CN-121975039-A - Synthesis method of haemophilus influenzae type b polysaccharide

CN121975039ACN 121975039 ACN121975039 ACN 121975039ACN-121975039-A

Abstract

The invention relates to the field of bioengineering and polysaccharide synthesis, and discloses a method for synthesizing haemophilus influenzae type b polysaccharide, which comprises the steps of carrying out phosphorylating polymerization reaction of ribose and ribitol phosphate monomers in a liquid phase reaction system, obtaining a primary reaction liquid, adding a polarity regulator until the light transmittance of the system reaches a metastable state threshold, carrying out variable-temperature phase annealing treatment on the system in the metastable state threshold, alternately carrying out swelling diffusion and compact rearrangement operation, reducing entanglement density by utilizing solvation effect, removing amorphous impurities by utilizing supersaturation difference, maintaining annealing treatment until solid phase particles are formed, carrying out solid-liquid separation, and constructing an in-situ grading purification field by periodically regulating the solubility parameter of the system by utilizing variable-temperature phase annealing treatment.

Inventors

XU NA
XU CHUNFANG
YUAN XIUJUAN
LIANG HONGYE
WANG GANG
JIANG TAO

Assignees

长春百克生物科技股份公司

Dates

Publication Date: 20260505
Application Date: 20260408

Claims (9)

1. The method for synthesizing the haemophilus influenzae type b polysaccharide is characterized by comprising the following steps of: step S100, carrying out phosphorylation polymerization reaction of ribose and ribitol phosphate monomer in a liquid phase reaction system to obtain primary reaction liquid containing polyribosyl ribitol phosphate chain segment, and adding a polarity regulator into the primary reaction liquid until the system light transmittance reaches a preset metastable state threshold; Step S200, carrying out variable-temperature phase annealing treatment on a system at a metastable state threshold, wherein the variable-temperature phase annealing treatment comprises a swelling diffusion substep and a compact rearrangement substep which are alternately carried out, and in the swelling diffusion substep, the temperature of the system is increased to a first process temperature to enable polysaccharide aggregates to undergo volume swelling so as to remove physical embedding of amorphous impurities; And step S300, maintaining variable-temperature phase annealing treatment until the polysaccharide chain segments complete lattice reconstruction and form solid-phase particles, and carrying out solid-liquid separation operation to collect purified haemophilus influenzae type b polysaccharide, wherein the variable-temperature phase annealing treatment drives the polysaccharide chain segments to carry out reversible conversion between a swelling state and a compact state by periodically adjusting the temperature of a system.
2. The method for synthesizing haemophilus influenzae type b polysaccharide according to claim 1, wherein in the step S100, absolute ethyl alcohol is selected as the polarity regulator, and the specific operation of adding the polarity regulator until the system light transmittance reaches a preset metastable state threshold comprises pumping the polarity regulator into a primary reaction liquid under a constant stirring working condition, collecting online light transmittance data of the system in real time, and generating a pumping stop instruction when a first derivative of the online light transmittance data is detected to generate a decreasing inflection point signal.
3. The method according to claim 1, wherein in step S200, the swelling and diffusing substep and the densification and rearrangement substep form an annealing cycle, the variable-temperature phase annealing process is configured to continuously complete 3 to 5 annealing cycles, and the operation logic of the annealing cycle follows the sequence of the swelling and diffusing substep performed before the densification and rearrangement substep is performed, and the temperature is continuously changed between two adjacent annealing cycles.
4. The method for synthesizing haemophilus influenzae type b polysaccharide according to claim 1, wherein the temperature difference between the first process temperature and the second process temperature is determined based on the characteristic of a double-node curve of the polar regulator at the volume percentage concentration corresponding to the metastable state threshold in the step S100, the temperature difference is regulated to be within a preset range of 3-5 ℃, the temperature difference is realized by controlling the flow of a heat exchange medium in a jacket of a reactor, and the temperature of a driving system is switched between the first process temperature and the second process temperature at a preset temperature change rate.
5. The method for synthesizing haemophilus influenzae type b polysaccharide according to claim 4, wherein the temperature difference is accurately set within a preset interval according to a thermodynamic relationship of DeltaT= K.R swell /R compact , wherein DeltaT is a temperature difference between a first process temperature and a second process temperature, R swell is a hydrodynamic radius of a polysaccharide chain segment at the first process temperature, R compact is a hydrodynamic radius of the polysaccharide chain segment at the second process temperature, and K is a thermal response characteristic coefficient, and the value of the temperature difference is determined by a slope obtained by linear regression analysis of a temperature difference and a hydrodynamic radius ratio in an analog solvent system.
6. The method according to claim 1, wherein the densification rearrangement step in step S200 follows a nucleation control mode based on molecular weight selectivity, and the nucleation control mode is used to control the cooling rate at the second process temperature to reach supersaturation state of long chains of high molecular weight polysaccharide over short chains of low molecular weight polysaccharide and form solid phase nuclei.
7. The method for synthesizing H.influenzae type b polysaccharide according to claim 1, wherein in step S300, after the variable temperature phase annealing treatment is maintained, an end point solidification sub-step is further included, wherein the end point solidification sub-step comprises adding the rest amount of the polarity regulator into the system after the last densification rearrangement sub-step is finished, and further reducing the temperature of the system to an end point temperature lower than the second process temperature, and the end point solidification sub-step is used for locking an ordered lattice structure formed after the variable temperature phase annealing treatment.
8. The method for synthesizing haemophilus influenzae type b polysaccharide according to claim 1, wherein the haemophilus influenzae type b polysaccharide is polyribosyl ribitol phosphate, the phosphorylating polymerization is performed in an anhydrous environment and sodium cyanoborohydride is used as a reducing agent in step S100, and the primary reaction solution is subjected to one-step concentration pretreatment based on tangential flow filtration before entering the variable-temperature phase annealing treatment to remove unreacted small molecular monomers and adjust the polysaccharide concentration to a preset interval suitable for phase separation.
9. The method according to claim 1, further comprising a step of stabilizing the solid phase particles, wherein the solid phase particles are subjected to displacement washing and vacuum drying with absolute ethanol after the solid-liquid separation operation, and the solid phase particles are formed into a form having a predetermined bulk density.

Description

Synthesis method of haemophilus influenzae type b polysaccharide Technical Field The invention relates to a method for synthesizing haemophilus influenzae type b polysaccharide, belonging to the technical fields of bioengineering and polysaccharide synthesis. Background In the existing preparation process of the H.influenzae type b capsular polysaccharide, the liquid phase synthesis method is widely adopted because of the controllability of reaction conditions, after the coupling and oxidation reaction of ribitol and ribitol monomers are completed, a polar regulator such as ethanol or isopropanol is added into a homogeneous reaction solution to reduce the dielectric constant of the system and destroy the solvation layer of polysaccharide molecules, thereby realizing the precipitation and separation of products by utilizing the solubility difference of long-chain polysaccharide and small molecular impurities in a mixed solvent, however, when the long-chain poly-ribitol phosphate with high polymerization degree is prepared, because the molecular skeleton carries high-density negative charge, the chain segment can generate relatively fast hydrophobic shrinkage and conformational collapse in the phase change process of switching a good solvent to a poor solvent, and the shrinkage rate is generally higher than the mass transfer rate of residual monomers and salt impurities in the reaction solution from the inside of a high molecular coil, and the diffusion mass transfer rate and the dissimilarity between the shrinkage rates of the chain segment can cause that the polysaccharide chain segment which is not escaped rapidly in time easily to lock the solvent and the impurities dissolved in the solvent in time in the formed precipitation and the formed precipitate particles, thereby forming the impurities which are difficult to remove the embedded impurities on the surface by washing. In order to reduce the impurity residues introduced in the precipitation process, the prior art generally needs to connect purification processes such as ion exchange chromatography or gel filtration chromatography after precipitation, but the introduced chromatography operation has the following limitations in engineering practice that 1, the chromatographic elution process causes a great deal of dilution of the product, the subsequent long-time decompression concentration operation can cause hydrolysis and fracture of the thermosensitive phosphate bond due to the action of heat and shear, so that the average polymerization degree of polysaccharide is reduced, 2, repeated dissolution, chromatography and re-concentration steps prolong the production period and increase the consumption and recovery load of organic solvents, and 3, the conventional precipitation lacks a fractional screening mechanism for molecular weight, so that the final product is mixed with a low molecular weight truncated sequence, and the molecular weight distribution index cannot meet the requirement of high uniformity. Therefore, how to eliminate the impurity embedding effect by regulating and controlling the phase change kinetics of the precipitation process on the premise of not depending on the column chromatography purification steps which are easy to degrade polysaccharide and complicated in procedure, and directly obtain the solid polysaccharide with high purity and uniform molecular weight distribution becomes the technical problem to be solved by the invention. Disclosure of Invention In order to solve the problems in the background art, the technical scheme of the invention is as follows: A method for synthesizing haemophilus influenzae type b polysaccharide comprises the following steps: step S100, carrying out phosphorylating polymerization reaction of ribose and ribitol phosphate monomer in a liquid phase reaction system to obtain primary reaction liquid containing polyribosyl ribitol phosphate chain segment, and adding a polarity regulator into the primary reaction liquid until the system light transmittance reaches a preset metastable state threshold, wherein the metastable state threshold corresponds to a physical state that polysaccharide molecular chain segment is at a solid-liquid phase separation critical point but no integral precipitate is formed; Step S200, carrying out variable-temperature phase annealing treatment on a system at a metastable state threshold, wherein the variable-temperature phase annealing treatment comprises a swelling diffusion substep and a compact rearrangement substep which are alternately carried out, in the swelling diffusion substep, the temperature of the system is increased to a first process temperature to enable polysaccharide aggregates to undergo volume swelling so as to remove physical embedding of amorphous impurities; And step S300, maintaining variable-temperature phase annealing treatment until the polysaccharide chain segments complete lattice reconstruction and form solid-phase particles, and carrying o