Method for measuring content of free polysaccharide in polysaccharide-protein conjugate

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a method for determining the content of free polysaccharides in a polysaccharide-protein conjugate. The method is suitable for single polysaccharides (such as certain uniform linear polysaccharides) with regular structures and good cholate and acid tolerance and glycan systems (such as highly branched alpha-glucan, arabinomannan, beta-glucan and the like) with complex branch structures or special spatial conformation, and has relatively high stability and recovery rate on free polysaccharides; the method has good applicability and popularization prospects for various glycoconjugation drugs (such as glycoconjugation vaccines), glycosylation drugs and sugar modified biological materials.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for determining the content of free polysaccharides in polysaccharide-protein conjugates. Background Technology

[0002] Polysaccharides are widely present on the surface structures of various bacteria, fungi, and viruses, serving as key mediators in microbial-host interactions, particularly in processes such as adhesion, invasion, and immune evasion. Some polysaccharides possess adhesin-like properties, recognizing host cell surface receptors and inducing a series of biological responses, thus being widely used in the construction of vaccine antigens or glycoconjugated drugs. To enhance their immunogenicity, polysaccharides are often coupled with proteins to form glycoprotein conjugates for use in vaccine preparation, biomaterial modification, or targeted drug design.

[0003] Currently, the NaDOC (sodium deoxycholate)-HCl (hydrochloric acid) precipitation method is a commonly used technique for detecting free sugars in the field of polysaccharide vaccines. Its basic principle is to utilize the binding of NaDOC with proteins to form bile salt complexes. Under acidic conditions, the protein and its coupled structures are precipitated, thereby separating the unbound free sugars. This method performs well in traditional monopolysaccharide systems with homogeneous structures and well-defined repeating units. Commonly used conditions are NaDOC concentrations of 1–10% (w / v) and HCl concentrations of 0.05–1 mol / L.

[0004] However, in practical applications, it has been found that the traditional NaDOC-HCl system has significant limitations for certain complex glycans with significant spatial branching or highly heterogeneous glycan composition. These glycans are prone to non-specific co-precipitation during precipitation, resulting in a significant decrease in the content of free glycans in the supernatant, or even making them undetectable, thus severely underestimating the actual residual amount. Their sensitivity to bile salts or acidic environments makes them difficult to distinguish effectively from coupled proteins, reducing the accuracy and reliability of detection.

[0005] Currently, there is no publicly available method to systematically and accurately determine the free sugars in complex polysaccharide conjugates. Traditional precipitation methods lack adaptability and have become a technical bottleneck in the product development and registration of polysaccharide conjugate systems. Therefore, there is an urgent need to establish a detection method suitable for complex spatial structure polysaccharide-protein conjugates, possessing both high polysaccharide recovery rate and selective protein precipitation capability, to promote the standardization of quality analysis for related products. Summary of the Invention

[0006] To address the problem of accurately determining the free sugar content of certain polysaccharides with large molecular weights, complex structures, and unique characteristics in coupling / conjugates, the technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a combination for processing polysaccharide samples to obtain free polysaccharides, the combination comprising 0.4-1% (w / v, g / mL) of NaDOC (sodium deoxycholate) and 0.01-0.05 mol / L HCl (hydrochloric acid); preferably, the combination comprises 0.6-0.9% of NaDOC and 0.02-0.04 mol / L HCl; more preferably, the combination comprises 0.8% of NaDOC and 0.02 mol / L HCl.

[0007] In some embodiments, the concentration of polysaccharides in the polysaccharide sample is 100 μg / ml to 300 μg / ml or any value between them, preferably 200 μg / ml.

[0008] In some embodiments, the mass concentration of the sodium deoxycholate is 0.4% to 1% or any value therebetween, for example: 0.4%, 0.6%, 0.8% or 1%; further, 0.6% to 1%; even further, 0.7% to 0.9%; and still further, 0.8%.

[0009] In some embodiments, the concentration of the hydrochloric acid is 0.01 to 0.05 mol / L or any value therebetween, for example: 0.01, 0.015, 0.02, 0.025, 0.03, 0.04 or 0.05 mol / L; further, 0.015 to 0.03 mol / L; even further, 0.019 to 0.021 mol / L; and still further, 0.02 mol / L.

[0010] In some embodiments, the NaDOC (sodium deoxycholate) and HCl (hydrochloric acid) are present independently.

[0011] In some embodiments, the volume ratio of NaDOC (sodium deoxycholate) to HCl (hydrochloric acid) is (0.8-1.2):(0.8-1.2) or any value between them, preferably 1:1.

[0012] In some embodiments, the polysaccharide sample is selected from a solution of polysaccharides or a solution of polysaccharide-protein conjugates.

[0013] The purpose of this invention is to detect free polysaccharides in polysaccharide samples, mainly in the field of conjugate-conjugated vaccines, where polysaccharides bind to proteins, and the free polysaccharides present in the polysaccharide samples are detected. Therefore, this invention does not require a carrier protein for binding polysaccharides.

[0014] In some embodiments, the polysaccharide includes at least one selected from pneumococcal capsular polysaccharide, Neisseria meningitidis capsular polysaccharide, α-glucan, arabinomannan, and β-glucan; and further includes pneumococcal capsular polysaccharide, Neisseria meningitidis capsular polysaccharide, α-glucan, arabinomannan, or β-glucan.

[0015] In some embodiments, the protein in the polysaccharide-protein conjugate is a carrier protein; further, it is a vaccine carrier protein; even further, it includes at least one of the following: detoxified Pseudomonas aeruginosa exotoxin A, Escherichia coli flagellin, CRM197, maltose-binding protein, diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, agglutination factor A, agglutination factor B, Escherichia coli heat-labile enterotoxin, detoxified Escherichia coli heat-labile enterotoxin variant, cholera toxin B subunit, cholera toxin, detoxified cholera toxin variant, Escherichia coli Sat protein, the guest-carrying domain of Escherichia coli Sat protein, Streptococcus pneumoniae hemolysin, keyhole hemocyanin, Pseudomonas aeruginosa PcrV, Neisseria meningitidis outer membrane protein, and untyped Haemophilus influenzae protein D; and even further, it is tetanus toxoid.

[0016] In some embodiments, the polysaccharide-protein conjugate is selected from α-glucan-tetanus toxoid conjugates, arabinomannan-tetanus toxoid conjugates, or β-glucan-tetanus toxoid conjugates.

[0017] In a second aspect, the present invention discloses a free polysaccharide detection kit, the kit containing the combination described in the present invention.

[0018] In a third aspect, the present invention discloses the application of the combination or kit described herein in vaccine preparation.

[0019] In a fourth aspect, the present invention discloses a method for extracting free polysaccharides from polysaccharide-protein conjugates, comprising the following steps: (1) Prepare polysaccharide-protein conjugate samples, (2) The combination described in this invention is used to extract free polysaccharides. Step (2) specifically includes adding NaDOC to the solution of the polysaccharide-protein conjugate, reacting, then adding HCl, separating the solid and liquid, and obtaining the supernatant.

[0020] In some embodiments, during the extraction of free polysaccharides using the combination described in this invention, the theoretical final concentration of polysaccharides in the polysaccharide-protein conjugate sample is 80-250 ug / ml, the theoretical final concentration (mass concentration) of NaDOC is 0.03-0.1%, and the theoretical final concentration of HCl is 0.001-0.005 mol / L. Furthermore, the theoretical final concentration of polysaccharide in the polysaccharide-protein conjugate sample is 100~200 ug / ml, the theoretical final concentration of NaDOC is 0.05%~0.1%, and the theoretical final concentration of HCl is 0.001~0.003mol / L; Furthermore, the theoretical final concentration of polysaccharide in the polysaccharide-protein conjugate sample is 160~170 ug / ml, the theoretical final concentration of NaDOC is 0.07%~0.08%, and the theoretical final concentration of HCl is 0.0015~0.002mol / L.

[0021] On the other hand, the present invention provides a method for extracting free polysaccharides from polysaccharide-protein conjugates, comprising the following steps: (1) Prepare polysaccharide-protein conjugate samples, (2) Using the combination described in this invention, free polysaccharides are extracted by sodium deoxycholate precipitation. Step (2) specifically includes adding NaDOC to the solution of the polysaccharide-protein conjugate, reacting, then adding HCl, separating the solid and liquid, and obtaining the supernatant.

[0022] In some embodiments, during the process of extracting free polysaccharides by precipitation with sodium deoxycholate using the combination described in this invention, the theoretical final concentration of polysaccharides in the polysaccharide-protein conjugate sample is 80~250ug / ml, the theoretical final concentration (mass concentration) of NaDOC is 0.03~0.1%, and the theoretical final concentration of HCl is 0.001~0.005mol / L; Furthermore, the theoretical final concentration of polysaccharide in the polysaccharide-protein conjugate sample is 100~200 ug / ml, the theoretical final concentration of NaDOC is 0.05%~0.1%, and the theoretical final concentration of HCl is 0.001~0.003mol / L; Furthermore, the theoretical final concentration of polysaccharide in the polysaccharide-protein conjugate sample is 160~170 ug / ml, the theoretical final concentration of NaDOC is 0.07%~0.08%, and the theoretical final concentration of HCl is 0.0015~0.002mol / L.

[0023] In a fifth aspect, the present invention provides a method for determining the free polysaccharide content in a polysaccharide-protein conjugate, comprising the following steps: S1: Prepare solutions of polysaccharide-protein conjugates and polysaccharides as samples; S2: The solutions of the polysaccharide-protein conjugate and the polysaccharide were subjected to sodium deoxycholate precipitation using the combination described in this invention, and were respectively designated as the conjugate treatment group and the polysaccharide treatment group; The solutions of the polysaccharide-protein conjugate and the polysaccharide were respectively subjected to control treatments and were designated as the untreated conjugate group and the untreated polysaccharide group. in, The method for precipitating sodium deoxycholate is as follows: NaDOC is added to the solution of the polysaccharide-protein conjugate and the solution of the polysaccharide, respectively, and the reaction is carried out. Then HCl is added, and solid-liquid separation is performed to obtain the supernatant. The control treatment method is as follows: a control solution is added to the solution of the polysaccharide-protein conjugate and the solution of the polysaccharide respectively to obtain a mixture. The control solution includes buffer and / or physiological saline, and the volume of the control solution is the sum of the volumes of NaDOC and HCl. S3: The polysaccharide content in the supernatant of the conjugate-treated group and the polysaccharide-treated group were detected respectively and recorded as Sb (sugar concentration in the supernatant after conjugate treatment, Supernatant from binding sample) and Sr (sugar concentration in the supernatant after (uncoupled) polysaccharide treatment, Supernatant from reference sample). The polysaccharide content in the mixtures of the untreated conjugate group and the untreated polysaccharide group was measured separately and denoted as Cb (concentration of binding sample) and Cr (concentration of reference sample) respectively. S4: Calculate the free polysaccharide content Fp (free polysaccharide percentage) in the polysaccharide-protein conjugate: Wherein, Rc is the recovery coefficient, which is the actual recovery efficiency of free polysaccharides under the conditions of sodium deoxycholate precipitation treatment;

[0024] In some embodiments, the volume of NaDOC is 5-20% of the solution of the polysaccharide-protein conjugate and the solution of the polysaccharide, and the volume of HCl is 5-20% of the solution of the polysaccharide-protein conjugate and the solution of the polysaccharide.

[0025] In some embodiments, the volume of NaDOC is 10-15% of the solution of the polysaccharide-protein conjugate and the solution of the polysaccharide; further, it is 10-12%; and even further, it is 11.1%.

[0026] In some embodiments, the volume of the HCl is 10-15% of the solution of the polysaccharide-protein conjugate, or 10-12% of the solution of the polysaccharide; more particularly, it is 11.1%.

[0027] In some embodiments, the volume of NaDOC is the same as the volume of HCl.

[0028] In some embodiments, the volume of the polysaccharide-protein conjugate solution and the polysaccharide solution in the method of sodium deoxycholate precipitation treatment and the method of control treatment is 200-600 μL; further, 400-500 μL; even further, 440-460 μL; and still further, 450 μL.

[0029] In some embodiments, the volume of the polysaccharide-protein conjugate solution and the polysaccharide solution are the same in the deoxycholate sodium precipitation treatment method and the control treatment method.

[0030] In some embodiments, the concentration of the polysaccharide-protein conjugate solution and the polysaccharide solution in the method of sodium deoxycholate precipitation treatment and the method of control treatment is 100-300 μg / mL, further 150-250 μg / mL, even further 190-210 μg / mL, and still further about 200 μg / mL; wherein the concentration of the polysaccharide-protein conjugate solution is based on the polysaccharide.

[0031] In some embodiments, the concentrations of the polysaccharide-protein conjugate solution and the polysaccharide solution are the same in the method of sodium deoxycholate precipitation treatment and the method of control treatment, wherein the concentration of the polysaccharide-protein conjugate solution is based on the polysaccharide.

[0032] In some embodiments, during the sodium deoxycholate precipitation process, the theoretical final concentration of the polysaccharide-protein conjugate solution and the polysaccharide solution is 80~250ug / ml, the theoretical final concentration (mass concentration) of NaDOC is 0.03~0.1%, and the theoretical final concentration of HCl is 0.001~0.005mol / L; Furthermore, the theoretical final concentration of the polysaccharide in the polysaccharide-protein conjugate solution and the polysaccharide in the polysaccharide solution is 100~200 ug / ml, the theoretical final concentration of NaDOC is 0.05%~0.1%, and the theoretical final concentration of HCl is 0.001~0.003mol / L; Furthermore, the theoretical final concentration of the polysaccharide in the polysaccharide-protein conjugate solution and the polysaccharide in the polysaccharide solution is 160~170 ug / ml, the theoretical final concentration of NaDOC is 0.07%~0.08%, and the theoretical final concentration of HCl is 0.0015~0.002mol / L; The "theoretical final concentration" mentioned in this invention refers to the initial concentration divided by the dilution factor, where the dilution factor is the final volume divided by the initial volume. It does not consider reagent consumption during the reaction and is not the actual final concentration; it is mainly used for ease of description. For example, the initial polysaccharide concentration of 450 μL at 200 ug / ml in this invention has a dilution factor of final volume 550 μL (450 μL + 50 μL + 50 μL) / initial volume 450 μL, which is 11 / 9. Therefore, the theoretical final concentration is (200 × 9) / 11, approximately 163 ug / ml.

[0033] In some embodiments, the addition of NaDOC to the solutions of the polysaccharide-protein conjugate and the polysaccharide is carried out under ice bath conditions.

[0034] In some embodiments, a mixing step is included before the reaction.

[0035] In some embodiments, the reaction is carried out under ice bath conditions.

[0036] In some embodiments, the reaction is allowed to proceed under the following conditions: 20-40 minutes of rest; 25-35 minutes of rest; and approximately 30 minutes of rest.

[0037] In some embodiments, the solid-liquid separation is centrifugation.

[0038] In some embodiments, the centrifugation conditions are: centrifugation at 8000-12000g for 5-40 min; further, centrifugation at 8000-10000g for 25-35 min; and even further, centrifugation at 9000g for 30 min.

[0039] In some embodiments, the centrifugation is performed at 4–8°C.

[0040] In some embodiments, the control treatment is performed under ice bath conditions.

[0041] In some embodiments, the control solution is physiological saline.

[0042] In some implementations, chemical or immunological methods are used to detect the polysaccharide content in the supernatant of the conjugate-treated group and the polysaccharide-treated group, as well as in the mixture of the untreated conjugate group and the untreated polysaccharide group.

[0043] In some embodiments, the chemical method is selected from the anthrone-sulfuric acid method, the ribose determination method, the phosphorus content determination method, or the sialic acid determination method; more specifically, the anthrone-sulfuric acid method.

[0044] In some embodiments, the immunological method is enzyme-linked immunosorbent assay (ELISA) or immunoturbidimetry.

[0045] In some embodiments, the protein is a carrier protein; further, a vaccine carrier protein; and even further, at least one of the following: detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose-binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, agglutination factor A, agglutination factor B, Escherichia coli heat-labile enterotoxin, detoxified Escherichia coli heat-labile enterotoxin variant, cholera toxin B subunit (CTB), cholera toxin, detoxified cholera toxin variant, Escherichia coli Sat protein, the guest-carrying domain of Escherichia coli Sat protein, Streptococcus pneumoniae hemolysin, keyhole hemocyanin (KLH), Pseudomonas aeruginosa PcrV, Neisseria meningitidis outer membrane protein (OMPC), and protein D from untyped Haemophilus influenzae; and even further, tetanus toxoid.

[0046] In some embodiments, the polysaccharide includes at least one of natural polysaccharides and synthetic polysaccharides (synthetic sugar polymers).

[0047] In some embodiments, the polysaccharide is a single polysaccharide with regular structure and good bile salt and acid tolerance, such as pneumococcal capsular polysaccharide or Neisseria meningitidis capsular polysaccharide.

[0048] In some embodiments, the polysaccharide is a polysaccharide with a branched structure or complex conformation, preferably a polysaccharide with a complex branched structure or a special spatial conformation, or a polysaccharide with a complex structure, significant branching of spatial configuration, or strong heterogeneity in sugar chain composition; more preferably, it includes at least one of α-glucan, arabinomannan, and β-glucan.

[0049] In some embodiments, the polysaccharide-protein conjugate includes at least one of α-glucan-tetanus toxoid conjugate, arabinomannan-tetanus toxoid conjugate, and β-glucan-tetanus toxoid conjugate.

[0050] In some embodiments, the polysaccharide-protein conjugate may be a glycoconjugated drug (e.g., a glycoconjugated vaccine), a glycosylated drug, or a glycomodified biomaterial, encompassing forms such as covalent conjugation and non-covalent modification.

[0051] A sixth aspect of the present invention provides a method comprising the steps of the determination method of the fifth aspect of the present invention; The method is used for any one of a1)-a4): a1) Quality control in the production of the polysaccharide-protein conjugate; a2) Process optimization in the production of the polysaccharide-protein conjugate; a3) Batch consistency evaluation in the production of the polysaccharide-protein conjugate; a4) Evaluation of the binding (coupling / conjugation) efficiency of the polysaccharide-protein conjugate.

[0052] The beneficial effects of this invention are: This invention provides a combination for processing polysaccharide samples to obtain free polysaccharides. This combination is suitable for obtaining free polysaccharides from processed polysaccharide samples. The polysaccharides can be single polysaccharides with regular structures and good bile salt and acid tolerance (such as pneumococcal capsular polysaccharide or Neisseria meningitidis capsular polysaccharide), as well as polysaccharide systems with complex branched structures or special spatial conformations (such as highly branched α-glucan, arabinomannan, β-glucan, etc.). It has high stability and recovery rate for free polysaccharides and can be used for detecting free polysaccharides, preparing vaccines, extracting free polysaccharides from polysaccharide-protein conjugates, and determining the free polysaccharide content in polysaccharide-protein conjugates to be tested.

[0053] Furthermore, this invention provides a method for determining the free polysaccharide content in polysaccharide-protein conjugates. This method is applicable to single polysaccharides with regular structures and good tolerance to bile salts and acids (such as pneumococcal capsular polysaccharide or Neisseria meningitidis capsular polysaccharide), as well as polysaccharide systems with complex branched structures or special spatial conformations (such as highly branched α-glucan, arabinomannan, β-glucan, etc.). It exhibits high stability and recovery rate for free polysaccharides and has good applicability and promotion prospects for various glycoconjugated drugs (such as glycoconjugated vaccines), glycosylated drugs, and glycomodified biomaterials. It is applicable to various types of polysaccharide-protein conjugates, including but not limited to the binding forms of natural polysaccharides, synthetic sugar polymers, and common carrier proteins (such as tetanus toxoid). It can be used for quality control, process optimization, batch consistency evaluation, and assessment of the binding (coupling / conjugation) efficiency of polysaccharide-protein conjugates in production. Attached Figure Description

[0054] Figure 1 The graph shows the results of the protein removal capability verification of the NaDOC-HCl system.

[0055] Figure 2 The graph shows the verification results of the NaDOC-HCl system's ability to remove conjugates. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0057] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0058] the term As used herein, the term "polysaccharide sample" refers to a sample containing polysaccharides, which may also be called polysaccharides. The sample containing polysaccharides refers to a sample containing free polysaccharides, with the content of the free polysaccharides ranging from 0% to 100%. In some embodiments, the content of the free polysaccharides is 0% to 20%. In some embodiments, the content of the free polysaccharides is 80% to 100%. In some embodiments, the content of the free polysaccharides is 20% to 50%. In some embodiments, the content of the free polysaccharides is 50% to 80%.

[0059] The term "free polysaccharide" as used in this article refers to polysaccharides that exist freely in solution and are not bound or coupled with other substances.

[0060] In this article, 1% w / v means 1g of solute dissolved in 100mL of solvent.

[0061] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value. In some embodiments, the term "about" indicates a range of ±1% of the following value.

[0062] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0063] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0064] This invention first uses α-glucan, which has a complex structure and large molecular weight, as a model. By systematically optimizing the NaDOC-HCl precipitation system, it achieves effective separation of free polysaccharides and protein conjugates, and the applicability of the method is verified stepwise. Subsequent experiments further extended the method to arabinomannan with many branched chains and β-glucan with high structural specificity, both of which showed good method compatibility.

[0065] α-Glucan, arabinomannan, carrier protein tetanus toxoid, and rabbit anti-tetanus toxoid polyclonal antibody can be prepared by existing methods. Among them, the preparation method of α-glucan is referenced in: Schwebach JR, Glatman-Freedman A, Gunther-Cummins L, Dai Z, Robbins JB, Schneerson R, Casadevall A. Glucan is a component of the Mycobacterium tuberculosis surface that is expressed in vitro and in vivo. Infect Immun. 2002 May;70(5):2566-75.doi: 10.1128 / IAI.70.5.2566-2575.2002. PMID: 11953397; PMCID: PMC127896; The preparation method of arabinomannan is referenced in: Hamasur B, Haile M, Pawlowski A, Schröder U, Williams A, Hatch G, Hall G, Marsh P, Källenius G, Svenson SB. Mycobacterium tuberculosis arabinomannan-protein conjugates protect against tuberculosis. Vaccine. 2003 Sep 8;21(25-26):4081-93. doi: 10.1016 / s0264-410x(03)00274-3.PMID: 12922145; The preparation method of the carrier protein tetanus toxoid is based on Example 1 of patent document CN114805504A; The preparation method of rabbit anti-tetanus toxoid polyclonal antibody is based on textbook: Medical Immunology, 5th edition, page 45, edited by Jin Boquan, People's Medical Publishing House. Rabbits were injected with tetanus toxoid, and then the antibody was collected and purified by chromatography.

[0066] Example 1. Evaluation of the recovery rate of uncoupled polysaccharides under conventional NaDOC-HCl treatment conditions This embodiment uses structurally complex polysaccharides, represented by α-glucan, as a model to verify the applicability of the traditional NaDOC-HCl method (NaDOC mass concentration of 1~10%, HCl concentration of 0.05~1mol / L) in processing uncoupled polysaccharide samples and to evaluate the recovery rate of free polysaccharides by this method.

[0067] 1) Sample preparation For the treatment group, 450 μL of uncoupled α-glucan solution at a concentration of 200 μg / mL was prepared (i.e., 450 μL of α-glucan solution with a concentration of 200 μg / mL). Another equal volume of sample (concentration of 200 μg / mL) was prepared for the untreated control group, and physiological saline of the same volume as NaDOC and HCl was added to standardize the total volume.

[0068] 2) NaDOC-HCl treatment Add 50 μL of 1-10% (w / v) NaDOC solution to the treatment group sample and let it stand for 30 minutes in an ice-water bath; then add 50 μL of 0.05-1 mol / L HCl solution, mix well, and centrifuge at 9000g for 30 minutes at 4℃.

[0069] 3) Supernatant collection and quantitative sugar detection Supernatants from both the untreated and treated groups were collected, and sugar concentrations were determined using the anthrone-sulfuric acid method. The concentration measured in the untreated sample was denoted as Cr, and the concentration measured in the treated supernatant was denoted as Sr. The recovery rate Rc was calculated. 4) Experimental Results As shown in Table 1, under traditional NaDOC-HCl treatment conditions, polysaccharide samples generally precipitated, resulting in a significant decrease in the polysaccharide content in the supernatant. The highest recovery rate of supernatant polysaccharides was only 13.92%. Under some treatment conditions, the samples were gel-like without precipitation, thus failing to obtain a usable supernatant for detection. These results indicate that this method suffers from significant recovery loss when processing certain structurally complex or environmentally sensitive polysaccharides, limiting its application in the accurate detection of free polysaccharides. Therefore, further optimization of the precipitation system is needed to improve the detection efficiency and broad-spectrum adaptability of the method.

[0070] Table 1. Recovery rate of uncoupled polysaccharides under conventional NaDOC-HCl concentration combination treatment conditions

[0071] Example 2. Effect of different combinations of NaDOC and HCl concentrations on the recovery rate of uncoupled polysaccharides This embodiment uses structurally complex polysaccharides, represented by α-glucan, as a model to screen suitable NaDOC-HCl treatment conditions for polysaccharides. By setting different combinations of NaDOC mass concentration (1%~3%) and HCl concentration (0.005~0.03mol / L), the recovery effect on free polysaccharides is evaluated, and a preliminary applicable range is identified.

[0072] 1) Sample preparation For the treatment group, 450 μL of uncoupled α-glucan solution at a concentration of 200 μg / mL was prepared (i.e., 450 μL of α-glucan solution with a concentration of 200 μg / mL). Another equal volume of sample (concentration 200 μg / mL) was prepared for the untreated control group, and physiological saline of the same volume as NaDOC and HCl was added to standardize the total volume.

[0073] 2) NaDOC-HCl treatment Add 50 μL of 1-3% (w / v) NaDOC solution to the treatment group sample and let it stand for 30 minutes in an ice-water bath; then add 50 μL of 0.005-0.03 mol / L HCl solution, mix well, and centrifuge at 9000g for 30 minutes at 4℃.

[0074] 3) Supernatant collection and quantitative sugar detection Supernatants from both the untreated and treated groups were collected, and sugar concentrations were determined using the anthrone-sulfuric acid method. The concentration measured in the untreated sample was denoted as Cr, and the concentration measured in the treated supernatant was denoted as Sr. The recovery rate Rc was calculated. 4) Experimental Results As shown in Table 2, this experiment is the first to clearly demonstrate that excessively high NaDOC concentrations (≥2%) will significantly reduce the retention of polysaccharides in the supernatant, suggesting that this polysaccharide is far more sensitive to bile salts than other common vaccine polysaccharides.

[0075] Within the test range, the optimal recovery rate of free polysaccharides (approximately 50%) was achieved when the NaDOC concentration was 1% and the HCl concentration was 0.02–0.03 mol / L. This indicates that this concentration combination can serve as preliminary applicable conditions for polysaccharide recovery, providing a basis for further optimization.

[0076] Table 2. Recovery rates of uncoupled polysaccharides under different NaDOC-HCl concentration combinations

[0077] Example 3. Optimization of the recovery rate of uncoupled polysaccharides under different NaDOC and HCl concentrations. Based on Example 2, this embodiment further narrows the NaDOC concentration range and evaluates the recovery effect of low-mass-concentration NaDOC (0.4~0.8%) at 0.02~0.03 mol / L HCl concentration to screen out the most suitable treatment conditions for the polysaccharide.

[0078] 1) Sample preparation For the treatment group, 450 μL of uncoupled α-glucan solution at a concentration of 200 μg / mL was prepared (i.e., 450 μL of α-glucan solution with a concentration of 200 μg / mL). Another equal volume of sample (concentration of 200 μg / mL) was prepared for the untreated control group, and physiological saline of the same volume as NaDOC and HCl was added to standardize the total volume.

[0079] 2) NaDOC-HCl treatment Add 50 μL of 0.4-0.8% (w / v) NaDOC solution to the treatment group sample and let it stand for 30 minutes in an ice-water bath; then add 50 μL of 0.02-0.03 mol / L HCl solution, mix well, and centrifuge at 9000g for 30 minutes at 4℃.

[0080] 3) Supernatant collection and quantitative sugar detection Supernatants from both the untreated and treated groups were collected, and polysaccharide concentrations were determined using the anthrone-sulfuric acid method. The concentration measured in the untreated sample was denoted as Cr, and the concentration measured in the treated supernatant was denoted as Sr. The recovery rate Rc was calculated. 4) Experimental Results As shown in Table 3, the recovery rate of free polysaccharides was highest, reaching approximately 90%, when the NaDOC concentration was 0.8% and the HCl concentration was 0.02 mol / L. Under these conditions, the supernatant was clear with minimal precipitation, indicating that the polysaccharide structure was not excessively precipitated. These are the recommended treatment conditions for subsequent determination of free sugars in the conjugate.

[0081] When the NaDOC concentration is below 0.8% (e.g., 0.6% or 0.4%), the sugar concentration in the supernatant after treatment decreases significantly. The likely reason is that under insufficient bile salt concentration, the polysaccharide is difficult to disperse stably in an acidic environment, leading to a decrease in recovery rate. Therefore, an appropriate NaDOC concentration is crucial for maintaining the polysaccharide's solubility; while excessively low concentrations reduce precipitation, they are detrimental to the complete retention of free sugars in the supernatant.

[0082] Table 3. Recovery rate of uncoupled polysaccharides under optimized NaDOC-HCl concentration combination treatment conditions

[0083] Example 4. Investigating the effect of different HCl concentrations on free sugar recovery rate under a fixed NaDOC concentration. Based on the established NaDOC mass concentration of 0.8%, this embodiment further optimizes the hydrochloric acid (HCl) concentration parameter to investigate its impact on the recovery rate of free polysaccharides and precipitation formation, in order to clarify the key parameter range of the NaDOC-HCl combination.

[0084] 1) Sample preparation For the treatment group, 450 μL of uncoupled α-glucan solution at a concentration of 200 μg / mL was prepared (i.e., 450 μL of α-glucan solution with a concentration of 200 μg / mL). Another equal volume of sample (concentration of 200 μg / mL) was prepared for the untreated control group, and physiological saline of the same volume as NaDOC and HCl was added to standardize the total volume.

[0085] 2) NaDOC-HCl treatment Add 50 μL of 0.8% (w / v) NaDOC solution to the treatment group sample and let it stand for 30 minutes in an ice-water bath; then add 50 μL of 0.01~0.02 mol / L HCl solution, mix well, and centrifuge at 9000g for 30 minutes at 4℃.

[0086] 3) Supernatant collection and quantitative sugar detection Supernatants from both the untreated and treated groups were collected, and sugar concentrations were determined using the anthrone-sulfuric acid method. The concentration measured in the untreated sample was denoted as Cr, and the concentration measured in the treated supernatant was denoted as Sr. The recovery rate Rc was calculated. 4) Experimental Results As shown in Table 4, when the HCl concentration was 0.02 mol / L, a small amount of white flocculent precipitate was observed, and the sugar concentration in the supernatant was significantly increased, with a recovery rate of over 90%.

[0087] It is worth noting that when the HCl concentration is below 0.02 mol / L, although no precipitate forms after sample treatment, and the concentration of sugar in the supernatant is basically the same as that of the untreated sample, this indicates that the polysaccharide does not precipitate or lose under these conditions. However, in practical applications for determining the free sugar content in conjugates, a key prerequisite is the effective precipitation of proteins or conjugates, so that the free sugars are retained in the supernatant for accurate detection.

[0088] Therefore, although low-concentration hydrochloric acid does not precipitate glycans, whether it has sufficient precipitation ability to remove proteins from the conjugates still needs further verification. To this end, in Example 5 of this invention, protein was used as a sample to investigate the effect of different hydrochloric acid concentrations on protein precipitation at the same NaDOC concentration, in order to verify the necessity and selectivity of 0.02 mol / L hydrochloric acid in this system.

[0089] Table 4. Effect of different HCl concentrations on the recovery rate of uncoupled polysaccharides under fixed NaDOC treatment conditions

[0090] Example 5: Verification of protein removal capability of the NaDOC-HCl system To further verify the effectiveness of the treatment system composed of 0.8% NaDOC and different concentrations of HCl selected in this invention in protein precipitation, and to evaluate its feasibility for removing protein components from conjugates, given that tetanus toxoid (TT) is a commonly used protein carrier in actual vaccine conjugates, this embodiment selects TT protein as the test object to verify the protein precipitation performance of the NaDOC-HCl system.

[0091] 1) Sample preparation For the treatment group, 450 μL of TT protein solution with a concentration of 200 μg / mL was prepared (i.e., 450 μL of TT protein solution with a concentration of 200 μg / mL). Another equal volume of sample (concentration of 200 μg / mL) was prepared for the untreated control group, and physiological saline of the same volume as NaDOC and HCl was added to standardize the total volume.

[0092] 2) NaDOC-HCl treatment Add 50 μL of 0.8% (w / v) NaDOC solution to the treatment group sample and let it stand for 30 minutes in an ice-water bath; then add 50 μL of 0.01~0.02 mol / L HCl solution, mix well, and centrifuge at 9000g for 30 minutes at 4℃.

[0093] 3) Sample collection and testing The processed samples were centrifuged and separated into layers. The supernatant was used for subsequent analysis, while the precipitate was retained and fully reconstituted with 300 μL of 0.1 mol / L NaOH. Subsequently, all supernatants and the reconstituted precipitate samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein distribution was detected by Coomassie brilliant blue staining.

[0094] 4) Experimental Results like Figure 1 As shown in the results, this experiment clearly demonstrates that when the NaDOC concentration is 0.8%, TT protein can only be effectively precipitated and completely removed from the supernatant when the HCl concentration reaches 0.02 mol / L. HCl solutions below 0.02 mol / L do not possess sufficient acidity to synergize with NaDOC in protein precipitation, and a large amount of protein remains in the supernatant, making separation impossible.

[0095] In summary, the combination of 0.8% NaDOC and 0.02 mol / L HCl is the key processing condition of this invention, which can selectively precipitate proteins. In the aforementioned examples, it was also verified that it has no precipitation effect on free α-glucan, making it an ideal system for the accurate determination of free polysaccharides in the complex.

[0096] Example 6: Determination of free polysaccharides in α-glucan-TT conjugates and verification of method suitability To further verify the applicability of the NaDOC-HCl separation system established in this invention in the detection of actual vaccine conjugates, this embodiment uses α-glucan-TT as the test object to evaluate the method's ability to accurately determine the content of free glycans in the conjugates. The protein precipitation effect is verified by Western blotting (WB), thereby comprehensively evaluating the reliability and practicality of the treatment system.

[0097] 1) Sample preparation Three different batches of α-glucan-TT conjugate samples, each 450 μL in volume, were collected. The sugar concentration was 200 μg / mL (i.e., 450 μL of α-glucan-TT conjugate sample with a concentration of 200 μg / mL based on α-glucan concentration). Each batch was divided into a treated group and an untreated group. An equal volume of uncoupled α-glucan solution (200 μg / mL) from the corresponding batch was also taken as a reference sample and divided into a treated group and an untreated group.

[0098] 2) NaDOC-HCl treatment The treatment group was subjected to optimized treatment conditions: 50 μL of 0.8% (w / v) NaDOC solution was added, allowed to stand for 30 minutes, and then 50 μL of 0.02 mol / L HCl solution was added. After mixing, the mixture was centrifuged at 4°C and 9000g for 30 minutes. The untreated group was supplemented with physiological saline of the same volume as NaDOC and HCl to ensure a uniform total volume.

[0099] 3) Sample collection and testing The processed samples were centrifuged to separate layers. The supernatant was used for subsequent analysis, while the precipitate was retained and fully reconstituted with 300 μL of 0.1 mol / L NaOH.

[0100] Sugar concentration was determined using the anthrone-sulfuric acid method. The sugar concentration measured in the untreated, uncoupled α-glucan sample was denoted as Cr, and the sugar concentration measured in the supernatant after treatment was denoted as Sr. The sugar recovery rate Rc was calculated based on these values ​​to assess whether free sugars were lost during treatment. The calculation formula is as follows: The sugar concentration measured in the untreated α-glucan-TT conjugate sample is denoted as Cb, and the sugar concentration measured in the supernatant after treatment is denoted as Sb. Based on these, the percentage of free sugar in the conjugate, Fp, is calculated, reflecting the residual level of uncoupled glucans in the conjugate sample. The calculation formula is as follows: The results are shown in Table 5. After the same treatment, the sugar concentration in the supernatant of the uncoupled polysaccharide in the reference group remained above 90%, confirming that free sugars did not precipitate under these conditions and could accurately reflect the content of unbound sugars in the conjugate samples. Using the calculation formula defined in the invention, the percentage of free sugars in the three batches of conjugate samples were 10.13%, 6.55%, and 9.20%, respectively.

[0101] Table 5. Results of determination of α-glucan-TT conjugate samples from three batches

[0102] 4) Protein removal validation (Western Blot) To verify the ability of the NaDOC-HCl treatment system used in this invention to remove protein carriers (TT) from the conjugate, the original conjugate solution before treatment, the supernatant after treatment, and the precipitate reconstituted solution were analyzed by Western blotting. Rabbit anti-tetanus toxoid polyclonal antibody was used as the primary antibody to detect protein distribution.

[0103] The results are as follows Figure 2 As shown: The untreated conjugate contained protein bands with molecular weights much higher than that of the TT bulk (approximately 150 kDa), indicating that α-glucan had been successfully coupled to TT to form a large molecular complex; no conjugate protein bands were detected in the supernatant after treatment, while clear bands were visible in the precipitated and reconstituted sample, indicating that the treatment system could fully precipitate the conjugate, and no conjugate components remained in the supernatant.

[0104] Example 7: Validation of the recovery rate of arabinomannan in an optimized NaDOC-HCl treatment system This embodiment aims to evaluate the applicability of the optimized NaDOC-HCl treatment system (0.8% NaDOC + 0.02 mol / L HCl) in treating arabinomannan (AM) samples with unique structures, and to further verify the compatibility and broad applicability of this method to polysaccharides with different configurations.

[0105] 1) Sample preparation For the treatment group, 450 μL of purified uncoupled arabinomannan solution with a concentration of 200 μg / mL was taken (i.e., 450 μL of arabinomannan solution with a concentration of 200 μg / mL). Another equal volume of the same batch of sample (concentration of 200 μg / mL) was taken as the untreated control group, and physiological saline with an equal volume of NaDOC and HCl was added to standardize the total volume.

[0106] 2) NaDOC-HCl treatment Add 50 μL of 0.8% (w / v) NaDOC solution to the treatment group sample and let it stand in an ice-water bath for 30 minutes; then add 50 μL of 0.02 mol / L HCl solution, mix well, and centrifuge at 4℃ and 9000g for 30 minutes.

[0107] 3) Supernatant collection and quantitative sugar detection Supernatants from both the untreated and treated groups were collected, and sugar concentrations were determined using the anthrone-sulfuric acid method. The concentration measured in the untreated sample was denoted as Cr, and the concentration measured in the treated supernatant was denoted as Sr. The recovery rate Rc was calculated. 4) Experimental Results As shown in Table 6, under optimized NaDOC-HCl treatment conditions, no obvious precipitation occurred in the arabinomannan sample, and the supernatant was clear and transparent. Quantitative sugar analysis results showed that the recovery rate of this system in the arabinomannan sample was approximately 90%, with no significant loss. This indicates that the optimized treatment system also has good compatibility and separation accuracy for this type of neutral polysaccharide with small molecular weight and spatially branched structure (multiple branched chains).

[0108] Table 6. Recovery rate of uncoupled arabinomannan under optimized NaDOC-HCl concentration combination treatment conditions

[0109] Example 8: Determination of free polysaccharides in arabinomannan-TT conjugates and verification of method suitability This embodiment, based on Example 7, further verifies the applicability and detection accuracy of the optimized NaDOC-HCl precipitation system (0.8% NaDOC + 0.02 mol / L HCl) in actual arabinomannan-TT protein conjugate samples.

[0110] 1) Sample preparation Three batches of 450 μL arabinomannan-TT protein conjugate samples from different sources were collected, with a sugar concentration of 200 μg / mL (i.e., 450 μL of arabinomannan-TT conjugate sample with a concentration of 200 μg / mL based on arabinomannan). Each batch was divided into a treated group and an untreated group. An equal volume of uncoupled arabinomannan solution (200 μg / mL) was also taken as a reference sample and divided into a treated group and an untreated group.

[0111] 2) NaDOC-HCl treatment In the treatment group, 50 μL of 0.8% (w / v) NaDOC solution was added to both the conjugate sample and the reference sample, and the mixture was incubated in an ice-water bath for 30 minutes. Then, 50 μL of 0.02 mol / L HCl solution was added, and after mixing, the mixture was centrifuged at 9000g for 30 minutes at 4°C, and the supernatant was collected. In the untreated group, physiological saline of the same volume as NaDOC and HCl was added to equalize the total volume.

[0112] 3) Sample collection and testing The processed samples were centrifuged to separate layers. The supernatant was used for subsequent analysis, while the precipitate was retained and fully reconstituted with 300 μL of 0.1 mol / L NaOH.

[0113] Sugar concentration was determined using the anthrone-sulfuric acid method. The sugar concentration measured in the untreated, uncoupled arabinomannan sample was denoted as Cr, and the sugar concentration measured in the supernatant after treatment was denoted as Sr. The sugar recovery rate Rc was calculated based on these values ​​to assess whether free sugars were lost during treatment. The calculation formula is as follows: The sugar concentration measured in the untreated arabinomannan-TT conjugate sample is denoted as Cb, and the sugar concentration measured in the supernatant after treatment is denoted as Sb. Based on these values, the percentage of free sugar in the conjugate, Fp, is calculated, reflecting the residual level of uncoupled polysaccharides in the conjugate sample. The calculation formula is as follows: 4) Experimental Results As shown in Table 7, the recovery rate (Rc) of the reference sugar sample remained above 90% under optimized NaDOC-HCl treatment conditions, indicating that free arabinomannan maintained good stability in this system. The sugar concentration in the supernatant of the conjugate sample was significantly reduced after treatment. Combined with the concentration measured in the untreated group and the calculated Fp value, the free sugar content in the sample was successfully quantified with good repeatability.

[0114] The results further validated the applicability of the method of the present invention in low molecular weight, spatially branched (multiple branched chains) glycan coupling systems, and supported the feasibility of its application in the quality control of different types of glycan-protein conjugates.

[0115] Table 7. Results of determination of arabinomannan-TT conjugate samples from three batches

[0116] Example 9: Validation of β-glucan recovery rate in an optimized NaDOC-HCl treatment system This embodiment aims to verify the applicability of the established 0.8% NaDOC + 0.02 mol / L HCl precipitation system in processing β-glucan samples, evaluate the recovery efficiency of this method for β-glucan, and further expand the applicability of this method in different types of polysaccharides.

[0117] 1) Sample preparation For the treatment group, 450 μL of uncoupled β-glucan (Aladdin, G304913) solution at a concentration of 200 μg / mL (i.e., 450 μL of 200 μg / mL arabinomannan solution) was prepared. An equal volume of sample (200 μg / mL) was prepared as the untreated control group, and physiological saline of equal volume to NaDOC and HCl was added to standardize the total volume.

[0118] 2) NaDOC-HCl treatment Add 50 μL of 0.8% (w / v) NaDOC solution to the treatment group sample and let it stand in an ice-water bath for 30 minutes; then add 50 μL of 0.02 mol / L HCl solution, mix well, and centrifuge at 4℃ and 9000g for 30 minutes.

[0119] 3) Supernatant collection and quantitative sugar detection Supernatants from both the untreated and treated groups were collected, and sugar concentrations were determined using the anthrone-sulfuric acid method. The concentration measured in the untreated sample was denoted as Cr, and the concentration measured in the treated supernatant was denoted as Sr. The recovery rate Rc was calculated. 4) Experimental Results As shown in Table 8, under optimized NaDOC-HCl treatment conditions, no obvious precipitation was formed in the treated samples, the supernatant was clear, and the recovery rate of β-glucan was higher than 90%, indicating that the glucan did not undergo non-specific precipitation in this precipitation system and has good detection stability and adaptability.

[0120] These results further verify that the method of the present invention has good applicability to glycans of different sources and structural types. In particular, it can maintain a high recovery rate in glycan systems with irregular spatial configurations or high branching (high structural specificity), providing a methodological basis for the subsequent detection of free sugars in multiple types of glycan-protein conjugates.

[0121] Table 8. Recovery rate of uncoupled β-glucan under optimized NaDOC-HCl concentration combination treatment conditions

[0122] Example 10: Determination of free polysaccharide content in β-glucan-TT conjugates and verification of method suitability This embodiment, based on Example 9, further verifies the applicability and detection accuracy of the established 0.8% NaDOC + 0.02 mol / L HCl treatment system in β-glucan-TT protein conjugates.

[0123] 1) Sample preparation Three batches of β-glucan-TT protein conjugate samples, each 450 μL in size, were collected. The sugar concentration was 200 μg / mL (i.e., 450 μL of β-glucan-TT conjugate sample with a concentration of 200 μg / mL based on α-glucan concentration). Each batch was divided into a treated group and an untreated group. An equal volume of uncoupled β-glucan solution (200 μg / mL) was also taken as a reference sample and divided into a treated group and an untreated group.

[0124] 2) NaDOC-HCl treatment In the treatment group, 50 μL of 0.8% (w / v) NaDOC solution was added to both the conjugate sample and the reference sample, and the mixture was incubated in an ice-water bath for 30 minutes. Then, 50 μL of 0.02 mol / L HCl solution was added, and after mixing, the mixture was centrifuged at 9000g for 30 minutes at 4°C, and the supernatant was collected. In the untreated group, physiological saline of the same volume as NaDOC and HCl was added to equalize the total volume.

[0125] 3) Sample collection and testing The processed samples were centrifuged to separate layers. The supernatant was used for subsequent analysis, while the precipitate was retained and fully reconstituted with 300 μL of 0.1 mol / L NaOH.

[0126] Sugar concentration was determined using the anthrone-sulfuric acid method. The sugar concentration measured in the untreated, uncoupled β-glucan sample was denoted as Cr, and the sugar concentration measured in the supernatant after treatment was denoted as Sr. The sugar recovery rate Rc was calculated based on these values ​​to assess whether free sugars were lost during treatment. The calculation formula is as follows: The sugar concentration measured in the untreated β-glucan-TT conjugate sample is denoted as Cb, and the sugar concentration measured in the supernatant after treatment is denoted as Sb. The percentage of free sugar in the conjugate, Fp, is calculated based on these values, reflecting the residual level of uncoupled glucans in the conjugate sample. The calculation formula is as follows: 4) Experimental Results As shown in Table 9, under the optimized treatment system, the uncoupled β-glucan maintained a good recovery rate (Rc ≥ 90%), indicating that the free sugar has high stability; the sugar concentration was significantly reduced after the conjugate treatment, and the percentage of free sugar was obtained by calculation, which showed good reproducibility.

[0127] These results demonstrate that the 0.8% NaDOC + 0.02 mol / L HCl treatment system is suitable not only for α-glucan and arabinomannan, but also for the detection of β-glucan conjugates with different spatial structures and origins. This further proves that the method has good adaptability and detection accuracy in a wide range of glycan-protein systems, and is suitable for application in the quality control analysis of various types of glycan conjugated vaccines.

[0128] Table 9. Results of determination of β-glucan-TT conjugate samples from three batches

[0129] 5) Summary of the applicability verification of the NaDOC-HCl method in various polysaccharide systems To verify the applicability and stability of the NaDOC-HCl treatment system described in this invention in different types of glycan-protein conjugates, this invention selected three types of glycans with significant structural differences—α-glucan, arabinomannan, and β-glucan—and systematically conducted experiments to determine the sugar recovery rate and free sugar content after treatment in their uncoupled form and in the form of conjugates coupled with tetanus toxoid, respectively.

[0130] α-Glucan is a typical high-molecular-weight, unbranched glycan with a molecular weight of approximately 120 kDa, representing a well-structured macromolecular glycan system. Arabinomannan has a lower molecular weight (approximately 25 kDa) but is richly branched, exhibiting a highly branched and spatially twisted structure, representing a glycan system with many branched chains. β-Glucan, on the other hand, has a characteristic structure linked by β-1,3 / 1,6 bonds, representing a natural glycan with a unique configuration and wide range of sources (high structural specificity). These three glycans exhibit significant differences in physicochemical properties, spatial configuration, and protein binding mechanisms, effectively representing different types of glycan systems.

[0131] Under the treatment conditions of 0.8% NaDOC + 0.02 mol / L HCl established in this invention, the uncoupled samples of the three types of glycans all showed good recovery rates (≥90%), with no obvious precipitation or signal loss, indicating that the treatment system has good stability for free sugars. At the same time, the corresponding glycan-TT conjugates achieved effective differentiation between bound and unbound glycans after treatment, the calculated percentage of free sugar content was accurate and repeatable, the protein components were completely precipitated, and the system was clearly stratified, verifying the wide applicability of this method in various glycan-protein coupled structures.

[0132] In summary, the NaDOC-HCl precipitation system established in this invention combines the selective precipitation capability of proteins with the high recovery rate of free glycans. It is applicable to various glycan types with complex spatial structures, large molecular weight differences, and varying degrees of branching, demonstrating good versatility and practicality. This method provides stable and reliable technical support for the quality analysis of structurally complex glycan-conjugated vaccines and sugar-modified biomaterials, and has significant application and promotion value.

[0133] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for processing polysaccharide samples to obtain free polysaccharides, characterized in that, The combination comprises 0.4-1% NaDOC and 0.01-0.05 mol / L HCl by mass concentration. Preferably, the combination comprises 0.6-0.9% NaDOC and 0.02-0.04 mol / L HCl by mass concentration.

2. The combination according to claim 1, characterized in that, The polysaccharide sample includes a solution of polysaccharide or a solution of polysaccharide-protein conjugate.

3. The combination according to claim 2, characterized in that, The polysaccharide includes at least one of the following: pneumococcal capsular polysaccharide, Neisseria meningitidis capsular polysaccharide, α-glucan, arabinomannan, and β-glucan; Preferably, the protein in the polysaccharide-protein conjugate is a carrier protein; further, it is a vaccine carrier protein; and even further, it includes at least one of the following: detoxified Pseudomonas aeruginosa exotoxin A, Escherichia coli flagellin, CRM197, maltose-binding protein, diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, agglutination factor A, agglutination factor B, Escherichia coli heat-labile enterotoxin, detoxified Escherichia coli heat-labile enterotoxin variant, cholera toxin B subunit, cholera toxin, detoxified cholera toxin variant, Escherichia coli Sat protein, the guest-carrying domain of Escherichia coli Sat protein, Streptococcus pneumoniae hemolysin, keyhole hemocyanin, Pseudomonas aeruginosa PcrV, Neisseria meningitidis outer membrane protein, and untyped Haemophilus influenzae protein D; Preferably, the polysaccharide-protein conjugate is selected from α-glucan-tetanus toxoid conjugate, arabinomannan-tetanus toxoid conjugate, or β-glucan-tetanus toxoid conjugate.

4. A free polysaccharide detection kit, characterized in that, The kit contains the combination as described in any one of claims 1-3.

5. The use of the combination of any one of claims 1-3 or the kit of claim 4 in vaccine preparation.

6. A method for extracting free polysaccharides from polysaccharide-protein conjugates, comprising the following steps: (1) Prepare polysaccharide-protein conjugate samples, (2) Extracting free polysaccharides using any combination as described in claims 1-3, Step (2) specifically includes adding NaDOC to the solution of the polysaccharide-protein conjugate, reacting, then adding HCl, separating the solid and liquid, and obtaining the supernatant.

7. A method for determining the free polysaccharide content in a polysaccharide-protein conjugate, comprising the following steps: S1: Prepare solutions of polysaccharide-protein conjugates and polysaccharides as samples; S2: The solution of the polysaccharide-protein conjugate and the solution of the polysaccharide are respectively subjected to sodium deoxycholate precipitation treatment according to any combination as described in claims 1-3, and are respectively referred to as the conjugate treatment group and the polysaccharide treatment group; The solutions of the polysaccharide-protein conjugate and the polysaccharide were respectively subjected to control treatments and were designated as the untreated conjugate group and the untreated polysaccharide group. in, The method for sodium deoxycholate precipitation is as follows: NaDOC is added to the solution of the polysaccharide-protein conjugate and the solution of the polysaccharide, respectively, and the reaction is carried out. Then HCl is added, and solid-liquid separation is performed to obtain the supernatant. The control treatment method is as follows: a control solution is added to the polysaccharide-protein conjugate solution and the polysaccharide solution respectively to obtain a mixture. The control solution includes buffer and / or physiological saline, and the volume of the control solution is the sum of the volumes of NaDOC and HCl. S3: The polysaccharide content in the supernatant of the conjugate treatment group and the polysaccharide treatment group were detected respectively and denoted as Sb and Sr; The polysaccharide content in the mixture of the untreated group and the untreated group was determined and denoted as Cb and Cr, respectively. and S4: Calculate the free polysaccharide content Fp of the polysaccharide-protein conjugate: Where Rc is the recovery coefficient; 。 8. The method according to claim 6 or the determination method according to claim 7, characterized in that, The polysaccharides include pneumococcal capsular polysaccharide, Neisseria meningitidis capsular polysaccharide, α-glucan, arabinomannan, or β-glucan; Preferably, the protein in the polysaccharide-protein conjugate is a carrier protein; further, it is a vaccine carrier protein; and even further, it includes at least one of the following: detoxified Pseudomonas aeruginosa exotoxin A, Escherichia coli flagellin, CRM197, maltose-binding protein, diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, agglutination factor A, agglutination factor B, Escherichia coli heat-labile enterotoxin, detoxified Escherichia coli heat-labile enterotoxin variant, cholera toxin B subunit, cholera toxin, detoxified cholera toxin variant, Escherichia coli Sat protein, the guest-carrying domain of Escherichia coli Sat protein, Streptococcus pneumoniae hemolysin, keyhole hemocyanin, Pseudomonas aeruginosa PcrV, Neisseria meningitidis outer membrane protein, and untyped Haemophilus influenzae protein D; Preferably, the polysaccharide-protein conjugate is selected from α-glucan-tetanus toxoid conjugate, arabinomannan-tetanus toxoid conjugate, or β-glucan-tetanus toxoid conjugate.

9. The method according to claim 6 or the determination method according to claim 7, characterized in that, The reaction conditions are: standing for 20-40 minutes; further, standing for 25-35 minutes. Preferably, the solid-liquid separation is centrifugation; Preferably, the centrifugation conditions are centrifugation at 8000~12000g for 5~40min; further, centrifugation at 8000~10000g for 25~35min.

10. A method comprising the steps of the determination method according to any one of claims 7-9, said method being used in any one of a1)-a4): a1) Quality control in the production of the polysaccharide-protein conjugate; a2) Process optimization in the production of the polysaccharide-protein conjugate; a3) Batch consistency evaluation in the production of the polysaccharide-protein conjugate; a4) Evaluation of the binding efficiency of the polysaccharide-protein conjugate.

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