Biochemical reaction control method in chromatographic column and application thereof

Abstract

The invention relates to a control and purification method for biochemical reaction in a chromatographic column and application of the control and purification method for the biochemical reaction in the chromatographic column, and in the biochemical reaction, a first reactant and a second reactant are in contact in the chromatographic column for reaction, the first reactant is subjected to the reaction in the process of chasing and passing through the second reactant area in the chromatographic column, the reaction is finished when the first reactant is separated from the second reactant area, the first reactant comprises but is not limited to biological macromolecular particles, and the second reactant comprises but is not limited to a chemical small molecule reagent. According to the implementation method, the biochemical reaction process is transferred into the chromatographic column to be dynamically carried out, and the expected biochemical reaction conditions are realized by pre-calculating and designing chromatographic operation parameters such as sample loading amount and proportion of two reactants, working flow velocity and the like through a'tracing problem 'data model, so that the biochemical reaction process is transferred into the chromatographic column to be carried out. Due to the characteristics of the chromatography technology, the reaction can be performed uniformly and timely in a contact manner for reaction or temperature exchange, the control of entering or leaving the reaction environment is more timely and accurate, and the purification effect of separation according to the time sequence of the generation of reaction products can be possibly generated; and a chromatography system can be used for carrying out process and automatic operation.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical engineering, and in particular to a biochemical reaction control method in a chromatography column and application thereof. Background Art

[0002] Chromatography, also known as chromatography technology, is the most widely used separation, purification, detection and identification technology in the biological industry. The chromatography technology commonly used in the pharmaceutical industry belongs to liquid chromatography technology, which is the most mainstream separation and purification process method in industrial pharmaceutical manufacturing.

[0003] In molecular exclusion chromatography, biological macromolecules flow faster than inorganic small molecules because they cannot enter smaller medium spaces. Therefore, after a mixed sample flows through a chromatography column, it can be separated and purified according to particle size. The separation effect is related to the chromatography medium, the size difference of sample particles, and the length of the chromatography column. In the process optimization of chromatography purification, the mobile phase pH value and conductivity adjustment methods are often used to change the charge state and polymerized bonding state of the sample and impurities to achieve better separation effects.

[0004] The chromatography process is a process of continuously pumping mobile phase (solution, liquid) into the chromatography column. While the solution flows into the upper end of the chromatography column, the lower end will also flow out to redistribute the liquid. With the help of the monitor, the useful components are collected, which is called sampling. In molecular exclusion chromatography, this process can be broken down into five steps: equilibrium, loading, washing, sampling (unloading), and cleaning and regeneration. Equilibrium refers to pumping in buffer to fill the chromatography column, loading refers to pumping in the sample to be purified, washing refers to continuing to pump in buffer to flush the sample out of the chromatography column, sampling (collecting, unloading) is to collect the useful liquid flowing out of the chromatography column, and cleaning and regeneration is to pump in cleaning solution.

[0005] Chromatography work usually starts with the loading of samples as the formal start of purification work. The recorder takes this as the zero point and records the cumulative volume of liquid entering or the working time according to the pump speed. In this way, the "working volume" can be viewed instantly through the record. The sample loading volume (sample liquid volume) and sample collection volume (sample loading volume, collected liquid volume) can be viewed or calculated from the recorded "working volume" and mark. The sample loading volume and sample collection volume can also be measured with measuring instruments such as measuring cylinders.

[0006] In the development of the lysis process of influenza vaccine, the current verification method of the lysis process is limited to qualitative tests such as electron microscopy observation and two-way immune diffusion, which cannot distinguish the differences between similar lysis conditions. The degree of influenza virus lysis is related to the detection rate of hemagglutinin antigen, so the hemagglutinin test results are often used as a reference in research. In addition, different researchers choose different sample protein concentrations when conducting lysis process research. Choosing different concentrations of detergent may only better meet the reaction dose requirements of different protein amounts, rather than the concentration requirements of lysis (the reaction dose in this article refers to the feed ratio, the molar ratio or mass ratio of the feed material).

[0007] In the production process of influenza vaccine subunit vaccine, a process of separation and lysis in a sugar density gradient is applied, which is to mix the lysis agent into the sucrose density zone layer. In the continuous flow ultraspeed zone centrifugation process, the whole influenza virus will pass through the low and medium concentration sugar layer into the high concentration sugar density layer, but due to the presence of the lysis agent, the surface spikes (HA, NA) of the influenza virus will fall off and stay in the medium and low concentration sugar layer, and the matrix protein spheres without surface spikes or incompletely lysed virus fragments will enter the higher sugar density layer. However, this method is limited by the volume of the centrifugal chamber, and the dosage of the lysis agent can only be increased by increasing the concentration of the lysis agent. The dosage of the lysis agent will affect the total amount of protein that can be processed in one centrifugal operation, and the excessive amount of protein loaded will not be lysed. At the same time, the increase in the concentration of the lysis agent may affect the requirements for lysis-the virus that enters early in the high concentration and high dose of the lysis agent environment may be completely broken instead of just the surface spikes falling off. At the same time, the sample demand for these process details in the continuous flow zone is too large, and the cost of one test is too high. Summary of the invention

[0008] Based on the above problems, the present invention proposes a method for performing biochemical reactions in a chromatography column. Conventional chromatography operations can also perform biochemical reactions in a chromatography column by adding chemical reactants to a pre-equilibrated buffer, but a large amount of chemical reactants will be wasted in the equilibrium flow washing process and will not participate in the reaction; in addition, the requirements for general chromatography purification are different from those for biochemical reactions in chromatography columns. The purification requirement is to purify the target sample without denaturation, while the purpose of the biochemical reaction is to transform and denature the target sample. Therefore, chromatography purification operations generally only start with simple equilibrium reaction control, and by adjusting the pH value, ionic strength, complexing ions, etc. of the buffer, the equilibrium reaction is allowed to proceed in a direction that is more conducive to depolymerization and separation.

[0009] How to use the movement law of chromatography to carry out dynamic biochemical reactions and how to apply the chromatography purification process to more complex and more intense biochemical reactions are the problems that the present invention needs to solve. The present invention provides a cross-border technology for the simultaneous mixing of biochemical reactions and purification separation, a separation technology for screening reaction products according to the sequence of reactions, and a continuous biochemical reaction control technology for separation and reaction.

[0010] The present invention provides a method for controlling a biochemical reaction in a chromatography column. In the biochemical reaction, a first reactant and a second reactant contact and react in the chromatography column. The reaction is carried out when the first reactant chases and passes through the second reactant region in the chromatography column, and the reaction ends when the first reactant leaves the second reactant region.

[0011] Furthermore, the first reactant includes but is not limited to biological macromolecule particles, the second reactant includes but is not limited to chemical small molecule reagents, and the second reactant is a reactant that cooperates with the first reactant to produce a biochemical reaction.

[0012] Furthermore, in the biochemical reaction, the process in which the first reactant catches up with and passes through the area of ​​the second reactant in the chromatographic column is constructed as a "pursuit problem" mathematical model, and the amount and proportion of the reactants, the working flow rate, the effective height of the chromatographic column, etc. are calculated and designed according to the mathematical model to control or set the biochemical reaction conditions to meet the expected requirements. The reaction conditions include reaction time, reaction concentration, reaction dose, etc.

[0013] Due to the characteristics of chromatography technology, reactions can be carried out evenly and timely, or temperature exchange can be carried out, and the control of entering or leaving the reaction environment is more timely and accurate.

[0014] Furthermore, according to the "pursuit problem" mathematical model, the reaction time is controlled by adjusting the working flow rate during chromatography and the volume of the second reactant, which can be used to adjust the biochemical reaction time; the amount of the second reactant loaded is positively correlated with the pursuit distance, and the pursuit distance is the effective height of the chromatography column.

[0015] Furthermore, according to the mathematical model of the "pursuit problem", the reaction time is also related to the flow rate coefficients of the first reactant and the second reactant. The flow rate coefficients of the first reactant and the second reactant are important parameters. The flow rate coefficients of the reactants are related to their own particle size and the resolution of the chromatographic medium. Selecting chromatographic media with different resolutions can change the flow rate coefficients.

[0016] Furthermore, the biochemical reaction time (t x ) is calculated as:

[0017] Among them, t xThe unit is h, d2 is the component length of the second reactant, the unit is cm, S d1 is the component flow rate of the first reactant, in cm / h, S d2 is the component flow rate of the second reactant, in cm / h, S d0 is the working flow rate, in cm / h, θ1 is the flow rate coefficient of the first reactant, θ2 is the flow rate coefficient of the second reactant, and the flow rate coefficient refers to the ratio between the flow rate of different components and the working flow rate;

[0018] The component flow rate (S d ) is calculated by: S d =θ*S d0 ; where S d0 is the working flow rate, θ is the flow rate coefficient, S d0 , S d The unit is cm / h.

[0019] Furthermore, the calculation formula of the flow rate coefficient (θ) is: θ = CV / V 出 , where CV is the column volume in ml, V 出 The working volume of the reactant from loading to flowing out of the chromatography column, in ml;

[0020] The calculation formula of the component length (d) is: x =θ*(V x / M), where V x is the loading amount of the reactant, M is the cross-sectional area of ​​the column, θ is the flow rate coefficient of the reactant, V x The unit is ml, the unit of d is cm, and the unit of M is cm 2 .

[0021] Furthermore, the reaction dosage control can be adjusted by changing the loading amount ratio of the first reactant and the second reactant when the concentration of the reactant is determined.

[0022] Furthermore, in the case of continuous multi-step biochemical reactants, after the first reactant leaves the second reactant region, the first reactant or its product continues to contact with more reactants for reaction.

[0023] In the case of continuous multi-step biochemical reactants, after the first reactant leaves the second reactant area, the first reactant or its product continues to contact with other reactants for reaction, and each biochemical reaction is controlled by the above control method; the number of other reactants is determined according to the actual needs of the biochemical reaction. In order to accommodate more reaction processes or reactants, the reaction batch size can be reduced or the length (height) of the chromatography column can be increased.

[0024] The present invention also provides a novel purification method for separating biochemical reaction products according to the order in which they are generated. When the properties of the biochemical reaction products are similar, they may be separated according to the order in which the reaction products are generated. The distribution range of different products can be determined after identification through continuous segmented collection, thereby achieving the purpose of purifying the reaction products.

[0025] The present invention provides a production process for influenza subunit vaccines based on biochemical reactions of chromatography columns, which is characterized in that, by using the above-mentioned control method, the influenza virus (first reactant) is allowed to pass through a lysing agent (second reactant) layer in the chromatography column, and the surface antigens and matrix proteins of the influenza virus are gradually stripped off, thereby achieving separation and purification of the surface antigens and the matrix proteins, thereby preparing a subunit vaccine that theoretically only contains surface antigens.

[0026] The present invention provides a virus inactivation process based on a biochemical reaction of a chromatography column, which is characterized in that the live virus (first reactant) is allowed to pass through an inactivator (second reactant) layer in the chromatography column through the above-mentioned control method, and the passed virus bodies lose their activity after the reaction, and is suitable for the viral vaccine products used.

[0027] Compared with the prior art, the advantages of the present invention are:

[0028] 1. The present invention dynamically conducts biochemical reactions in a chromatography column, and can more accurately control the biochemical reaction conditions, uniformly and instantaneously contact and mix, instantaneously exchange temperature, separately control the reaction concentration and dosage ratio, and accurately calculate and determine the reaction time. At the same time, chromatography technology can usually perform trace test analysis, and chromatography technology is a mature industrial production technology, which also makes the biochemical reaction control method in the chromatography column provided by the present invention suitable for laboratory trace tests, and the reaction conditions of trace tests can be easily scaled up for production.

[0029] 2. The present invention can realize separation according to the sequence of biochemical reactions in the chromatography column. Since the chromatography technology itself has a certain ability to test and analyze, after separation according to the sequence of chemical reactions, the progress of the reaction can be directly analyzed, and the separated samples can also be sampled and tested for more accurate and rapid analysis to obtain the best reaction conditions.

[0030] 3. The present invention transfers the biochemical reaction to the chromatography system, and can utilize the automation program of the chromatography system for automated operation. Purification, inactivation and other more production process flows are integrated in the chromatography system to help promote the automation of production based on the chromatography system.

[0031] 4. When the conventional method of mixing and reacting in a container is affected by excessively high local concentrations, when there is an effect of slow temperature control, when there is a conflict between the reaction concentration and the dosage requirement, when there are by-products of the same nature that are difficult to purify, when there is a problem of difficulty in batch-scale biochemical reactions, when there is a need for automated reactions for multiple reactions... the method of the present invention can be used to improve or resolve the problem. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the separation and purification process of size exclusion chromatography;

[0033] Figure 2 Chromatogram of whole influenza virus (first reactant);

[0034] Figure 3 Chromatographic purification diagram of influenza virus lysate mixture;

[0035] Figure 4 Chromatographic purification diagram of influenza virus in-column cleavage reaction.

[0036] Specific implementation method

[0037] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below. However, it should be understood that the description here is only used to explain the present application and is not used to limit the scope of the present application.

[0038] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present application, and the terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0039] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0040] Example 1

[0041] This embodiment provides a method for controlling a biochemical reaction in a chromatography column. In the biochemical reaction, a first reactant and a second reactant contact and react in the chromatography column. The reaction is controlled to be carried out in the process of the first reactant chasing and passing through the second reactant area in the chromatography column. The reaction ends when the first reactant leaves the second reactant area.

[0042] Preferably, the first reactant includes but is not limited to biological macromolecular particles, and the second reactant includes but is not limited to chemical small molecule reagents.

[0043] Since biochemical reactions and chromatography purification have a wide range of applications, the present invention only provides a method for transferring biochemical reactions to chromatography columns, and does not involve the types and reaction parameters of biochemical reactions. When the mixing reaction in a conventional container is affected by excessively high local concentrations, when there is an effect of slow temperature control, when there is a conflict between the reaction concentration and the dosage requirement, when there are byproducts of the same nature that are difficult to purify, when there is a problem of difficulty in batch-scale biochemical reactions, and when there is a need for automated reactions of multiple reactions, the method of the present invention can be considered.

[0044] As a further preferred embodiment, the process in which the first reactant catches up with and passes through the area of ​​the second reactant in the chromatography column is constructed as a "pursuit problem" mathematical model, and the amount and proportion of the reactants, the working flow rate, and the effective height of the chromatography column are calculated and designed according to the mathematical model to control or set the biochemical reaction conditions to meet the expected requirements, and the reaction conditions include reaction time, reaction concentration, and reaction dosage.

[0045] As a further preferred implementation mode, according to the "pursuit problem" data model, the reaction time is controlled by adjusting the working flow rate during chromatography and the loading amount of the second reactant, and the loading amount of the second reactant is positively correlated with the pursuit distance, and the pursuit distance is the effective height of the chromatography column.

[0046] Only by accurately grasping the distribution and movement of samples in the chromatography column during the chromatography operation can the contact reaction and reaction separation of the reactants be precisely controlled, which is the key to the biochemical reaction in the chromatography column. According to chromatography theory and work practice, the following phenomena can be found:

[0047] During the column equilibrium process, the mobile phase with new components pushes the old mobile phase out of the column like pushing a box, and the new mobile phase gradually occupies the entire column to complete the replacement. However, the new mobile phase will penetrate each other at the interface where the mobile phases are replaced, forming a concentration slope.

[0048] During the chromatographic purification process (size exclusion chromatography), particles of different sizes in the sample move forward at different flow rates. The larger the molecule, the faster the flow rate. Only the small molecule buffer continues to push the previous buffer in the chromatography column forward in a box-pushing manner. Large molecules all transcend their own buffer system and enter the new buffer. This is also the principle of chromatographic desalting or buffer replacement.

[0049] If air is introduced into the chromatography process, the air will pass through the chromatography column in a very short time, a dozen or tens of seconds, depending on the size of the chromatography column and the flow rate. After the air is introduced, the large and small particles in the chromatography column remain in place and need the solvent to move forward, which is consistent with the separation of the sample before the air is introduced, but the air bubble will hinder the sample in the bubble from moving forward. Based on this, the microscopic laws of chromatography are inferred, which will be the theoretical basis for biochemical reactions in the chromatography column, as follows:

[0050] 1. Movement of particles in the chromatography column

[0051] The flow rate of water (solvent) in the chromatography column is very fast. It can pass through the 80-90 cm long chromatography column in a very short time just like air. The particles in the solution can move under the scouring of this high-speed water (solvent) flow. The macromolecules in the outer water volume are the fastest moving ones detected, but their speed is less than 1 / 30 of the water flow. After being blocked by the chromatography medium, the speed of water entering the inner water volume slows down, driving the movement speed of small molecules in the inner water volume to be slower. Just like sand and stone deposits in the river bend, although small molecules can also move in the outer water volume, they can always be washed into the infinite "river bend", thus slowing down their movement. In the dense "river bend" environment of the chromatography medium, small molecules can hardly escape the "river bend" constraint, so they can maintain their established flow rate.

[0052] 2. Relationship between sample dilution and column efficiency during size exclusion chromatography

[0053] In the process of molecular exclusion chromatography, the monitoring peak of the sample is similar to the normal distribution curve, and the monitoring curve reflects the dilution change of the sample concentration. This is because when the sample contacts different mobile phases before and after, due to Brownian motion, samples of different concentrations or types tend to mix with each other. In the slender pipes and chromatographic media, this mutual mixing process is extremely long, thus forming a peak monitoring diagram with gradual dilution of the contact surface. There is a local siphon effect in the chromatographic medium, which accelerates the mixing and makes the monitoring peak wider. As a result, the average column efficiency value calculated in the process of measuring column efficiency is lower. It is generally believed that low column efficiency has poor separation effect, but in fact, the sample peak is wider and the impurity peak is also wider. The concentration slope caused by dilution requires a longer moving distance to separate. More actual column efficiency can be obtained by extending the chromatography column, and the average column efficiency only reflects the quality of the chromatography column filling, and the quality of the chromatography column filling only reflects the peak width level of particles in different "channels" during the chromatography process. In the chromatographic medium, Brownian motion is also restricted because the particles can only move forward under the impact of extremely high-speed water flow. This means that the peak width of the sample does not change much when it flows through a longer or shorter chromatographic column. The volume of the peak width of chromatographic columns of different thicknesses varies greatly, but when converted into linear width, the difference is not significant.

[0054] 3. Theoretical differential "channel"

[0055] Without considering the concentration slope at the intersection of the mobile phase, the sample appears to be advancing in a box-like manner in a square state of equal concentration when entering the chromatography column. This is because the speed is the same and cannot be exceeded or delayed. Due to different chromatographic media, there can be countless speed "channels" of this box-like manner, which are determined by the pore size distribution of the chromatographic medium. Here, the highest flow rate, the lowest flow rate and the middle flow rate are selected for analysis. Figure 1 As shown in the figure, under the action of flow rate, every time a unit volume of mobile phase enters, the samples in the three channels will advance a unit distance, but the unit distances of the three channels are different, which is determined by the flow rate difference. Figure 1 It not only shows the separation process of molecular exclusion chromatography, but also shows the distribution length of different flow rate components of the sample in the chromatography column, which are all calculated by speed. The length of the square peak reflects the length of the component in the chromatography column, and the height of the square peak reflects the change in the content of the component in the chromatography column (the concentration may decrease due to volume stretching due to fast speed, or the concentration may remain unchanged due to the stretching of spatial barrier length. In either case, the high flow rate component in the external water volume is more dispersed in the unit column volume, and the content is reduced). In the process of flowing out of the chromatography column, the three channel components can be restored to the original concentration and volume (ignoring the cross slope dilution).

[0056] 4. Dispersion state of the sample in the chromatography column

[0057] Without considering the combination and adsorption of the chromatography process, the components in the external water volume are very uniform in the process of balancing the ionic environment of the chromatography column, and there is almost no local excessive concentration. Due to the existence of slope dilution, it is a process from low concentration contact to normal concentration contact, which is very beneficial for biochemical reaction control. As long as the chromatography liquid is mixed before entering, the two reactants will react in the chromatography column in an instant and uniform state.

[0058] To carry out biochemical reaction in the chromatography column, the chemical small molecule (second reactant) and biological macromolecule (first reactant) samples are sequentially loaded as samples to be purified for chromatography operation. The other steps are the same as the chromatography purification operation method. The specific steps are as follows:

[0059] 1) Balance: flush the column with chromatography buffer for 1 to 3 column volumes to fully balance the medium and stabilize the column bed;

[0060] 2) Loading 1: Pump a certain volume of chromatography buffer containing the second reactant into the chromatography column (the volume of the second reactant affects the reaction time and reaction dosage). The loading amount of the second reactant is determined by the experimenter according to the reaction concentration, dosage (reactant feed ratio) requirements and the first reactant loading amount requirements. Generally, the reaction concentration is determined by referring to books or literature, and the reactant theoretical feed ratio is determined according to the reaction equation. Finally, the optimal conditions are determined by analyzing the results of multiple experiments, converted into the ratio of the second reactant to the first reactant, and then the loading amount of the second reactant is determined.

[0061] 3) Sample loading 2: Pump in a certain volume of the first reactant, set the loading time to 0, and mark the volume at the loading time as 0 (the loading amount ratio of the first reactant to the second reactant can adjust the amount of the reactant). The loading amount of the first reactant refers to the principle of the chromatography medium and the recommended loading amount for the purification effect determined by the experiment, which is generally between 1% and 30% of the column volume. After selecting the chromatography column, make adjustments based on the calculation results of the mathematical model of the "pursuit problem". In addition, if the loading amount is too small, it may cause the limit problem of subsequent sample analysis, and it is necessary to adjust the effective height of the chromatography column (the filling height of the chromatography medium in the chromatography column).

[0062] 4) Flow washing: Continuously pumping in the chromatography buffer, during which the first reactant will pass through and leave the second reactant area, contacting the reaction until the reaction is completed;

[0063] 5) Sample collection: Continuous flow washing, starting from about 1 / 3 column volume after the start of sample loading, the separated and / or purified substances are continuously collected for inspection and analysis to determine the collection position and range of the target product. Online monitoring data can also provide reference.

[0064] 6) The entire chromatography operation is carried out at a fixed pump speed (working flow rate). The flow rate has a great influence on the reaction time. If the pump speed needs to be changed, the impact of the reaction progress on the local reaction time must be comprehensively considered.

[0065] The medium in the chromatography column refers to the special substance filled inside the chromatography column. The present invention mainly adopts molecular exclusion gel medium. Since other gel media are mostly made by adding groups with adsorption effect to molecular exclusion gel media, under certain conditions, other media such as ion exchange chromatography media, hydrophobic chromatography media, reverse phase chromatography media, affinity chromatography media, etc. can also be used to carry out biochemical reactions in the chromatography column.

[0066] As a further preferred embodiment, the biochemical reaction time is controlled by adjusting the working flow rate during chromatography loading. When performing chromatography biochemical reaction operations, the only thing that can be controlled is the working flow rate. Other control methods are all calculated and determined before the chromatography operation or controlled by external equipment, such as:

[0067] The biochemical reaction time can also be controlled by adjusting the loading amount of the second reactant during chromatography; the loading amount of the second reactant is positively correlated with the pursuit distance, which is the effective height of the chromatography column. The reaction temperature is controlled by external temperature control support equipment (column oven, circulating chiller, etc.); the reaction dosage is controlled by the loading amount ratio of the first reactant to the second reactant, etc. Other reaction environments such as reaction concentration, pH, ionic strength, etc. are determined during the preparation and preparation of reactants and buffer solutions. The selected range is also the scope of protection of this application based on the actual experimental operation.

[0068] As a further preferred embodiment, according to the "pursuit problem" mathematical model, the reaction time is also related to the flow rate coefficients of the first reactant and the second reactant. The flow rate coefficients of the first reactant and the second reactant are important parameters. The flow rate coefficients of the reactants are related to their own particle size and the resolution of the chromatographic medium. Selecting chromatographic media with different resolutions can change the flow rate coefficient.

[0069] The following mainly explains the mathematical model and related mathematical formulas of the "pursuit problem" of chromatographic column biochemical reactions.

[0070] To calculate the contact reaction time of two reactants in a chromatography column, it is necessary to determine the width they occupy in the chromatography column and their respective movement speeds. In this way, the reaction time becomes a problem of catching up between two line segments. The flow rate of the reactants can be calculated by measuring the time difference between their entry and exit of the chromatography column. After calculating the reaction time (Where S d0 is the working flow rate, d2 is the length of the second reactant small molecule, θ is the speed coefficient (constant), from the formula, we can know that the control of reaction time can be achieved by changing the amount of the second reactant or changing the working flow rate. The specific calculation process is as follows:

[0071] The linear velocity and flow rate coefficient (ratio of reactant component flow rate to working flow rate) of each reactant in the chromatographic column are calculated by pre-loading the reactants for chromatography purification on the chromatographic column. The flow rate coefficient is converted by the formula equal to the column volume divided by the working volume when the component is sampled (the sampling time is taken as the volume 0 point). Secondly, the component length of different reactants in the chromatographic column is calculated according to the linear velocity. The component length of the reactant is equal to the reactant loading amount divided by the cross-sectional area of ​​the chromatographic column multiplied by the flow rate coefficient.

[0072] Figure 1 The model is obtained by calculation, and it has all the data for the biochemical reaction control study. This is the calculation method summarized after multiple loadings on a Sepharose 4FF gel column (XK 16 / 100) with a height of about 87 cm. The calculation method is as follows.

[0073] 1. Calculation of linear velocity of component “channel”

[0074] In a chromatography column with good column efficiency and symmetry, the sample loading volume is approximately equal to the volume before loading plus the dilution volume before and after. By excluding the dilution volume, the sample discharge time point can be found. In simulated chromatography loading, the loading time is set to 0, and then the discharge time or working volume is found. The flow rate of the components in the "channels" with different flow rates can be calculated based on the column length.

[0075] The loading time is marked as 0, that is, the working volume is 0 when loading. When discharging, record the working volume and time of starting discharging. After eliminating the dilution volume, find the discharging time and working volume when discharging in the record. The discharging of the sample indicates that the sample just passes through the entire chromatography column, that is, the moving distance is equal to the column height, and the column volume (1CV) = cross-sectional area * column height. Therefore:

[0076]

[0077] In the chromatography process, the working flow rate affects the sample time, while the working volume during sample discharge is relatively fixed and is usually expressed in column volumes (CV), i.e., V 出 =r*CV or V 出 = rCV represents the working volume when the sample is discharged. The r value is relatively fixed in the same component at different working flow rates or different specifications of the chromatographic column. The chromatography process can be paused or carried out discontinuously. The above formula should be the continuous working time, which is often calculated by dividing the working volume by the working flow rate, that is, T = V 出 / S v0 =rCV / S v0 ,but

[0078]

[0079] The reciprocal of r, 1 / r, can be described as the flow rate coefficient, which reflects the multiple relationship between the component flow rate and the working flow rate. For example, if a component with an external water volume flows out of the chromatography column at about 1 / 3CV, then the flow rate coefficient of the component is 3. Component flow rate S d It can be expressed as the working flow rate multiplied by the flow rate coefficient, that is,

[0080]

[0081] The working volume V of the target sample was tested by chromatography loading. 出 Then, calculate the θ value (θ=1CV / V 出 ), the linear velocity S of the sample in the chromatography column can be calculated d =θ*S d0 , different components can be represented by different numerical subscripts:

[0082]

[0083] The θ value is relatively fixed for the same component in the same chromatographic medium and will not change due to changes in column type or working flow rate.

[0084] 2. Calculation of the length occupied by sample components in the chromatography column

[0085] When the sample enters the chromatography column, components of different sizes in the sample will flow at different flow rates until all the samples enter the chromatography column. The extension length of each component no longer changes. The length of the component is related to the amount of sample loaded and the flow rate of the component:

[0086]

[0087]

[0088] The length of a component in a chromatographic column can be expressed as the linear height of the sample load multiplied by the flow rate coefficient.

[0089] The above measurement plus calculation method can more accurately calculate the flow rate and flow rate coefficient of different flow rate channels. In addition, the flow rate coefficient can be estimated based on the approximate peak position in the purification experience. For example, for mixed samples loaded at the same time, the large particle component with a fast flow rate in the external water volume generally begins to flow out at a working volume of 1 / 3CV, while the inorganic small molecules that can carry all the internal water volume need about 1CV to flow out. In this way, it can be estimated that the flow rate coefficient of the inorganic small molecules is θ=1, that is, the component flow rate of the inorganic small molecules is approximately equal to the working flow rate, and the component flow rate coefficient θ of the external water macromolecules is 3, and the component flow rate of the external water macromolecules is about 3 times the working flow rate.

[0090] 3. Calculation of biochemical reaction time

[0091] In the control of biochemical reactions in chromatography columns, biological macromolecules with faster flow rates are generally allowed to catch up with chemical small molecules and gradually come into contact and react. This is similar to the calculation of the "overtaking problem" of a high-speed passenger train chasing another low-speed train. When the front of the macromolecule "passenger train" contacts the rear of the small molecule "train" and starts to react, the rear of the "passenger train" has not yet started to react. Therefore, the macromolecule "passenger train" can be treated as a point to calculate the time required for the point to cross the "train" line segment, which is the time of the biochemical reaction.

[0092]

[0093] 4. Calculation of the length of the “catch-up” route

[0094] The above calculations are all performed under the condition that the "pursuit" route is long enough. However, in actual work, the length (height) of the chromatography column is limited. In this way, there may be a situation where the chemical reactants flow out of the chromatography column before "catching up" with the front and cannot participate in the reaction. This requires calculating the length of the "pursuit" route and ensuring a certain distance for the biological macromolecules to escape from the reaction conditions (the small molecule "line segment" area).

[0095] The calculation is based on the fact that chemical small molecules and biological macromolecules are loaded in sequence without any gap, that is, the distance between the head of the "passenger car" and the tail of the "train" is zero:

[0096]

[0097]

[0098] Since large molecules will surpass small molecules, that is, L1>L2 is inevitable, there is no need to calculate L2, and the column length can be greater than L1. When the column length is fixed, the length of the "train" and "bus" needs to be shortened, that is, the amount of sample loaded needs to be reduced to ensure the path of the reaction process.

[0099] The control of reaction time is based on the previous calculation formula: When the reaction substrate remains unchanged, θ1 and θ2 are fixed values. Therefore, the reaction time is only related to the length of the "line segment" of the chemical small molecule reactant and the working flow rate. The reaction time can be controlled over a large range by adjusting the working flow rate. The loading amount of the chemical small molecule reactant is also related to the dosage of the biochemical reaction and should be considered comprehensively when adjusting the reaction time.

[0100] As a further preferred embodiment, according to the "pursuit problem" mathematical model, the reaction dosage is controlled by controlling the loading ratio of the first reactant and the second reactant when the concentration of the reactants is determined.

[0101] The reaction dose is determined by the experimenter who conducts the biochemical reaction. It is generally determined by the required ratio of several reactants at the molecular level, and is finally selected through analysis of the test results. In conventional mixed biochemical reactions, the adjustment of the dose often changes the reaction concentration. In the method of the present invention, the two reactants are fed separately, which provides another dosage adjustment method that does not affect the concentration, that is, adjusting the feeding (sampling) volume of the two reactants.

[0102] In conventional biochemical reaction control, reactant concentration is a necessary condition to ensure the efficiency of biochemical reactions and even the occurrence of reactions. At the same time, concentration is also the only way to adjust the dosage of reactants. When conducting biochemical reactions in a chromatographic column, the optimal reaction concentration can be selected, and the reaction dosage can be adjusted by adjusting the loading amount of chemical small molecules. Since biological macromolecules gradually enter the range of chemical small molecules to react, the chemical small molecules always maintain an excess dosage and an appropriate concentration during the reaction process.

[0103] Set the reaction substrate concentration and dosage ratio, and finally inject the samples in sequence according to the principle of slow flow rate entering the column first. The reactant with fast flow rate surpasses the slow reactant to produce contact reaction, and the reaction time is calculated as a mathematical "catch-up problem" algorithm.

[0104] As a further preferred embodiment, the reaction temperature is controlled by a circulating water machine or a column temperature box.

[0105] The chromatography column can be stored in a column oven for temperature control. Large-scale chromatography columns can also be temperature controlled by a circulating water machine. The chromatography column and reactants can be pre-cooled or pre-heated for high reaction temperatures. The chromatography medium itself can be used as a heat exchange medium to allow the reactants entering to quickly reach a temperature close to that of the medium. As for the reaction temperature, in general, the conventional temperature range used in existing operations of such reactions can be used. As long as the temperature does not significantly affect the reaction, it can be used. Generally speaking, the temperature of the relevant reactions is room temperature or slightly higher, such as 2 to 60°C. Due to the influence of the chromatographic medium's tolerance temperature, it generally does not exceed 70°C. For example, when the influenza split vaccine reaction is controlled using the present invention, the temperature can be 2 to 50°C, that is, basically room temperature.

[0106] As a further embodiment, all reaction conditions are controlled by entering and leaving the reaction environment. The advantage of molecular sieve chromatography purification is that it can quickly change the ion environment. The condition control described above is related to this. Because the second reactant is quickly separated, the reaction time is controlled. The reaction buffer is consumed and a new buffer environment is quickly entered. Therefore, the reaction dosage is controlled. The temperature requirement is changed, and another chromatography column controlled at a different temperature can be quickly entered. Through precise calculations, the entry and exit of the reaction environment can be completed in a very short time.

[0107] Take XK16 / 100 chromatography column as an example for calculation. The inner diameter of the column is 1.6 cm and the cross-sectional area S is about 2 cm. 2 , filling height 87cm, column volume 1CV=174ml:

[0108] ① Linear velocity measurement: The amount of mobile phase (working volume) for inorganic small molecules from entering the chromatography column to flowing out is about 1CV, 174mL. Calculated at a flow rate of 1mL / min, it takes 174min to pass through 87cm of chromatography filler. The linear velocity is 0.5cm / min (S d2 =30cm / h); for biomacromolecules that can only pass through the external water volume, the amount of mobile phase (working volume) flowing out of the chromatography column is about 60mL, and the flow rate is 1mL / min for 60min, that is, the biomacromolecule only takes 60min to pass through the 87cm chromatography column, and its linear velocity is 87cm / h (S d1 =87cm / h); the working flow rate is 1ml / min (S d0 =30cm / h).

[0109] ② Calculation of the length of the sample in the chromatography column: The length of the small molecule buffer in the chromatography column is calculated by the measured linear flow rate of the inorganic small molecule, that is, the length of 60mL of the lysing agent buffer pumped in is about 30cm. The length of 12mL of the biomacromolecule sample after entering the chromatography column at a speed of 87cm / h is 17.4cm.

[0110] ③ Calculation of reaction time: How long does it take for a biomacromolecule to pass through a 30cm long chemical reactant line at a speed of 87cm / h, while the line itself moves at a speed of 30cm / h? How long does it take for a 17.4cm long biomacromolecule to completely pass through the lysing agent line? Is it still in the chromatography column at that time? The calculation result is that regardless of the length of the biomacromolecule solution, the time it takes for the biomacromolecule to pass through the chemical reactant is 31.58min (t x =31.58min).

[0111] ④ The time it takes for all biomacromolecules to pass through the lysing agent is about 49.89 min (t y =49.89min), at 49.58min the macromolecule advances to the position of 71.9cm and does not flow out of the 87cm column;

[0112] ⑤ The effect of the change in working flow rate: The amount of mobile phase for inorganic small molecules to flow out of the chromatography column is still 1CV, 174mL, but the time is shorter. The working flow rate is changed to 60cm / h, while the amount of mobile phase for large molecules with external water volume is still 60ml, and the flow rate is 174cm / h. The speed is doubled and the time is halved. x =15.5min,t y =24.5min, the macromolecule's forward position is still 71.9cm, which does not affect the pursuit journey, but can conveniently control the biochemical reaction time.

[0113] ⑥ Actual dilution slope Considering the above calculation is a theoretical state, in actual work, the interface of different solutions will diffuse and penetrate each other and dilute, and the length of the solution will be longer. For example, after 12mL of sample enters the chromatography column, a normal curve peak of about 18mL will flow out. The extra volume of 6mL is caused by the dilution of the front and back interfaces, that is, the dilution length of 1.5cm is added to the front and back. At the same time, the original 12mL sample also decreases in concentration due to dilution, and only the peak tip is close to the initial concentration. If the sample volume is reduced, the peak tip cannot maintain the initial concentration. After 60mL of the second reactant (small molecule) solution enters the chromatography column, 48mL can maintain the initial concentration. Diffusion and penetration at the interface are inevitable, but the length of dilution increase is related to the column efficiency. The higher the column efficiency, the shorter the dilution layer, and vice versa. The lower the column efficiency, the longer the dilution layer. In terms of dilution volume, it is related to the thickness of the chromatography column. The thinner the chromatography column, the smaller the dilution volume, and vice versa, the thicker the chromatography column, the larger the dilution volume. In practice, the dilution slope can be applied to chromatography columns of different thicknesses: without considering the influence of column efficiency, the linear length of the dilution slope is almost constant in chromatography columns of different thicknesses, and the larger the cross-sectional area, the larger the dilution volume.

[0114] As a further preferred embodiment, the biochemical reaction also includes a situation in which there are continuous multi-step biochemical reactants, and after the first reactant leaves the second reactant area, the first reactant or its product continues to contact with more reactants for reaction.

[0115] As the amount of the second reactant loaded decreases (the length becomes smaller), or the length of the chromatography column increases, it is possible to add the third and fourth reactants to participate. For example, the 60ml second reactant in the above example can be changed to 30ml second reactant plus 30ml third reactant. In the same chromatography process, the time of contact reaction with the second reactant is halved, and the time is divided for the third reactant. However, by halving the working flow rate, the reaction time can be doubled to the time before the change. Similarly, if the length of the chromatography column is doubled, the 60ml loading of the second reactant can be maintained, and 60ml of the third reactant can be added. It should be noted that the serial number of the reactant only represents a solution with similar solute size, which can be a mixture of multiple solutes. No matter how many solutes are involved in a one-step biochemical reaction process, two reactant solutions can be prepared. The reason for considering the third and fourth reactant solutions is generally that continuous multi-step reactions need to be carried out in a certain order.

[0116] In a specific experiment, when there are multiple biochemical reactions, the order in which the reactants are pumped into the chromatography column is that the small molecule reactant that reacts last is pumped in first, for example, the fourth reactant is pumped in first, followed by the third reactant, then the second reactant, and finally the first reactant. Refer to the specific experimental steps.

[0117] Example 2

[0118] As the simplest application of chromatography column biochemical reaction, the present invention also provides a virus inactivation process method, through the control method described above, the live virus can pass through the inactivator layer in the chromatography column, and the passed virus body loses activity after the reaction, which is suitable for the viral vaccine products used. The first reactant is the purified virus liquid, and the second reactant is the inactivator.

[0119] The virus inactivation process is generally carried out by mixing in a container. Inactivation often fails because the container wall or dead corners of the container are contaminated with live viruses and cannot contact the inactivator. Therefore, the operation of connecting the inactivation container in the middle of the inactivation process is derived, which increases the complexity of the process operation. The main operation process of inactivation in the chromatography column is: pumping buffer to balance the chromatography column, pumping a certain volume of buffer containing inactivator, pumping in virus sample loading, and collecting the virus particle components that pass through the chromatography column, which is the virus inactivation liquid. In this process, the inactivator layer is countless chemical barriers. Passing through them means inactivating the virus, and those that cannot move in the dead corners will not pass through the inactivator layer. More importantly, chromatography purification is the most commonly used purification method for viral vaccines. After introducing the inactivation process into the chromatography column, it is possible to integrate all downstream processes of production into the chromatography system, which lowers the threshold for system integration of fully automatic production lines and accelerates the promotion of automated production lines in the vaccine industry.

[0120] The size of the virus particles will not change significantly during the inactivation reaction, so the purification effect of separation based on reaction time cannot be added.

[0121] Example 3

[0122] A novel purification method for separating reaction products in the order of their generation. Through the control method described in the above claims, when the properties of biochemical reaction products are similar, they may be separated according to the order of their generation. The solutions that flow out successively are segmented and identified to determine the distribution range of different products, thereby achieving the purpose of purifying the reaction products.

[0123] As an application of separating products simultaneously during biochemical reaction in a chromatographic column, the present invention provides a chromatographic lysis surface antigen subunit separation process of an influenza subunit vaccine. The influenza virus is allowed to pass through a lysis agent layer in a chromatographic column through the control method described above, and the surface antigen and matrix protein of the influenza virus are gradually stripped off to achieve separation and purification of the surface antigen and matrix protein, thereby obtaining a subunit vaccine containing only the surface antigen; the products are separated in the order of their generation, and then identified and collected. The first reactant is a purified whole virus solution, and the second reactant is a lysis agent.

[0124] Traditional influenza split vaccines use a general mixed reaction method, and the purification process cannot specifically separate the virus's surface antigens, matrix protein fragments, and ribonucleoprotein fragments, so they can only be called split vaccines. The theoretical assumption of splitting and separating by biochemical reaction in the chromatography column is that the influenza virus flows through the buffer layer containing the splitting agent in the chromatography column, flowing forward while undergoing a splitting reaction. The surface antigens HA and NA are the first to contact the splitting agent, so they fall off first. The HA and NA that fall off are small molecules, and the flow rate is limited and they are separated from the large virus particles. Then the virus gradually breaks up, releasing the matrix protein M, core protein NP, etc. Although the size of the HA, NA, M, and NP protein particles after the splitting is not much different, there is a difference in order. In this way, the first to flow out is the whole virus large particle with the fastest flow rate, followed by the M and NP proteins that are split later, and then the HA and NA proteins that fall off earlier, and finally the original buffer ions in the virus sample. After the segmented components are identified and collected, the HA and NA surface antigens can be harvested to make subunit vaccines.

[0125] Chromatographic cleavage studies were performed using an XK 16 / 100 column filled with 87 cm of sepharose 4FF chromatography medium. The column cross-sectional area was approximately 2 cm 2 , column volume 1CV = 174ml (cm 3 ), the test steps are as follows:

[0126] First, through ordinary chromatography loading, it can be calculated that in the chromatography column used, the working volume of the mobile phase from the sample loading to the outflow of the external water volume channel is about 60ml, and the flow rate of this channel can be calculated to be 87cm / h (working flow rate 1ml / min). At the same time, it can be seen that the whole influenza virus sample used is of high purity, without impurity peaks, and the collection range of the whole virus is 60-82ml. Figure 2 shown.

[0127] Secondly, 0.7% lysing agent was used to lyse for 2 hours in a mixed reaction mode, and then chromatographic purification was performed, that is, separation and purification was performed according to molecular size. Collection began from 60 ml, and 6 tubes were collected in sequence. It was found that HA antigen was mainly distributed in the 6th tube, and 1 to 5 tubes were also distributed, indicating that the cleaved HA antigen was very close to the small molecule ion peak in the chromatographic medium, which met the separation condition that the molecule became smaller and the flow rate slowed down after shedding and separated from the large virus group; according to the different degrees of lysis, fragments of various sizes existed, and there was also a whole virus peak, such as Figure 3 shown.

[0128] Finally, a lysis reaction scheme in a chromatography column was selected based on the calculation: the virus was allowed to pass through a 0.7% lysis buffer layer (the lysis working volume was 5 times that of the sample, and the lysis time was about 1 hour), such as Figure 4As shown in Table 1, the results show that the antigen is concentrated in tubes 5 to 6, with a small amount distributed in tube 4. No HA antigen is detected in tubes 1 to 3 but there is a higher UV monitoring peak, indicating that the flow rate slows down after the HA antigen falls off and finally flows out of the chromatography column. The virus matrix balls continue to flow rapidly to tube 1, and the broken matrix balls are distributed in tubes 2 to 3.

[0129] Table 1 Distribution of hemagglutinin antigens cleaved in the column

[0130] Tube No. 1 2 3 4 5 6 Hemagglutinin <10 13.09 <10 88.79 399.71 303.66 Collection volume 15 15 15 15 15 19.5 Total hemagglutinin <150 196.35 <150 1331.85 5995.65 5921.37 Antigen distribution <1.1％ 1.4％ <1.1％ 9.7％ 43.6％ 43.1％

[0131] The advantage of the present invention is that there is a cleavage agent penetration process from the surface to the inside from the peeling of the surface antigen to the crushing of the matrix protein, and there is a time difference, and there is also a difference between the action time of peeling the surface antigen and the time required for crushing the matrix protein (breaking the matrix protein may require more cleavage agents to participate and provide more action). The present invention utilizes this time difference. When the surface antigen falls off and separates in the chromatography, the matrix protein has not been crushed, and the ribonucleoprotein has not been released, so that the component solution containing only the surface antigen subunits in theory is collected.

[0132] Compared with zonal centrifugal lysis, which also performs simultaneous lysis and separation, firstly, the processing capacity of chromatography is more flexible, while centrifugation is limited by the capacity of the centrifuge rotor, and large batches can only be performed in multiple times; secondly, the use of zonal centrifugal continuous flow equipment has improved the processing capacity of a single operation to a certain extent, but the continuous loading volume is still limited by the dosage of the lysis agent, and the dosage of the lysis agent is still limited by the capacity of the rotor. At the same time, the continuous sampling method has the problem of sample mixing, resulting in the actual surface antigen subunit purity after lysis and purification is not high; finally, the reaction time of simultaneous purification and lysis is determined by the movement speed of the virus particles. The movement power of the virus particles in the zonal centrifugal lysis process is provided by the centrifugal force. Under overspeed conditions (above 25,000 rpm), the virus particles need about 4 hours to complete the separation. If you want to shorten the time, you can only increase the centrifugal speed, but it is difficult to increase the speed under overspeed conditions; at the same time, the flow rate of continuous loading is also very slow. If it is too fast, it will be lost from the supernatant, so only a milder lysis agent can be selected. Chromatographic lysis is easier to control, with lysis times ranging from a few minutes to infinity, and equipment options available for sample loading ranging from microliters to more than ten liters, allowing for a wider range of control over reaction conditions.

[0133] Example 4

[0134] As an application of continuous multi-step biochemical reactions, Example 3 can be modified as follows:

[0135] The first reactant is influenza virus liquid, the second reactant is general buffer, the third reactant is dispersant, the fourth reactant is lysing agent, and the fifth reactant is inactivating agent. The virus liquid passes through the general buffer, which is equivalent to adding a step of chromatography to remove general impurities before chromatography lysis, and then passes through the dispersant to remove impurities adsorbed on the surface. The high-purity complete virus continues to pass through the lysing agent layer to strip the surface antigen, and then passes through the inactivating agent layer to inactivate the residual infection activity, and the inactivated purified stock solution can be obtained.

[0136] Secondly, based on molecular exclusion chromatography in different buffer environments, the adsorption and dissociation of biomacromolecules in buffer is a very mild equilibrium reaction. According to the principles of ion exchange chromatography and hydrophobic chromatography, the ionic force in a low-salt environment is stronger and the ions are more tightly bound. In a high-salt environment, the ionic force will be weakened, which may break the ionic bonds and make the biomolecules and impurities free. On the contrary, the hydrophobic force in a low-salt environment is weaker, which can make the proteins and impurities polymerized based on hydrophobic forces free, and the high-salt environment is more prone to hydrophobic polymerization. Different pH environments can adjust the charged properties of biomolecules and impurities, making them uncharged or repelling and dissociating with the same charge. If conditions permit, other dispersants, endonucleases, etc. can be used for desorption to improve the purification effect.

[0137] In molecular exclusion chromatography purification, it is very common that the target molecule adsorbs impurities and the purification efficiency is not high. Traditional chromatography methods can only screen an optimal buffer for purification. Purification using multiple buffers requires multiple purifications, which has no practical value due to large losses. According to the method of the present invention, the sample can be sequentially passed through a low-salt environment, a high-salt environment, and a variety of buffers with different pH values. When passing through a low-salt environment, impurities adsorbed by hydrophobic effects are separated, and when passing through a high-salt environment, impurities adsorbed by ions are separated, thereby improving the purification effect of molecular exclusion chromatography. One purification achieves multiple chromatography effects of multiple buffer environments, and can basically maintain a chromatography loss level. In order to use more types of buffers, the effective column length of the molecular exclusion chromatography column for chromatography purification can be extended to more than 2 meters in series. The advantage of the present invention is that it reacts and separates while it is reacting, and it will not be re-adsorbed back due to changes in the conditions of the buffer.

[0138] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for controlling a biochemical reaction in a chromatography column, wherein the biochemical reaction is characterized in that: The first reactant and the second reactant contact in the chromatography column to react. The reaction is carried out when the first reactant chases and passes through the second reactant area in the chromatography column, and the reaction ends when the first reactant leaves the second reactant area.

2. The control method according to claim 1, characterized in that: The first reactant includes but is not limited to biological macromolecule particles, and the second reactant includes but is not limited to chemical small molecule reagents.

3. The control method according to claim 1, characterized in that: The process in which the first reactant catches up with and passes through the area of ​​the second reactant in the chromatography column is constructed as a "catch-up problem" mathematical model. The amount and proportion of the reactants, the working flow rate, and the effective height of the chromatography column are calculated and designed according to the mathematical model to control or set the biochemical reaction conditions to meet the expected requirements. The reaction conditions include reaction time, reaction concentration, and reaction dosage.

4. The control method according to claim 3, characterized in that: According to the "pursuit problem" data model, the reaction time is controlled by adjusting the working flow rate during chromatography and the loading amount of the second reactant. The loading amount of the second reactant is positively correlated with the pursuit distance, and the pursuit distance is the effective height of the chromatography column.

5. The control method according to claim 3, characterized in that: According to the mathematical model of the "pursuit problem", the reaction time is also related to the flow rate coefficients of the first reactant and the second reactant. The flow rate coefficients of the first reactant and the second reactant are important parameters. The flow rate coefficients of the reactants are related to their own particle size and the resolution of the chromatographic medium. Selecting chromatographic media with different resolutions can change the flow rate coefficients.

6. The control method according to claim 3, characterized in that: According to the mathematical model of the "pursuit problem", the reaction dosage is adjusted by changing the loading ratio of the first reactant and the second reactant when the concentration of the reactant is determined.

7. The control method according to claim 1, characterized in that: The biochemical reaction also includes the situation where there are continuous multi-step biochemical reactants, and after the first reactant leaves the second reactant area, the first reactant or its product continues to contact with more reactants to react.

8. A novel purification method for separating reaction products in the order of their generation, characterized in that: By using the control method described in any one of claims 1 to 7, when the properties of the biochemical reaction products are similar, they may be separated according to the order in which the reaction products are produced, and the distribution ranges of different products can be determined after the solutions flowing out successively are segmented and identified, thereby achieving the purpose of purifying the reaction products.

9. A process for producing influenza subunit vaccines based on chromatographic column biochemical reactions, characterized in that: By using the control method described in any one of claims 1 to 7, the influenza virus is allowed to pass through the lysing agent layer in the chromatography column, and the surface antigens and matrix proteins of the influenza virus are gradually stripped off, thereby achieving separation and purification of the surface antigens and matrix proteins, thereby obtaining a subunit vaccine containing only the surface antigens.

10. A virus inactivation process based on chromatographic column biochemical reaction, characterized in that: By using the control method described in any one of claims 1 to 7, the live virus is allowed to pass through the inactivator layer in the chromatography column, and the passed virus bodies lose their activity after the reaction, which is suitable for the viral vaccine products used.

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