High-concentration dNTP monomer solution and preparation process thereof

Abstract

The invention relates to the technical field of preparation of high-concentration dNTP, in particular to a high-concentration dNTP monomer solution with the concentration larger than 100 mM and a preparation technology of the high-concentration dNTP monomer solution. The method comprises the following steps: freezing a dNTP monomer solution with an initial concentration of less than or equal to 100mM into a solid at a temperature at which the dNTP monomer solution can be frozen into the solid by adopting a freeze-thaw method, then standing and dissolving under the condition of not higher than 30 DEG C, and taking a solution with the height less than half of the height of a container, so as to obtain a dNTP monomer aqueous solution with the concentration greater than the initial concentration. According to the application, the dNTP refers to dATP, dCTP, dTTP, dGTP and dUTP. The prepared high-concentration dNTP monomer aqueous solution is good in stability, and the method is simple, convenient and easy to operate.

Description

Technical Field

[0001] The present application relates to the technical field of compound purification, and specifically designs a method for preparing a high-concentration deoxynucleoside triphosphate monomer solution by utilizing freeze-thaw technology. Background Art

[0002] The basic substance of DNA synthesis is deoxyribonucleoside triphosphate (dNTP). According to its structure, dNTP can be divided into dATP (2'-deoxyadenosine 5'-triphosphate), dCTP (2'-deoxycytidine-5'-triphosphate), dTTP (2'-deoxythymidine-5'-triphosphate), dGTP (2'-deoxyguanosine-5'-triphosphate), dUTP (2'-deoxyuridine-5'-triphosphate). Artificially synthesized DNA fragments have been widely used in life science fields such as genetic engineering and molecular biology (CN102168123A). Deoxynucleoside triphosphate (dNTP) has various uses in the fields of biochemistry and molecular biology, such as PCR polymerase chain reaction and IVT in vitro transcription, which require high-purity deoxynucleoside triphosphate monomers as raw materials. At present, most of the dNTP aqueous solutions sold on the market are 10mM to 100mM. This may be because there are currently two methods for preparing dNTP aqueous solutions. One is to prepare directly through high-pressure preparative liquid phase, and then concentrate with nanofiltration membrane to directly obtain dNTP aqueous solution products. However, after the concentration of dNTP aqueous solution prepared by rolled nanofiltration membrane reaches about 100mM, it cannot be further concentrated. In the process of nanofiltration, there will be different degrees of loss depending on the retention rate, resulting in less total material and significantly increased costs. Another method is to freeze-dry the high-purity dNTP aqueous solution prepared by high-pressure preparative liquid phase to obtain dNTP freeze-dried powder, and then prepare it into a dNTP aqueous solution of corresponding concentration according to needs when using it. Generally speaking, the stability of compound aqueous solutions is lower than that of solids, such as the stability of lyophilized powders. What is strange is that the stability of dNTP lyophilized powders is lower than that of dNTP aqueous solutions during the preparation and storage process. For example, test data shows that the purity of dNTP lyophilized powders decreased by about 2% after being placed at -20°C for one month, and the impurities produced include deoxycytidine diphosphate and deoxycytidine tetraphosphate. Therefore, what is sold on the market are dNTP aqueous solutions with a concentration of less than 100mM. Therefore, preparing dNTP aqueous solutions with a concentration greater than 100mM while ensuring purity has long been a technical bottleneck that has been difficult to break through in this field. Summary of the invention

[0003] The high concentration dNTP monomer aqueous solution described in this application has two meanings. One is that the high concentration dNTP The concentration of the monomer aqueous solution is greater than or equal to 100 mM. Secondly, the concentration of the dNTP monomer aqueous solution after freezing and thawing is higher than the initial concentration. The unsaturated dNTP monomer aqueous solution is also called the initial dNTP aqueous solution, and its concentration is also called the initial concentration.

[0004] The dNTP monomers described in this application refer to dATP, dCTP, dTTP, dGTP, and dUTP.

[0005] In order to solve the above technical problems, the present application first provides an aqueous solution with a concentration greater than or equal to 100 mM. The dNTP monomer aqueous solution with a concentration greater than or equal to 100 mM is obtained by freezing and thawing a dNTP monomer aqueous solution less than 100 mM; more specifically, the dNTP monomer aqueous solution with a concentration less than 100 mM is placed at a temperature that allows it to be frozen into a solid and is frozen into a solid. The solid formed by freezing the dNTP monomer aqueous solution is called dNTP-H 2 O solid, the unsaturated dNTP aqueous solution is also called the initial dNTP aqueous solution, and its concentration is also called the initial concentration, and then 2 After standing at a temperature at which the solid melts, an aqueous dNTP solution having a concentration greater than the initial concentration is obtained.

[0006] It can be expanded that if we want to store or obtain an aqueous solution with a higher concentration than the initial concentration simply, quickly and at a low cost, we can choose to obtain it by freeze-thawing, just like obtaining a dNTP aqueous solution with a concentration greater than or equal to 100mM, the dNTP monomer aqueous solution with a concentration less than 100mM can be placed below 0°C, and in order to improve efficiency, it can be frozen into a solid at below -10°C, and then left to melt in an environment where it can be restored to a dNTP aqueous solution. Such freezing temperature may vary with changes in the environment or the concentration of the dNTP monomer aqueous solution, or we can further improve the efficiency of obtaining solid dNTP-H 2 O speed, choose a lower temperature, such as -20℃, -50℃, -80℃ or even lower. Similarly, make dNTP-H 2 The temperature at which the solid is allowed to melt may also vary depending on the environment or initial concentration. For example, we can choose 0°C, 10°C, room temperature or higher, as long as we ensure that no unwanted dNTP decay reactions occur during the melting process. That is, as long as such a temperature ensures that the icy dNTP aqueous solution can be restored to a liquid state without other decay reactions occurring, the preferred melting temperature is 0-37°C.

[0007] The applicant believes that such a description is understandable to those skilled in the art, that is, those skilled in the art can obtain a dNTP monomer aqueous solution with a concentration higher than the initial concentration, or conveniently store the dNTP monomer aqueous solution during use without creative work. Although the present application is a method obtained by the inventor based on the preparation of a dNTP monomer aqueous solution with a concentration greater than or equal to 100 mM, it has been proved by experiments that it is feasible within the concentration range of 10 mM to 350 mM.

[0008] Although the method of the present application is conventional, it is not easy to achieve. This is because the characteristics of the aqueous solution of dNTP monomers are contrary to common sense. In the pharmaceutical field, it is a common practice to make drugs that are unstable in water into freeze-dried powder or other solid states. It is convenient to transport and store liquids into solids. Compared with the shelf life of liquids or solutions, solids usually have a longer shelf life. However, dNTP is an exception.

[0009] The inventors of the present application also discovered another characteristic of the dNTP monomer aqueous solution during the experiment, that is, after freezing and thawing, the aqueous solution is a homogeneous liquid in the concentration gradient, not a heterogeneous phase. Especially after freezing and thawing, in the specific experiment, we have selected multiple concentrations such as 20mM, 50mM, 80mM, 100mM and the like for the experiment. After freezing and thawing, the concentration of the dNTP monomer aqueous solution increases uniformly from the top to the bottom of the container, and the liquid surface concentration is 10mM-20mM. After two-thirds of the liquid surface height, the concentration must be greater than the initial concentration, and the bottom layer concentration is 300mM-350mM. It should be noted that the concentration of the dNTP monomer aqueous solution is close to 350mM, which is near saturation, and there will be no solid precipitation, and it is a uniform liquid.

[0010] Based on the above exceptions, the applicant infers that this is the reason why those skilled in the art have not broken through the bottleneck of dNTP aqueous solution products with a concentration greater than 100 mM for a long time.

[0011] According to the experimental tests of multiple initial concentrations and multiple freeze-thaw heights, it is known that from the liquid surface to the bottom of the solution, the concentration after half of the liquid surface height must be greater than the initial concentration. It is worth noting that such a gradient distribution is distributed from top to bottom in a regular pattern from low to high concentration. Therefore, if you want to obtain a relatively large volume of dNTP monomer aqueous solution with a concentration higher than the initial concentration at one time, you can use the law of conservation of energy combined with testing to obtain it. For example: For example, the total volume of the dNTP monomer aqueous solution with the initial concentration is called V, and the concentration is called C 初 The volume of the dNTP monomer aqueous solution taken out from the top of the container after freezing and thawing is called V 检 After mixing, the uniform concentration is called C 检 ; Take out V 检The remaining volume is called V 剩 The remaining solution is mixed to a uniform concentration called C 剩 According to the principle of conservation of matter, C 初 *V=C 检 ×V 检 +C 剩 ×V 剩 , where C 初 , V, C 检 、V 检 , C 剩 and V 剩 can be measured, so if you want to get a dNTP monomer aqueous solution with a concentration greater than the initial concentration, you want C 剩 Greater than C 初 For example, if C is initially 80 mM, the volume of the upper layer solution taken out after freeze-thawing is half of the total volume, that is, V 检 and V 剩 Both are half V, C 检 is 30 mM, so C 剩 Equal to (80mM×V-30mM×1 / 2V)÷1 / 2V=130mM greater than C 初 Of course, if you want to obtain a larger volume of dNTP monomer aqueous solution with a concentration greater than the initial concentration, you can also repeatedly freeze and thaw or take out the upper solution in small amounts multiple times to test more points.

[0012] The present invention adopts a freeze-thaw method to freeze a dNTP monomer solution with an initial concentration of less than or equal to 100 mM into a dNTP-H 2 O solidifies at a temperature that allows dNTP-H 2 The dNTP monomer aqueous solution having a concentration greater than the initial concentration can be obtained by standing and melting at a temperature at which the solid melts, especially at a temperature not higher than 30°C, and taking a solution less than half the height of the container. For example, a dNTP monomer aqueous solution greater than 100 mM, which is difficult to obtain with the current existing technology, can be selected based on the absorbance value and volume.

[0013] The method for preparing a high-concentration dNTP monomer aqueous solution provided in the present application is simple and easy to operate, especially a dNTP aqueous solution with a concentration greater than 100 mM, or even a dNTP monomer aqueous solution with a concentration as high as 300 mM, which provides a fast, simple and easy method for experiments requiring a high-concentration dNTP monomer aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Attached Figure 1 Synthesis process 100mM aqueous solution of dCTP, HPLC purity spectrum at T0 point; Attached Figure 2 Synthesis process HPLC spectrum of 100mM aqueous solution of dCTP after being frozen at -20℃ for two months and then allowed to thaw; Attached Figure 3 HPLC purity spectrum of dCTP lyophilized powder produced by synthetic process at T0 point; Attached Figure 4 HPLC purity profile of dCTP lyophilized powder produced by synthetic process after freezing at -20℃ for two months; Attached Figure 5 HPLC purity spectrum of dCTP lyophilized powder produced by fermentation process at T0 point; Attached Figure 6 HPLC purity profile of dCTP lyophilized powder produced by fermentation process after being frozen at -20℃ for two months; Attached Figure 7 dCTP diphosphate impurity LCMS spectrum; Attached Figure 8 LCMS spectrum of dCTP tetraphosphate impurity. DETAILED DESCRIPTION

[0015] The specific embodiments are merely explanations of the present application and are not limitations of the present application. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, as long as they are within the scope of the claims of the present application, they are protected by the patent law.

[0016] In order to clearly and briefly illustrate the implementation scheme of the present application, the present application takes one or two examples of a certain type with similar results as representatives. For example, in the present application, dNTP can be divided into dATP, dCTP, dTTP, dGTP, and dUTP. The impurities they produce are of the same type, which are the corresponding diphosphate and tetraphosphate impurities. Therefore, the present application takes dCTP as an example for explanation.

[0017] For example, the long-term stability comparison of dCTP aqueous solution and dCTP lyophilized powder represents the stability of all dNTP monomer aqueous solutions. The specific example of the long-term stability comparison of dCTP aqueous solution and dCTP lyophilized powder only takes the data of freezing at -20°C for two months as an example to explain why dCTP aqueous solution and dCTP lyophilized powder are unstable under these conditions and what kind of impurities will be generated. By comparison, it can be seen that the degradation products of dCTP aqueous solution and dCTP lyophilized powder are the same, both of which are deoxycytidine diphosphate and / or deoxycytidine tetraphosphate, but the purity of dCTP aqueous solution is basically stable and has not decreased significantly, while the deoxycytidine diphosphate impurity produced by dCTP lyophilized powder is obvious, and its purity has been significantly reduced. In addition, during the preparation of freeze-dried powder from dCTP solution, due to the low vacuum degree, its degradation impurities include not only deoxycytidine diphosphate but also deoxycytidine tetraphosphate. It is speculated that the reason may be that when deoxycytidine triphosphate is degraded into deoxycytidine diphosphate, a molecule of phosphate is released. This active free phosphate can react with deoxycytidine triphosphate under high vacuum to eventually become deoxycytidine tetraphosphate.

[0018] For another example, the influence of freezing temperature, by examining that dNTP monomer aqueous solutions all have the same characteristics as dCTP, that is, after being frozen into a solid, they melt again, and the concentration gradient increases from the liquid surface to the bottom of the container. By examining that the temperature at which the dNTP monomer aqueous solution can be frozen into a solid is similar to pure water, 0°C is the critical point, and at this temperature, the dNTP monomer aqueous solution can be frozen into a solid, the difference is that the freezing time is different, the lower the temperature, the longer the freezing time. Since those skilled in the art can implement the freezing step without any labor, only the above investigation results of the freezing temperature are described here, and no other details are given.

[0019] Another example is the effect of melting temperature. Between 0-37°C, dNTP-H 2 After the O solid is melted into an aqueous solution, the purity of the melted dNTP solution will not decrease, but if it is left at 37° C. for one day after melting, the purity of the dNTP solution will decrease by about 1%. Those skilled in the art can select the storage conditions after melting as needed.

[0020] Each example or comparative example of the present application has at least 3 parallel samples, and the average value is taken, that is, each data has statistical significance.

[0021] The liquid phase conditions for dCTP purity detection in this application are as follows: Example 1 Effect of different container volumes and melting temperatures on the purity of dCTP aqueous solution The dCTP aqueous solution with a purity of 99.73% and a concentration of 100 mM was frozen as dCTP-H 2 O solid, in order to investigate the influence of containers of different volumes, a small bottle of 100mL (10cm in height) and a large bottle of 1L (22cm in height) were used to hold the corresponding volume of solution, respectively, and placed in a -10℃ refrigerator overnight to freeze and freeze. After being taken out and naturally placed until all ice cubes become liquid, about 100uL of the upper, middle and lower layers of solution were immediately taken with a pipette to test the absorbance, and then their concentrations were calculated. The results showed that the concentration of the upper layer solution could be as low as about 10mM, and the concentration of the lower layer solution could be as high as about 350mM, forming a very obvious concentration gradient. Moreover, as the bottle height increases, the concentration of the upper layer solution shows a decreasing trend, but the concentration of the bottom layer solution remains basically unchanged. The samples melted at 10℃, 25℃ and 37℃, respectively. Each melting temperature and each sample of the same volume were grouped into three. After melting, the purity and the concentration of the page layer, the half-height layer and the bottom layer were detected. The conditions and calculation method for the dCTP concentration detection in this embodiment are as follows: The test wavelength of dCTP is 272nm, and the molar absorption coefficient at this wavelength is 9.1mM -1 ·cm, take a small sample of 100uL, dilute it 2000 times, and calculate the absorbance of the UV 1cm test according to the formula of concentration = absorbance * dilution factor / molar absorption coefficient / optical path. The molar concentration results of the calculated solutions are shown in Tables 1-3.

[0022] Table 1 Standing melting temperature is 10°C Table 2 Standing melting temperature is 25°C Table 3 Standing melting temperature is 37°C According to Table 1-3, we can draw the following conclusions: 1. All purity values ​​in Tables 1-3 are not significantly different from the initial purity, indicating that dNTPs will not degrade or decay when left to melt in the range of 0-37°C.

[0023] 2. The concentration test results in Table 1-3 show that the concentration of the upper solution can be as low as about 10mM, and the concentration of the lower solution can be as high as about 350mM, forming a very obvious concentration gradient. Moreover, as the bottle height increases, the concentration of the upper solution shows a decreasing trend, but the concentration of the bottom solution remains basically unchanged.

[0024] 3. Method for obtaining dCTP with a concentration greater than the initial concentration: Taking the large bottle in Table 1 as an example, C 初The upper layer solution was 100 mM. After freezing and thawing, the volume of the upper layer solution was half of the total volume. The absorbance value was 0.166 and the calculated concentration was 36.484 mM, that is, V 检 and V 剩 Both are half V, C 检 is 36.484 mM, so the concentration of the remaining half of the bottom layer V is (100 mM × V - 36.484 mM × 1 / 2 V) ÷ 1 / 2 V = 163.516 mM, which is greater than C 初 100mM, after mixing at the bottom layer, the absorbance value was 0.735, and the calculated concentration was 161.538mM. The difference between the theoretical and actual test values ​​was less than 2%, which means that the theoretical and actual test values ​​were basically consistent.

[0025] Example 2 Effect of initial concentration This example uses dCTP with initial concentrations of 10 mM and 161 mM for verification. The conditions and calculation method for concentration detection are as follows: The test wavelength of dCTP is 272 nm, and the molar absorption coefficient at this wavelength is 9.1 mM -1 ·cm, take a small sample of 100uL, dilute 2, the absorbance and calculated concentration of UV 1cm test are shown in Table 4, the calculation formula concentration = absorbance * dilution factor / molar absorption coefficient / optical path, after calculating the molar concentration of the top and bottom solutions, see Table 4: source Dilution multiple Test absorbance A Calculate concentration in mM 10mM-DCTP-top layer 20 0.261 0.574 10mM-DCTP-bottom layer 1000 0.305 33.516 161mM-DCTP-top layer 2000 0.264 58.022 161mM-DCTP-bottom layer 2000 1.483 325.934 The following conclusions were drawn from Example 2 and the test results of Example 1: The initial concentration was in the range of 10mM to 161mM, and the test results after freeze-thaw showed that the concentration of the solution from the top layer to the bottom layer was linearly distributed. The lower the initial concentration, the lower the concentration of the top solution and the bottom solution, and the higher the initial concentration, the higher the concentration of the top solution and the bottom solution. The concentration of the top dilute solution after freeze-thaw of the 10mM dilute solution was 0.574mM, and the concentration of the bottom solution was 33.516mM; the concentration of the top dilute solution after freeze-thaw of the 161mM concentrated solution was 58.022, and the concentration of the bottom concentrated solution was 325.934mM.

[0026] It is worth emphasizing that when the concentrated solution becomes more concentrated, for example, the initial concentration is 300mM, after freezing and thawing, the solution at the bottom of the container reaches saturation and no solid will precipitate. The concentration still shows a gradient distribution from low to high from the top to the bottom.

[0027] Example 3 Long-term stability study The fermentation process dCTP freeze-dried powder in this example was purchased from Hangzhou Hunyi Biotechnology Co., Ltd. In order to investigate the stability of dCTP solution and lyophilized powder, the homemade dCTP solution prepared by the synthesis process, the lyophilized powder prepared by the lyophilization of the homemade dCTP solution prepared by the synthesis process, and the purchased lyophilized powder prepared by the fermentation process were placed at -20°C for 2 months. 2 The solid was allowed to stand and melt, and the lyophilized powder was dissolved in pure water to prepare a 5 mM aqueous solution and then tested for purity changes.

[0028] Synthesis process dCTP 100mM aqueous solution, T0 point HPLC spectrum purity is 99.73%, see attached Figure 1 HPLC spectrum after 2 months, purity is 99.67%, see attached Figure 2 ; The dCTP aqueous solution produced by the synthesis process was freeze-dried to obtain a freeze-dried powder, and the HPLC spectrum purity at T0 point was 99.24%, see attached Figure 3 HPLC spectrum after 2 months, purity is 94.77%, see attached Figure 4 ; The purity has been significantly reduced.

[0029] The dCTP freeze-dried powder produced by the external fermentation process has a purity of 92.34% according to the HPLC spectrum at T0. Figure 5 HPLC spectrum after 2 months, purity is 90.37%, see attached Figure 6 ; The purity has been significantly reduced.

[0030] Figure 1 and Figure 2 contrast, Figure 3 and Figure 4 Compare and Figure 5 and Figure 6 By comparison: 1. The HPLC purity of dCTP solution was not reduced substantially under the condition of -20°C, because the purity before and after freezing was 99.73% and 99.67%, respectively, with no significant difference.

[0031] 2. The synthesis process dCTP aqueous solution was freeze-dried to obtain a freeze-dried powder. The HPLC purity at T0 was 99.24%, which was significantly different from the HPLC purity at 2 months of 94.77%. Therefore, the freeze-dried powder obtained by freeze-drying the dCTP aqueous solution in the synthesis process was significantly degraded to generate diphosphate impurities and tetraphosphate impurities, respectively.

[0032] 3. There is also a statistically significant difference between the HPLC purity of 92.34% at T0 of the fermentation process dCTP freeze-dried powder and the HPLC purity of 90.37% two months later. The impurities generated by decay are the same as those of the synthetic process dCTP freeze-dried powder, both of which are diphosphate impurities and tetraphosphate impurities.

[0033] 4. From the conclusions (1) to (3), it can be seen that for freeze-dried powder, whether it is dCTP produced by the synthesis process or the fermentation process, the HPLC purity is significantly reduced after being frozen at -20°C for 2 months, indicating that the stability of the dCTP aqueous solution is higher than that of the dCTP freeze-dried powder.

[0034] 5. The LCMS test of dCTP freeze-dried powder HPLC degradation impurities showed that the MS result of the negative ion before dCTP was 386, so it was inferred to be a diphosphate impurity with the following structure The LCMS detection spectrum is attached. Figure 7 Among the generated impurities, the negative ion MS result after dCTP is 546, so it is inferred to be a tetraphosphate impurity, and its structure is as follows: The LCMS detection spectrum is attached. Figure 8 From this, we can judge that the degradation impurities of dCTP are mainly diphosphate impurities with one phosphate missing in front of dCTP and tetraphosphate impurities with one phosphate added in the back of dCTP.

Claims

1. A high concentration dNTP monomer aqueous solution, characterized in that, The concentration of the high-concentration dNTP monomer aqueous solution is greater than 100 mM and less than or equal to the concentration of its corresponding saturated solution.

2. A method for preparing a dNTP monomer aqueous solution having a concentration higher than the initial concentration, characterized in that: The method for preparing a dNTP monomer aqueous solution with a concentration higher than the initial concentration comprises the steps of freezing and standing to thaw, wherein the dNTP monomer aqueous solution with the initial concentration is an unsaturated dNTP monomer aqueous solution; the freezing step is to place the non-enclosed dNTP monomer aqueous solution at a temperature that can freeze it into a solid and freeze it into a solid, and the solid formed by freezing the dNTP monomer aqueous solution is called a dNTP-H2O solid; and the thawing step is to stand to thaw at a temperature that can melt the dNTP-H2O solid.

3. The method for preparing a high concentration dNTP monomer aqueous solution according to claim 2, wherein: The temperature at which the unsaturated dNTP monomer aqueous solution can be frozen into a solid is less than or equal to -10°C.

4. The method for preparing a high concentration dNTP monomer aqueous solution according to claim 3, wherein: The temperature at which the unsaturated dNTP monomer aqueous solution can be frozen into a solid is between -10°C and -80°C.

5. A method for preparing a high concentration dNTP monomer aqueous solution according to claim 2, characterized in that: The temperature capable of melting the solid dNTP-H2O solid is greater than 0°C.

6. A method for preparing a high concentration dNTP monomer aqueous solution according to claim 5, characterized in that: The temperature capable of melting the solid dNTP-H2O solid is greater than 0°C and less than or equal to 30°C.

7. The method for preparing a dNTP monomer aqueous solution having a concentration higher than the initial concentration according to claim 2, characterized in that: The method for preparing a dNTP monomer aqueous solution having a concentration higher than the initial concentration further comprises the step of detecting the concentration of the dNTP monomer aqueous solution after melting.

8. The method for preparing a dNTP monomer aqueous solution having a concentration higher than the initial concentration according to claim 7, characterized in that: The step of detecting the concentration of the dNTP monomer aqueous solution after melting includes detecting a top solution and a bottom solution.

9. A method for preparing a dNTP monomer aqueous solution having a concentration higher than the initial concentration according to claim 2, characterized in that: The initial concentration is less than or equal to 100 mM.

10. The use of the high concentration dNTP monomer aqueous solution according to claim 1, characterized in that: The high-concentration dNTP monomer aqueous solution according to claim 1 is directly or indirectly used for the preparation of DNA; or for the preparation of dNTP monomer lyophilized powder; or for the preparation of dNTP monomer aqueous solution below 100 mM.

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