Preparation method of biomarker preservation solution and biomarker lyophilized product

Abstract

The present invention discloses a biomarker preservation solution and a method for preparing a biomarker lyophilized product. The biomarker preservation solution comprises the following raw materials: buffer solution; protein; preservative; polysaccharide; antioxidant; filler; thiol reagent and high molecular surfactant. The thiol reagent and high molecular surfactant of the present invention significantly improve the protective effect of conventional lyophilization protective agents on some small molecule protein markers.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic reagents, and in particular to a method for preparing a biomarker preservation solution and a biomarker freeze-dried product. Background Art

[0002] Biomarkers are a very important component of diagnostic reagents. They contain bioactive substances such as enzymes, antigens, and antibodies. They are particularly sensitive to temperature, very unstable, and easily react with each other. In order to maintain the stability of biomarkers, freeze-drying technology is generally used to freeze-dry biomarkers for preservation. When freeze-drying biomarkers, a preservation solution is added to protect the biomarkers to avoid damage to the biomarkers during the freeze-drying process. Although the current preservation solution can better preserve the large molecular antibody proteins in biomarkers, it has poor protection performance for some small molecule active protein markers. Summary of the invention

[0003] The main purpose of the present invention is to propose a biomarker preservation solution and a method for preparing a biomarker lyophilized product, aiming to solve the problem that although the current preservation solution can better preserve the large molecular antibody protein in the biomarker, it has poor protection performance for some small molecular active protein markers.

[0004] To achieve the above-mentioned purpose, the present invention provides a biomarker preservation solution, which includes the following raw materials: buffer; protein; preservative; polysaccharide; antioxidant; filler; thiol reagent and polymer surfactant.

[0005] Optionally, the polymer surfactant includes at least one of polyvinyl pyrrolidone, hydroxypropyl methylcellulose and polyethylene glycol 20000; and / or the mass concentration of the polymer surfactant is 5 to 25 g / L.

[0006] Optionally, the thiol reagent includes 2-mercaptoethanol; and / or the mass concentration of the thiol reagent is 0.2-1 g / L.

[0007] Optionally, the protein includes casein, bovine serum albumin or whey protein; and / or, the mass concentration of the protein is 5 to 25 g / L.

[0008] Optionally, the preservative includes at least one of sodium azide, thimerosal, gentamicin and ProClin 300; and / or, the amount of the preservative added is 0.01 to 0.05% of the total mass of the preservation solution.

[0009] Optionally, the polysaccharide includes trehalose; and / or, the mass concentration of the trehalose is 20-80 g / L.

[0010] Optionally, the antioxidant includes ethylenediaminetetraacetic acid; and / or the mass concentration of the antioxidant is 1 to 5 g / L.

[0011] Optionally, the buffer comprises Tris-HCl; and / or the concentration of the buffer is 50 to 150 mmol / L.

[0012] Optionally, the filler comprises amino acids and / or sugar alcohols, wherein the amino acids comprise glycine; and the sugar alcohols comprise mannitol.

[0013] The present invention provides a method for preparing a lyophilized product of a biomarker, comprising the following steps:

[0014] S10, diluting the biomarker mother solution with any of the biomarker storage solutions described above to obtain a marker semi-finished product;

[0015] S20, freeze-drying the labeled semi-finished product to obtain a biomarker freeze-dried product.

[0016] In the technical solution provided by the present invention, a thiol reagent and a high molecular surfactant are added to the raw materials. The thiol reagent can prevent some cysteine-containing proteins from being oxidized to subsulfonic acid or disulfide. The high molecular surfactant can change the thermodynamic properties of the solution, thereby enhancing the stability of the protein, having a good stabilizing effect on the non-polar surface of the protein, and inducing the self-polymerization of protein subunits and having the effects of reducing wall adsorption and antifreeze. The thiol reagent and high molecular surfactant of the present invention significantly improve the protective effect of conventional freeze-drying protective agents on some small molecular protein markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention provides a flow chart of the preparation method of the lyophilized product of the biomarker. DETAILED DESCRIPTION

[0018] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0019] Biomarkers are a very important component of diagnostic reagents. They contain bioactive substances such as enzymes, antigens, and antibodies. They are particularly sensitive to temperature, very unstable, and easily react with each other. In order to maintain the stability of biomarkers, freeze-drying technology is generally used to freeze-dry biomarkers for preservation. When freeze-drying biomarkers, a preservation solution is added to protect the biomarkers to avoid damage to the biomarkers during the freeze-drying process. Although the current preservation solution can better preserve the large molecular antibody proteins in biomarkers, it has poor protection performance for some small molecule active protein markers.

[0020] In view of this, the present invention proposes a biomarker preservation solution, aiming to solve the problem that although the current preservation solution can better preserve the large molecular antibody protein in the biomarker, it has poor protection performance for some small molecular active protein markers.

[0021] The biomarker preservation solution provided by the present invention comprises the following raw materials: buffer solution, protein, preservative, polysaccharide, antioxidant, filler, thiol reagent and high molecular surfactant.

[0022] In the above technical scheme, adding protein to the biomarker preservation solution can inhibit protein unfolding caused by freezing, and by increasing the initial concentration of protein, the tolerance of protein preparations to damage during freezing and thawing can be enhanced; polysaccharides are the most commonly used excipients, because they have many hydroxyl groups in their structure, so they can provide a stabilizing effect by replacing the hydrogen bonds between protein and water. The high viscosity of polysaccharides such as sucrose and trehalose can also reduce the rate of protein denaturation during drying at a temperature above the glass transition temperature (Tg'); thiol reagents can prevent some proteins containing cysteine ​​on the surface from being oxidized to subsulfonic acid or disulfide; the polymer surfactant will change the thermodynamic properties of the solution, so that the stability of the protein is enhanced, and it has a good stabilizing effect on the non-polar surface of the protein, and can also induce the self-polymerization of protein subunits and has the effect of reducing wall adsorption and antifreeze. At the same time, these polymer surfactants can produce steric hindrance between protein molecules and can increase the viscosity of the solution. The present invention adds thiol reagents and polymer surfactants, which significantly improves the protective effect of conventional lyophilization protective agents on some small molecule protein markers.

[0023] Furthermore, the polymer surfactant includes at least one of polyvinyl pyrrolidone, hydroxypropyl methylcellulose and polyethylene glycol 20000; and / or, the mass concentration of the polymer surfactant is 5 to 25 g / L. In the present invention, the polymer surfactant can be polyvinyl pyrrolidone, a mixture of polyvinyl pyrrolidone and hydroxypropyl methylcellulose or other compositions. When the polymer surfactant is used, the mass concentration of the polymer surfactant is limited. For example, it can be 5 g / L, 10 g / L, 15 g / L or 25 g / L. When the mass concentration of the polymer surfactant is lower than 5 g / L, there is not enough polymer surfactant to fully contact with the marker, thereby reducing the protective effect on the protein. When the mass concentration of the polymer surfactant is higher than 25 g / L, due to the excessive amount of the polymer surfactant, some of the polymer surfactants cannot fully contact with the marker, so the effect is not further improved, and adding too much polymer surfactant wastes the material.

[0024] Furthermore, the thiol reagent includes 2-mercaptoethanol; and / or, the mass concentration of the thiol reagent is 0.2-1g / L, 2-mercaptoethanol is also called β-mercaptoethanol. When used, the present invention limits the concentration of 2-mercaptoethanol, for example, it can be 0.2g / L, 0.5g / L, 0.8g / L, 1g / L, 1.5g / L or 2g / L. When the concentration of 2-mercaptoethanol is too low, it cannot fully protect the protein. When the concentration is too high, it will cause the disulfide bonds of the protein to break and the peptide chain to stretch, which will destroy the structure of the protein.

[0025] Furthermore, the protein includes casein, bovine serum albumin or whey protein; and / or, the mass concentration of the protein is 5-25 g / L. Casein, bovine serum albumin and whey protein can inhibit protein unfolding caused by freezing. By increasing the initial concentration of the protein, the tolerance of the protein preparation to damage during the freezing and thawing process can be enhanced. In some embodiments, the concentration of the protein is limited. For example, its concentration can be 5 g / L, 8 g / L, 10 g / L, 15 g / L, 18 g / L, 20 g / L, 22 g / L, 25 g / L. When the protein is too much, it will affect other proteins in the marker, so it should not be too much.

[0026] Furthermore, the preservative includes at least one of sodium azide, thimerosal, gentamicin and ProClin 300; and / or, the amount of the preservative added is 0.01-0.05% of the total mass of the preservation solution. The preservative can inhibit the growth of microorganisms in the solution and extend the preservation time of the preservation solution. In some embodiments, the amount added is 0.01-0.05% of the total mass of the preservation solution, for example, the amount added is 0.01%, 0.02%, 0.04%, or 0.05%.

[0027] Furthermore, the polysaccharide includes trehalose; and / or, the mass concentration of the trehalose is 20-80 g / L. Trehalose can not only be used as an excipient, but its high viscosity can also reduce the protein denaturation rate during drying at a temperature higher than the glass transition temperature (Tg'). In some embodiments, the mass concentration of trehalose is 20-80 g / L, for example, it can be 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L. The higher the content of polysaccharide, the better the protection of protein. Too much polysaccharide will cause the overall viscosity of the preservation solution to be too high, which is not conducive to its own preservation.

[0028] Furthermore, the antioxidant includes ethylenediaminetetraacetic acid; and / or the mass concentration of the antioxidant is 1 to 5 g / L. Ethylenediaminetetraacetic acid is a commonly used antioxidant that can delay the oxidation of the preservation solution.

[0029] Furthermore, the buffer solution includes Tris-HCl; and / or the concentration of the buffer solution is 50 to 150 mmol / L, and the buffer solution can be used to maintain the acid-base balance of the preservation solution.

[0030] Further, the filler includes amino acids and / or sugar alcohols, wherein the amino acids include glycine; the sugar alcohols include mannitol. In some embodiments, amino acids such as glycine are added. Glycine has advantages when used as a filler because it is non-toxic, has a high eutectic / eutectic temperature (Teu), and can achieve efficient freeze-drying and high solubility. The mass concentration range of amino acids is 2-10 g / L, and the mass concentration range of sugar alcohols is 1-5 g / L.

[0031] The present invention provides a method for preparing a lyophilized product of a biomarker, comprising the following steps:

[0032] S10, diluting the biomarker mother solution with the biomarker preservation solution to obtain a marker semi-finished product;

[0033] S20, freeze-drying the labeled semi-finished product to obtain a biomarker freeze-dried product.

[0034] Specifically, in some implementations, freeze drying is performed by a freeze dryer, and the specific conditions for freeze drying are: the marker semi-finished product is precooled at a temperature of -80 to -60°C for 5 to 8 hours; after the vacuum freeze dryer is precooled to -60°C to -45°C, it is dried once, and the freeze dryer is simultaneously evacuated to 1 to 30 Pa, and enters a sublimation program for a duration of 1 to 3 hours to complete the first drying, and then gradually warms to -30 to -20°C, and keeps warm at this temperature for 2 to 5 hours; then warms to 0°C, and keeps warm at this temperature for 2 to 8 hours; warms to 10 to 15°C within 1.5 to 2 hours, and keeps warm at this temperature for 1 to 2 hours; warms to 25°C within 1 to 2 hours, and keeps warm at this temperature for 3 to 8 hours, to obtain a freeze-dried product of the biomarker.

[0035] By adopting the above technical solution, the labeled semi-finished product can be preserved by freeze-drying, which can extend its preservation time. In addition, since thiol reagents and high molecular surfactants are added to the preservation solution, the protection effect on some small molecule proteins is improved.

[0036] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0037] The types and contents of the components of the biomarker preservation solutions of Examples 1-6 and Comparative Examples 1-3 are shown in Table 1.

[0038] Table 1

[0039]

[0040]

[0041] Note: ProClin 300 weight is a percentage of the total preservation solution weight.

[0042] Performance testing and results

[0043] The intrinsic factor protein and folate binding protein biomarkers were respectively placed in the preservation solution prepared by Examples 1-6 and Comparative Examples 1-3 in Table 1 to obtain semi-finished marker products, which were then freeze-dried. The specific steps of freeze-drying were as follows: the semi-finished marker products were pre-cooled at a temperature of -80 to -60°C for 5 to 8 hours; after the vacuum freeze dryer was pre-cooled to -60°C to -45°C, they were dried once, and the freeze dryer was simultaneously evacuated to 1 to 30 Pa, and entered the sublimation program for a duration of 1 to 3 hours to complete the first drying, and then gradually heated to -30 to -20°C, and kept at this temperature for 2 to 5 hours; then heated to 0°C, and kept at this temperature for 2 to 8 hours; heated to 10 to 15°C within 1.5 to 2 hours, and kept at this temperature for 1 to 2 hours; heated to 25°C within 1 to 2 hours, and kept at this temperature for 3 to 8 hours, to obtain biomarker freeze-dried products 1-9, respectively. The biomarker lyophilized products 1-9 were tested by dry immunoassay analyzer, and the results are shown in Table 2.

[0044] Table 2

[0045]

[0046] Note: In Table 2, acceleration refers to the effect after one week of accelerated placement.

[0047] It can be seen from Table 2 that adding thiol reagents and high molecular surfactants to the preservation solution can effectively improve the protection effect on some small molecule proteins.

[0048] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A biomarker preservation solution, characterized in that: It is composed of the following raw materials: buffer, protein, preservative, polysaccharide, antioxidant, filler, thiol reagent and high molecular surfactant; The buffer is Tris-HCl with a concentration of 50 to 150 mmol / L; The protein is casein with a mass concentration of 5 to 25 g / L; The preservative is ProClin 300 with a mass fraction of 0.01 to 0.05%; The polysaccharide is trehalose with a mass concentration of 20 to 80 g / L; The antioxidant is ethylenediaminetetraacetic acid with a mass concentration of 1 to 5 g / L; The filler is composed of glycine with a mass concentration of 2 g / L, or of glycine with a mass concentration of 8 to 10 g / L and mannitol with a mass concentration of 3 to 5 g / L; The polymer surfactant is polyvinyl pyrrolidone with a mass concentration of 5 to 27 g / L; The thiol reagent is 2-mercaptoethanol with a mass concentration of 0.1 to 2 g / L.

2. A method for preparing a lyophilized product of a biomarker, characterized in that: The following steps are involved: S10, diluting the biomarker mother solution with the biomarker preservation solution of claim 1 to obtain a marker semi-finished product; S20, freeze-drying the labeled semi-finished product to obtain a biomarker freeze-dried product.

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