Pine needle extract scalp health restorer and method of making same

Abstract

This invention relates to the field of biopharmaceutical manufacturing technology, and discloses a pine needle extract scalp health repair agent and its preparation method, comprising: using a fixed-bed bioreactor to set an average residence time that simultaneously satisfies the desired reaction and avoids the generation of impurities; and by actively applying transient flow rate disturbances and analyzing the product concentration response, diagnosing the inactivation state of the bioconversion enzyme system online, and compensatingly reducing the flow rate accordingly. This invention avoids the generation of immunogenic impurities; at the same time, by actively diagnosing and compensating for enzyme inactivation, it solves the fundamental problem of yield decline in continuous production, ensuring the absolute stability of the process in long-term operation.

Description

Technical Field

[0001] This invention relates to a pine needle extract scalp health repair agent and its preparation method, belonging to the field of biopharmaceutical manufacturing technology. Background Technology

[0002] In the current biomanufacturing process of natural product active ingredients, such as through enzymatic hydrolysis or fermentation, the reaction system is essentially a complex multi-pathway reaction network. The desired target active substance generation pathway and the defective pathway that leads to further degradation or side reactions that generate undesirable impurities have different reaction rate time scales in chemical kinetics. Currently, such biotransformation generally adopts batch reactor technology, in which all materials and enzymes are added to the reaction vessel at once for a long-term mixing reaction. This process has been widely accepted in long-term production practice because it is relatively simple to operate and the equipment is mature. However, when it is applied to the manufacture of active ingredients with extremely high requirements for purity and safety, such as immunogenicity, its fundamental and inherent physical defects become apparent, namely, the residence time of all molecules in the reactor is forced to be uniform and the average value is too long.

[0003] This process, characterized by prolonged and homogeneous mixing, leads to technological limitations that directly contradict the core concerns of biopharmaceutical manufacturing: even if the desired active ingredient (fast reaction) is efficiently generated in a short time, it must remain in the reactor for several more hours until the entire batch is finished. This passively extended time provides the most abundant reaction conditions for slower, defective pathways, such as oxidation, degradation, or side reactions, inevitably leading to the large-scale generation and accumulation of critical immunogenic impurities in the reactor. As a result, manufacturers are forced to weigh product yield against impurity risk and rely heavily on complex and costly downstream purification steps to passively remove these impurities already generated at the source. Existing technologies also have limitations in other areas of bio-extract preparation, where the focus is often on the active ingredient. The approach focuses on compounding rather than addressing the impurity control issues in complex reaction networks at the source of the process. For example, Chinese invention patent CN107550934B discloses a skin repair agent prepared by mixing stem cell extracts and traditional Chinese medicine extracts and its application. The technical solution in this document focuses on mixing stem cell extracts with specific traditional Chinese medicine extracts such as reed rhizome and blue lotus to achieve synergistic effects. However, in the preparation process, both stem cell extracts, such as ultrasonic lysis, and traditional Chinese medicine extracts, such as ethanol soaking, adopt traditional batch processing methods. This approach does not address how to actively avoid side reactions in continuous production by precisely controlling the reaction time scale. Therefore, it is also subject to the inherent defects of traditional batch processing and cannot fundamentally solve the technical problem of the desired product and undesirable impurities being forcibly bound together in terms of reaction time.

[0004] Therefore, the technical problem to be solved by this invention is how to provide a preparation method that, based on the differences in reaction kinetics, can achieve efficient conversion of the desired substance while actively avoiding the generation of pathogenic impurities through precise control of the process timing. Summary of the Invention

[0005] This invention provides a pine needle extract scalp health repair agent and its preparation method. Its main purpose is to solve the problem that in existing batch processing processes, the generation of desired products and the accumulation of immunogenic impurities cannot be decoupled due to reaction time, resulting in the difficulty in ensuring product purity and safety from the source.

[0006] To achieve the above objectives, the present invention provides a method for preparing a pine needle extract scalp health repair agent, comprising: Provide a fixed-bed bioreactor containing a biotransformation enzyme system; The feed solution containing pine needles is pumped into a fixed-bed bioreactor at a constant reference flow rate for biotransformation. The constant reference flow rate is set so that the average residence time of the feed solution in the fixed-bed bioreactor, when the biotransformation enzyme system is in its initial healthy state, simultaneously meets the following two conditions in terms of timing: a. The average residence time is longer than the preset expected reaction time, which is used to generate the target active ingredient; b. The average residence time is shorter than the preset defect reaction time, which is used to generate key immunogenic impurities; and the method further includes the following steps to compensate for the progressive inactivation of the bioconverting enzyme system during continuous operation: during the continuous pumping of the feed solution, a preset transient flow rate disturbance is periodically applied to the reference constant flow rate; the response characteristics of the target active ingredient concentration in the product liquid at the outlet of the fixed-bed bioreactor due to this transient flow rate disturbance are detected online; the response characteristics are compared with the reference response characteristics corresponding to the initial healthy state; when the difference between the response characteristics and the reference response characteristics indicates that the bioconverting enzyme system has been inactivated, the reference constant flow rate is automatically and compensatorily reduced to prolong the average residence time in order to offset the effect of bioconverting enzyme system inactivation on the target active ingredient generation rate.

[0007] Preferably, the preset expected reaction time and the preset defective reaction time are determined based on the characterization of the kinetic parameters of the biotransformation reaction under standard raw material batches; the baseline response characteristics are the pre-calibrated target active ingredient concentration response patterns exhibited by the biotransformation enzyme system in its initial healthy state to transient flow rate disturbances of a preset form.

[0008] Preferably, before the feed solution enters the fixed-bed bioreactor, the method further includes: diverting a portion of the feed solution to a parallel calibration microfluidic reactor, the calibration microfluidic reactor containing the same biotransformation enzyme system as the fixed-bed bioreactor; detecting online parameters characterizing the total reaction activity at the outlet of the calibration microfluidic reactor to obtain a real-time reaction rate; comparing the real-time reaction rate with a baseline reaction rate set based on a standard batch of feed; and dynamically adjusting the calibration value of the baseline response characteristics in the step of comparing the response characteristics with the baseline response characteristics based on the comparison results to compensate for the influence of batch-to-batch fluctuations in the feed solution on the response characteristics.

[0009] Preferably, the method further includes: establishing a health characteristic relationship between a baseline constant flow rate and the corresponding inlet pressure when the fixed-bed bioreactor is in a healthy hydraulic state; monitoring the inlet pressure in real time during continuous feed pumping; comparing the current operating point of the baseline constant flow rate and the real-time monitored inlet pressure with the health characteristic relationship to diagnose the hydraulic state of the fixed-bed bioreactor online; and automatically triggering a self-healing cleaning step with pulse flow rate or reverse flow rate when a progressive blockage state is diagnosed.

[0010] Preferably, the fixed-bed bioreactor includes at least two reaction units connected in series, and the at least two reaction units connected in series have different volumes; and the combination of a reference constant flow rate and different volumes provides an average residence time for the feed liquid in each reaction unit for the preset biotransformation steps within that unit, so as to realize the timing arrangement control of multi-step series biotransformation reactions.

[0011] Preferably, the method further includes: real-time monitoring of the actual operating temperature of the fixed-bed bioreactor; determining a dynamic safe residence time that ensures both conditions a and b are met based on the actual operating temperature and a preset Arrhenius compensation model characterizing the relationship between biotransformation reaction kinetics and temperature; and compensatingly reducing the baseline constant flow rate, the goal of which is to match the extended average residence time with the dynamic safe residence time.

[0012] Preferably, the step of comparing the response characteristics with the baseline response characteristics specifically includes: obtaining the maximum value of the target active ingredient concentration in the response characteristics. and minimum value And the baseline concentration of the target active ingredient before the application of the transient flow rate disturbance. ; Calculate the normalized response amplitude ,in The characterization of inactivation of the biotransformation enzyme system is based on the normalized response amplitude. Exceeding the preset inactivation threshold .

[0013] Preferably, the transient flow rate disturbance of the preset form is a pulse signal, the amplitude of the pulse signal changes by 1% to 20% relative to the reference constant flow rate, and the duration of the pulse signal is shorter than the average residence time. The bioconversion enzyme system is fixed on a solid carrier in the fixed bed bioreactor by physical adsorption or covalent bonding. The method also includes the step of quenching or pH adjustment immediately after the product liquid flows out of the fixed bed bioreactor to terminate any residual enzyme activity.

[0014] Preferably, an ultraviolet-visible spectrophotometer or polarimeter is installed at the outlet of the calibrated microfluidic reactor for online detection of parameters characterizing the overall reaction activity.

[0015] A pine needle extract scalp health repair agent, which is directly obtained by a method for preparing a pine needle extract scalp health repair agent.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention transforms the biotransformation process from a traditional batch processing method to a continuous flow process. By setting a constant flow rate for the material, the average residence time in the reactor is limited to a specific kinetic window. This time window provides sufficient reaction time for the desired active substance generation pathway, while ensuring that the product leaves the reactor with the fluid immediately after generation. This avoids subsequent degradation or side reactions, especially the reaction time necessary for slow reactions that lead to the generation of immunogenic impurities. This achieves the acquisition of high-purity products at the reaction stage, avoiding the technical limitation of the inevitable simultaneous accumulation of active substances and impurities in traditional batch processing processes.

[0017] 2. Before feeding into the main reactor, a new batch of feed liquid is tested online using a parallel calibrated microfluidic reactor. This design utilizes a simple online detector, such as an ultraviolet detector, to obtain relative parameters characterizing the overall reactivity, rather than attempting to measure specific material components. These relative parameters are used for comparison with a baseline rate, and the material flow rate of the main reactor is dynamically adjusted accordingly. As a result, without relying on complex and expensive sensors, the residence time control logic of the main scheme has a feedforward adaptive capability, enabling it to actively compensate for the reaction kinetic drift caused by batch-to-batch fluctuations in natural feed, ensuring that the core timing gating remains effective in the face of real-world operating condition fluctuations.

[0018] 3. The present invention further provides a method for online diagnosis of enzyme activity status inside a reactor. During continuous production, a transient, preset flow rate disturbance is periodically applied to a constant flow pump. By analyzing the response characteristics of the product concentration signal at the outlet to this disturbance (e.g., whether it remains stable or fluctuates), the current inactivation status of the immobilized enzyme system can be deduced. When the inactivation characteristics are decoded, the system automatically and compensatorily reduces the reference flow rate. This design reuses the material pump (actuator) as an active diagnostic tool, realizing the perception of the internal health status of the system without stopping production or intruding into the reactor. By extending the residence time through closed-loop regulation, the decrease in enzyme activity is compensated, ensuring the product yield within the continuous production cycle. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the process control of integrating online diagnostics and feedforward calibration in this invention. Figure 2 This is a response characteristic diagram of enzyme activity diagnosis based on transient flow velocity perturbation according to the present invention; Figure 3 This is a schematic diagram of the physical system architecture for integrating multiple online monitoring systems according to the present invention; Figure 4 This is a use case diagram of the interaction between the process engineer and the system automation diagnosis in this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0021] This invention provides a pine needle extract scalp health repair agent and its preparation method. The core of this method is the use of a continuous flow biotransformation process to replace the traditional batch reactor process. This method utilizes one or more fixed-bed bioreactors containing biotransformation enzyme systems, and achieves kinetic separation of the target product formation reaction and impurity side reactions by precisely controlling the physical parameter of average residence time. Furthermore, this method integrates an online enzyme activity diagnosis and compensation mechanism based on transient disturbance response analysis, a parallel microfluidic feedforward calibration system for compensating for batch-to-batch fluctuations in raw materials, and a hydraulic state monitoring system for diagnosing physical blockages in the reactor. Together, these mechanisms ensure a long-cycle biotransformation process with high purity and high yield. Stable operation is required. In specific implementation methods, the execution of this method first involves offline calibration of core reaction kinetic parameters, which forms the basis for setting process control logic. Those skilled in the art use standard batches of pine needle raw materials (i.e., feed solution) in a laboratory-scale batch reactor, adding biotransformation enzyme systems, such as complex glycosidases and hydrolases, to carry out the reaction. Samples are taken at different time points, such as every minute, and analytical techniques such as high-performance liquid chromatography (HPLC) are used to monitor the changes in the concentrations of target active substances (such as specific repair factors) and key immunogenic impurities (such as sensitizing terpene oxides) over time. By analyzing this kinetic data, two key time thresholds can be determined: one is the desired reaction time. Defined as the minimum time required for the production of the target active ingredient to reach 95% of the plateau phase (e.g., The other is defect response time (seconds). This is defined as the time point at which the concentration of a key immunogenic impurity begins to accumulate (e.g., exceeding the safety threshold of 0.01%). (minutes); this calibration process establishes the dynamic window required for control in this method, namely Less than Based on the above calibration, the main process of this method is carried out in one or more industrial-grade fixed-bed bioreactors (PBRs), which are filled with solid carriers, such as calcium alginate gel microspheres or porous ceramic particles, and the bioconversion enzyme system is immobilized on the solid carriers by physical adsorption or covalent bonding.

[0022] A high-precision constant flow pump delivers slurry containing pine needle raw materials at a constant reference flow rate. Continuously pumped into the PBR; this The setting of this parameter is the first core control point of this method, and it is based on the effective empty bed volume of the reactor. It is precisely calculated to set a specific average stay time. ;Should For the enzyme system to be in its initial healthy state, two conditions must be met simultaneously in time: condition a, Must be better For example, setting minutes, ensure (seconds) to ensure sufficient generation of the target active ingredient; condition b, Must be shorter than ,For example The residence time should be less than 15 minutes to ensure that the product leaves the reactor with the liquid flow immediately after formation, thus depriving the reaction of the time necessary for impurity formation (slow reaction). Through this kinetic selection mechanism based on residence time gating, this method actively avoids the formation of immunogenic impurities at the source of the reaction. Given that the gradual inactivation of immobilized enzymes is inevitable in long-cycle continuous production, this will lead to practical... Extend, if If the flow rate remains unchanged, the yield will decrease. To address this issue, this method introduces an active online diagnostic and compensation cycle. During the continuous pumping of the feed liquid, the control system periodically, for example, every 30 minutes, adjusts the feed flow rate. A transient flow rate disturbance of a preset form is applied; this disturbance can be a pulse signal with a small amplitude, specifically increasing the flow rate by 5% within 30 seconds and then returning it to the baseline value within 30 seconds, with the amplitude change ranging from 1% to 20%, here 5%, and the duration being 60 seconds, shorter than the average residence time (5 minutes). At the PBR outlet, an online detector, such as a UV-Vis spectrophotometer, monitors in real time the response characteristics of the target active ingredient concentration in the product solution caused by this disturbance. The system compares this response characteristic with a pre-calibrated baseline response characteristic corresponding to the initial healthy state. In the initial healthy state, enzyme activity is sufficient, the reaction is kinetically saturated, and the 5% small disturbance has almost zero impact on the outlet concentration. The baseline response is characterized by a nearly flat line; however, as the enzyme becomes inactive, the reaction enters a kinetically sensitive state, and the same 5% flow rate pulse ( A momentary shortening will cause an observable negative fluctuation in the outlet concentration (i.e., a decrease in concentration); the control system acquires the maximum concentration value in this response characteristic in real time. Minimum value and the baseline concentration before the disturbance And calculate the normalized response amplitude. When the The value exceeds the preset inactivation threshold. (For example When the enzyme system reaches a certain point, it determines that inactivation requiring compensation has occurred; at this time, the control system will automatically and compensatorily reduce the reference constant flow rate. (For example, lower it by 0.5%) to extend the period accordingly. This is used to counteract the effect of decreased enzyme activity on the rate of formation of the target active ingredient.

[0023] To address the interference of batch-to-batch fluctuations in natural raw materials such as pine needles on reaction kinetics and the aforementioned enzyme activity diagnostic model, this method may also include a feedforward calibration mechanism. Before the feed solution enters the main PBR, a portion of the feed solution, for example, 1% of the total flow rate, is diverted to a parallel calibration microfluidic reactor. This microfluidic reactor is small in volume but contains the same immobilized biotransformation enzyme system as the main PBR. An online detector, such as a UV-Vis spectrophotometer or polarimeter, is also installed at its outlet to detect parameters characterizing the total reaction activity online, such as the rate of change of absorbance at a specific wavelength, to obtain the real-time reaction rate. The control system compares this real-time reaction rate with a baseline reaction rate set based on standard raw material batches. If the real-time reaction rate of the new batch of feed solution is lower than the baseline, such as decreasing by 8%, this indicates that the reaction activity of the new batch is low. The system will then dynamically adjust, for example, by correspondingly increasing the calibration value of the baseline response characteristic used for comparison in the aforementioned enzyme activity diagnostic step, based on this 8% deviation, thereby compensating for the impact of batch-to-batch fluctuations on the enzyme activity diagnostic response characteristics and ensuring the accuracy of the enzyme activity compensation action. Furthermore, to ensure... The physical effectiveness of the control logic is also considered in this method, which includes online diagnosis of the PBR's hydraulic state. Natural product feed solutions easily lead to progressive clogging of the PBR, resulting in channeling or short-circuiting, causing the actual residence time to be much shorter than the theoretically calculated time. Therefore, when the PBR is in a healthy hydraulic state, immediately after startup or cleaning, the system automatically executes a calibration procedure: maintaining a constant flow rate at multiple different reference points. Record the corresponding stable inlet pressures below. Thus establishing and The relationship of health characteristics between them, namely During continuous operation, the system monitors the current status in real time. and Working points, and compare their relationship with health characteristics; when diagnosed as consistently higher When the corresponding value reaches the clogging threshold above 20%, the system determines that a progressive clogging state has occurred. At this time, the system can automatically trigger a self-healing cleaning step, for example, pausing the feed and applying a brief (e.g., 10 seconds) high-intensity pulse flow rate or reverse flow rate to use hydraulic shear force to remove the blockage at the bed inlet, and then resume normal operation. This design ensures... It is physically real and effective.

[0024] In some implementations, the biotransformation process may involve a multi-step tandem reaction, such as A B (Fast Response) C (slow reaction), where B and C are both desired products, but the formation rate of impurity D is between the two; in this case, a single It is difficult to balance both; this method can achieve time-series orchestration control through structural design; a fixed-bed bioreactor may include at least two connected in series with different volumes ( , ) reaction unit; due to In a series system, the amount of material is constant, and the material is in the first reaction unit ( Obtain a shorter length from a smaller (smaller) (Used to complete fast reaction A) B), then enters the second reaction unit (B), and then enters the second reaction unit ( (Larger) to obtain a longer (Used to complete slow reaction B) C), while the total stay time The reaction time is still set to be shorter than the defect time of impurity D, thus achieving precise matching of the multi-step reaction sequence. In other embodiments, to cope with fluctuations in reaction temperature during the process (which have a significant impact on enzyme reaction kinetics), this method can also introduce temperature compensation logic. The system monitors the actual operating temperature of the PBR in real time through a temperature sensor. The control unit contains a pre-stored compensation model calibrated based on the Arrhenius equation, which characterizes... and Follow The precise relationship of change; based on this model, the system calculates in real time the current... The dynamic safe dwell time that can simultaneously satisfy conditions a and b At this point, all the aforementioned actions to compensate for enzyme inactivation by compensating for the reduction of the baseline constant flow rate will have the same target after the reduction. It will no longer be a fixed value, but will dynamically change with... The method is matched to achieve feedforward compensation for temperature disturbances. Finally, after the product liquid flows out of the fixed-bed bioreactor, in order to ensure the termination of the reaction and the stability of the product, the method also includes an immediate post-processing step, namely, rapid quenching of the product liquid, such as rapid cooling through a plate heat exchanger, or pH adjustment treatment, such as adding acid or base to make the pH deviate from the optimal range of the enzyme, so as to terminate any possible residual enzyme activity.

[0025] Example 1: This example demonstrates the specific application of the technical solution in a particular industrial operation scenario, showcasing the adaptive compensation process for two simultaneous implicit process disturbances occurring during biopharmaceutical manufacturing when multiple mechanisms work together. In a continuous production task for preparing a pine needle extract scalp health repair agent, a fixed-bed bioreactor (PBR) system containing an immobilized complex glycosidase is set to operate continuously for 168 hours. This system, based on the aforementioned specific implementation method, also integrates a mechanism based on average residence time. The system employs time-series gating, feedforward calibration based on bypass microfluidic reactors, and online enzyme activity diagnosis and compensation methods based on transient flow rate perturbations; the system's initial reference constant flow rate... It was set to 100.0 mL / min to produce, under initial healthy conditions. The average stay time is 1 minute. The value satisfies Second A kinetic window of minutes; at the 72-hour mark when the system is running smoothly, two parallel process drift events occur: Event 1, the upstream feed tank switches to a new batch of pine needle slurry. Due to differences in origin, this batch of slurry contains a higher concentration of natural enzyme inhibitors than the standard batch, leading to actual... Extended; Event 2: After 72 hours of continuous operation, the immobilized biotransformation enzyme system in the PBR began to show slight but gradual inactivation, resulting in a decline in its catalytic ability.

[0026] When this new batch of feed solution enters the system, the feedforward calibration mechanism of the microfluidic reactor is triggered first. A portion of the feed solution is diverted to the parallel microfluidic reactor, and the real-time reaction rate measured by the UV-Vis spectrophotometer at its outlet immediately shows a reading that is 9% lower than the baseline reaction rate. Based on this, the control system determines that the current biotransformation activity of the feed solution is low and immediately dynamically adjusts the calibration value of the baseline response characteristic used for enzyme activity diagnosis in the main control logic. That is, when the system performs subsequent diagnosis, it has anticipated that the reaction rate will be inherently slower due to the raw material problem. 9%; then, at hour 72.5, the system automatically performed an online enzyme activity diagnosis based on a transient flow rate disturbance, as scheduled. The control unit applied a transient flow rate disturbance of +5% for 30 seconds to the constant flow pump of the main PBR. Since the enzyme system in the PBR had indeed undergone slight inactivation (Event 2) at this time, the reaction had entered a kinetically sensitive state. The online detector at the PBR outlet captured the observable response characteristics caused by this disturbance, namely, a transient and measurable decrease in the concentration of the target active ingredient. The control system calculated the normalized response amplitude. At that time, what it used The baseline has been corrected based on the results of the feedforward calibration. This collaborative operation demonstrates a composite control logic of feedforward and feedback: the feedforward calibration mechanism originates from the microfluidic reactor, firstly eliminating the apparent phenomena caused by raw material fluctuations, allowing the subsequent transient disturbance diagnosis mechanism, i.e., the analysis of response characteristics, to accurately and undisturbedly reflect the internal health status of the system—enzyme activity inactivation; based on this accurate inactivation judgment, the control system automatically compensates by maintaining a constant baseline flow rate. Reducing the flow rate from 100.0 mL / min to 99.0 mL / min increases the average residence time. This single adjustment action simultaneously compensates for disturbances from two different sources, thus prolonging the duration of the disturbance. To adapt to the slowed-down This is caused by a combination of raw material inhibitors and decreased enzyme activity, thus ensuring the yield of the target active ingredient and extending the recovery period. Still strictly in a state of being much smaller than Within the safety window, the generation of key immunogenic impurities is avoided.

[0027] Example 2: This example includes a comparative experiment to objectively verify the technical effectiveness of the method of the present invention compared to the traditional batch processing process and the simplified continuous flow process without integrated online compensation function in terms of product purity and long-term yield stability. The experiment consisted of three groups: Control Group 1, simulating the traditional batch processing process; Control Group 2, using the simplified continuous flow process; and the sample group of the present invention, employing the complete technical solution of the present invention. Control Group 1 used a 10L standard jacketed stirred batch reactor; Control Group 2 and the sample group of the present invention both used fixed-bed bioreactors (PBRs) with an effective bed volume of 1L. Both PBRs were filled with the same batch and the same loading of immobilized composite glycosidase prepared according to the specific implementation method. All three experimental groups used the same batch (batch number A) of pine needle raw material solution, which was offline calibrated, and its kinetic parameters were: expected reaction time... Seconds, defect response time minutes; control group 1, after being fed according to the standard process, was at 30 minutes. Samples were taken after 8 hours of stirring reaction; control group 2 was controlled at a constant reference flow rate. Continuous operation, the flow rate is calculated and set to ensure the average residence time Fixed at 5 minutes (300 seconds), this The value satisfies the dynamic window, 90 seconds. Second minutes, and control group 2 did not perform any online diagnosis or flow rate compensation; the initial baseline constant flow rate of the sample group of the present invention. Similar to control group 2, the initial The test duration was also 5 minutes, but the control system of the sample group of this invention integrated the enzyme activity online diagnosis and compensation method based on transient flow rate disturbance disclosed in the aforementioned specific embodiments. This method was set to be executed automatically once every 1 hour. After the test was started, both control group 2 and the sample group of this invention ran continuously for 168 hours. Samples were taken from the PBR outlet at the nodes of the 2nd hour, 72nd hour and 168th hour of operation, respectively. Control group 1 was sampled after running for 8 hours. All samples were immediately quenched and the same high performance liquid chromatography (HPLC) method was used to quantitatively detect the concentration (mg / L, quantification rate) of the target active ingredient and the concentration (%, characterizing purity) of the key immunogenic impurities in the samples according to the standard curve method. The test data are recorded as shown in Table 1.

[0028] Table 1: Performance Comparison of Different Preparation Methods in Long-Term Operation

[0029] Referring to the data in Table 1, first compare the purity indicators; control group 1, due to its reaction time of 480 minutes, is much longer. (15 minutes) led to the accumulation of key immunogenic impurities at a concentration of 1.52%; while control group 2 and the sample group of this invention, due to their average residence time The time was controlled to approximately 5 minutes (far less than 15 minutes), showing very low impurity concentrations (below 0.02%) at all time points; this data confirms that the use of PBR and The time-gated continuous flow process can mechanistically avoid the generation of impurities. Further comparison of yield stability indicators showed that in the initial stage of operation (2 hours), the enzyme activity of both control group 2 and the sample group of this invention was in an initial healthy state, with the target active ingredient concentration of both at 98.6 mg / L. However, after long-term operation (168 hours), control group 2 experienced enzyme inactivation, leading to... Extended, and its The reaction time was kept fixed at 5 minutes, resulting in incomplete reaction and a yield decrease to 40.3 mg / L. In contrast, the control system of this invention periodically performs transient flow rate disturbances and analyzes the response characteristics to diagnose enzyme inactivation and automatically compensate by lowering the flow rate. , and The time limit was gradually increased from 5.0 minutes to 6.4 minutes; this extension... This compensated for the decrease in enzyme activity, allowing the yield to remain at 98.2 mg / L for 168 hours.

[0030] To further verify the synergistic effect of the parallel microfluidic feedforward calibration system used to compensate for batch-to-batch fluctuations in raw materials in the method of the present invention, the following comparative examples are set up.

[0031] Example 3: Comparative Example 1 was set up, with process conditions basically the same as the sample group of the present invention in Example 2. Both used a PBR reactor, immobilized enzyme, and an online enzyme activity diagnosis and compensation method based on transient flow rate disturbance. The difference was that the system of Comparative Example 1 did not integrate the calibration microfluidic reactor and the associated feedforward calibration logic, and its enzyme activity compensation control relied only on the concentration feedback at the main PBR outlet. The sample group of the present invention still adopted the complete scheme in Example 2, that is, it simultaneously integrated the online enzyme activity diagnosis and compensation method and the feedforward calibration mechanism. Both groups of experiments were started using batch A feed solution from Example 2, with an initial constant flow rate. All are set to generate Minutes; after 72 hours of stable operation, both systems simultaneously switched to using batch B pine needle raw material solution; this batch B raw material solution, after offline calibration, contains a high concentration of natural inhibitors, and its expected reaction time is [not specified]. Extending the reaction time to 100 seconds corresponds to a decrease of approximately 10% in the reaction rate of the raw materials. Both systems continued to run for 168 hours after the raw materials were switched. During this period, the enzyme system in the PBR underwent the same progressive inactivation as in Example 2. The experimental data are recorded in Table 2.

[0032] Table 2: Performance Comparison of Different Compensation Strategies in Coping with Raw Material Batch Fluctuations

[0033] Referring to the data in Table 2, after switching to batch B feedstock, the control system of Comparative Example 1, which only has enzyme compensation logic, detected a decrease in the total reaction rate caused by the combined effects of enzyme inactivation and reduced feedstock activity during transient flow rate disturbance diagnosis at hour 73. Because it could not distinguish between these two disturbance sources, the system attributed the entire decrease in the total rate to enzyme inactivation, thus performing excessive compensatory adjustments. Extended to 8.2 minutes; as this misattribution continued to accumulate, by the 168th hour, the system had... The cumulative dwell time has increased to 16.5 minutes; this dwell time exceeds the defect response time. (15 minutes) This resulted in the accumulation of critical immunogenic impurities to 2.85%, causing product safety failure. In contrast, when the sample group of this invention switched to batch B raw material solution, its calibrated microfluidic reactor first identified a 10% reduction in the raw material activity itself and immediately updated the reaction baseline of the control system. Subsequently, its transient flow rate disturbance during the 73-hour run was diagnosed based on the calibrated baseline, thus enabling the separation of the rate decrease caused solely by enzyme inactivation and implementing corresponding compensatory adjustments. It remained at 5.7 minutes; up to 168 hours, The time was extended to 6.4 minutes only as a compensation, which ensured the yield while remaining within a safe window of less than 15 minutes, keeping the impurity concentration at 0.02%.

[0034] Example 4: This example combines Figures 1 to 4 Description of pine needle extract scalp health repair agent and its preparation method, such as... Figure 1 As shown, pine needle feedstock liquid from upstream is pumped into a fixed-bed bioreactor (PBR) at a constant reference flow rate by a high-precision constant flow pump for biotransformation. The core function of the PBR is to control the average residence time. The PBR product liquid sequentially passes through an online detector at the outlet to measure the concentration of the target active ingredient and a post-treatment unit for quenching or pH adjustment to terminate any residual enzyme activity, ultimately obtaining a high-purity pine needle extract remedial agent, thereby avoiding pathogenic impurities. To achieve long-term stable operation, the system also includes a parallel calibration microfluidic reactor, which measures the real-time reaction rate through an online calibration detector to compensate for batch-to-batch fluctuations in raw materials. The output signal of this parallel calibration system, together with the signal from the PBR outlet online detector, is input to an online enzyme activity diagnosis and compensation mechanism. This mechanism analyzes the transient disturbance response characteristics and sends the diagnostic results to the main control unit. The main control unit is responsible for calculating and compensatingly reducing the flow rate. This compensation signal is fed back to the high-precision constant flow pump to adjust its constant reference flow rate in a closed loop.

[0035] like Figure 2 As shown, the horizontal axis represents time (seconds), the left vertical axis represents the target active ingredient concentration (mg / L), and the right vertical axis represents the flow rate (%). The preset transient flow rate disturbance is applied at 0 seconds, and its form is that the flow rate jumps instantaneously from 100% to 105%, remains at this high level for 30 seconds, and then instantly drops back to 100% at 30 seconds. The data shows that when the enzyme system is in an initial healthy state with a baseline of 98.5 mg / L, the outlet concentration is basically unaffected by this disturbance and remains stable. However, when the enzyme system is in a slightly inactivated state with a baseline of 97.8 mg / L or a moderately inactivated state with a baseline of 96.7 mg / L, the reaction enters a kinetically sensitive state. The same flow rate disturbance will cause a clearly observable decrease in the outlet target active ingredient concentration, which is related to the degree of inactivation. This difference in response characteristics is used to diagnose the inactivation state of the enzyme system online.

[0036] like Figure 3As shown, in this architecture, the material from the pine needle slurry in the raw material tank is diverted to the main reaction unit, namely the fixed-bed bioreactor (PBR), and a parallel calibrated microfluidic reactor via a high-precision constant flow pump. The outlet of the PBR is connected to the post-processing unit for quenching / pH adjustment, and finally flows into the product tank for high-purity extract. To achieve closed-loop control, the main control system integrates compensation and diagnostic logic. The system receives input signals from multiple sensors, including: calibration detectors such as UV / polarization detectors for monitoring the parallel reactors, inlet pressure monitoring and actual operating temperature monitoring for monitoring the PBR status, and an online outlet detector for monitoring the quality of the final product. The main control system performs calculations based on these input signals and sends its control output signal to the high-precision constant flow pump to dynamically regulate the material flow rate of the entire system.

[0037] like Figure 4 As shown, the process engineer, as the operator, can perform a series of core operations, including performing system calibration before production to calibrate kinetic, hydraulic, and response benchmarks, as well as starting continuous production and triggering self-healing cleaning. During continuous system operation, the process engineer's main task is to monitor process stability, while the system automatically performs a series of diagnostic functions in the background, including online diagnosis of enzyme inactivation status, feedforward calibration of raw material fluctuations, and diagnosis of progressive blockage status. These automated diagnostic functions provide decision support for the engineer's monitoring tasks and form the basis of the system's automatic compensation logic.

[0038] Example 5: This example describes a standardized engineering procedure for calibrating key control parameters and a baseline model before the method of the present invention is put into continuous production, in order to eliminate parameter black boxes and model black boxes. After the system containing the fixed-bed bioreactor (PBR) is installed and commissioned, and it is confirmed that the immobilized biotransformation enzyme system is filled in an initial healthy state, a two-stage system calibration procedure is initiated. The first stage is hydraulic calibration, the purpose of which is to establish a health characteristic relationship for online diagnosis of progressive clogging. In this stage, an inert buffer solution (pH 6.5) that does not participate in the biological reaction is used as the calibration medium. The control system drives a constant flow pump to pump into the PBR at multiple preset step-increasing flow rate points (e.g., 50.0 mL / min, 75.0 mL / min, 100.0 mL / min, 125.0 mL / min). At each flow rate point, the system inlet pressure is... Once the flow rate stabilizes (e.g., fluctuations less than 1% lasting for 60 seconds), record the flow rate. and Stable operating point data pairs; these data points ( Fitting, for example, fitting to a polynomial curve using the least squares method. The resulting functional relationship is stored as a health characteristic relationship in the control logic, which is used for real-time comparison of the blockage status in subsequent production.

[0039] The second stage is the calibration of the biotransformation control logic, which is executed after hydraulic calibration and replacement of the buffer solution with feed solution from the standard raw material batch (batch number A). First, the system operates at an initial baseline constant flow rate, such as 100.0 mL / min. Once the target active ingredient concentration at the PBR outlet reaches stability, it enters the initial healthy state. At this point, the control system automatically applies a transient flow rate disturbance of a preset form, such as a 30-second pulse with a +5% amplitude. The online detector records the response pattern of the target active ingredient concentration at the outlet at this moment. This pattern should be close to zero fluctuation at this time, digitized, and stored as a baseline response characteristic, serving as a healthy template for subsequent diagnosis of enzyme inactivation. Next, the process for determining the inactivation threshold is executed. The calibration procedure is implemented by simulating known, slight deactivation conditions; the control unit adjusts the actual operating temperature of the PBR from the optimal 30°C. Adjust downwards to a slightly off-center 28. This temperature reduction, in the art, can stably simulate a slight decrease in enzyme activity, until the system reaches 28°C. After reaching a new steady-state reaction, the exact same transient flow rate perturbation is applied again; since enzyme activity is at 28... The concentration has already decreased slightly, and the system has entered a kinetically sensitive state. This disturbance will cause the outlet concentration to produce a normalized response amplitude that can be clearly measured, for example, calculated from... Meanwhile, the system is at 30 The reference response amplitude (i.e., background noise) measured under healthy conditions is: Based on this, the control system sets the inactivation judgment threshold. Set a value between the two that can distinguish between true inactivation and background noise, for example, It is set to 0.03; this calibration procedure will The settings are transformed from empirical values ​​into engineering parameters that can be reproduced through standardized experimental procedures.

[0040] Example 6: This example describes the specific application of the method of the present invention in a multi-step tandem biotransformation reaction, which involves converting raw material A into intermediate product B (fast reaction, (minutes), then B is converted into the target product C (slow reaction, minutes), Minutes); simultaneously, there exists a defective pathway where C degrades to generate the key immunogenic impurity D ( (minutes); In this scenario, a system consisting of two PBR reaction units with different volumes connected in series, as described in the specific implementation, is used; the effective empty bed volume of the first reaction unit (PBR-1) is... The effective empty bed volume of the second reaction unit (PBR-2) is set to 2L. The system was set to 8L; both units were filled with the corresponding immobilized biotransformation enzyme system; the system was operated at a baseline constant flow rate. Continuous pumping of feed liquid at L / min; based on Calculate the average residence time of the feed liquid in PBR-1. L / (1.0L / min) = 2.0 minutes, this time is comparable to a fast reaction time. (2 minutes) Matching ensures that raw material A is almost completely converted into intermediate product B at the unit outlet; subsequently, the feed solution rich in B enters PBR-2, where its average residence time is... L / (1.0L / min) = 8.0 minutes, this time is related to slow reaction (8 minutes) matching, so that intermediate product B has been almost completely converted into target product C at the unit outlet; the total average residence time of the system minutes; this The value is strictly controlled to be shorter than the defect response time. Within a kinetic window of 12 minutes, this timing arrangement control using series reaction units of different volumes enables the system to provide the required residence time for each of the two desired reaction steps with different kinetic rates at a single constant flow rate, while ensuring that the final product C leaves the system immediately after its formation, avoiding the formation of impurity D, thereby obtaining the target product C with high purity at a high conversion rate.

[0041] At the 80th hour of continuous production operation, the actual operating temperature of the PBR was... Due to environmental fluctuations, from the baseline of 30 Rising to 31.5 The control system monitors the temperature change in real time and immediately calls the preset Arrhenius compensation model for calculation; the model is based on 31.5 The reaction kinetics at this point shorten, It was also shortened, and a new dynamic safe stay time was determined. The time was reduced to 5.6 minutes, down from 5.8 minutes; the control system then switched to the baseline constant flow rate. Automatically increased by approximately 3.6% to adjust the extended... and This matching ensures the reaction yield while mitigating the risk of excessive impurities due to temperature increases; at the 95th hour of system operation, the control system, during routine hydraulic condition diagnostics, detected that the current ( The working point, compared to the calibrated health characteristic relationship, its The value has remained 22% high, exceeding the 20% clogging threshold; based on this, the system determines that the PBR has entered a progressive clogging state; the control system immediately and automatically triggers a self-healing cleaning step, which includes: pausing the feed and pumping buffer in reverse at a 200% pulse flow rate for 15 seconds; after the cleaning step is completed, the system returns to the adjusted state. (correspond (minutes) running, at which time the monitored data... The data has returned to the health characteristic relationship curve, indicating that the physical blockage has been cleared, ensuring subsequent... The physical effectiveness of control.

[0042] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method of preparing a pine needle extract scalp health restorer, characterized by, include: Provide a fixed-bed bioreactor containing a biotransformation enzyme system; The feed solution containing pine needles is pumped into a fixed-bed bioreactor at a constant reference flow rate for biotransformation. The constant reference flow rate is set so that the average residence time of the feed solution in the fixed-bed bioreactor, when the biotransformation enzyme system is in its initial healthy state, simultaneously meets the following two conditions in terms of timing: a. The average residence time is longer than the preset expected reaction time, which is used to generate the target active ingredient; b. The average residence time is shorter than the preset defect reaction time, which is used to generate key immunogenic impurities; and the method further includes the following steps to compensate for the progressive inactivation of the bioconverting enzyme system during continuous operation: during the continuous pumping of the feed solution, a preset transient flow rate disturbance is periodically applied to the reference constant flow rate; the response characteristics of the target active ingredient concentration in the product liquid at the outlet of the fixed-bed bioreactor due to this transient flow rate disturbance are detected online; the response characteristics are compared with the reference response characteristics corresponding to the initial healthy state; when the difference between the response characteristics and the reference response characteristics indicates that the bioconverting enzyme system has been inactivated, the reference constant flow rate is automatically and compensatorily reduced to prolong the average residence time in order to offset the effect of bioconverting enzyme system inactivation on the target active ingredient generation rate.

2. The method of claim 1, wherein the pine needle extract scalp health restorer is prepared by the steps of: a) extracting pine needles with water; b) filtering the pine needle extract; c) concentrating the pine needle extract; d) adding a preservative to the pine needle extract; and e) sterilizing the pine needle extract. The preset expected reaction time and the preset defective reaction time are determined based on the characterization of the kinetic parameters of the biotransformation reaction under standard raw material batches; the baseline response characteristics are the pre-calibrated target active ingredient concentration response of the biotransformation enzyme system in the initial healthy state to the transient flow rate disturbance of the preset form.

3. The method for preparing a pine needle extract scalp health repair agent according to claim 1, characterized in that, Before the feed solution enters the fixed-bed bioreactor, the method further includes: diverting a portion of the feed solution to a parallel calibration microfluidic reactor containing the same biotransformation enzyme system as the fixed-bed bioreactor; detecting online parameters characterizing the total reaction activity at the outlet of the calibration microfluidic reactor to obtain a real-time reaction rate; comparing the real-time reaction rate with a baseline reaction rate set based on a standard batch of feedstock; and dynamically adjusting the calibration value of the baseline response characteristics in the step of comparing the response characteristics with the baseline response characteristics based on the comparison results.

4. The method for preparing a pine needle extract scalp health repair agent according to claim 1, characterized in that, The method also includes: establishing a health characteristic relationship between a baseline constant flow rate and the corresponding inlet pressure when the fixed-bed bioreactor is in a healthy hydraulic state; monitoring the inlet pressure in real time during continuous feed pumping; comparing the current operating point of the baseline constant flow rate and the real-time monitored inlet pressure with the health characteristic relationship to diagnose the hydraulic state of the fixed-bed bioreactor online; and automatically triggering a self-healing cleaning step with pulse flow rate or reverse flow rate when a progressive clogging state is diagnosed.

5. The method for preparing a pine needle extract scalp health repair agent according to claim 1, characterized in that, The fixed-bed bioreactor comprises at least two reaction units connected in series, the at least two reaction units having different volumes; and the combination of a baseline constant flow rate and different volumes provides an average residence time for the feed liquid in each reaction unit for the preset biotransformation steps within that unit.

6. The method for preparing a pine needle extract scalp health repair agent according to claim 1, characterized in that, The method also includes: real-time monitoring of the actual operating temperature of the fixed-bed bioreactor; determining a dynamic safe residence time that ensures both conditions a and b are met based on the actual operating temperature and a pre-set Arrhenius compensation model characterizing the relationship between biotransformation reaction kinetics and temperature; and compensatingly reducing the baseline constant flow rate, with the goal of matching the extended average residence time with the dynamic safe residence time.

7. The method for preparing a pine needle extract scalp health repair agent according to claim 1, characterized in that, The step of comparing the response characteristics with the baseline response characteristics specifically includes: obtaining the maximum value of the target active ingredient concentration in the response characteristics. and minimum value And the baseline concentration of the target active ingredient before the application of the transient flow rate disturbance. ; Calculate the normalized response amplitude ,in The characterization of inactivation of the biotransformation enzyme system is based on the normalized response amplitude. Exceeding the preset inactivation threshold .

8. The method for preparing a pine needle extract scalp health repair agent according to claim 1, characterized in that, The transient flow rate disturbance of the preset form is a pulse signal. The amplitude of the pulse signal changes by 1% to 20% relative to the reference constant flow rate, and the duration of the pulse signal is shorter than the average residence time. The bioconversion enzyme system is fixed on a solid carrier in the fixed bed bioreactor by physical adsorption or covalent bonding. The method also includes the step of quenching or pH adjustment immediately after the product liquid flows out of the fixed bed bioreactor.

9. The method for preparing a pine needle extract scalp health repair agent according to claim 3, characterized in that, At the outlet of the calibrated microfluidic reactor, a UV-Vis spectrophotometer or polarimeter is installed for online detection of parameters characterizing the overall reaction activity.

10. A scalp health repair agent based on pine needle extract, characterized in that, The pine needle extract scalp health repair agent is obtained by the preparation method of the pine needle extract scalp health repair agent as described in claim 1.

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