Chromatographic purification method of haemophilus influenzae type b vaccine

Abstract

The invention relates to the technical field of colloidal chemistry and physical separation, and discloses a chromatographic purification method of a type b haemophilus influenzae vaccine, which comprises the following steps: preparing a synergistic desolvation mobile phase containing 0.45 mol / L to 0.55 mol / L sodium chloride and 45 mmol / L to 55 mmol / L sodium citrate, and establishing an equilibrium field in pores of a chromatographic medium; adjusting the ion environment of the crude liquid to be consistent with that of the mobile phase, so that the free polysaccharide is subjected to conformation collapse; according to the method, a critical solvation environment in which high salt and specific lyophilic ions coexist is constructed, electrostatic repulsive force of polysaccharide chain segments is shielded, a hydration membrane of the polysaccharide chain segments is stripped, stretched line clusters are converted into compact spheres, and the high salt-specific lyophilic ions are separated from the compact spheres, so that the high salt-specific lyophilic ions in the polysaccharide chain segments are separated. Therefore, the volume difference with a conjugate is enlarged, the separation problem caused by volume pseudo-overlapping in conventional chromatography is solved, and high-resolution separation on a low-cost medium is realized.

Description

Technical Field

[0001] This invention relates to a chromatographic purification method for Haemophilus influenzae type b vaccine, belonging to the field of colloidal chemistry and physical separation technology. Background Technology

[0002] The core of current Haemophilus influenzae type b vaccine preparation is to covalently couple Hib capsular polysaccharide with carrier proteins such as tetanus toxoid and remove unreacted free polysaccharide from the reaction mixture. Industrially, gel filtration chromatography based on the principle of size exclusion is commonly used for purification, and isotonic buffer that simulates physiological conditions is commonly used as the mobile phase. In a conventional ionic environment, free polysaccharide, as a flexible polyelectrolyte with high charge density, is driven by strong electrostatic repulsion between chain segments to form a highly extended random coil state, with an abnormally expanded apparent hydrodynamic radius.

[0003] Solvation causes volume expansion, resulting in a nonlinear overlap between the hydrodynamic volume of low-molecular-weight free polysaccharides and high-molecular-weight conjugates. Conventional chromatography media cannot distinguish between components with similar physical scales but different topological structures, leading to low separation. To maintain purity, existing processes are forced to reduce sample loading or cut off extremely narrow elution windows, resulting in low production efficiency, large yield loss, and insufficient media utilization. To avoid the inherent difficulties of separation based on physical volume, the industry has attempted to introduce separation methods based on charge differences. For example, Chinese invention patent CN106397537B discloses an efficient and rapid method for purifying and analyzing polysaccharide-protein conjugate vaccines. It uses ultrafiltration to remove unbound proteins and DEAE anion exchange resin chromatography. By utilizing the charge difference between the conjugates and free polysaccharides, the conjugates are adsorbed onto the column while the free polysaccharides permeate and elute. The product is obtained by high-salt elution.

[0004] Therefore, how to regulate the physical state of components in a dispersion system, break the hydrodynamic volume degeneracy between polysaccharides and conjugates, and achieve physical baseline separation between the two has become the technical problem to be solved by this invention. Summary of the Invention

[0005] To address the problems mentioned in the background section, the technical solution of this invention is as follows:

[0006] A chromatographic purification method for Haemophilus influenzae type b vaccine, the method comprising the following steps:

[0007] Step S101: Prepare a synergistic desolvation mobile phase by dissolving sodium chloride and sodium citrate in an aqueous solution and adjusting the pH value to the range of 7.0 to 7.4 using an inorganic acid, wherein the molar concentration of sodium chloride is limited to 0.45 mol / L to 0.55 mol / L and the molar concentration of sodium citrate is limited to 45 mmol / L to 55 mmol / L, forming a composite ionic environment that can simultaneously shield electrostatic repulsion and competitively strip the polysaccharide hydration membrane;

[0008] Step S102: Establish a pore equilibrium field in the medium. The synergistic desolvation mobile phase is introduced into a chromatography column packed with cross-linked agarose gel at a linear flow rate of 30 cm / h to 60 cm / h. The column is continuously flushed until the conductivity of the effluent is consistent with that of the injection mobile phase. A thermodynamic equilibrium state in which electrostatic shielding and hydrophilic stripping coexist is established inside the micropores of the chromatography medium.

[0009] Step S103: Perform sample ion environment assimilation. Add solid sodium chloride and sodium citrate to the crude solution containing Haemophilus influenzae type b capsular polysaccharide, tetanus toxoid conjugate, and free capsular polysaccharide to adjust the ion environment of the crude solution to be completely consistent with the synergistic desolvation mobile phase, so that the free capsular polysaccharide undergoes conformational collapse before entering the chromatography column.

[0010] Step S104: Perform differential hydrodynamic elution. Load the crude liquid processed in step S103 onto the chromatography column and perform isocratic elution using a synergistic desolvation mobile phase. Utilize the difference in hydrodynamic volume between the conjugate and the free capsular polysaccharide in the equilibrium field, and collect the first elution peak after the external water volume as the target product.

[0011] Preferably, in step S101, the molar concentration of sodium chloride is preferably 0.5 mol / L, the molar concentration of sodium citrate is preferably 50 mmol / L, the inorganic acid is phosphoric acid or hydrochloric acid, and the conductivity of the synergistic desolvation mobile phase is controlled between 53 mS / cm and 57 mS / cm.

[0012] Preferably, the cross-linked agarose gel used in step S102 is a spherical particle with a cross-linking degree of 4% to 6%, and the grading range covers the molecular weight range of 10kDa to 4000kDa.

[0013] Preferably, the method further includes a pretreatment step prior to step S103, in which cetyltrimethylammonium bromide and ethanol are added to the crude liquid to precipitate and collect the polysaccharide and conjugate components, and the precipitate is re-dissolved using a high ionic strength solution to remove nucleic acid and non-target protein impurities.

[0014] Preferably, in step S104, the sample loading volume is controlled at 3% to 5% of the chromatography column bed volume, and the linear flow rate during elution is controlled at 30 cm / h to 45 cm / h, so as to ensure that the collapsed free capsular polysaccharide diffuses fully into the micropores of the chromatography medium.

[0015] Preferably, in step S104, the separation effect of differential fluid dynamics elution is determined by the change in the partition coefficient, wherein the ionic strength of the synergistic desolvation mobile phase is adjusted to cause the partition coefficient K of the free capsular polysaccharide in the chromatography medium to change. avThe following relationship must be satisfied: 0.30 ≤ (V e -V0) / (V t -V0)≤0.45, where V e V0 is the retention volume of the pure free capsular polysaccharide reference material determined by single injection under synergistic desolvation mobile phase conditions, where V0 is the external water volume of the chromatography column. t The total volume of the chromatography column represents the free capsular polysaccharide that has moved from the exclusion zone to the partial permeation zone.

[0016] Preferably, the collection operation in step S104 is performed based on the synchronous monitoring of the ultraviolet absorption signal and the differential refractive index signal; the collection start and end points are set as follows: collection begins when the absorbance value of the ultraviolet detector at a wavelength of 280 nm rises to 5% of the peak value, and collection stops when the absorbance value drops to 5% of the peak value and the differential refractive index detector signal does not show a second rising edge.

[0017] Preferably, in step S103, the mixing process after adding solid sodium chloride and sodium citrate is carried out in a constant temperature environment of 20 to 25 degrees Celsius, and the standing time after mixing is not less than 30 minutes to ensure that the colloidal conformational transformation reaches thermodynamic equilibrium.

[0018] Preferably, after the synergistic desolvation mobile phase is prepared, it is filtered and degassed under vacuum through a filter membrane with a pore size of 0.22 micrometers. The entire chromatographic purification process is performed in a sterile and closed fluid pathway.

[0019] Preferably, the synergistic desolvation mobile phase configured in step S101 does not contain any organic solvents, surfactants, or denaturants other than sodium chloride, sodium citrate, and inorganic acids used to adjust the pH value. The method relies on the physical solvation effect of inorganic ions to achieve separation.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. In Haemophilus influenzae type b vaccine, a composite electrolyte environment with anisotropic solvation characteristics is constructed to achieve the physical discretization of the hydrodynamic volume of the polydisperse colloidal system. By utilizing the high ionic strength double-layer compression effect and the competitive stripping mechanism of a specific lyophilic ion hydration membrane, the conformational collapse of the flexible chain-like impurities in the extended state is specifically induced, transforming them from random coils into dense spheres. The target binder with a rigid core maintains a relatively stable volume. The differential colloidal conformation remodeling increases the difference in hydrodynamic radius between the target product and the impurities, eliminating the volume pseudo-overlap phenomenon in conventional isotonic elution environments. This enables the chromatography medium based on the pore size exclusion principle to identify and separate components that are originally similar in physical scale, achieving baseline separation.

[0022] 2. By altering the thermodynamic partitioning behavior of impurity components in porous gel media, an elution window is established that achieves both high yield and high purity. Under the synergistic desolvation of the mobile phase, the partition coefficient of volume-shrinking impurities in the medium is fundamentally shifted from the completely exclusion zone to the partially permeable zone, resulting in delayed elution. The target product maintains its rapid migration characteristics near the exclusion volume. The directional control of partitioning behavior avoids the traditional process of cutting the main peak to remove tailing impurities, ensuring deep removal of impurities while fully preserving the elution fraction of the target product, thus resolving the engineering contradiction of balancing yield and purity in chromatography.

[0023] 3. Based on the high resolution capability of general-purpose, low-cost chromatography media for processing complex biomacromolecule systems, the core separation driving force comes from the active reconstruction of the physical state of the solute by the mobile phase environment, rather than simply relying on the pore size distribution accuracy of the stationary phase medium. This allows conventional cross-linked agarose media to achieve the purification effect that originally required high-resolution media, reducing the dependence on special and expensive packing materials in industrial production, improving process economy and media compatibility, and making it suitable for low-cost deployment and long-term stable operation of large-scale production lines. Attached Figure Description

[0024] Figure 1 This is a purification process flow diagram of the synergistic desolvation and conformational collapse mechanism of the present invention.

[0025] Figure 2 This is a trend graph showing the quantitative effect of sodium citrate concentration on the yield and purity of the conjugate in this invention.

[0026] Figure 3 This is a diagram of the tomography system architecture that integrates assimilation preprocessing and dual signal monitoring according to the present invention. Detailed Implementation

[0027] The following explains the principles and implementation methods of the present invention and provides specific embodiments. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0028] This invention proposes a chromatographic purification method for Haemophilus influenzae type b vaccine, comprising four core process stages: construction of a synergistic desolvation mobile phase, establishment of a pore thermodynamic field in the chromatographic medium, assimilation of the ion environment of the sample to be separated, and elution and interception based on differential fluid dynamics volume. In the construction stage of the synergistic desolvation mobile phase, an aqueous solution-based composite electrolyte system is configured as the mobile phase. The solute components of this mobile phase include sodium chloride and sodium citrate, wherein the molar concentration of sodium chloride is limited to the range of 0.45 mol / L to 0.55 mol / L. This high-concentration electrolyte environment is used to purify the Debye length κ of the solution system. -1Compressing to below 1 nm shields the electrostatic repulsion between polysaccharide molecular segments and between the polysaccharide and the solvent, eliminating segment rigidity and swelling caused by the polyelectrolyte effect. Simultaneously, the molar concentration of sodium citrate is limited to the range of 45 mmol / L to 55 mmol / L, utilizing citrate ions... 3- As a structured ion with strong lyophilic properties in the Hofmeister sequence, it competitively captures water molecules from the hydration layer surrounding the polysaccharide molecule within this concentration range, and this concentration is below the critical threshold that causes salting-out precipitation of the carrier protein. During the preparation process, the pH of the solution is adjusted to between 7.0 and 7.4 using inorganic acids such as phosphoric acid or hydrochloric acid, and the conductivity of the final system is maintained in the range of 53 mS / cm to 57 mS / cm. After preparation, the solution is filtered through a 0.22 μm pore size filter membrane and subjected to vacuum degassing.

[0029] Regarding the dynamic calibration and judgment criteria for the critical solvation environment, considering the potential fluctuations in the molecular weight distribution of Haemophilus influenzae type b capsular polysaccharides and the surface properties of impurity proteins in crude solutions from different batches or sources, this invention provides a standardized calibration method based on dynamic light scattering (DLS) coupled with turbidity to ensure the accurate construction of the critical solvation environment. This method specifically includes the following steps: gradient system construction: maintaining a constant sodium chloride concentration between 0.45 mol / L and 0.55 mol / L (e.g., 0.5 mol / L), and preparing a series of gradient buffers with sodium citrate concentrations covering 20 mmol / L to 100 mmol / L (step size recommended not to exceed 10 mmol / L); thermodynamic equilibrium simulation: taking samples of the current batch of crude solution, mixing them with the above gradient buffers according to the process ratio, and allowing them to stand at 20-25°C for at least 30 minutes to simulate the equilibrium state within the chromatography column; and two-parameter anisotropy characterization: measuring the hydrodynamic radius (Ri) of the polysaccharide component in the mixed system using a dynamic light scattering (DLS) instrument. h ) and polydispersity index (PDI); simultaneously monitor the turbidity changes of the system using a turbidimeter or UV 350-400nm absorbance; critical point determination and window locking, defining the collapse initiation point (point A): determined to be polysaccharide R h The minimum sodium citrate concentration at which the PDI shows a significant decrease (typically >50%, e.g., from 50 nm to 15 nm) and narrows to a plateau indicates that the polysaccharide conformation has completed the transition from coils to dense spheres; define the precipitation critical point (point B): the minimum sodium citrate concentration at which the system turbidity begins to show a non-linear upward trend; determine the process setpoints, the optimal sodium citrate concentration should be set within the range [C]. A 0.8×C B Within the range.

[0030] In the stage of establishing the thermodynamic field of the chromatographic medium pores, spherical cross-linked agarose gel with a cross-linking degree of 4% to 6% is selected as the chromatographic medium. Its fractionation range covers the molecular weight range of 10 kDa to 4000 kDa, such as Sepharose 4FF. A synergistic desolvation mobile phase is introduced into the chromatography column packed with this medium at a linear flow rate of 30 cm / h to 60 cm / h. Continuous rinsing continues until the conductivity and pH of the effluent are completely consistent with the injection mobile phase, thereby establishing a thermodynamic equilibrium state where electrostatic shielding and hydrophilic exfoliation coexist within the micropores of the chromatographic medium. In the ion environment assimilation stage of the sample to be separated, a pre-equilibration procedure is performed to address the risk of local conformational disturbances that may occur due to abrupt changes in the solvent environment when the sample enters the chromatography column. Before chromatographic loading, the ion environment is assimilated. In a crude liquid containing Haemophilus influenzae type b capsular polysaccharide, tetanus toxoid conjugate, and free capsular polysaccharide, calculated amounts of solid sodium chloride and sodium citrate were directly added to adjust the ionic environment of the crude liquid to be completely consistent with the synergistic desolvation mobile phase. This mixing process was carried out at a constant temperature of 20°C to 25°C, and the settling time after mixing was no less than 30 minutes to ensure that the colloidal conformational transformation reached thermodynamic equilibrium. In this environment, the free capsular polysaccharide molecules underwent conformational collapse from extended random coils to dense spheres due to the dual effects of high salt shielding electrostatics and hydrophilic ion water-removing, resulting in a decrease in their hydrodynamic volume. Meanwhile, the target conjugate, due to the rigid protein core support and the difference in the properties of the surface hydration layer, experienced a smaller volume shrinkage than the free polysaccharide, thus widening the difference between the two on a physical scale.

[0031] In the elution and cut-off stage based on differential fluid dynamics volume, the crude solution assimilated by the ion environment is loaded onto the equilibrated chromatography column, with the loading volume controlled at the column bed volume V. t Between 3% and 5%, isocratic elution was performed using a synergistic desolvation mobile phase. The linear flow rate during elution was controlled between 30 cm / h and 45 cm / h to ensure that the collapsed free capsular polysaccharides fully diffused into the micropores of the chromatography medium. The bound polysaccharides, due to their relatively large volume, mainly migrated between the medium particles, exhibiting exclusion characteristics; while the collapsed free polysaccharides, due to their reduced volume, could enter more micropores, exhibiting retention characteristics. This separation effect was measured by the partition coefficient K. av Characterization, the ionic strength of the mobile phase affects the partition coefficient K of free capsular polysaccharides in the chromatography medium. av Satisfying 0.30≤K av The relation is ≤0.45, where K av Defined as (V) e -V0) / (V t -V0), where V eV0 is the elution volume of free capsular polysaccharide, and V0 is the volume of water outside the chromatography column, indicating that the free polysaccharide has shifted from the complete exclusion zone to the partial permeation zone under normal conditions. The collection operation is based on the simultaneous monitoring of UV absorption and differential refractive index (DRI) signals. The collection start and end points are set as follows: collection begins when the absorbance value of the UV detector at 280 nm rises to 5% of the peak value, and stops when the absorbance value drops to 5% of the peak value and the DRI signal does not show a second rising edge. The target product is thus obtained from the first elution peak eluting after the volume of water outside the chromatography column. This method is performed entirely in a sterile and closed fluidic pathway. The mobile phase contains only sodium chloride, sodium citrate, and an inorganic acid for pH adjustment; no other organic solvents, surfactants, or denaturants are introduced. Regarding K... av Procedure for Determination and Verification of Partition Coefficient: For differential fluid dynamics elution, in order to accurately obtain K... av V in the calculation formula e The value is determined using the external standard method, as specified in this invention. The specific operation is as follows: Before purifying the crude solution, use the same chromatography column and synergistic desolvation mobile phase as in step S102; take a small amount (approximately 0.5% of the column volume) of pure free capsular polysaccharide with a purity greater than 90% as a reference for separate analysis; record the volume corresponding to the elution peak of the reference, which is defined as V. e ; Using this V e Value calculation K av This is to confirm whether the ionic strength of the current mobile phase is sufficient to satisfy 0.30 ≤ K for the free polysaccharide. av The conformational collapse requirement is ≤0.45. After confirming that everything is correct, the mixture of crude liquid is then loaded for separation. At this point, the free polysaccharide component in the crude liquid will strictly conform to the calibrated V. e Location leaked.

[0032] Example 1: In large-scale chromatographic purification of Haemophilus influenzae type b vaccine conjugates, free capsular polysaccharide impurities exhibit a strong solvation effect due to their high charge density. In conventional isotonic buffer systems, this results in a highly expanded state, causing significant overlap between the hydrodynamic volume and the target conjugate, leading to the failure of separation based on the pore size exclusion principle. This invention addresses this challenge by employing a critical anisotropic solvation sieving strategy. A synergistic desolvation mobile phase containing 0.5 mol / L sodium chloride and 50 mmol / L sodium citrate at pH 7.2 is prepared. Thermodynamic equilibrium is established within the micropores of the Sepharose 4FF chromatographic medium. In this composite electrolyte environment, the high concentration of sodium chloride reduces the Debye length κ of the solution system. -1 Compressing to below 1 nm shields the electrostatic repulsion between polysaccharide molecular chain segments, eliminating swelling caused by their conformational rigidity.

[0033] Simultaneously, citrate ions 3-Utilizing the strong lyophilic properties of the Hofmeister sequence, the hydration membrane on the polysaccharide surface is competitively exfoliated. The synergistic effect of these two mechanisms causes the free polysaccharide to undergo conformational collapse from an extended coil to a dense sphere before entering the chromatography column. Meanwhile, the target conjugate, supported by a rigid protein core, experiences only a small volume shrinkage. The crude solution, pretreated with ion-environment assimilation, is loaded and isocratically eluted at a linear flow rate of 30 cm / h. During this process, the conformationally collapsed free polysaccharide, due to its reduced hydrodynamic radius, can diffuse deeply into the micropores of the medium, resulting in a partition coefficient K0 within the medium. av When the concentration jumps to above 0.30, it shifts from the resistance zone to a partial permeation zone, while the target binder still mainly migrates between the medium particles, maintaining rapid outflow in the resistance zone.

[0034] Example 2: This example constructs a confirmatory experiment to investigate the influence of the concentration of key components in the mobile phase on the hydrodynamic behavior and final separation effect of free polysaccharides through a control system containing gradient changes. The experiment was conducted on a fully automated chromatography system equipped with a high-precision online ultraviolet (UV) detector and a differential refractive index (RI) detector to monitor the elution behavior of different components in real time. The chromatography column was 10cm × 100cm, packed with Sepharose 4FF gel medium, and the column bed volume (CV) was calibrated to 7.85L. The crude solution used in the experiment was taken from the same batch of Haemophilus influenzae type b conjugate reaction solution, the main components of which were polysaccharide-protein conjugates (target product), unreacted free capsular polysaccharides, and free tetanus toxoid. All reagents used to prepare the mobile phase were of analytical grade and filtered through a 0.22μm filter membrane before use. The experimental design followed the principle of multidimensional control, setting up four parallel chromatography processes, with each group separated only in the mobile phase. The phase composition differs to isolate variables and verify the influence of specific parameters; Control group: a conventional 0.15 mol / L NaCl phosphate buffer (PBS, pH 7.2) was used to simulate the separation performance of existing technologies under isotonic conditions; Sample group 1 of this invention: a synergistic desolvating mobile phase (pH 7.2) containing 0.5 mol / L NaCl and 50 mmol / L sodium citrate was used to verify the effect of the preferred technical solution of this invention; Sample group 2 of this invention (lower limit of parameters): the concentration of sodium citrate was reduced to 20 mmol / L, while the concentration of NaCl was kept at 0.5 mol / L, to examine the separation efficiency when the concentration of lyophilic ions was insufficient; Sample group 3 of this invention (upper limit of parameters): the concentration of sodium citrate was increased to 100 mmol / L, while the concentration of NaCl was kept at 0.5 mol / L, to examine the boundary effect that may be caused by excessively high concentration of lyophilic ions. The loading volume of all groups was uniformly controlled at 4% CV, and the elution flow rate was kept constant at 30 cm / h.

[0035] During the experiment, differences were observed through real-time chromatograms. In the control group, the UV absorption peak and the polysaccharide peak detected by RI showed a large overlap, and their elution volumes were extremely close, indicating that the hydrodynamic volumes of free polysaccharide and conjugates could not be effectively distinguished under these conditions. In sample group 1 of this invention, the elution peak of free polysaccharide shifted backward, forming a clear baseline separation with the main peak of the conjugate. Furthermore, the RI signal showed that the polysaccharide peak became sharper, suggesting a more compact conformation. Although sample group 2 of this invention showed a certain degree of separation trend, there was still a significant saddle between the polysaccharide peak and the conjugate peak, and baseline separation was not achieved. Although sample group 3 of this invention achieved separation, a non-linear rapid increase in column pressure was detected, and trace amounts of protein aggregates were detected in the effluent, suggesting that excessively high concentrations of structured ions may induce non-specific protein precipitation. To quantitatively characterize the above phenomena, Table 1 summarizes the key process data of each experimental group, where K av The distribution coefficient, as a core indicator for measuring the permeability of a component in the pores of a medium, is calculated using the formula K. av =(V e -V0) / (V t -V0), Table 1 shows the chromatographic separation performance data under different mobile phase systems.

[0036] Table 1: Chromatographic Separation Performance Data under Different Mobile Phase Systems

[0037] Experimental group Key components of the mobile phase <![CDATA[Free polysaccharide K av > <![CDATA[Resolution R S > Bound product yield (%) Conjugate purity (%) Remark control group 0.15 mol / L NaCl 0.05 0.72 65.2 88.5 The peaks overlap significantly, requiring substantial cutting. Sample 1 of the present invention 0.5 mol / L NaCl + 50 mmol / L Citrate 0.38 2.15 92.4 99.1 Baseline separation, peak symmetry Sample 2 of the present invention 0.5 mol / L NaCl + 20 mmol / L Citrate 0.18 1.25 78.5 94.2 Incomplete separation, insufficient polysaccharide collapse Sample 3 of this invention 0.5 mol / L NaCl + 100 mmol / L Citrate 0.45 2.40 81.6 98.8 Abnormally high column pressure, slight protein precipitation.

[0038] See Table 1, K for the control group av The value is only 0.05, indicating that the free polysaccharide is almost completely excluded from the micropores of the medium, verifying the physical fact of volume pseudo-overlap under normal conditions. With the increase of sodium citrate concentration, the Kc of the free polysaccharide... av It exhibits a non-linear growth trend: increasing to 0.18 at 20 mmol / L, and jumping to 0.38 at 50 mmol / L. This indicates that when the concentration of lyophilic ions reaches a certain threshold, the desolvation effect of polysaccharide molecules triggers a rapid conformational collapse, allowing them to penetrate deeply into the micropores of the medium. However, when the concentration is further increased to 100 mmol / L, although K... av The value was increased further to 0.45, but the yield of the complex decreased, accompanied by an increase in column pressure.

[0039] Example 3: This example combines Figures 1 to 3 The chromatographic purification method for a Haemophilus influenzae type b vaccine is described, as follows: Figure 1As shown, the auxiliary flow includes step S101, which involves preparing a synergistic desolvation mobile phase containing 0.5 mol / L NaCl and 50 mmol / L sodium citrate at pH 7.2 to shield electrostatic repulsion and competitively strip the hydration membrane. Step S102 involves using Sepharose 4FF media to establish a thermodynamic equilibrium field where electrostatic shielding and hydrophilic stripping coexist. The main flow path uses a crude solution containing conjugates, free capsular polysaccharides, and tetanus toxoid as input. Step S103 performs sample ion environment assimilation, i.e., by adding solid salt to adjust the ion environment to match the mobile phase, causing the free polysaccharides to undergo conformational collapse from coils to spheres. The sample volume is 3% to 5% CV, and the flow proceeds to step S104 for differential fluid dynamics elution. The fluid dynamic volume difference between the conjugates and the collapsed polysaccharides is utilized to elute the K+. av The range is controlled within 0.30≤K av Between ≤0.45, after the final eluent flows out, it is determined based on the simultaneous monitoring of UV absorption and differential refractive index, that is, collection begins when the UV absorbance at 280nm rises to 5% of the peak value and stops when the UV drops to 5% and there is no second rising edge of RI.

[0040] like Figure 2 As shown, the horizontal axis represents the concentration of sodium citrate in mmol / L, and the vertical axis represents the percentage in %. The solid line in the graph represents the change in the yield of the conjugate, and the dashed line represents the change in the purity of the conjugate. As the concentration on the horizontal axis increases from 0 to 50 mmol / L, the yield of the conjugate shows an upward trend and reaches a peak. At the same time, the purity of the conjugate also rises and remains at a high level. However, when the concentration is further increased to 80 mmol / L and 100 mmol / L, although the purity curve remains stable, the yield curve shows a clear downward inflection point. Figure 3 As shown, the system structure includes a co-current phase preparation tank and a crude liquid storage tank, both of which are connected to an assimilation pretreatment reactor. This reactor has the function of inducing the spheroidization of free polysaccharides. The pretreated material flows to the core chromatography column, which is filled with Sepharose 4FF medium and controlled by a PC automated central control system. The outlet of the chromatography column is connected to a dual-signal monitoring station containing ultraviolet (UV) and differential emission (RI) signals. Here, the effluent is split according to the signal feedback. The tail peak is discharged to the waste liquid tank to remove impurities, while the main peak is intercepted and flows to the finished product collection tank to obtain the conjugate. The entire flow path is controlled by the dotted line connection of the PC automated central control system, forming a complete closed-loop purification hardware facility.

[0041] Example 4: In the large-scale industrial production of Haemophilus influenzae type b vaccine, the stability of the chromatographic purification process depends not only on the configuration of the mobile phase, but also on the batch-to-batch quality fluctuations of the crude raw material and the systematic errors caused by the aging of the chromatographic medium. Therefore, this example constructs a standardized engineering calibration procedure. By dynamically determining the critical anisotropic solvation point, the optimal working concentration of sodium citrate is determined for each batch of production. This calibration procedure is based on gradient experimental design, preparing a series of buffer systems with gradient sodium citrate concentrations, keeping the sodium chloride concentration constant at 0.5 mol / L, and setting the sodium citrate concentration from 20 mmol / L to 100 mmol / L in 10 mmol / L increments. A small amount of the crude liquid sample to be treated is taken and mixed with each of the above gradient buffers according to the actual process ratio, and allowed to stand at 25°C for 30 minutes to reach thermodynamic equilibrium. Dynamic light scattering (DLS) technology is used to measure the hydrodynamic radius R of the polysaccharide component in each mixture. h The polydispersity index (PDI) and the turbidity changes of the system were monitored using a turbidimeter to characterize protein stability.

[0042] By correlating DLS measurement data with turbidity data, two key physical threshold points were identified. The first is the collapse initiation point, defined as polysaccharide R... h The minimum sodium citrate concentration at which the PDI value decreases and narrows to a plateau indicates that the polysaccharide conformation has completed the transition from coils to spheres. The second is the precipitation critical point, defined as the minimum sodium citrate concentration at which the system's turbidity begins to show a non-linear upward trend, indicating that high concentrations of lyophilic ions begin to induce non-specific protein aggregation. In the typical calibration experiments of this invention, data show that when the sodium citrate concentration reaches 40 mmol / L, the polysaccharide R... h The turbidity dropped sharply from an initial 50 nm to 15 nm, indicating that conformational collapse had fully occurred. When the concentration exceeded 60 mmol / L, the turbidity of the system slowly increased from the baseline of 0.05 NTU and then increased sharply to 1.2 NTU at 80 mmol / L, clearly indicating the risk boundary of protein precipitation. Based on the above calibration results, the calculation logic of the optimal working window was established: the collapse initiation point was selected as the lower limit of the process window, and 80% of the precipitation critical point was selected as the upper limit of the process window to leave sufficient safety margin. Under the specific operating conditions of this embodiment, the optimal concentration range derived by this logic is 40 mmol / L to 64 mmol / L. Finally, combining the principles of convenience and robustness of engineering operation, the production process parameters were locked at 50 mmol / L.

[0043] Example 5: This example establishes a standardized on-site pre-calibration and dynamic compensation procedure. It involves performing a baseline ion strength measurement step, specifically, before each batch of chromatography runs, using a metrologically calibrated high-precision conductivity meter to measure the initial conductivity data of the crude solution online. This measured value is then used as a dynamic input parameter to a preset compensation model. Based on the principle of charge conservation and Debye-Hückel theory, and according to the deviation between the initial conductivity and the target critical solvation environment parameters, the required amounts of sodium chloride and sodium citrate to achieve the set ion strength and hydrophilic / hydrophobic equilibrium are precisely calculated. A nonlinear conductivity-concentration response matrix K is constructed, with a fixed molar ratio constraint γ=n. NaCl / n Cit =10, calibrating the composite ion strength response coefficient Φ mix Execute the quality compensation calculation instruction: Where β is the Debye-Hückel interaction constant, ξ=0.98 is the overshoot damping factor, and Δm is calculated simultaneously. Cit =Δm NaCl· M Cit / 10M NaCl The interlocked PID controller sets the solid feeding rate threshold v. feed ≤0.05·S(T) min .

[0044] In the specific implementation phase, the control system automatically adjusts the addition rate and dissolution process of the solid salt based on the above calculation results. During this period, the conductivity and pH value of the mixed system are monitored in real time through a closed-loop feedback loop until the real-time measured value is completely matched with the set parameters of the synergistic desolvation mobile phase, and the control deviation is limited to within ±0.5%.

[0045] Example 6: This example constructs and validates a standardized dual-ion strength gradient elution process. By dynamically adjusting the concentration of hydrophilic ions in the elution environment in stages, it ensures sufficient conformational collapse of the polysaccharide while minimizing the risk of protein aggregation, thereby improving process yield without sacrificing separation. On the Sepharose 4FF chromatography validation platform of the same specifications as the previous examples, a control experiment with a step-wise elution procedure was designed. The control group used the single high-concentration sodium citrate (100 mmol / L) isocratic elution mode of sample group 2 in Example 2. This example group introduces a segmented gradient elution logic: In the first stage, isocratic elution is performed using an initial buffer containing 0.5 mol / L NaCl and 50 mmol / L sodium citrate, with the elution volume set to 0.8 CV. In this stage, the appropriate hydrophilic ion strength induces the initial collapse of the polysaccharide and elutes the conjugates; the column bed aging parameter HETP is introduced. rel Dynamic correction was applied to the elution volume threshold, and the theoretical plate number N of the initial cycle was determined. currCalculate the volume correction factor Corrected first-stage cutoff volume: V cut =0.8CV·Ψ+τ·(d(UV 280 )) / dt, where τ is the peak tailing compensation coefficient, which is used when the first derivative of the ultraviolet signal crosses zero (peak apex) and the cumulative elution amount reaches V. cut The flow path is switched on time to eliminate the risk of retention time drift caused by media compression; in the second stage, the flow path is switched to a subsequent buffer containing 0.5 mol / L NaCl and 20 mmol / L sodium citrate for elution, and the elution volume is set to 2.0 CV to quickly reduce the concentration of lyophilic ions in the environment and prevent the precipitation of retained proteins.

[0046] The experimental results showed that in this example group, which used dual ion intensity gradient elution, the column pressure remained within the normal range of below 0.15 MPa throughout the entire process, and no pressure spikes were observed in the control group. More importantly, the elution time of the main peak of the conjugate was not affected by the low-concentration buffer in the second stage, and it remained at around 0.3 CV. The peak symmetry improved from 1.4 in the control group to 1.1. At the same time, SEC-HPLC analysis of the effluent showed that the removal rate of free polysaccharides in this example group remained at a high level of 98.5%, while the protein yield increased from 81.6% in the control group to 94.2%.

[0047] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for the chromatographic purification of a Haemophilus influenzae type b vaccine, characterized in that, The method comprises the following steps: Step S101, configure a synergistic desolvation mobile phase, dissolve sodium chloride and sodium citrate in an aqueous solution, and use an inorganic acid to adjust the pH value to the range of 7.0 to 7.4, wherein the molar concentration of sodium chloride is limited to 0.45 mol / L to 0.55 mol / L, and the molar concentration of sodium citrate is limited to 45 mmol / L to 55 mmol / L, forming a complex ion environment that can shield electrostatic repulsion and competitively strip the water film of polysaccharides at the same time; Step S102, establish a medium pore balance field, pass the synergistic desolvation mobile phase into the chromatography column filled with cross-linked agarose gel at a linear flow rate of 30 cm / h to 60 cm / h, continue to flush until the conductivity of the effluent is consistent with that of the sample mobile phase, and establish a thermodynamic equilibrium state of coexistence of electrostatic shielding and lyophilic stripping inside the micropores of the chromatography medium; Step S103, perform sample ion environment assimilation, add solid sodium chloride and sodium citrate to the crude liquid containing b-type Haemophilus influenzae capsular polysaccharide and tetanus toxoid conjugate and free capsular polysaccharide, and adjust the ion environment of the crude liquid to be completely consistent with the synergistic desolvation mobile phase, so that the free capsular polysaccharide collapses in conformation before entering the chromatography column; Step S104, implement differential fluid dynamics elution, load the crude liquid treated in step S103 into the chromatography column, and use the synergistic desolvation mobile phase for isocratic elution, use the difference in fluid dynamics volume of the conjugate and free capsular polysaccharide in the balance field, and collect the first elution peak after the outer water volume as the target product.

2. The process for the chromatographic purification of a Haemophilus influenzae type b vaccine according to claim 1, characterized in that, In step S101, the molar concentration of sodium chloride is preferably 0.5 mol / L, the molar concentration of sodium citrate is preferably 50 mmol / L, the inorganic acid is phosphoric acid or hydrochloric acid, and the conductivity of the synergistic desolvation mobile phase is controlled between 53 mS / cm and 57 mS / cm.

3. The process for the purification of Haemophilus influenzae type b vaccine by chromatography as claimed in claim 1 wherein, The cross-linked agarose gel used in step S102 is a spherical particle with a cross-linking degree of 4% to 6%, and the classification range covers a molecular weight interval of 10 kDa to 4000 kDa.

4. The process for the purification of Haemophilus influenzae type b vaccine by chromatography as claimed in claim 1 wherein, The method further comprises a pretreatment step before step S103, adding cetyltrimethylammonium bromide and ethanol to the crude liquid, precipitating and collecting the polysaccharide and conjugate components, and resuspending the precipitate with a high ionic strength solution to remove nucleic acid and non-target protein impurities.

5. The process for the purification of Haemophilus influenzae type b vaccine by chromatography as claimed in claim 1 wherein, In step S104, the loading volume is controlled at 3% to 5% of the chromatography column bed volume, and the linear flow rate during elution is controlled at 30 cm / h to 45 cm / h.

6. The process for the purification of Haemophilus influenzae type b vaccine by chromatography as claimed in claim 1 wherein, In step S104, the separation effect of the differential hydrodynamic elution is judged by the change in the distribution coefficient, wherein the adjustment of the ionic strength of the synergic desolvation mobile phase causes the free capsular polysaccharide to change its distribution coefficient K av satisfies the following relationship: 0.30≤(V e -V0) / (V t -V0)≤0.45, wherein V e is the retention volume of a free capsular polysaccharide pure reference determined in a single injection under the conditions of the synergic desolvation mobile phase, V0 is the external water volume of the chromatographic column, V t is the total volume of the chromatographic column, and this relationship indicates that the free capsular polysaccharide has entered the partial permeation zone from the complete exclusion zone.

7. The process for the purification of a Haemophilus influenzae type b vaccine according to claim 1, characterized in that, The collection operation in step S104 is performed based on the synchronous monitoring of ultraviolet absorption signals and differential refractive signals; the start and end points of collection are set as: when the absorbance value of the ultraviolet detector at 280 nm wavelength rises to 5% of the peak value, collection starts; when the absorbance value drops to 5% of the peak value and the differential refractive detector signal does not appear a second rising edge, collection stops.

8. The process for the purification of a Haemophilus influenzae type b vaccine according to claim 1, characterized in that, In step S103, the mixing process after adding solid sodium chloride and sodium citrate is carried out in a constant temperature environment of 20 to 25 degrees Celsius, and the standing time after mixing is not less than 30 minutes.

9. The process for the purification of a Haemophilus influenzae type b vaccine according to claim 1, characterized in that, The synergistic desolvation mobile phase, after being prepared, is filtered through a filter membrane with a pore size of 0.22 microns and vacuum degassed, and the entire chromatographic purification process is performed in a sterile and closed fluid path.

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