Phosphate buffer stable at ph in hypothermic conditions, process for its preparation and use

Abstract

The application discloses a phosphate buffer stable in pH under an ultralow-temperature environment, a preparation method and application thereof. The buffer uses water as a solvent, and solutes include a phosphate component, an alkaline amino acid protective agent component and an osmotic pressure regulator component. The phosphate component includes hydrogen phosphate and dihydrogen phosphate, and the total concentration is 0.5-5 mmol / L, preferably 1-2.38 mmol / L. The alkaline amino acid protective agent is selected from at least one of histidine, arginine and lysine, and the concentration is 0.2-1 mmol / L. The buffer effectively inhibits the pH deviation caused by the selective precipitation of hydrogen phosphate during ultralow-temperature freezing by reducing the phosphate concentration and adding the alkaline amino acid protective agent, so that the pH of the buffer is maintained in a neutral range during the cooling and freezing process and the freezing preservation period, and the buffer is suitable for the ultralow-temperature preservation of biological samples such as cells, tissues, proteins, nucleic acids, virus vectors, exosomes and nanoliposomes.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a pH-stable phosphate buffer solution under ultra-low temperature conditions, its preparation method, and its uses. Background Technology

[0002] Phosphate-buffered saline (PBS) is a widely used buffer system in the biotechnology field, commonly used in cell culture, biological sample preservation, and protein solution preparation. In cryopreservation applications, PBS is typically used as the base solvent for cryopreservation solutions. A standard PBS has a total phosphate concentration of approximately 9.53 mmol / L, and at room temperature, the pH is maintained within the range of 7.2-7.4 through a buffer pair of hydrogen phosphate and dihydrogen phosphate.

[0003] However, traditional phosphate buffer solutions exhibit a severe pH shift when frozen at ultra-low temperatures ranging from -60°C to -80°C. Because the solubility of disodium hydrogen phosphate decreases significantly with decreasing temperature, it preferentially crystallizes out before sodium dihydrogen phosphate, leading to a sharp increase in the hydrogen ion concentration in the solution and a significant drop in pH from 7.4 at room temperature to approximately 4.0. This pH shift causes irreversible damage to cell membrane integrity, protein spatial structure, and nucleic acid stability, significantly reducing the resuscitation activity of frozen samples.

[0004] CN113519504A discloses a serum-free and protein-free cryopreservation solution for direct liquid nitrogen cryopreservation, including a PBS buffer containing osmotic protectants, cell membrane protectants, and cell stabilizers, and additives. However, this patent still uses conventional concentrations of PBS as the base buffer, failing to address the pH shift problem caused by hydrogen phosphate precipitation at ultra-low temperatures. CN113170778B discloses a cell cryopreservation solution and its preparation method and application, including mannose-erythritol ester, trehalose, and phosphate buffer. Although a cryoprotectant is added, the composition and concentration of the phosphate buffer are not optimized for ultra-low temperature pH stability, and there is still a risk of significant pH shift during ultra-low temperature freezing.

[0005] In addition, existing methods for detecting the pH value of solutions in ultra-low temperature environments usually require the frozen sample to be warmed to room temperature before being measured with a pH meter. It is not possible to directly observe the pH value in the frozen state, which is cumbersome and may lead to inaccurate measurement results due to changes in chemical equilibrium during the warming process.

[0006] Tris buffer does not undergo pH shift under cryogenic freezing conditions, maintaining solution pH stability. This characteristic makes it a common reference system in cryopreservation research. However, the effective buffering range of Tris buffer is alkaline, with commonly prepared pH values ​​typically above 7.5, which does not meet the physiological pH range (7.2-7.4) required for the preservation of most biological samples. Furthermore, Tris buffer requires relatively high concentrations to achieve effective buffering. In addition, Tris is not a naturally occurring buffering component in the human body and may inhibit certain enzymes, posing some biocompatibility issues and limiting its use in clinical applications such as in vivo drug delivery and cell therapy. In contrast, phosphate buffer systems are naturally present in various human body fluids (such as blood and intracellular fluid) and are an important component of the human physiological buffer system. They offer superior biocompatibility and safety, are suitable for various routes of administration, and have wider applicability in the cryopreservation of biological products such as cell therapy, protein drugs, and nucleic acid drugs.

[0007] Therefore, there is an urgent need to develop a phosphate buffer system specifically designed for ultra-low temperature environments that can effectively maintain pH stability, and to provide a simple and reliable method for ultra-low temperature pH detection. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a pH-stable phosphate buffer solution under ultra-low temperature conditions, its preparation method, and its uses.

[0009] To achieve the above objectives, the present invention provides the following technical solution: One aspect of the present invention provides a pH-stable phosphate buffer solution under ultra-low temperature conditions. The solvent of the buffer solution is water, and the solute comprises a phosphate component, a basic amino acid protectant component, and an osmotic pressure regulating component. The phosphate component is selected from any one or more of hydrogen phosphate or dihydrogen phosphate. The basic amino acid protectant is selected from any one or more of histidine, arginine, and lysine. The total concentration of the phosphate component is 0.5-5 mmol / L, and the concentration of the basic amino acid protectant component is 0.2-1 mmol / L.

[0010] Furthermore, the hydrogen phosphate is selected from any one or two of Na2HPO4 and K2HPO4, and the dihydrogen phosphate is selected from any one or two of KH2PO4 and NaH2PO4.

[0011] Furthermore, the buffer solution also contains an osmotic pressure regulating component, such that the osmotic pressure of the buffer solution is 200-400 mOsm / L.

[0012] Furthermore, the osmotic pressure regulating component is selected from any one or two of NaCl and KCl.

[0013] Furthermore, the total concentration of the phosphate component is 1-2.38 mmol / L.

[0014] Furthermore, the concentration of the basic amino acid protective agent component is 0.2-0.5 mmol / L or 0.5-1 mmol / L.

[0015] Further, the ultra-low temperature refers to -60℃ to -80℃; the pH stability refers to the pH value being maintained between 6.5 and 7.4. Preferably, the pH value is maintained between 7.2 and 7.4.

[0016] Another aspect of the present invention provides a method for preparing the above-mentioned buffer solution, the method comprising the following steps: 1) Obtain an aqueous solution containing phosphate components; 2) Add osmotic pressure regulating components; 3) Add the alkaline amino acid protective agent component under stirring conditions; 4) Adjust the pH value to 7.0-7.4; 5) Use water to bring the volume to the target level, then filter and sterilize using a filter membrane.

[0017] Another aspect of the present invention provides a product comprising the above-described buffer solution.

[0018] Furthermore, the product may be a kit containing the aforementioned buffer solution, and the kit may be used for the following purposes: cell cryopreservation, tissue preservation, protein preservation, nucleic acid preservation, viral vector preservation, exosome preservation, or nanoliposome preservation.

[0019] Another aspect of the present invention provides applications of the above-mentioned buffer solution, the applications being selected from any of the following: cell cryopreservation, tissue preservation, protein preservation, nucleic acid preservation, viral vector preservation, exosome preservation, or nanoliposome preservation.

[0020] Compared with the prior art, the beneficial effects of the present invention are: This invention effectively suppresses pH shift caused by selective precipitation of hydrogen phosphate during cryogenic freezing by reducing phosphate concentration and adding alkaline amino acid protectants. This keeps the pH of the buffer solution in the neutral range during the cooling and freezing process and cryopreservation, making it suitable for cryogenic preservation of biological samples such as cells, tissues, proteins, nucleic acids, viral vectors, exosomes, and nanoliposomes. Attached Figure Description

[0021] Figure 1 Fluorescence images of 293T cells infected with lentiviruses in different buffer solutions.

[0022] Figure 2The results are fluorescence analysis of 293T cells infected with lentiviruses in different buffer solutions. Detailed Implementation

[0023] To address the above problems, the present invention provides a method for preparing a buffer solution, the method comprising the following steps: 1) Obtain an aqueous solution containing phosphate components; 2) Add osmotic pressure regulating components; 3) Add the alkaline amino acid protective agent component under stirring conditions; 4) Adjust the pH value to 7.0-7.4; 5) Use water to bring the volume to the target level, then filter and sterilize using a filter membrane.

[0024] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0025] Unless otherwise specified, all raw materials used in the following examples are commercially available products purchased from Aladdin, Thermo Fisher Scientific, or the Discovery Platform. Unless otherwise specified, all solvents used are ultrapure water. pH values ​​were measured using a Mettler FE pH meter, calibrated and measured at 25°C. The methyl red indicator used for visual pH verification in each example is an added reagent and is not a component of the phosphate buffer solution formulation of this invention. The methyl red indicator colorimetric test was performed visually under frozen conditions: a yellow color at pH not lower than 6.5 indicates neutrality; an orange color at pH 5.5-6.0 indicates weak acidity; and a pink or purplish-red color at pH not higher than 5.0 indicates strong acidity.

[0026] Example 1: Effects of different phosphate concentrations and histidine on ultra-low temperature pH stability This experiment aimed to compare the effects of different phosphate concentrations and the presence or absence of histidine on pH stability under ultra-low temperature conditions, and to verify the synergistic effect of reducing phosphate concentration and adding basic amino acid preservatives. Two control groups and three experimental groups were set up. pH changes were observed by colorimetric testing with methyl red indicator after freezing at -60℃ for 72 hours. The osmotic pressure of the solution was set to approximately 280 mOsm / L.

[0027] Table 1: Formulations of each group in Experiment 1

[0028] All phosphate buffer groups were used with ultrapure water as solvent and filtered through a 0.22 μm filter membrane. Methyl red indicator (1 g / L, 0.5 mL / 10 mL buffer) was added to each group, and the mixture was sealed and frozen at -60°C or below for 72 hours. The color was observed and photographed immediately after the samples were removed.

[0029] Table 2: Methyl red color development results of each group of buffer solutions in Experiment 1 after being frozen at -60℃ for 72 hours.

[0030] Table 2 shows that control group 1 (Tris buffer) turned yellow after being frozen at -60℃ for 72 hours, and the pH remained in the neutral range, verifying the characteristic that Tris buffer does not undergo pH shift at ultra-low temperatures. Control group 2 (DPBS, 9.53 mmol / L phosphate) turned purple-red, and the pH dropped to the strongly acidic range (≤ 5.0), consistent with the phenomenon of significant pH drop in traditional phosphate buffers at ultra-low temperatures reported in the literature. Experimental group 1 (9.53 mmol / L phosphate + 1 mmol / L histidine) also turned purple-red, with no significant difference from control group 2, indicating that under high phosphate concentration (9.53 mmol / L), simply adding 1 mmol / L histidine is insufficient to effectively inhibit the large amount of hydrogen phosphate precipitation during ultra-low temperature freezing and the resulting sharp pH drop. Experimental group 2 (phosphate 2.38 mmol / L, no amino acids) showed a pale orange color and a weakly acidic pH (5.5-6.0), slightly better than control group 2, but still not reaching the neutral range. This indicates that simply reducing the phosphate concentration to 2.38 mmol / L can alleviate the pH shift to some extent, but the effect is insufficient. Experimental group 3 (phosphate 2.38 mmol / L + histidine 1 mmol / L) showed a yellow color and maintained a neutral pH (≥ 6.5), significantly better than control group 2 and experimental groups 1 and 2, and close to control group 1 (Tris buffer). This indicates that reducing the phosphate concentration to 2.38 mmol / L and simultaneously adding 1 mmol / L histidine can effectively inhibit the precipitation of hydrogen phosphate during cryogenic freezing, thereby maintaining the solution pH in the neutral range.

[0031] Example 2: Effects of different phosphate concentrations and histidine concentration gradients on ultra-low temperature pH stability This experiment aimed to investigate the effects of different phosphate and histidine concentrations on pH stability during cryopreservation. Building upon Experiment 1, the phosphate concentration gradient was further refined, and the effect of histidine concentration was verified. Two control groups and five experimental groups were set up. pH changes were observed by colorimetric testing with methyl red indicator after 72 hours of cryopreservation at -60°C or below. The osmotic pressure of the solution was set to approximately 280 mOsm / L.

[0032] Table 3: Comparison of formulations in each group of Experiment 2

[0033] The preparation methods for control groups 1 and 2 were the same as those for experiment 1. All phosphate buffer groups used ultrapure water as solvent and were filtered through a 0.22 μm filter membrane before use. Methyl red indicator (1 g / L, 0.1 mL / 10 mL buffer) was added to each group and then sealed and stored in a freezer at -60℃ or below for 72 hours. The color was observed and photographed immediately after being taken out.

[0034] Table 4: Methyl red colorimetric results of each group of buffer solutions in Experiment 2 after being frozen at -60℃ for 72 hours.

[0035] Table 4 shows that both experimental group 1 (phosphate 2.38 mmol / L, no histidine) and experimental group 2 (phosphate 1.92 mmol / L, no histidine) showed a pale orange color and were determined to be weakly acidic (5.5-6.0). There was no significant difference between the two groups, indicating that without the addition of an amino acid preservative, further reducing the phosphate concentration from 2.38 mmol / L to 1.92 mmol / L did not significantly improve pH stability; simply reducing the phosphate concentration was insufficient to maintain a neutral pH at ultra-low temperatures. Experimental group 3 (phosphate 2.38 mmol / L + histidine 1 mmol / L) and experimental group 4 (phosphate 1 mmol / L + histidine 1 mmol / L) both showed a yellow color and were determined to be neutral (≥ 6.5). The results were consistent, indicating that with the addition of 1 mmol / L histidine, the phosphate concentration in the range of 1-2.38 mmol / L had little effect on pH stability; both concentrations could maintain a neutral pH at ultra-low temperatures. Experimental group 5 (1 mmol / L phosphate + 0.5 mmol / L histidine) showed a pale orange color and was determined to be weakly acidic (5.5-6.0). Compared with experimental group 4 (1 mmol / L phosphate + 1 mmol / L histidine, yellow, neutral), this indicates that reducing the histidine concentration from 1 mmol / L to 0.5 mmol / L significantly weakened the pH stabilization effect, suggesting that 1 mmol / L histidine is the lowest concentration threshold for use alone. Combining the results of experiments 1 and 2, the preferred formulation of the phosphate buffer solution of this invention is confirmed to be: 1-2.38 mmol / L phosphate + 1 mmol / L histidine. This combination can effectively maintain the solution pH in the neutral range under ultra-low temperature cryopreservation conditions below -60℃.

[0036] Example 3: Effects of different amino acids and combinations on pH protection capability during cryopreservation This experiment aimed to further investigate the protective effect of different amino acid types and combinations on solution pH during cryopreservation, comparing the effects of histidine, arginine, and lysine alone or in combination, as well as the effect of glucose as a control. Two control groups and nine experimental groups were set up. All experimental groups used 2.38 mM phosphate buffer as a base, and pH changes were observed by colorimetric testing with methyl red indicator after freezing at -60°C for 72 hours. The osmotic pressure of the solution was set at approximately 280 mOsm / L.

[0037] Table 5: Comparison of formulations in each group of Experiment 3

[0038] The preparation methods for control groups 1 and 2 were the same as those for experiment 1. All phosphate buffer groups used ultrapure water as solvent and were filtered through a 0.22 μm filter membrane before use. Methyl red indicator (1 g / L, 0.5 mL / 10 mL buffer) was added to each group and then sealed and stored in a freezer at -60℃ or below for 72 hours. The color was observed and photographed immediately after the samples were taken out.

[0039] Table 6: Methyl red colorimetric results of each buffer group after being frozen at -60℃ for 72 hours

[0040] Table 6 shows that glucose (0.1%) alone or in combination with 0.5 mM histidine did not increase the protective effect: Experimental group 3 (0.1% glucose) showed a purple-red color and a pH of strongly acidic (≤ 5.0), comparable to control group 2 (DPBS), indicating that glucose has no protective effect against pH shift of phosphate buffer at ultra-low temperatures. Experimental group 5 (0.1% glucose + 0.5 mM histidine) showed the same result as experimental group 4 (0.5 mM histidine), both showing a pale pink color and a pH of weakly acidic (close to pH 5.5), further confirming that glucose does not have a synergistic protective effect. 0.2 mM arginine or 0.2 mM lysine alone had no protective effect: Experimental groups 6 (0.2 mM arginine) and 7 (0.2 mM lysine) both showed a purple-red color and a pH of strongly acidic (≤ 5.0), comparable to control group 2 (DPBS), indicating that at this concentration, arginine or lysine alone is insufficient to maintain pH stability at ultra-low temperatures. The combined use of 0.5 mM histidine with 0.2 mM arginine or lysine can enhance the protective effect to a certain extent: both experimental groups 8 (0.5 mM histidine + 0.2 mM arginine) and experimental groups 9 (0.5 mM histidine + 0.2 mM lysine) showed an orange-yellow color and were determined to be weakly acidic (5.5-6.0) at pH 4, which was better than 0.5 mM histidine alone (pale pink, close to pH 5.5, experimental group 4), indicating that the combined use of arginine or lysine with histidine has a certain synergistic protective effect; however, the effect of both was not as good as that of 1 mM histidine alone (experimental group 2, yellow, neutral, pH ≥ 6.5), indicating that increasing the concentration of histidine is more crucial for improving the pH protection effect. Based on the combined results of the three experiments, histidine is the most effective basic amino acid protectant in the phosphate buffer system of this invention. 1 mM histidine can effectively maintain pH stability at ultra-low temperatures at phosphate concentrations ranging from 1 mM to 2.38 mM. Arginine and lysine are ineffective when used alone at low concentrations (0.2 mM), but they can exert a certain synergistic effect when combined with histidine.

[0041] Example 4: Effect of the improved buffer solution on the stability of lentiviral vectors under cryogenic conditions This experiment aimed to verify the protective effect of the improved phosphate buffer (low-concentration phosphate + histidine formulation) of this invention on the preservation of lentiviral vector activity under ultra-low temperature conditions. Conventional DPBS was used as a control, and the infectivity of the lentivirus was assessed by the green fluorescence expression signal after infecting HEK293T cells. The osmotic pressure of the solution was set to approximately 280 mOsm / L.

[0042] Table 7: Comparison of Formulas in Each Group

[0043] Lentiviral viruses simultaneously expressing ZsGreen fluorescent protein were prepared at the same concentration in both the control group (DPBS) and experimental group buffers, with three replicates prepared for each buffer. After preparation, three replicates from each group were used to infect HEK293T cells in 96-well plates. Seventy-two hours post-infection, green fluorescence was observed under a fluorescence microscope, and fluorescence images were captured as baseline data before cryopreservation. The remaining samples were sealed and stored at -60°C or below for 14 days. After 14 days, they were thawed and used to infect HEK293T cells using the same method, and fluorescence signals were detected. The fluorescence images were converted to grayscale using ImageJ for grayscale analysis to compare lentiviral infectivity.

[0044] Experimental results: Comparison of the activity of lentiviruses in infecting HEK293T cells before and after cryogenic storage is shown in the figure. Figure 1 , Figure 2 . Figure 2 In the ns (no significance) range, there was no significant difference. Figures 1-2 As can be seen, before cryopreservation, there was no significant difference in the ZsGreen fluorescence signal after lentivirus infection of HEK293T cells in the control group (DPBS) and the experimental group, proving that the initial infectivity of lentivirus in the two buffers was comparable, thus ruling out the influence of the buffer itself on viral activity. After 14 days of storage in an ultra-low temperature freezer below -60°C, the fluorescence signal after lentivirus infection in the experimental group was significantly higher than that in the control group, proving that the improved buffer of this invention can better maintain the infectivity of lentiviral vectors during ultra-low temperature storage and has a better lentivirus preservation effect. The above results further verify the application value of the buffer of this invention in the field of ultra-low temperature preservation of biological products.

[0045] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A pH-stable phosphate buffer solution under ultra-low temperature conditions, characterized in that, The buffer solution is water as the solvent, and the solute comprises a phosphate component, a basic amino acid protectant component, and an osmotic pressure regulating component; the phosphate component is selected from any one or more of hydrogen phosphate or dihydrogen phosphate; the basic amino acid protectant is selected from any one or more of histidine, arginine, and lysine; the total concentration of the phosphate component is 0.5-5 mmol / L; and the concentration of the basic amino acid protectant component is 0.2-1 mmol / L.

2. The buffer solution according to claim 1, characterized in that: The hydrogen phosphate is selected from any one or both of Na2HPO4 and K2HPO4, and the dihydrogen phosphate is selected from any one or both of KH2PO4 and NaH2PO4.

3. The buffer solution according to claim 1, characterized in that: The osmotic pressure regulating component makes the osmotic pressure of the buffer solution 200-400 mOsm / L.

4. The buffer solution according to claim 1, characterized in that: The osmotic pressure regulating component is selected from any one or two of NaCl or KCl.

5. The buffer solution according to claim 1, characterized in that: The total concentration of the phosphate component is 1-2.38 mmol / L.

6. The buffer solution according to claim 1, characterized in that: The concentration of the alkaline amino acid protective agent component is 0.2-0.5 mmol / L or 0.5-1 mmol / L.

7. The buffer solution according to claim 1, characterized in that: The ultra-low temperature refers to -60℃ to -80℃; the pH stability refers to the pH value being maintained between 6.5 and 7.

4.

8. The method for preparing the buffer solution according to claim 1, wherein the method comprises the following steps: 1) Obtain an aqueous solution containing phosphate components; 2) Add osmotic pressure regulating components; 3) Add the alkaline amino acid protective agent component under stirring conditions; 4) Adjust the pH value to 7.0-7.4; 5) Use water to bring the volume to the target level, then filter and sterilize using a filter membrane.

9. A product for preserving organisms, characterized in that, The product contains the buffer solution as described in claim 1.

10. The application of the buffer solution as described in claim 1, wherein the application is selected from any of the following: cell cryopreservation, tissue preservation, protein preservation, nucleic acid preservation, viral vector preservation, exosome preservation, or nanoliposome preservation.

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