Polypeptide solid phase synthesis on-line monitoring method and on-line monitoring system thereof

Abstract

The invention provides a polypeptide solid-phase synthesis online monitoring method and an online monitoring system thereof. The polypeptide solid-phase synthesis on-line monitoring method comprises the steps that a polypeptide solid-phase synthesis reaction is conducted in a synthesis reaction column, and the polypeptide solid-phase synthesis reaction has a deprotection stage and a coupling stage; monitoring the conductivity of the liquid discharged from the synthetic reaction column in real time by using a conductivity detector; a coupling judgment step of judging whether the conductivity change of the conductivity in a preset time period is smaller than a preset value or not; if yes, it is judged that the coupling stage of the polypeptide solid-phase synthesis reaction is completed, and if not, the polypeptide solid-phase synthesis reaction continues to be conducted. According to the technical scheme, the problems that a polypeptide solid-phase synthesis instrument in the prior art is difficult to judge whether a coupling stage is completed or not, time and labor are wasted in judgment, and the accuracy of a test judgment result is influenced are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide solid phase synthesis, and in particular to an online monitoring method and an online monitoring system for polypeptide solid phase synthesis. Background Art

[0002] Peptides are a class of compounds formed by connecting multiple amino acids through peptide bonds. With the rapid development of technologies related to peptide solid phase synthesis (such as the emergence of solid phase peptide solid phase synthesis technology), equipment and processes, the cost of peptide drug research and development and production costs have been greatly reduced, and the development of peptide drugs has continued to heat up. In the process of peptide solid phase synthesis, in order to achieve reaction only at the target position, the groups that do not participate in the reaction are usually protected and then removed after the reaction. Peptide solid phase synthesis is based on its synthesis process. The most common is the protection of amino groups. The Fmoc protecting group is a commonly used protecting group for amino acids. In peptide solid phase synthesis, amino acids with Fmoc protecting groups are used as synthetic reactants. The entire synthesis process involves two processes, namely amino acid deprotection and coupling. Amino acid deprotection is the removal of the Fmoc group from the amino acid, and coupling is the dehydration condensation of the carboxyl group of the previous amino acid and the amino group that has just removed the protecting group to form a peptide bond. After both steps, cleaning is required to wash away the residual reagents.

[0003] In the prior art, although the synthesis of peptide solid phase synthesis instruments can greatly save manpower, each peptide solid phase synthesis requires two steps of deprotection and coupling for synthesizing one amino acid. In manual synthesis, samples are taken after the two steps to perform Kaiser test to determine whether the reaction is complete. However, for commercially available peptide solid phase synthesis instruments, it is difficult to achieve resin sampling and detection, and it is difficult to determine the progress of the synthesis, that is, it is difficult to determine whether the coupling stage is completed; even if sampling can be performed, it will be time-consuming and laborious to determine whether the coupling stage is completed, and it will affect the accuracy of the test results. Summary of the invention

[0004] The main purpose of the present invention is to provide an online monitoring method and an online monitoring system for polypeptide solid phase synthesis, so as to solve the problems in the prior art that the polypeptide solid phase synthesis instrument is difficult to judge whether the coupling stage is completed, the judgment is time-consuming and labor-intensive, and affects the accuracy of the test judgment results.

[0005] In order to achieve the above-mentioned object, the present invention provides an online monitoring method for polypeptide solid phase synthesis, comprising: performing a polypeptide solid phase synthesis reaction in a synthesis reaction column, the polypeptide solid phase synthesis reaction having a deprotection stage and a coupling stage; using a conductivity detector to monitor the conductivity of a liquid discharged from the synthesis reaction column in real time; a coupling judgment step of judging whether a conductivity change within a preset time period is less than a preset value; if yes, judging that the coupling stage of the polypeptide solid phase synthesis reaction has been completed, and if not, continuing the polypeptide solid phase synthesis reaction.

[0006] Furthermore, before the coupling judgment step, the polypeptide solid phase synthesis online monitoring method also includes: obtaining the conductivity of the liquid discharged from the synthesis reaction column at a first time; obtaining the conductivity of the liquid discharged from the synthesis reaction column at a second time, wherein the second time-the first time=the preset time period; calculating the ratio between the conductivity at the second time and the conductivity at the first time, so as to output the conductivity change within the preset time period.

[0007] Furthermore, the preset value is 1%, and the preset time period is 5 minutes.

[0008] Furthermore, the method for online monitoring of polypeptide solid phase synthesis also includes: using an ultraviolet monitor to determine whether there is a deprotection judgment step of the Fmoc group in the liquid discharged from the synthesis reaction column; if so, it is determined that the deprotection stage is in progress, and a washing deprotection step is performed, and then the deprotection judgment step is performed; if not, it is determined that the deprotection stage is completed.

[0009] Furthermore, the cleaning and deprotection step includes: injecting a cleaning reagent into the synthesis reaction column to flush the inside of the synthesis reaction column; and injecting a deprotection reagent into the synthesis reaction column.

[0010] Furthermore, the deprotection judgment step includes: setting the wavelength of the ultraviolet monitor to a preset wavelength value; using the ultraviolet monitor to monitor whether the liquid discharged from the synthesis reaction column has an ultraviolet absorption peak at the preset wavelength value, if yes, the Fmoc group exists, if not, the Fmoc group does not exist.

[0011] Furthermore, the ultraviolet monitor is a dual-wavelength detector.

[0012] Furthermore, the preset wavelength values ​​are 289 nm and 301 nm.

[0013] Furthermore, the method for online monitoring of polypeptide solid phase synthesis also includes: using a temperature detector to detect the temperature of the liquid discharged from the synthesis reaction column in real time; judging in real time whether the temperature obtained by the temperature detector is within a preset range, if so, maintaining the current reaction temperature; if not, controlling the heat exchange device to heat or cool the synthesis reaction column.

[0014] Furthermore, the liquid discharged from the synthesis reaction column includes amino acid, activator, DIC and solvent DMF, and the amino acid, activator and DIC are dissolved in the solvent DMF.

[0015] According to another aspect of the present invention, the present invention provides an online monitoring system for polypeptide solid phase synthesis, which uses the above-mentioned online monitoring method for polypeptide solid phase synthesis to monitor the polypeptide solid phase synthesis reaction.

[0016] By applying the technical solution of the present invention, the consumption of reactants (mainly amino acids and activators) in the coupling stage can be accurately captured by real-time monitoring of conductivity. Since the change in conductivity directly reflects the change in ion concentration in the solution, when the change in conductivity within a preset time period is less than a preset value, this indicates that the reactants have been consumed to a dynamic equilibrium, and then it can be judged that the coupling stage has been completed. In this way, compared with traditional manual sampling and analysis, not only can the completion of the coupling stage be accurately judged, but the labor intensity of operators can also be reduced, unnecessary waiting time can be reduced, the process of solid-phase synthesis of polypeptides can be accelerated, and production efficiency can be significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 A flow chart showing an embodiment of the method for online monitoring of polypeptide solid phase synthesis of the present invention; and

[0019] Figure 2 A schematic diagram showing the structure of an embodiment of the polypeptide solid phase synthesis online monitoring system of the present invention;

[0020] Figure 3 The conductivity test results of the on-line monitoring method for solid-phase synthesis of polypeptides of the present invention for on-line monitoring of 10 peptide synthesis processes of the complete process are shown;

[0021] Figure 4 The UV1 test results of the online monitoring method for solid phase synthesis of polypeptides of the present invention for online monitoring of 10 peptide synthesis processes of the complete process are shown;

[0022] Figure 5 The UV2 test results of the online monitoring method for solid phase synthesis of polypeptides of the present invention for online monitoring of 10 peptide synthesis processes of the complete process are shown;

[0023] Figure 6 The temperature monitoring diagram shows the temperature of the liquid discharged from the synthesis reaction column in real time using a temperature detector in the on-line monitoring method for solid phase synthesis of a polypeptide of the present invention.

[0024] The above drawings include the following reference numerals:

[0025] 1. First injection valve; 2. Fourth switching valve; 3. Conductivity detector; 4. Second injection valve; 5. Third switching valve; 6. First switching valve; 7. Fifth switching valve; 8. Activation reactor; 9. Synthesis reaction column; 10. Second switching valve; 11. UV detector; 13. Current limiter; 14. Waste liquid outlet valve; 15. First pump body; 16. Second pump body. DETAILED DESCRIPTION

[0026] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0027] like Figure 1 As shown, an embodiment of the present invention provides an online monitoring method for polypeptide solid phase synthesis, comprising: performing a polypeptide solid phase synthesis reaction in a synthesis reaction column, the polypeptide solid phase synthesis reaction having a deprotection stage and a coupling stage; using a conductivity detector to monitor the conductivity of a liquid discharged from the synthesis reaction column in real time; a coupling judgment step of judging whether a conductivity change within a preset time period is less than a preset value; if yes, judging that the coupling stage of the polypeptide solid phase synthesis reaction has been completed, and if not, continuing the polypeptide solid phase synthesis reaction.

[0028] In the above technical solution, by real-time monitoring of conductivity, the consumption of reactants (mainly amino acids and activators) in the coupling stage can be accurately captured. Since the change in conductivity directly reflects the change in ion concentration in the solution, when the change in conductivity within a preset time period is less than a preset value, this indicates that the reactants have been consumed to a dynamic equilibrium, and then it can be judged that the coupling stage has been completed. In this way, compared with traditional manual sampling and analysis, it can not only accurately judge the completion of the coupling stage, but also reduce the labor intensity of operators, reduce unnecessary waiting time, speed up the process of peptide solid phase synthesis, and significantly improve production efficiency.

[0029] Furthermore, by analyzing the conductivity change rate under different conditions and comparing it with the preset value, the process conditions can be optimized to find the most suitable reaction temperature, pH value, reaction time, etc., so as to improve the yield and purity of the peptide solid phase synthesis.

[0030] like Figure 1 As shown, in an embodiment of the present invention, before the coupling judgment step, the polypeptide solid phase synthesis online monitoring method further includes: obtaining the conductivity of the liquid discharged from the synthesis reaction column at a first time; obtaining the conductivity of the liquid discharged from the synthesis reaction column at a second time, wherein the second time-the first time=the preset time period; calculating the ratio between the conductivity at the second time and the conductivity at the first time, so as to output the conductivity change within the preset time period.

[0031] In the above technical solution, by obtaining the conductivity of the liquid discharged from the synthesis reaction column at the first time and the second time and calculating the ratio between the two, the change trend of the conductivity during the reaction process can be monitored in real time. In this way, instant feedback on the reaction dynamics is provided, so that the operator or control system can respond quickly, adjust the reaction conditions or judge the reaction progress according to the change in conductivity, thereby achieving more precise control of the coupling stage.

[0032] Furthermore, the ratio of conductivity changes within a preset time period is calculated. Compared with monitoring only the absolute change in conductivity, this method can more accurately reflect the consumption of reactants and the reaction rate. Especially when the reaction is close to the end point, the absolute value change may be small, but the change in the rate of change (ratio) can more clearly indicate whether the reaction is complete.

[0033] Furthermore, by calculating the ratio of conductivity changes, it is possible to automatically determine whether the coupling stage is completed. This avoids the uncertainty of manual intervention, reduces the labor intensity of operators, and also reduces the error of manual judgment, thereby improving the reliability and consistency of reaction judgment.

[0034] Preferably, in an embodiment of the present invention, the preset value is 1%, and the preset time period is 5 minutes.

[0035] In the above technical solution, by monitoring the change in conductivity within 5 minutes, when the change is less than 1%, it can be judged that the coupling stage has been completed. This is because the rate of change of conductivity reflects the consumption of the reactants. When the reaction approaches the end point and the reactants are consumed, the rate of change of conductivity will significantly decrease and reach a dynamic equilibrium state, which provides an objective standard for judging whether the reaction is completed.

[0036] Furthermore, setting a clear preset time period and preset value can avoid overreaction and washing, ensure that the coupling reaction of each amino acid is both sufficient and not excessive, thereby improving the efficiency of the reaction. While ensuring the complete reaction, it also avoids waste of resources and reduces production costs.

[0037] It should be noted that, in the embodiments of the present invention, for judging the synthesis process, the solution in the synthesis (the liquid discharged from the synthesis reaction column) includes amino acids, activators, DIC (N, N'-diisopropylcarbodiimide) and solvent DMF (Dimethylformamide), amino acids, activators and DIC are dissolved in the solvent DMF, and the DMF solution is a non-electrolyte solution. Therefore, the main source of conductivity is the amino acids, activators and DIC dissolved in the DMF solution. At the same time, during the entire synthesis process, the amino acids free in the solution are coupled to the resin and are continuously consumed. Therefore, the conductivity of the synthesis system solution will continue to change until the reaction is completed, the ions in the solution reach a dynamic equilibrium, and the conductivity no longer changes. The progress of the synthesis can be judged by monitoring the conductivity of the solution in the synthesis system. Among them, DIC is a condensing agent, which also has certain applications in polypeptide synthesis. The advantage of DIC is that it has good selectivity for amino acids and mild reaction conditions.

[0038] like Figure 1As shown, in an embodiment of the present invention, the method for online monitoring of polypeptide solid phase synthesis further includes: using an ultraviolet monitor to determine whether there is a deprotection judgment step of the Fmoc group in the liquid discharged from the synthesis reaction column; if so, it is determined that the deprotection stage is in progress, and a washing deprotection step is performed, and then the deprotection judgment step is performed; if not, it is determined that the deprotection stage is completed.

[0039] In the above technical solution, by using a UV monitor for real-time monitoring, the completion of the deprotection stage can be accurately determined. When the presence of Fmoc groups in the liquid is detected, the system can immediately recognize that the deprotection stage is in progress, thereby performing a cleaning step to ensure complete deprotection and avoid residual Fmoc groups in the subsequent coupling stage.

[0040] Furthermore, combining UV monitoring with automated cleaning steps can achieve automated control of the deprotection stage during peptide solid phase synthesis. The system can automatically determine and execute the decision of cleaning or continuing deprotection based on the monitoring results, greatly reducing the need for manual intervention and improving production efficiency and process continuity.

[0041] Furthermore, real-time monitoring and accurate judgment of the completion of the deprotection stage reduces the number of unnecessary cleaning times and the use of cleaning solvents, avoiding waste of resources. This not only reduces the cost of chemical reagents and solvents, but also reduces the energy consumption during the cleaning process.

[0042] Furthermore, the reaction conditions in the deprotection stage, such as reaction time, temperature, pH value, etc., can be further optimized through the data fed back by the UV monitor to achieve the best reaction efficiency and product purity.

[0043] like Figure 1 As shown, in an embodiment of the present invention, the cleaning and deprotection step includes: inputting a cleaning reagent into the synthesis reaction column to flush the inside of the synthesis reaction column; and inputting a deprotection reagent into the synthesis reaction column.

[0044] In the above technical solution, after confirming that the deprotection stage is completed and performing the cleaning step, the deprotection reagent is input again and the deprotection judgment step is performed to ensure the thoroughness of the deprotection reaction. Even if the previous deprotection has reached the standard, the reagent is input again to verify whether there are still unremoved Fmoc groups, thereby ensuring the integrity and quality of the peptide solid phase synthesis reaction.

[0045] Furthermore, by combining the determination step with the automated input of cleaning and deprotection reagents, the need for manual operation can be significantly reduced, the degree of automation and continuity of the peptide solid phase synthesis process can be improved, the uncertainty caused by manual operation can be reduced, and production efficiency can be improved.

[0046] Furthermore, automated determination and control of the input of cleaning and deprotection reagents can accurately control the amount of reagents used, avoid excessive use of reagents and solvents, effectively save chemical reagent and solvent resources, and reduce production costs.

[0047] Furthermore, by combining online monitoring with automated cleaning and deprotection, the process flow can be optimized, the transition time from one stage to the next can be shortened, ineffective waiting can be reduced, and the efficiency of the entire peptide solid phase synthesis process can be improved.

[0048] Preferably, in an embodiment of the present invention, the cleaning reagent is the solvent DMF.

[0049] like Figure 1 As shown, in an embodiment of the present invention, the deprotection judgment step includes: setting the wavelength of the ultraviolet monitor to a preset wavelength value; using the ultraviolet monitor to monitor whether the liquid discharged from the synthesis reaction column has an ultraviolet absorption peak at the preset wavelength value, if yes, the Fmoc group exists, if not, the Fmoc group does not exist.

[0050] In the above technical solution, by setting the wavelength of the UV monitor to a specific preset wavelength value, the presence of the Fmoc group can be specifically detected. The Fmoc group has a significant UV absorption peak at these wavelengths. Therefore, the detection of the UV absorption peak indicates that the deprotection stage has not been completed, and the disappearance of the absorption peak means that the Fmoc group has been completely removed and the deprotection stage is over. This precise monitoring method ensures that the deprotection reaction of each amino acid in the solid phase synthesis of the peptide is complete, thereby improving the purity of the peptide product.

[0051] Furthermore, this solution can monitor the deprotection stage in real time during the whole process of peptide solid phase synthesis, and provide timely feedback on the removal of the Fmoc group. This helps operators or automated control systems to adjust the reaction process in real time, ensuring that the reaction conditions are always in the best state, thereby improving reaction efficiency and product quality.

[0052] Furthermore, automated monitoring replaces manual sampling and testing, which not only reduces the workload of operators, but also reduces errors and inconsistencies caused by human factors, and improves the standardization and reproducibility of the process.

[0053] Furthermore, by monitoring the complete removal of the Fmoc group, the most appropriate cleaning time can be determined to avoid over-cleaning or under-cleaning, saving cleaning reagents and reducing unnecessary waiting time, thereby improving production efficiency and resource utilization.

[0054] Furthermore, the presence of the Fmoc group may have an adverse effect on subsequent coupling reactions. Monitoring its removal can ensure the safety of reaction conditions, prevent abnormal reactions caused by residual Fmoc groups, and improve the controllability and reliability of the peptide solid phase synthesis process.

[0055] Furthermore, the collected UV monitoring data can be used to analyze the dynamic changes of the deprotection reaction and provide a basis for the adjustment of process parameters, such as reaction time, temperature and pH value, so as to continuously optimize the peptide solid phase synthesis process and improve the reaction efficiency and product purity.

[0056] Preferably, in an embodiment of the present invention, the ultraviolet monitor is a dual-wavelength detector.

[0057] In the above technical solution, the dual-wavelength detector can effectively reduce interference by simultaneously monitoring ultraviolet absorption at two wavelengths, thereby improving the accuracy and specificity of detection of target substances (such as Fmoc groups) and avoiding impurity interference problems that may be encountered in single-wavelength ultraviolet detection.

[0058] Furthermore, the dual-wavelength detector can be set near the wavelength where the target substance has the strongest absorption, thereby improving the sensitivity of the detection. This is particularly important for monitoring the removal process of the Fmoc group, because the completion mark of the deprotection stage is the complete removal of the Fmoc group. The dual-wavelength setting allows even trace amounts of Fmoc groups to be accurately detected, ensuring the accuracy of the reaction endpoint judgment.

[0059] Furthermore, by using a dual-wavelength UV monitor, the reliance on other detection methods (such as chromatography, mass spectrometry, etc.) can be reduced, simplifying the online monitoring system for peptide solid phase synthesis. This not only reduces equipment costs and maintenance complexity, but also improves the overall operating efficiency of the system and reduces the time for data processing and analysis.

[0060] Preferably, in an embodiment of the present invention, the preset wavelength values ​​are 289 nm and 301 nm.

[0061] In the above technical solution, the Fmoc group has significant ultraviolet absorption peaks near 289nm and 301nm. After one deprotection is completed, the deprotection reagent is added again, and then the two wavelengths are used for detection, which can specifically identify and quantify the presence of the Fmoc group, thereby accurately determining whether the deprotection stage in the solid phase synthesis of the peptide is completed. This specific detection reduces the interference of background signals and improves the accuracy and reliability of the detection.

[0062] Furthermore, the maximum absorption wavelength of the Fmoc group is selected for detection, so that the detector can capture the signal of the Fmoc group with the highest sensitivity. Even if the concentration of the Fmoc group is very low, it can be detected, which is crucial for judging whether the cleaning process is thorough and whether the deprotection is complete.

[0063] Furthermore, by comparing the detection results at two wavelengths, 289 nm and 301 nm, the signal of the Fmoc group can be effectively distinguished from the background signal, reducing false positive or false negative results caused by the absorption of other substances at specific wavelengths, thereby improving the accuracy of reaction monitoring.

[0064] Furthermore, based on the changes in the Fmoc group signal at wavelengths of 289 nm and 301 nm, the cleaning and reaction conditions, such as the cleaning time and the amount of deprotection reagent, can be adjusted in real time to ensure the high efficiency and purity of the peptide solid phase synthesis reaction.

[0065] It should be noted that the solid phase synthesis of polypeptides involves more than twenty kinds of amino acids, and the difficulty of synthesis of each amino acid is different due to differences in structural properties. Therefore, different amino acids require different reaction times. During cleaning, reagent residues can easily cause cross contamination, affecting the quality of synthesis. Existing commercially available instruments cannot determine whether the cleaning is complete. Therefore, the online monitoring method for solid phase synthesis of polypeptides of the embodiment of the present invention is adopted. During the synthesis process, by setting the absorption characteristics of the reagents at two specific wavelengths, the dual-wavelength detector can monitor the cleaning process to ensure that all reagents are completely removed, avoid cross contamination and affect the purity of the final polypeptide product.

[0066] It should be noted that, in the embodiments of the present invention, the change in the solution in the deprotection stage is mainly that the Fmoc group and piperidine form a fluorene-piperidine adduct. The end point of the cleaning can be determined by selecting ultraviolet monitoring of the content of the fluorene-piperidine adduct. The fluorene-piperidine adduct has ultraviolet absorption peaks at wavelengths of 289nm and 301nm. The solution in the coupling stage is mainly unreacted amino acids and condensation reagents. The absorption peak of Fmoc can also be detected at 301nm. Therefore, it is very feasible to introduce ultraviolet online monitoring into the solid phase synthesis of polypeptides to determine the cleaning process.

[0067] Specifically, in the embodiments of the present invention, UV monitoring can be associated with reagent residues: by detecting the UV value at a specific wavelength, the residual amount of a specific reagent in the solution can be reflected. This is because the Fmoc group has unique ultraviolet absorption characteristics at wavelengths of 289nm and 301nm (where UV1 is the UV value of the Fmoc group at a wavelength of 301nm, such as Figure 4 As shown; UV2 is the UV value of the Fmoc group at a wavelength of 289nm, such as Figure 5As shown in the figure, in the deprotection stage, the Fmoc group exists in the fluorene-piperidine adduct, and in the coupling stage, Fmoc exists in the amino acid. Therefore, the UV value data in the deprotection cleaning stage corresponds to the piperidine content, and the UV value data in the coupling cleaning stage corresponds to the amino acid content. Among them, in the deprotection stage, the residual amount of piperidine (agent used for deprotection) is mainly monitored. The set judgment standard is: when the UV value of the solution after cleaning is lower than 400ppm, the corresponding residual amount of piperidine is below 400ppm, indicating that the deprotection cleaning process has been sufficient; in the coupling stage, the residual amount of unreacted amino acids needs to be monitored. The set judgment standard is: when the UV value of the solution after cleaning is reduced to below 800ppm, the residual amount of amino acids is controlled between 500-1000ppm, ensuring the cleanliness and efficiency of the coupling reaction.

[0068] Specifically, in the embodiments of the present invention, by comparing the UV value judgment index obtained by the online monitoring of the instrument with the laboratory synthesis process, the accuracy and practicality of the monitoring system can be verified. In laboratory synthesis, the judgment of the cleaning effect often relies on manual sampling and analysis, while the present invention realizes automated judgment through real-time UV monitoring. According to the comparison results, the cleaning conditions, such as the number of cleaning times, the type and amount of cleaning agent, etc., can be further optimized to ensure that the effect of polypeptide synthesis on the automated equipment is equivalent to or even better than that of the laboratory manual operation, while reducing the error and cost of manual operation.

[0069] like Figure 1 As shown, in an embodiment of the present invention, the method for online monitoring of solid phase synthesis of polypeptides further includes: a step of detecting the temperature of the liquid discharged from the synthesis reaction column by using a temperature detector in real time; judging in real time whether the temperature obtained by the temperature detector is within a preset range, if so, maintaining the current reaction temperature; if not, controlling the heat exchange device to heat or cool the synthesis reaction column.

[0070] In the above technical solution, the temperature of the liquid in the synthesis reaction column is monitored in real time and compared with the preset temperature range, which can ensure that the peptide solid phase synthesis reaction is carried out at the most suitable temperature. Temperature is one of the key factors affecting the efficiency of peptide solid phase synthesis and product purity. By adjusting the reaction temperature in real time, the reaction rate changes or side reactions caused by temperature fluctuations can be avoided, thereby improving the controllability and quality of the synthesis.

[0071] It should be noted that at different stages of the reaction, different temperatures are set in the preset range, and the temperature is monitored online using a temperature detector. When the temperature is higher than the set value, the system automatically cools down, and when it is lower than the preset value, the temperature is increased. The preset range can be set for the instrument within the range of the external temperature controller.

[0072] like Figure 6As shown, the temperature of the liquid discharged from the synthesis reaction column can be monitored online using a temperature detector and displayed in real time.

[0073] Furthermore, during the solid phase synthesis of peptides, the coupling and deprotection reactions of each amino acid may have different sensitivities to temperature. Real-time monitoring and instant temperature control by the temperature detector can maintain the temperature consistency of the entire synthesis process, ensuring that each reaction step is carried out under optimal conditions, improving the batch-to-batch consistency of peptide solid phase synthesis, and facilitating large-scale production.

[0074] Furthermore, by collecting the monitoring data of the temperature detector, the specific effect of temperature on the peptide solid phase synthesis reaction can be further analyzed to provide a scientific basis for the optimization of process conditions. For example, the optimal temperature range for the synthesis of different amino acids can be determined and the reaction conditions can be adjusted to achieve the best synthesis effect.

[0075] It should be noted that the polypeptide solid phase synthesis online monitoring method can change the wavelength to monitor the Fmoc group during synthesis; it can replace infrared spectrum monitoring and other spectra; and it can calculate the reagent content by peak area. An embodiment of the present invention provides a polypeptide solid phase synthesis online monitoring system, which uses the above-mentioned polypeptide solid phase synthesis online monitoring method to monitor the polypeptide solid phase synthesis reaction. The polypeptide solid phase synthesis online monitoring system mainly includes the following components: Figure 2As shown, the whole device includes a first injection valve 1, a fourth switching valve 2, a conductivity detector 3, a second injection valve 4, a third switching valve 5, a first switching valve 6, a fifth switching valve 7, an activation reactor 8, a synthesis reaction column 9, a second switching valve 10, a UV detector 11 (ultraviolet monitor); a flow limiter 13, a waste liquid outlet valve 14, a temperature detector, a first pump body 15 and a second pump body 16, wherein the temperature detector is embedded in the conductivity detector 3, monitors the ultraviolet absorption, conductivity and temperature in the solution online, interacts with the peptide solid phase synthesizer software, forms a monitoring curve on the synthesizer software, and monitors and judges the synthesis situation. Among them, the first injection valve 1 is an amino acid feeding valve, which can provide the import of 13 kinds of amino acids; the second injection valve 4 is a reagent feeding valve, which can provide the import of 6 kinds of reagents and the import of flushing; the first switching valve 6 is a three-point selection valve, which can choose to circulate through the path of the activation column-the second pump body 16 or the path of the synthesis column-the first pump body 15 or the path of the fifth switching valve 7-the waste liquid outlet valve 14; the second switching valve 10 is a column position valve, which provides three kinds of path functions to meet the function of material circulation or passing; the third switching valve 5 is a two-point selection valve, which can choose to couple the path of the second injection valve 4-the first pump body 15 or the path of UV-the first pump body 15 for circulation; the second pump body 16 and the first pump body 15 are feed pumps, which provide amino acid and reagent feeding, and also play the role of circulation; the conductivity detector 3 is a conductivity detector, which can test the conductivity signal and system solution temperature during the synthesis process online; UV detector 11 is an ultraviolet monitor, and the dual-wavelength ultraviolet monitor can detect ultraviolet absorption during the synthesis process online; the current limiter 13 can control the system pressure by giving an electrical signal to ensure the accuracy of the pump; solid phase synthesis control system, the control system is connected to the serial port server of the host through the Ethernet port, and the serial port server is respectively connected to multiple pumps and multiple detectors. The solid phase synthesis control system also includes a valve control device connected to the serial port server, and the valve control device includes a microcontroller unit, a communication module, a storage module, and multiple communication interfaces connected to the microcontroller unit, and an injection valve module, a valve power supply control module, a mixing component control module, and a pressure acquisition module respectively connected to the microcontroller unit. The microcontroller unit is connected to the serial port server through the communication module, thereby realizing signal interaction with the host. The data fed back by the online monitoring system is displayed on the instrument software operation interface, and the changes in ultraviolet absorption, conductivity and temperature can be intuitively seen, so as to judge and intervene in the synthesis process. The online monitoring system for solid phase synthesis of polypeptides of the embodiment of the present invention can adopt the existing technology, which will not be repeated here.

[0076] The above-mentioned polypeptide solid phase synthesis online monitoring system has all the advantages of the above-mentioned polypeptide solid phase synthesis online monitoring method, which will not be described in detail here.

[0077] The inventors used the peptide solid phase synthesis online monitoring method and peptide solid phase synthesis online monitoring system of this embodiment to conduct tests. The amino acid sequence synthesized in this test was Boc-Glu(OtBu)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Gly-OH. In terms of ultraviolet detection, the ultraviolet dual-wavelength detector selected the wavelengths of 289nm and 301nm to monitor the deprotection stage cleaning and coupling stage cleaning of the peptide solid phase synthesis, monitor the piperidine content in the deprotection stage, and monitor the three amino acids Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Asn(Trt)-OH in the coupling.

[0078] The residual reagent concentration is matched with the instrument UV value. The test results have a certain deviation due to the reaction delay in sampling. The test results are shown in Table 1 and Table 2:

[0079] Table 1

[0080]

[0081] Table 2

[0082]

[0083] From the results, we can see that although there are some deviations, there is a corresponding relationship between the washing UV value in the deprotection stage and the residual content of the piperidine reagent. Similarly, there is a corresponding relationship between the washing UV value in the coupling stage and the residual content of the amino acid reagent. When cleaning the residual reagents, the UV value can be used as a judgment indicator to guide the cleaning steps of the online monitoring system for peptide solid-phase synthesis. At present, according to the synthetic process of the participating laboratories, the deprotection cleaning piperidine residue is below 400ppm, and the corresponding cleaning UV must be below 400ppm. The amino acid residue is 500-1000ppm, so the instrument chooses to clean the UV to below 800ppm.

[0084] In terms of conductivity detection, the 10 peptide synthesis processes of the complete process are monitored online, mainly the solid phase synthesis peptide amino acid coupling stage of the peptide online monitoring system. The test results are as follows Figure 3 As shown. During the synthesis, the conductivity showed a trend of decreasing and gradually becoming flat. In order to select the appropriate end point, the change in conductivity within 5 minutes was selected as the basis for judgment. The synthesis results of each amino acid were collected and the judgment basis was finally obtained. When the conductivity changed less than 1% within 5 minutes, the reaction was completed. Subsequently, the ten-peptide product was synthesized through this monitoring method, and the purity could reach 93%-95%.

[0085] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: by real-time monitoring of conductivity, the consumption of reactants (mainly amino acids and activators) in the coupling stage can be accurately captured. Since the change in conductivity directly reflects the change in ion concentration in the solution, when the change in conductivity within a preset time period is less than a preset value, this indicates that the reactants have been consumed to a dynamic equilibrium, and then it can be judged that the coupling stage has been completed. In this way, compared with traditional manual sampling and analysis, it can not only accurately judge the completion of the coupling stage, but also reduce the labor intensity of operators, reduce unnecessary waiting time, speed up the process of polypeptide solid phase synthesis, and significantly improve production efficiency.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for online monitoring of polypeptide solid phase synthesis, characterized in that: include: Carrying out a polypeptide solid phase synthesis reaction in a synthesis reaction column, wherein the polypeptide solid phase synthesis reaction has a deprotection stage and a coupling stage; Using a conductivity detector to monitor the conductivity of the liquid discharged from the synthesis reaction column in real time; A coupling judgment step of judging whether the conductivity change within a preset time period is less than a preset value; if yes, judging that the coupling stage of the polypeptide solid phase synthesis reaction has been completed, if not, continuing the polypeptide solid phase synthesis reaction.

2. The method for online monitoring of polypeptide solid phase synthesis according to claim 1, characterized in that: Before the coupling judgment step, the polypeptide solid phase synthesis online monitoring method further comprises: Acquiring the conductivity of the liquid discharged from the synthesis reaction column at the first time; Acquiring the conductivity of the liquid discharged from the synthesis reaction column at a second time, wherein the second time minus the first time=the preset time period; The ratio between the conductivity at the second time and the conductivity at the first time is calculated to output the conductivity change within the preset time period.

3. The method for online monitoring of polypeptide solid phase synthesis according to claim 1, characterized in that: The preset value is 1%, and the preset time period is 5 minutes.

4. The method for online monitoring of polypeptide solid phase synthesis according to any one of claims 1 to 3, characterized in that: The polypeptide solid phase synthesis online monitoring method further comprises: A UV monitor is used to determine whether there is a deprotection determination step of the Fmoc group in the liquid discharged from the synthesis reaction column; if yes, it is determined that the deprotection stage is in progress, and a cleaning deprotection step is performed, and then the deprotection determination step is performed; if no, it is determined that the deprotection stage is completed.

5. The method for online monitoring of polypeptide solid phase synthesis according to claim 4, characterized in that: The cleaning and deprotection step comprises: Inputting a cleaning reagent into the synthesis reaction column to flush the interior of the synthesis reaction column; A deprotection reagent is introduced into the synthesis reaction column.

6. The method for online monitoring of polypeptide solid phase synthesis according to claim 4, characterized in that: The deprotection judgment step comprises: Setting the wavelength of the ultraviolet monitor to a preset wavelength value; The ultraviolet monitor is used to monitor whether the liquid discharged from the synthesis reaction column has an ultraviolet absorption peak at the preset wavelength value. If yes, the Fmoc group exists; if no, the Fmoc group does not exist.

7. The method for online monitoring of polypeptide solid phase synthesis according to claim 6, characterized in that: The ultraviolet monitor is a dual-wavelength detector.

8. The method for online monitoring of polypeptide solid phase synthesis according to claim 7, characterized in that: The preset wavelength values ​​are 289 nm and 301 nm.

9. The method for online monitoring of polypeptide solid phase synthesis according to any one of claims 1 to 3, characterized in that: The polypeptide solid phase synthesis online monitoring method further comprises: A step of detecting the temperature of the liquid discharged from the synthesis reaction column in real time by using a temperature detector; It is determined in real time whether the temperature obtained by the temperature detector is within a preset range. If yes, the current reaction temperature is maintained; if no, the heat exchange device is controlled to heat or cool the synthesis reaction column.

10. The method for online monitoring of polypeptide solid phase synthesis according to any one of claims 1 to 3, characterized in that: The liquid discharged from the synthesis reaction column includes amino acid, activator, DIC and solvent DMF, and the amino acid, the activator and the DIC are dissolved in the solvent DMF.

11. A polypeptide solid phase synthesis online monitoring system, characterized in that: The polypeptide solid phase synthesis reaction is monitored using the polypeptide solid phase synthesis online monitoring method according to any one of claims 1 to 10.

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