodiments, the cryoprotectant comprises 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, 0.01 wt%-0.05 wt% Tween, 1 g / L-1.5 g / L glycerol, and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
[0088] In some embodiments, the pH value of the cryoprotectant is 6 to 8, for example 6.5, 7, 7.5 or 8.
[0089] In a second aspect of this application, a viral preparation is provided, comprising a cryoprotectant as described in the first aspect, and a virus.
[0090] In some embodiments, the concentration of the virus in the viral preparation is 1e10 IFU / mL-2e12 IFU / mL.
[0091] Unless otherwise specified, the term IFU in this application refers to Infectious Units, which is a unit of viral concentration.
[0092] In a third aspect of this application, a method for freezing a virus is provided, the method comprising:
[0093] The virus to be frozen is mixed with a cryoprotectant as described in the first aspect to obtain a viral preparation;
[0094] The viral preparation is frozen.
[0095] In some embodiments, the concentration of the virus in the viral preparation is 1e10 IFU / mL-2e12 IFU / mL.
[0096] In some embodiments, the freezing method includes freezing at a temperature below -10°C for at least 0.5 hours.
[0097] In some embodiments, the freezing method includes freezing at temperatures below -15°C, below -20°C, below -25°C, below -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C, or any range or value between two of these values.
[0098] In some embodiments, the freezing time may be, for example, at least 0.5h, at least 1h, at least 2h, at least 3h, at least 4h, at least 5h, at least 6h, at least 7h, at least 8h, at least 9h, at least 10h, at least 11h, at least 12h, at least 13h, at least 14h, at least 15h, at least 16h, at least 17h, at least 18h, at least 19h, at least 20h, at least 21h, at least 22h, at least 23h, at least 24h, or a range or value between any two values.
[0099] In some implementations, the mixture is frozen at least once, for example, twice, three times, or four times.
[0100] In some embodiments, the method includes a freeze-thaw treatment of the virus. In the freeze-thaw treatment, the freezing conditions are as defined above; the thawing conditions may be conventional in the art.
[0101] In some implementations, the thawing condition in the freeze-thaw treatment of the virus is thawing at room temperature.
[0102] In this application, the term "room temperature" generally refers to 4°C to 35°C. In some embodiments, room temperature refers to 20°C ± 5°C. In some embodiments, room temperature refers to 20°C to 30°C.
[0103] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.
[0104] Example 1
[0105] Experimental procedure: The purified virus was aseptically aliquoted into cryovials (each tube contained approximately 1.61E+11 viruses), and betaine was added at mass fractions of 1%, 5%, 10%, and 20% of the cryoprotectant. The titers were measured after 1, 2, and 3 freeze-thaw cycles, respectively.
[0106] The cryoprotectant contains: 20 mM tris, 2 mM MgCl2, 5 wt% sucrose, 0.01 wt% Tween 80, 5 wt% betaine, 1.5 g / L glycerol and 50 mM NaCl, with water as the solvent.
[0107] The freeze-thaw conditions were: freezing at -80℃ for 24 hours, and the titer detection method was the hexazonal live virus titer detection method.
[0108] The results of the titer test are shown in Table 1 below.
[0109] Table 1
[0110] Conclusion and analysis: The data in the table above show that the freeze-thaw effect is best when the mass fraction is 5%-10%, and the titer decrease is minimal and stable after three freeze-thaw cycles.
[0111] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0112] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.
Claims
1. A cryoprotectant, characterized in that, The cryoprotectant contains 1 wt%-10 wt% sugar protectant, pH buffer, and 0.5 mM-3 mM Mg. 2+ 1 wt%-10 wt% zwitterionic donor and aqueous solvent; wherein the mass percentage is the mass percentage of the cryoprotectant, and the concentration of the pH buffer in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
2. The cryoprotectant as described in claim 1, characterized in that, The sugar protectant is selected from the group consisting of monosaccharides and disaccharides.
3. The cryoprotectant as described in claim 2, characterized in that, The sugar protectant satisfies one or more of the following characteristics: The monosaccharide includes one or more of glucose and galactose; The disaccharide includes one or more of trehalose and sucrose.
4. The cryoprotectant according to any one of claims 1-3, characterized in that, The pH buffer is selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, citrate, phosphate, acetate and borate.
5. The cryoprotectant according to any one of claims 1-4, characterized in that, The zwitterion donor satisfies one or more of the following characteristics: The zwitterion donor includes betaine; The concentration of the zwitterionic donor in the cryoprotectant is 5wt%-10wt%.
6. The cryoprotectant according to any one of claims 1-5, characterized in that, The cryoprotectant also contains polyols.
7. The cryoprotectant as described in claim 6, characterized in that, The polyol satisfies one or more of the following characteristics: The polyols are selected from the group consisting of glycerol, mannitol, sorbitol, xylitol, inositol and calendula alcohol; The concentration of the polyol in the cryoprotectant is 1 g / L to 1.5 g / L.
8. The cryoprotectant according to any one of claims 1-7, characterized in that, The cryoprotectant also contains a surfactant.
9. The cryoprotectant as described in claim 8, characterized in that, The surfactant satisfies one or more of the following characteristics: The surfactant is selected from the group consisting of Triton 100, Tween 20, Tween 80 and S9; The surfactant accounts for 0.01 wt% to 0.05 wt% of the mass percentage of the cryoprotectant.
10. The cryoprotectant according to any one of claims 1-8, characterized in that, The cryoprotectant also contains Na. + .
11. The cryoprotectant as described in claim 10, characterized in that, The Na + The concentration of the cryoprotectant is 10mM-100mM.
12. The cryoprotectant according to any one of claims 1-11, characterized in that, The Mg 2+ It is derived from one or more of MgCl2 and MgSO4.
13. The cryoprotectant according to any one of claims 1-12, characterized in that, The cryoprotectant comprises: 1) 1wt%-10wt% sucrose, Tris, 10mM-100mM NaCl, 2mM MgCl2, 5wt%-10wt% betaine, 0.01wt%-0.05wt% Tween and aqueous solvent; The mass percentage refers to the mass percentage of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM; 2) 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, 1 g / L-1.5 g / L glycerol, and an aqueous solvent; wherein the mass percentages are the mass percentages of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM; or, 3) 1 wt%-10 wt% sucrose, Tris, 10 mM-100 mM NaCl, 2 mM MgCl2, 5 wt%-10 wt% betaine, 0.01 wt%-0.05 wt% Tween, 1 g / L-1.5 g / L glycerol and an aqueous solvent; wherein the mass percentage is the mass percentage of the cryoprotectant, and the concentration of Tris in the cryoprotectant is greater than 0 and less than or equal to 100 mM.
14. The cryoprotectant according to any one of claims 1-13, characterized in that, The pH value of the cryoprotectant is 6 to 8.
15. A viral preparation, characterized in that, It contains a cryoprotectant as described in any one of claims 1-14, and a virus.
16. The viral preparation as claimed in claim 15, characterized in that, The concentration of the virus in the viral preparation is 1e10 IFU / mL-2e12 IFU / mL.
17. A method for freezing viruses, characterized in that, The method includes: The virus to be frozen is mixed with the cryoprotectant as described in any one of claims 1-14 to obtain a viral preparation; The viral preparation is frozen.
18. The method as described in claim 17, characterized in that, The concentration of the virus in the viral preparation is 1e10 IFU / mL-2e12 IFU / mL.
19. The method as described in claim 17 or 18, characterized in that, The freezing process must be repeated at least once.
20. The method according to any one of claims 17-19, characterized in that, The freezing conditions are to freeze at a temperature below -10°C for at least 0.5 hours.