A manufacturing process for targeted protein modification

Abstract

This invention relates to the field of gene-engineered drug manufacturing technology and discloses a preparation process for targeted protein modification, comprising: introducing a recombinant polypeptide matrix flow containing recombinant polypeptide molecules into a continuous flow pipeline equipped with a helical alternating flow guide component; reconstructing the flow rate of the recombinant polypeptide matrix using the radial velocity field generated by the helical alternating flow guide component to generate a uniform flow field with plug flow characteristics; forming a low-pressure vortex region with a local minimum static pressure region on the back flow side of the helical alternating flow guide component; constructing a physical self-excited suction source using the pressure difference fluctuation between the central static pressure and the component pressure; introducing the modification component into the vortex core center; and causing the modification component to undergo directional collision modification with the recombinant polypeptide molecules in an unsteady conformation. This invention relies on the evolution of fluid structure to autonomously take up the modification component, physically shields the contact path of non-target residues, effectively improves the homogeneity of modification sites, and avoids protein polymerization induced by mechanical shearing.

Description

Technical Field

[0001] This invention belongs to the field of gene-engineered drug manufacturing technology, and in particular relates to a preparation process for targeted protein modification. Background Technology

[0002] Currently, in the large-scale preparation of recombinant therapeutic protein drug formulations, such as antibody-drug conjugates or long-acting peptides, continuous flow tubular reaction systems are widely used to meet the stringent requirements of pharmacopoeia standards for formulation purity and homogeneity. In the downstream modification process of such genetically engineered drugs and vaccines, maintaining the three-dimensional spatial folding conformation of protein molecules and the stability of the surface hydration layer is fundamental to ensuring the biological activity and clinical safety of these pharmaceutical preparations, and is directly related to the pharmacokinetic performance of the drug in vivo and the final clinical efficacy. As the production scale increases, a time delay mismatch occurs between the macroscopic fluid mixing scale in the pipeline and the microscopic protein molecule conformation evolution window. Protein molecules have flexible tertiary structures and chemically similar reactive residues distributed on their surfaces. To achieve targeted modification of specific sites, the process needs to control the solvent environment within a sub-second time window to cause the target protein to undergo reversible local unfolding, thereby exposing the target site.

[0003] To increase the probability of collision between modifiers and protein target sites, the industry often uses methods such as increasing the global fluid Reynolds number to enhance radial mixing. This method, which relies on increasing global turbulent flow, induces local shear stress in the boundary layer of the pipe wall or the stagnation region of the mixing components. Under shear stress and solvent exposure, the target protein's natural three-dimensional conformation is prone to exceed the thermodynamic renaturation critical point, resulting in irreversible denaturation or aggregation. If the flow rate is reduced to avoid damage, the diffusion rate of the modifier lags behind the reconstruction rate of the protein hydration layer, leading to random binding of non-target residues and the risk of immunogenicity. In the optimization path of continuous flow reaction systems, not only are the limitations of the aforementioned hardware roller morphology difficult to overcome, but the accompanying software control methods also have shortcomings. For example, the authorization announcement number CN216172359 U's Chinese invention patent discloses a vortex continuous flow reactor that relies on motor-driven staggered stirring paddles to force mechanical mixing of materials in the reaction chamber. The core premise of the conventional control strategy based on global mixing intensity enhancement is that the reaction fluid indiscriminately withstands externally applied mechanical energy. Under the specific micro-environment constraints of recombinant peptide molecule targeted modification, the control method is fundamentally mismatched with the actual objective working conditions. Macroscopic strong mechanical stirring will inevitably be accompanied by local shear force overload near the pipe wall or the edge of the paddle, which is very easy to peel off the hydration layer on the surface of the protein, inducing the flexible tertiary structure to cross the thermodynamic renaturation critical point and produce irreversible denaturation and aggregation. The existing technology deviates from the objective physical and mechanical boundary control mode of macromolecular conformation evolution and cannot achieve the natural alignment of the spatial distribution of modified components with the temporal exposure of protein active sites.

[0004] Therefore, how to utilize the physical evolution laws of fluid structures to align the physical injection of modifiers with the conformational evolution of protein molecules, thereby ensuring the high homogeneity of pharmaceutical proteins during formulation, is the technical problem to be solved by this invention. Summary of the Invention

[0005] To address the problems mentioned in the background art, the technical solution of the present invention is as follows: A preparation process for targeted protein modification, comprising the following steps: Step 101: Introduce a recombinant polypeptide matrix flow containing recombinant polypeptide molecules into a continuous flow pipeline equipped with a helical alternating flow guide component. Utilize the radial velocity field generated by the helical alternating flow guide component during axial flow to reconstruct the momentum of the recombinant polypeptide matrix flow, transforming the velocity vector distribution of the recombinant polypeptide matrix flow from a parabolic shape to a uniform field with plug flow characteristics. Relying on the fluid stripping effect on the back flow side of the helical alternating flow guide component, a low-pressure vortex region with a solvent polarity gradient and accompanied by a local minimum static pressure region is formed. Step 102: The modified component delivery port is located at the stagnation center of the low-pressure vortex region, and the component pressure in the modified component delivery port is adjusted so that the component pressure is maintained on the time scale within a range that is greater than the transient minimum of the static pressure at the center of the low-pressure vortex region and less than the average static pressure of the recombinant polypeptide matrix flow. This ensures that the modified component is in a restricted physical blockade state in the modified component delivery port when the flow field is in a macroscopic laminar flow state. Step 103: A physical self-excited suction source is constructed by utilizing the dynamic pressure difference fluctuation between the central static pressure and the component pressure. The physical self-excited suction source automatically opens the physical blockade state and introduces the modified component into the vortex core center of the low-pressure vortex region. The flow field shear stress in the vortex core center induces the recombinant polypeptide molecules to undergo local unfolding, so that the modified component and the recombinant polypeptide molecules in the unsteady conformation and enriched in the vortex core center undergo site-specific directional collision modification.

[0006] Preferably, in step 101, the helical alternating flow guiding components are arranged in a stepped alternating manner along the axial direction of the continuous flow pipeline. The radial mixing intensity of the recombinant polypeptide matrix flow is adjusted by the helical angle jump between adjacent levels of the helical alternating flow guiding components. After the directional collision modification is completed, the three-dimensional conformation of the recombinant polypeptide molecules is guided to the thermodynamic steady state by the cooling gradient field set downstream of the continuous flow pipeline.

[0007] Preferably, the recombinant polypeptide matrix flow is selected from one or more of monoclonal antibodies, antibody fragments, and extended-acting polypeptides; the modifying component is selected from one or more of polyethylene glycol derivatives, linker-cytotoxic drug conjugates, and fluorescent markers, and the residence time distribution of the recombinant polypeptide matrix flow and the modifying component in the vortex core is controlled by the lead of the helical alternating flow guide component.

[0008] Preferably, in step 102, the equivalent diameter corresponding to the outlet cross-sectional area of ​​the modified component delivery port is in the range of 5μm to 50μm. By adjusting the equivalent diameter, a capillary force constraint adapted to the physical self-excited suction source is generated, so that the modified component is locked inside the modified component delivery port by the liquid-solid interfacial tension established at the equivalent diameter before the physical self-excited suction source is started.

[0009] Preferably, the spiral lead of the spiral alternating flow guide component gradually decreases from the inlet end to the outlet end of the recombinant polypeptide matrix flow according to a preset ratio, generating an axial acceleration field for the recombinant polypeptide matrix flow in the continuous flow pipeline. The axial acceleration field enhances the central static pressure drop slope of the low-pressure vortex region by changing the local Reynolds number, thereby shortening the response delay of the physical self-excited suction source.

[0010] Preferably, after step 103, the spectral detection module located at the pipeline outlet collects the site homogeneity parameters of the modified product and converts the site homogeneity parameters into control electrical signals, which are then fed back to the pressure regulating unit. The pressure regulating unit corrects the component pressure in step 102 according to the control electrical signals, so that the molar ratio during the directional collision modification process is maintained within the preset target range.

[0011] Preferably, the stagnation center is located at the geometric intersection of the central axis of the helical alternating flow guide component and the backflow surface. The injection vector of the modified component delivery port coincides with the radial vector of the recombinant polypeptide matrix flow at the stagnation center, so as to reduce the transverse shear dissipation generated by the modified component during its entry into the vortex core center and maintain the biological activity of the recombinant polypeptide molecule.

[0012] Preferably, the turbulent kinetic energy in the global flow field of the recombinant polypeptide matrix is ​​maintained within a range below a preset turbulent kinetic energy threshold. The preset turbulent kinetic energy threshold is determined by the critical shear stress of the recombinant polypeptide molecule. By adjusting the surface roughness of the helical alternating flow guide component, the Reynolds stress in the near-wall region is reduced, thereby inhibiting the irreversible polymerization and precipitation of the recombinant polypeptide molecule.

[0013] Preferably, the recombinant peptide matrix after directional collision modification is directly connected to the tangential flow filtration system. The tangential flow filtration system uses an ultrafiltration membrane with a pore size distribution in the range of 10kDa to 100kDa to perform solvent replacement and concentration on the recombinant peptide molecules, so that the recombinant peptide molecules are output as GMP-compliant recombinant peptide molecular formulations under a constant ionic strength environment.

[0014] Compared with existing technologies, the preparation process for targeted protein modification in this invention has the following advantages: 1. In the preparation of targeted protein modification, by embedding the supply end face of the modifier into the back flow surface of the static alternating spiral guide plate, a physical connection is established between the local vortex core low-pressure region generated by fluid separation and the modifier supply branch. The periodic separation vortex generated inside the flow field not only provides a micro-fluidic environment for peeling off the hydration layer on the protein surface, but the accompanying local minimum static pressure also acts as a power source for modifier uptake. This endogenous dynamic-driven material inflow method enables the physical injection action of the modifier and the transient unfolded state of the protein molecule to be automatically aligned in the spatiotemporal dimension, thereby ensuring the structural consistency of the active ingredient of the gene-engineered drug at the molecular level. This mechanism eliminates the injection timing deviation problem existing in the traditional active pumping process, blocks the random collision between the modifier and the protein in the natural folded state, and improves the homogeneity of site modification of the product.

[0015] 2. Since the uptake of the modifier depends entirely on the self-excited suction mechanism inside the separation vortex, this process eliminates the transverse shear stress generated by the forced injection of the modifier using an external injection pump. When the protein stream passes through the targeted collision zone, it only experiences sub-second solvent polarity disturbances within the microenvironment of the vortex core. The fluid as a whole is maintained at a low turbulent kinetic energy level. This precise limitation of the stress distribution in the micro-flow field allows the protein molecules to quickly return to a stable thermodynamic state through a stepped cooling zone after completing the modification at specific sites. This process maintains the biological activity of the recombinant protein while reducing the risk of irreversible polymerization caused by mechanically induced denaturation.

[0016] 3. The process utilizes static alternating spiral guide vanes to generate radial disturbances in the fluid within the pipeline, transforming the parabolic velocity field into plug flow characteristics. This narrows the residence time distribution of protein molecules in the disturbance region. Combined with a self-excited suction mechanism, the injection frequency of the modifier and the shedding frequency of the separation vortex are naturally synchronized. This process does not rely on the detection delay or computational feedback of the electronic control system. Even when there are flow velocity fluctuations in the pipe wall boundary layer, the minimal static pressure zone at the center of the vortex core accurately captures and encapsulates unfolded protein molecules. This physical adaptive alignment ensures the consistency of modification quality for each batch of drugs in continuous flow mass production, resolving the engineering contradiction between high-throughput processing and high-purity control. Attached Figure Description

[0017] Figure 1 This is a complete process flow diagram of the self-excited suction and pressure feedback targeted protein modification of the present invention; Figure 2 This is a diagram of the multi-stage cooling recovery and molar ratio control architecture for targeted collision modification of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0019] A preparation process for targeted protein modification includes the following steps: Step 101: Introduce a recombinant polypeptide matrix flow containing recombinant polypeptide molecules into a continuous flow pipeline equipped with a helical alternating flow guide component. Utilize the radial velocity field generated by the helical alternating flow guide component during axial flow to reconstruct the momentum of the recombinant polypeptide matrix flow, transforming the velocity vector distribution of the recombinant polypeptide matrix flow from a parabolic shape to a uniform field with plug flow characteristics. Relying on the fluid stripping effect on the back flow side of the helical alternating flow guide component, a low-pressure vortex region with a solvent polarity gradient and accompanied by a local minimum static pressure region is formed. Step 102: The modified component delivery port is located at the stagnation center of the low-pressure vortex region, and the component pressure in the modified component delivery port is adjusted so that the component pressure is maintained on the time scale within a range that is greater than the transient minimum of the static pressure at the center of the low-pressure vortex region and less than the average static pressure of the recombinant polypeptide matrix flow. This ensures that the modified component is in a restricted physical blockade state in the modified component delivery port when the flow field is in a macroscopic laminar flow state. Step 103: A physical self-excited suction source is constructed by utilizing the dynamic pressure difference fluctuation between the central static pressure and the component pressure. The physical self-excited suction source automatically opens the physical blockade state and introduces the modified component into the vortex core center of the low-pressure vortex region. The flow field shear stress in the vortex core center induces the recombinant polypeptide molecules to undergo local unfolding, so that the modified component and the recombinant polypeptide molecules in the unsteady conformation and enriched in the vortex core center undergo site-specific directional collision modification.

[0020] Preferably, in step 101, the helical alternating flow guiding components are arranged in a stepped alternating manner along the axial direction of the continuous flow pipeline. The radial mixing intensity of the recombinant polypeptide matrix flow is adjusted by the helical angle jump between adjacent levels of the helical alternating flow guiding components. After the directional collision modification is completed, the three-dimensional conformation of the recombinant polypeptide molecules is guided to the thermodynamic steady state by the cooling gradient field set downstream of the continuous flow pipeline.

[0021] Preferably, the recombinant polypeptide matrix flow is selected from one or more of monoclonal antibodies, antibody fragments, and extended-acting polypeptides; the modifying component is selected from one or more of polyethylene glycol derivatives, linker-cytotoxic drug conjugates, and fluorescent markers, and the residence time distribution of the recombinant polypeptide matrix flow and the modifying component in the vortex core is controlled by the lead of the helical alternating flow guide component.

[0022] Preferably, in step 102, the equivalent diameter corresponding to the outlet cross-sectional area of ​​the modified component delivery port is in the range of 5μm to 50μm. By adjusting the equivalent diameter, a capillary force constraint adapted to the physical self-excited suction source is generated, so that the modified component is locked inside the modified component delivery port by the liquid-solid interfacial tension established at the equivalent diameter before the physical self-excited suction source is started.

[0023] Preferably, the physical self-excited suction frequency of the modified component and the vortex shedding frequency in the low-pressure vortex region maintain phase coupling, and the shedding frequency f satisfies the following relationship: Where St is the Strouhal number of the fluid in the continuous flow pipeline, v is the average flow velocity of the recombinant polypeptide matrix flow, and d is the characteristic diameter of the helical alternating flow guide component.

[0024] Preferably, the spiral lead of the spiral alternating flow guide component gradually decreases from the inlet end to the outlet end of the recombinant polypeptide matrix flow according to a preset ratio, generating an axial acceleration field for the recombinant polypeptide matrix flow in the continuous flow pipeline. The axial acceleration field enhances the central static pressure drop slope of the low-pressure vortex region by changing the local Reynolds number, thereby shortening the response delay of the physical self-excited suction source.

[0025] Preferably, after step 103, the spectral detection module located at the pipeline outlet collects the site homogeneity parameters of the modified product and converts the site homogeneity parameters into control electrical signals, which are then fed back to the pressure regulating unit. The pressure regulating unit corrects the component pressure in step 102 according to the control electrical signals, so that the molar ratio during the directional collision modification process is maintained within the preset target range.

[0026] Preferably, the stagnation center is located at the geometric intersection of the central axis of the helical alternating flow guide component and the backflow surface. The injection vector of the modified component delivery port coincides with the radial vector of the recombinant polypeptide matrix flow at the stagnation center, so as to reduce the transverse shear dissipation generated by the modified component during its entry into the vortex core center and maintain the biological activity of the recombinant polypeptide molecule.

[0027] Preferably, the turbulent kinetic energy in the global flow field of the recombinant polypeptide matrix is ​​maintained within a range below a preset turbulent kinetic energy threshold. The preset turbulent kinetic energy threshold is determined by the critical shear stress of the recombinant polypeptide molecule. By adjusting the surface roughness of the helical alternating flow guide component, the Reynolds stress in the near-wall region is reduced, thereby inhibiting the irreversible polymerization and precipitation of the recombinant polypeptide molecule.

[0028] Preferably, the recombinant peptide matrix after directional collision modification is directly connected to the tangential flow filtration system. The tangential flow filtration system uses an ultrafiltration membrane with a pore size distribution in the range of 10kDa to 100kDa to perform solvent replacement and concentration on the recombinant peptide molecules, so that the recombinant peptide molecules are output as GMP-compliant recombinant peptide molecular formulations under a constant ionic strength environment.

[0029] Example 1: In the preparation of monoclonal antibody formulations based on continuous flow pipelines, the process faces the technical problem of a spatiotemporal mismatch between the overall fluid mixing scale of the pipeline and the non-steady-state conformational evolution window of protein molecules. Traditional intervention methods rely on increasing the global fluid Reynolds number to enhance radial mixing, which induces mechanical shear stress in the boundary layer region of the pipe wall that exceeds the thermodynamic renaturation critical point of recombinant peptide molecules, leading to irreversible denaturation and polymerization of recombinant peptide molecules. On the other hand, the control path of reducing the matrix flow rate causes the diffusion rate of the modified component to lag behind the reconstruction rate of the hydration layer on the surface of the recombinant peptide molecules, resulting in random collision mismatch between the modified component and non-target residues. The technical solution of this invention introduces a recombinant peptide matrix flow containing recombinant peptide molecules into a continuous flow pipeline equipped with a stepped alternating spiral flow guide component. The radial velocity field generated by the spiral alternating flow guide component during axial flow reconstructs the recombinant peptide matrix flow, transforming the velocity vector distribution of the recombinant peptide matrix flow from a parabolic shape to a push flow field, and relying on the back flow side of the spiral alternating flow guide component... The fluid stripping effect forms a low-pressure vortex region with a solvent polarity gradient and a local minimum static pressure zone. The delivery port of the modified component is located at the stagnation center of this low-pressure vortex region. By adjusting the component pressure, it is maintained within a range greater than the minimum static pressure at the center of the low-pressure vortex region and less than the average static pressure of the recombinant peptide matrix flow. Thus, when the flow field is in a laminar state, the liquid-solid interfacial tension established by the modified component at a specific equivalent diameter within the delivery port is in a restricted physical blockage state. The solvent polarity gradient here does not refer to a spontaneous physical abrupt change in the chemical properties of the aqueous buffer solution itself, but rather to the high-frequency micro-mixing effect generated by the low-pressure vortex region. This causes the specific modified component, such as a polyethylene glycol derivative with a hydrophobic carbon chain, to diffuse instantaneously inside the vortex core. It forms a transient concentration difference diffusion field with the aqueous matrix flow around the stagnation center. This diffusion field is physically manifested as a gradient distribution of the relative permittivity of the local system as the concentration of the modified component decreases, thereby constructing a transient polar microenvironment band that encapsulates the recombinant peptide molecules.

[0030] As the recombinant peptide matrix continues to flow through the helical alternating flow guide, the dynamic pressure difference fluctuation between the central static pressure and the component pressure constructs a physically self-excited suction source. At the instant the fluid separation vortex is generated, the aforementioned physical blockade state is opened, and the modified component is introduced into the vortex core center of the low-pressure vortex region. The flow field shear stress enriched within the vortex core center induces local unfolding of the recombinant peptide molecules, causing the drawn-in modified component to undergo site-specific directional collision modification with the recombinant peptide molecules in their unsteady conformation. The momentum reconstruction action of the helical alternating flow guide narrows the variance of the residence time distribution of the recombinant peptide molecules in the perturbation region. The associated low-pressure vortex region directly acts as the power source for the uptake of the modified component, maintaining a phase coupling between the physically self-excited suction frequency of the modified component and the vortex shedding frequency of the low-pressure vortex region. The vortex shedding frequency follows the formula... The definition is as follows: f is the vortex shear frequency, St is the Strouhal number of the fluid in the continuous flow pipeline, v is the average flow velocity of the recombinant peptide matrix, and d is the characteristic diameter of the helical alternating flow guide component. This physical quantification mapping mechanism transforms the problem of lateral shear dissipation and spatial distribution deviation caused by forced injection from an external injection pump into a process of autonomous uptake of modified components based on the evolution of the internal physical structure of the fluid. In physical space, it cuts off the random contact path between the modified components and the non-targeted recombinant peptide molecules in a folded steady state. In this process, the macroscopic flow field shear stress does not directly and precisely target microscopic sites in space, but achieves specific selection through a cross-scale energy transfer mechanism. Due to the inherent hierarchical differences in the thermodynamic stability of different structural domains on the surface of recombinant peptide molecules, the macroscopic fluid mechanical shear work controlled inside the vortex core just exceeds the unfolding activation energy barrier of the weak hydrophobic structural domain where the target residue is located, while being lower than the critical energy for the overall denaturation of the molecular secondary structure. This mechanical stripping effect based on energy selection makes the target site, as the stress weak point in the system, preferentially excited and exposed, thereby completing the subsequent specific collision binding under the premise of non-denaturation.

[0031] The recombinant peptide matrix, modified by directional collision, flows into a cooling gradient field located downstream of the pipeline. Its three-dimensional conformation returns to steady state according to a thermodynamic trajectory determined by latent heat release. It then connects to a tangential flow filtration system, where an ultrafiltration membrane with a pore size distribution in the 10kDa to 100kDa range replaces and concentrates the solvent of the recombinant peptide molecules. The turbulent kinetic energy in the overall flow field is maintained below a preset turbulent kinetic energy threshold determined by the critical shear stress of the recombinant peptide molecules. The resulting product is a recombinant peptide formulation that meets the standards for manufacturing genetically engineered drugs and vaccines. This preparation process relies on the minimal hydrostatic pressure of the separated vortex core. The self-excited suction mechanism transforms the generation of fluid separation vortices and the injection of modified components into a single, synchronously bound physical event. This eliminates the physical spatial misalignment between the distribution sequence of modified components and the exposure sequence of recombinant peptide active sites. While maintaining the basic pipeline hardware architecture, it simultaneously achieves the goals of inhibiting non-targeted mismatch modifications and maintaining the biological activity of recombinant macromolecules. To physically convert the critical shear stress parameter at the microstructural level into a turbulent kinetic energy threshold that can be monitored by pipeline sensors, the system incorporates a computational control chain based on the Boussinesq eddy viscosity assumption. The main control logic module utilizes the formula... Perform a conversion process, where k is the upper limit of the turbulent kinetic energy threshold of the converted output, and ρ is the current matrix flux density of the recombinant peptide. The upper limit of the critical shear stress is determined in advance. As the empirical constant for eddy current energy dissipation (with a built-in fixed value of 0.09), this control chain establishes a complete deterministic mathematical calculation channel between the molecular mechanical yield limit and the macroscopic momentum diffusion index.

[0032] Example 2: To address the issues of fluid spatiotemporal mismatch and mechanical shear denaturation during the mixing of recombinant peptide molecules and modified components in a continuous flow pipeline, a continuous flow validation platform was constructed, incorporating a particle image velocimeter and a dynamic light scattering instrument. A particle image velocimeter with a velocity resolution better than 0.1 mm / s was selected, along with a dynamic light scattering instrument covering a hydration dynamic path range of 1 nm to 1000 nm. The fluid conditions in a pharmaceutical manufacturing environment were reproduced. A low-frequency mechanical vibration with a frequency of 50 Hz and an amplitude of 2.0 mm was input to the supply pump source as a test disturbance source. Within this operational boundary, the equivalent diameter parameter corresponding to the outlet cross-sectional area of ​​the modified component was determined. This parameter was determined based on the mechanism of balancing the surface tension of the physically sealed modified component and the minimum static pressure suction pressure difference of the vortex core. The vortex shedding frequency was determined when the average flow velocity of the recombinant peptide matrix increased. When increasing, according to the formula By defining a specific proportional relationship, the characteristic diameter d is simultaneously reduced and adapted to a smaller equivalent diameter to maintain the ability of the vortex core transient pressure difference to overcome interfacial tension. Here, f is the vortex shedding frequency, St is the Strouhal number of the fluid in the continuous flow pipeline, v is the average flow velocity of the recombinant polypeptide matrix flow, and d is the characteristic diameter of the helical alternating flow guide component. 5 μm is selected as the absolute lower limit experimental group parameter, 25 μm is selected as the normal median experimental group parameter, and 50 μm is selected as the absolute upper limit experimental group parameter. 2 μm and 60 μm are selected respectively to construct the first out-of-range control group and the second out-of-range control group, which exceed the working window of these parameters. The helical alternating flow guide component is removed to construct a straight pipe hybrid architecture as a partially missing control group.

[0033] The continuous flow validation platform was activated, and monoclonal antibody matrix flow and polyethylene glycol (PEG) modified components were introduced into the pipeline. A particle image velocimeter captured the velocity vector state of the monoclonal antibody matrix flow in the pipeline of the normal median experimental group. When the monoclonal antibody matrix flow passed through the back flow side of the helical alternating guide component, the velocity vector distribution changed from a parabolic shape to a plug flow field, forming a low-pressure vortex region at the stagnation point where the central static pressure was less than the component pressure. This physically self-excited suction source drew the PEG modified component into the vortex core. Under the condition of superimposed 50Hz mechanical vibration disturbance, the monoclonal antibody modification homogeneity index of the normal median experimental group was measured to be 98.5% to 98.8%. Comparative data showed that the modification homogeneity index of the partially missing control group dropped to 61.2% under the same flow conditions, accompanied by multipolymer impurities. This result objectively reflects the impact of the missing momentum reconstruction and vortex suction characteristics of the helical alternating guide component on the dynamic... The disruption of the synergistic effect of mechanical elements was investigated. Data from the first out-of-range control group based on the equivalent diameter showed that the interfacial tension barrier induced by a 2μm aperture blocked the physical self-excited suction process, with a monoclonal antibody modification rate of 14.5%. Data from the second out-of-range control group showed that a 60μm aperture caused the collapse of the physical blockade state, resulting in continuous jet overflow of the modified component and triggering non-target mismatch. The initial concentration of the monoclonal antibody was set to increase in a gradient from 10mg / mL to 50mg / mL and then to 100mg / mL. The targeted modification yields of both the absolute lower limit and absolute upper limit test groups were greater than 95.0%, demonstrating numerical stability independent of fluid viscosity gradient fluctuations. After the equivalent diameter aperture exceeded the nonlinear inflection point of 50μm, the modification specificity index decreased exponentially. This data change determined that the equivalent diameter parameter range constitutes the boundary condition for maintaining microscopic interfacial constraints and self-excited suction.

[0034] The measurement results obtained based on a specific fluid disturbance environment reflect that the vortex suction mechanism of the helical alternating flow guide component physically cuts off the random contact path between the modified component and the natural residues in a folded steady state. This measurement process clarifies the specific index boundary of the liquid-solid interface tension blockade state in the equivalent diameter range of 5μm to 50μm. This targeted collision control method based on fluid dynamics structure maintains the thermodynamically stable conformation of peptide molecules under viscosity gradient fluctuations and mechanical noise interference without external high-frequency injection components, ultimately obtaining conjugated products that meet the standards for manufacturing genetically engineered drugs and vaccines. The helical alternating flow guide component is composed of multiple helical blades with different torsion angles connected in series along the axial direction of the continuous flow pipeline. The helical directions of adjacent helical blades are opposite, and the flow guide surfaces of adjacent helical blades are staggered and intersecting in the axial projection. The stagnation center is located on the back The flow surface refers to the physical interface where the fluid flows over the trailing edge of the helical blade and generates backflow vortices due to the separation of the fluid boundary layer. This forces the matrix flow to separate and generates stable vortices, forming a low-pressure vortex zone and the physical premise for the generation of a physical self-excited suction source. In terms of hardware physical structure implementation, the delivery port of the modification component is not suspended in the flow field. Instead, it is a concealed capillary tube that runs axially straight to the back flow surface of the trailing edge of the helical blade, directly drilled inside the central metal axis of the solid helical alternating flow guide component through microelectromechanical processing (MEMS). The physical end face of the delivery port is flush with and fixed to the metal surface of the trailing edge of the blade. There is no need for additional probes or flow-blocking supports. This ensures that the delivery port is accurately located at the geometric center of the stagnation point defined by fluid dynamics, while not causing secondary mechanical disturbance to the original fluid boundary layer peeling morphology on the back flow side of the helical blade.

[0035] During the establishment of the confined physical blockade state of the modified component, a miniature high-frequency dynamic pressure sensor is installed in a straight pipe section 2mm to 5mm away from the injection outlet in the branch where the modified component delivery port is located. In the calibration stage before formally introducing the modified component, a recombinant peptide matrix flow is introduced into the continuous flow pipeline, and tracer gas is introduced into the modified component delivery port. The branch gas pressure is adjusted until the particle image velocimeter observes that the tracer gas is intermittently drawn into the vortex nucleus without continuous bubble overflow. At this point, the envelope value under dynamic pressure is extracted from the miniature high-frequency dynamic pressure sensor and used as the reference for the transient minimum value of the central static pressure. During the directional collision modification process, the back pressure valve and the micro... The flow-coordinated regulating loop sets the component pressure control point within a safe dead zone of 1.5 kPa to 3.0 kPa above the transient minimum reference. It utilizes the liquid-solid interfacial tension established at a specific equivalent diameter to counteract the system's background mechanical noise, ensuring that the physical blockage is only activated at the moment of minimum static pressure generated by vortex shedding. An online viscometer is integrated upstream of the targeted collision modification point in the continuous flow pipeline to collect real-time apparent viscosity data of the recombinant peptide matrix flow. The main control logic module dynamically controls the output flow rate of the inlet supply pump based on the apparent viscosity data, compensating for local dynamic viscosity waves caused by the shear-thinning rheological properties of high-concentration recombinant peptide molecules. The surface roughness of the helical alternating flow guide component is controlled within an arithmetic mean deviation of 0.1 μm to 0.3 μm through polishing process to mitigate dynamic and Reynolds number drift. The surface morphology characteristics and the flow dynamic compensation loop jointly define the physical boundary of the microscopic shear stress in the flow field, blocking the flow field evolution path that exceeds the critical shear stress upper limit of the recombinant polypeptide molecules. This ensures that the physical self-excited suction frequency maintains a stable phase coupling with the vortex shedding frequency in the low-pressure vortex region under fluctuating physical properties. Here, the coexistence of laminar flow conditions and turbulence-related parameters (such as Reynolds stress and turbulent kinetic energy) in the system characteristics does not constitute a contradiction in the physical flow state. (Continuous flow pipeline) The global Reynolds number in the main flow region is controlled below the normal temperature laminar threshold to maintain the macroscopically stable transport of the matrix flow. However, in the microscopic geometric dead zone of the solid wall back surface of the helical alternating guide component, the local Reynolds number exceeds the critical point, causing the KAMAN vortex street to fall off and generating sub-millimeter-scale velocity fluctuations. The system essentially uses the Reynolds stress equation and turbulent kinetic energy algorithm in the turbulence model to perform high-fidelity engineering quantification of the microscopic velocity variance and energy dissipation in the local transition state wake. It uses turbulence mathematical tools to accurately calibrate the boundary of local microenvironment fluctuations. This is consistent with and operates independently of the macroscopic laminar nature of its external global flow field.

[0036] Example 3: This example combines Figures 1 to 2 A description of a preparation process for targeted protein modification, such as... Figure 1As shown, the recombinant peptide matrix flow is introduced sequentially, i.e., a matrix flow containing recombinant peptide molecules is introduced into a continuous flow pipeline equipped with a helical alternating flow guide component. Then, momentum reconstruction and low-pressure vortex region generation are completed, transforming the velocity vector distribution into a uniform field characteristic of plug flow. A low-pressure vortex region with a local minimum static pressure region is formed on the back flow side. The modified component delivery port is positioned at the stagnation center of the low-pressure vortex region. Based on this, a confined physical blockade state is established. By adjusting the component pressure between the transient minimum and the average static pressure, the component is placed in a confined physical blockade state. Then, a physical self-excited suction source is constructed, automatically opening the physical blockade using dynamic pressure difference fluctuations. The process involves locking the modified component into the vortex core, triggering directional collision modification. This modification relies on flow field shear stress to induce local unfolding, allowing the modified component to undergo site-specific modification with the unsteady conformation recombinant polypeptide molecule. The final output is a modified product that meets the standards. Simultaneously, the bottom of the process includes a detection module executed by a spectral detection module to collect the site homogeneity parameters of the modified product and convert these parameters into control electrical signals. These signals are then transmitted as feedback control electrical signals to a pressure regulation unit. This pressure regulation unit adjusts the component pressure based on the control electrical signals, maintaining the molar ratio within the target range during the directional collision modification process and sending dynamic pressure correction commands to the restricted physical lock-in state establishment stage.

[0037] like Figure 2 As shown, the recombinant peptide matrix is ​​introduced to the targeted collision modification point, where the recombinant peptide exhibits a locally unfolded conformation and merges with the material from the modification component delivery port to form a modification reaction flow, which further flows to the modification product output node. Outside the main body of the pipeline, there are sequentially arranged a first-stage cooling region providing an initial refolding temperature field, a second-stage cooling region providing an intermediate refolding temperature field, and an Nth-stage cooling region providing the final steady-state temperature field. Simultaneously, at the end of the modification product output pipeline, a spectral detection module is installed to perform online sampling of the modification product. This spectral detection module extracts site homogeneity detection data signals and transmits them to a pressure regulating unit responsible for controlling the molar ratio of the modification component. Finally, the pressure regulating unit applies control actions to the modification component delivery port to complete the overall logic closed loop.

[0038] Example 4: In the continuous flow modification process, during the targeted collision action, the recombinant polypeptide molecules with modified groups are in a locally unfolded, unsteady conformation. This leads to a physical phenomenon where a sudden temperature drop causes hydrophobic core mismatch aggregation. The recombinant polypeptide matrix containing the modified product is introduced into a tubular heat exchanger with three independent temperature control zones. The first temperature control zone is set to a temperature range of 20°C to 25°C, and the fluid residence time is limited to 10s to 15s. Utilizing the frictional heat generated by the fluid separation vortex in the first temperature control zone, the recombinant polypeptide matrix flows along the pipeline into the second temperature control zone. The fluid temperature in the second temperature control zone decreases linearly from the outlet temperature of the first temperature control zone to 10°C over time. The initial cooling rate is set to 0.5°C / s. A flow-through ultraviolet fluorescence detector continuously collects the emission spectrum data of the recombinant polypeptide matrix under 295nm excitation light. The main control logic module extracts the shift Δλ of the tryptophan characteristic peak in the emission spectrum within the wavelength range of 330nm to 350nm, and sets a characteristic peak shift threshold. It is 2.5nm.

[0039] When the characteristic peak shift Δλ of tryptophan is greater than the characteristic peak shift threshold At that time, the main control logic module calculates the cooling rate deviation and outputs control commands to adjust the supply flow rate of the cooling medium in the jacket of the second temperature control zone, reducing the cooling rate to 0.2℃ / s. This flow feedback adjustment step constructs a matching constraint condition between the temperature drop gradient and the reconstruction rate of the hydration layer on the surface of the recombinant polypeptide. The recombinant polypeptide matrix, which has undergone gradient cooling constraint, is introduced into the third temperature control zone with a set constant temperature of 4℃, locking the three-dimensional conformation of the recombinant polypeptide molecule. The acquisition step of fluorescence spectral characteristic peaks and the gradient cooling action of the multi-level temperature control zone construct the thermodynamic trajectory of the non-steady-state polypeptide molecule returning to the folded steady state, blocking the physical path of thermal stress-induced polymer generation, and outputting a conjugated modified product that meets the standards for gene engineering drug manufacturing.

[0040] Example 5: When the system faces the situation of changing different batches of recombinant peptide matrix stream materials, the main control logic module faces the physical condition of uncertain initial state when extracting the reference characteristics of spectral data. The process switching stage includes a static spectral baseline calibration procedure. Before injecting the modified component, the flow-through ultraviolet fluorescence detector continuously collects the basic emission spectrum of the recombinant peptide matrix stream in pure solvent buffer, extracts the initial wavelength of the tryptophan characteristic peak within a continuous preset time period, and calculates the arithmetic mean of the time series as the reference wavelength. The main control logic module, based on the acquired reference wavelength A wavelength mapping relationship is established with the standard characteristic wavelengths of the target-modified product to determine the characteristic peak shift threshold. The calibration starting point is a static spectral baseline calibration procedure that offsets the background spectral drift bias introduced by different batches of recombinant polypeptide molecules due to differences in fermentation source impurities or initial concentrations.

[0041] Before commissioning, a component pressure feedback control model matching the current fluid characteristics is constructed for the continuous flow pipeline. The continuous flow pipeline uses a high-frequency dynamic pressure sensor installed at the center of the stagnation point on the back flow side of the spiral alternating flow guide component to obtain the transient minimum static pressure sequence of the vortex core under the injection state of the unmodified component. The lower envelope value of the transient minimum static pressure sequence of the vortex core is extracted as the first boundary pressure. The average static pressure of the recombinant peptide matrix flow at the current pipeline cross section is measured simultaneously as the second boundary pressure. The main control logic module sets the initial back pressure of the supply pump at the modified component delivery port to the arithmetic mean of the first boundary pressure and the second boundary pressure. The main control logic module adjusts the initial back pressure of the supply pump step by step by step pressure increase test, and simultaneously monitors the fluid separation trace at the center of the vortex core captured by the particle image velocimeter. The maximum back pressure value of the fluid separation trace in a state of physical blockage and no jet overflow is selected as the optimization threshold and filled into the pressure control database to establish the working window in which the component pressure is controlled by the evolution of the internal physical structure of the fluid.

[0042] Example 6: When the system faces the challenge of establishing the geometry of the helical alternating flow guide component for recombinant peptide molecules with different thermodynamic stability before production, there is a technical problem of lacking quantitative input parameters for the fluid shear stress distribution. The system performs the critical shear stress calibration and helical angle mapping steps for recombinant peptide molecules. The test module extracts a sample solution of the target recombinant peptide molecule and injects it into a cone-plate rheometer. A step shear rate sequence is applied at a constant temperature, and a circular dichroism spectroscopy is used to monitor the ellipticity change of the protein secondary structure in the sample solution. The main control logic module records the shear stress value corresponding to the instant when the ellipticity deviates from the initial baseline and exceeds the preset safety limit as the upper limit of the critical shear stress. The shear stress values ​​corresponding to the modified sites that are exposed by local unfolding are recorded as the lower limit of the critical shear stress. The main control logic module calculates the upper limit of the critical shear stress. With the lower limit of critical shear stress The arithmetic mean of the target vortex core shear stress is set as the target vortex core shear stress. This step establishes the physical benchmark for subsequent geometric parameter optimization. The preset safety limit introduced in the circular dichroism spectroscopy measurement is specifically set as an irreversible deviation of 5% from the initial steady-state spectral baseline before shearing when the molar ellipticity detected at a wavelength of 222 nm is measured. This specific limit value is established based on the critical damage tolerance characteristics of the protein α-helix structure. Since the slight shift in ellipticity directly reflects the loss of molecular secondary structure content, when the change approaches this 5% limit, it physically characterizes that the polypeptide backbone hydrogen bond network begins to face systematic breakage and is about to induce irreversible aggregation and denaturation. Therefore, it is used as a safety melting parameter for calibrating the upper limit of critical shear stress.

[0043] The main control logic module uses the acquired target vortex core shear stress as a basis. A fluid dynamics model is constructed, and the dynamic viscosity μ and average velocity v of the recombinant peptide matrix flow are extracted as input parameters. The radial velocity gradient within the continuous flow pipeline is calculated based on the Navier-Stokes governing equations. The monotonically increasing matching relationship between the helix angle difference Δθ between adjacent levels and the shear stress at the vortex core center is calculated. The main control logic module retrieves the output shear stress based on this monotonically increasing matching relationship, determining that it equals the target vortex core shear stress. The system outputs a command for the corresponding helical helix angle difference Δθ to drive the servo forming equipment to process the helical alternating flow guide component. This calibration procedure transforms the protein conformation stability requirements into the quantitative geometric construction of the physical flow guide hardware, establishes the engineering physical boundary of the local unfolding strength of the vortex core center under laminar flow, and applies the incompressible steady-state laminar boundary assumption to the actual calculation flow diagram of the bottom controller in the process of establishing this monotonically increasing matching relationship. This reduces the dimensionality of the complex three-dimensional Navier-Stokes equations and uses the Rankine vortex one-dimensional physical approximation model to fit the parameters of the tangential velocity field inside the helical component. Based on the physical law of shear torque balance of fluid micro-elements, the main control logic module directly calls the built-in polynomial analytical function. Where K is a dimensionless constant of the flow field structure and D is the inner diameter of the continuous flow pipe section, under the given geometric pipe diameter and known physical property parameters, the unique deterministic solution between the shear stress and the difference in the helix angle is successfully output, thereby transforming the complex flow field calculation into a linear mapping operation that can be executed by an industrial controller in milliseconds.

[0044] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. A preparation process for targeted protein modification, characterized in that, Includes the following steps: Step 101: Introduce a recombinant polypeptide matrix flow containing recombinant polypeptide molecules into a continuous flow pipeline equipped with a helical alternating flow guide component. Utilize the radial velocity field generated by the helical alternating flow guide component during axial flow to reconstruct the momentum of the recombinant polypeptide matrix flow, transforming the velocity vector distribution of the recombinant polypeptide matrix flow from a parabolic shape to a uniform field with plug flow characteristics. Relying on the fluid stripping effect on the back flow side of the helical alternating flow guide component, a low-pressure vortex region with a solvent polarity gradient and accompanied by a local minimum static pressure region is formed. Step 102: The modified component delivery port is set at the stagnation center of the low-pressure vortex region, and the component pressure in the modified component delivery port is adjusted so that the component pressure is maintained on the time scale within a range that is greater than the transient minimum value of the static pressure at the center of the low-pressure vortex region and less than the average static pressure of the recombinant polypeptide matrix flow. When the flow field is in a macroscopic laminar flow state, the modified component is in a restricted physical blockage state in the modified component delivery port. Step 103: A physical self-excited suction source is constructed by utilizing the dynamic pressure difference fluctuation between the central static pressure and the component pressure. The physical self-excited suction source automatically opens the physical blockade state and introduces the modified component into the vortex core center of the low-pressure vortex region. The flow field shear stress in the vortex core center induces the recombinant polypeptide molecules to undergo local unfolding, so that the modified component and the recombinant polypeptide molecules in the unsteady conformation and enriched in the vortex core center undergo site-specific directional collision modification.

2. The preparation process for targeted protein modification according to claim 1, characterized in that, In step 101, the helical alternating flow guiding components are arranged in a stepped alternation along the axial direction of the continuous flow pipeline. The radial mixing intensity of the recombinant polypeptide matrix flow is adjusted by the helical angle jump between adjacent levels of the helical alternating flow guiding components. After the directional collision modification is completed, the three-dimensional conformation of the recombinant polypeptide molecules is guided to thermodynamic steady state by the cooling gradient field set downstream of the continuous flow pipeline.

3. The preparation process for targeted protein modification according to claim 1, characterized in that, The recombinant polypeptide matrix flow is selected from one or more of monoclonal antibodies, antibody fragments, and extended-acting polypeptides; the modifying component is selected from one or more of polyethylene glycol derivatives, linker-cytotoxic drug conjugates, and fluorescent markers, and the residence time distribution of the recombinant polypeptide matrix flow and the modifying component in the vortex core is controlled by the lead of the helical alternating flow guide component.

4. The preparation process for targeted protein modification according to claim 1, characterized in that, In step 102, the equivalent diameter range corresponding to the outlet cross-sectional area of ​​the modified component delivery port is 5μm to 50μm. By adjusting the equivalent diameter, a capillary force constraint adapted to the physical self-excited suction source is generated, so that the modified component is locked inside the modified component delivery port by the liquid-solid interfacial tension established at the equivalent diameter before the physical self-excited suction source is started.

5. The preparation process for targeted protein modification according to claim 1, characterized in that, The spiral lead of the spiral alternating flow guide component gradually decreases from the inlet end to the outlet end of the recombinant polypeptide matrix flow according to a preset ratio, generating an axial acceleration field for the recombinant polypeptide matrix flow in the continuous flow pipeline. The axial acceleration field enhances the central static pressure drop slope of the low-pressure vortex region by changing the local Reynolds number, thereby shortening the response delay of the physical self-excited suction source.

6. The preparation process for targeted protein modification according to claim 1, characterized in that, After step 103, the spectral detection module located at the pipeline outlet collects the site homogeneity parameters of the modified product and converts the site homogeneity parameters into control electrical signals, which are then fed back to the pressure regulating unit. The pressure regulating unit corrects the component pressure in step 102 according to the control electrical signals, so that the molar ratio during the directional collision modification process is maintained within the preset target range.

7. The preparation process for targeted protein modification according to claim 1, characterized in that, The stagnation center is located at the geometric intersection of the central axis of the helical alternating flow guide component and the backflow surface. The injection vector of the modified component delivery port coincides with the radial vector of the recombinant polypeptide matrix flow at this stagnation center, so as to reduce the transverse shear dissipation generated by the modified component during its entry into the vortex core center and maintain the biological activity of the recombinant polypeptide molecules.

8. The preparation process for targeted protein modification according to claim 1, characterized in that, The turbulent kinetic energy in the global flow field of the recombinant peptide matrix is ​​maintained within a range below a preset turbulent kinetic energy threshold, which is determined by the critical shear stress of the recombinant peptide molecule. By adjusting the surface roughness of the helical alternating flow guide component, the Reynolds stress in the near-wall region is reduced, thereby inhibiting the irreversible polymerization and precipitation of the recombinant peptide molecule.

9. The preparation process for targeted protein modification according to claim 1, characterized in that, After the recombinant peptide matrix is ​​modified by directional collision, it is directly connected to the tangential flow filtration system. The tangential flow filtration system uses an ultrafiltration membrane with a pore size distribution in the range of 10kDa to 100kDa to perform solvent replacement and concentration on the recombinant peptide molecules, so that the recombinant peptide molecules are output as GMP-compliant recombinant peptide molecular formulations under a constant ionic strength environment.

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