Adjuvants are non-specific immune enhancers that are indispensable components of a wide range of vaccines, including inactivated vaccines, subunit vaccines and recombinant protein vaccines. Adjuvants enhance the adaptive immune response by activating and stimulating the innate immune cells, thereby enhancing the immunogenicity of vaccines. Based on the action mechanism, adjuvants can be mainly divided into immunologic stimulants and delivery systems. This article reviews the action mechanisms and research progress of some currently approved human vaccine adjuvants and novel vaccine adjuvants, in order to guiding the rational use of existing adjuvants and the development of novel adjuvants.

Chikungunya virus infection can cause chikungunya hemorrhagic fever, which is prevalent in a wide range of tropical and temperate regions and has become a global public health problem. Chikungunya hemorrhagic fever is mainly treated by symptomatic treatment, and there is no specific antiviral drug. Vaccination is an economical and effective means to prevent chikungunya virus infection. Although two vaccines have been approved for use in local areas, they are far from meeting the demand for vaccines in high-risk areas. This article reviews the research and development status of chikungunya virus vaccine, focusing on vaccines on the market, candidate vaccines entering clinical trials and vaccines that have potential to enter the clinical trial.

Objective To explore the feasibility of using DEAE Sephadex A-50 gel adsorption to separate and purify human prothrombin complex concentrate (PCC) from cryoprecipitate reduced plasma supernatant (CRPS). Methods The gel adsorption method was used to continuously prepare 3 batches of PCC products from CRPS. The recovery rate of human coagulation factor Ⅸ (FⅨ) titer, the content of miscellaneous proteins, and the effect of dry heat virus inactivation were detected and analyzed in PCC bulks, final bulks, and final products of the gel adsorption process. The PCC final products were subjected to verification analysis and stability tests. Results With FⅨ titer in CRPS as 100.0%, the recovery rates of FⅨ titer of PCC bulks,final bulks, and final products in gel adsorption process were （65.0±2.1）%，（56.3±4.2）%，and（40.3±1.8）%, respectively. The content of miscellaneous proteins in PCC bulks was low, and all quality indicators before and after dry heat treatment were qualified. The verification of PCC final products showed that quality indicators such as physical and chemical properties, titer, FⅨspecific activity, and activated coagulation factor activity met the requirements of the Chinese pharmacopoeia 2020 edition volume Ⅲ. Although the detection results of various quality indicators in the accelerated and long-term stability tests fluctuated compared with those at 0 month, all were qualified. Conclusion The DEAE Sephadex A-50 gel adsorption method can successfully separate and purify PCC from CRPS.

National Medical Products Administration started the pre-accession application for the Pharmaceutical Inspection Co-operation Scheme (PIC/S) in September 2021, and became a formal applicant on November 8, 2023. PIC/S is an international organization for developing and promoting GMP standards, and its GMP guidelines are widely regarded as authoritative standards for quality management in drug production. This paper compares and studies the similarities and differences between PIC/S and Chinese GMP in main document structure and content, aseptic appendix and biological product appendix, aiming at finding out the gap, providing improvement measures for drug regulatory agencies and drug manufacturers in China, continuously improving the quality and safety of drugs in China and increasing international competitiveness.

Objective To establish and validate the detection method of coxsackievirus A6 (CV-A6) neutralizing antibody by micro-cytopathic effect (CPE) method.Methods A neutralizing antibody detection method based on CPE was established using human rhabdomyosarcoma (RD) cells and CV-A6 standard detection strains. It was verified for relative accuracy, precision, linearity, specificity, and durability.Results A determination method of neutralizing antibody titer for CV-A6 immune serum was established. Three serum reference samples of different dilutions (1×, 16×, and 256×) were measured 3 times with relative biases of ﹣2%, ﹣4%, and ﹣16%, respectively. The slope of the fitted linear regression was 1.065, indicating good relative accuracy. The geometric coefficients of variation (GCVs) were 19%-50%, and the repeatability was good. The GCVs of 10 mouse immune sera neutralizing antibody titers measured for 3 times on different days were 0%-75%, indicating good intermediate precision. The regression coefficient of best fit line for the neutralizing antibody titers of CV-A6 neutralizing antibody serum reference at 9 dilution levels measured 3 times was 0.93, and the linear regression was statistically significant (F=340.99, P<0.000 1). The neutralizing titers of negative sera were all less than 8, showing good specificity. The GCVs of different RD cell passages, different RD cell densities, different viral loads, different culture days, different number of repeated freeze-thaw times and short-term storage time range of serum to be tested were 23%-101%, indicating good durability.Conclusions The CV-A6 neutralizing antibody detection method has been successfully established with good relative accuracy, precision, linearity, specificity, and durability. It can be used to evaluate the neutralizing antibody levels of immune sera against CV-A6.

Chikungunya virus (CHIKV) is a type of alphavirus transmitted by Aedes mosquitoes. CHIKV infection can result in chikungunya fever, which presents with symptoms such as high fever, joint pain, and chronic arthritis, among other clinical manifestations.In recent years, it has caused multiple outbreaks globally, leading to serious public health issues.However, there are currently no specific antiviral drugs approved for marketing, and clinical treatments mainly focus on symptomatic and supportive treatments. With the expanding geographic range of CHIKV transmission and increasing disease burden, there is an urgent need to develop effective antiviral drugs. This article reviews the epidemiology, genomic structure, and function of CHIKV, as well as recent research progress on antiviral drugs against CHIKV, including direct-acting antiviral agents and host-targeted drugs. Additionally, this article discusses the challenges faced during the drug development process, such as the occurrence of drug-resistant mutations, drug safety, and clinical translation issues. It also looks forward to future research directions, including multi-target drug design, combination therapy strategies, and computer-aided drug design with artificial intelligence. By systematically summarizing existing research findings, this article aims to provide guidance and insights for the further development of antiviral drugs against CHIKV.

Objective To perform bioinformatic analysis of the viral protein 1 (VP1) of coxsackievirus B5 (CV-B5), in order to provide scientific basis for immune surveillance, epitope vaccine design and disease research.Methods Online websites including ProtParam, SignalP-6.0, DeepTMHMM-1.0, NetPhos-3.1, NetNGlyc1.0, SOPMA, SWISS-MODEL, ABCpred, BCPred, and IEDB were used to predict and analyze the physicochemical properties, structural characteristics and linear B-cell epitope of CV-B5 VP1.Results The CV-B5 VP1 appeared to be an unstable and alkaline hydrophilic protein with no signal peptide and one transmembrane domain. Thirty-three phosphorylation sites and 15 glycosylation sites were predicted in the protein. Random curling was the main secondary structure of VP1, and multiple potential dominant B-cell epitopes were identified.Conclusion Using bioinformatics tools to predict the CV-B5 VP1, the biological function and linear epitopes of VP1 are primarily identified.

Objective To evaluate the optimal mouse immunogenicity model of 13-valent pneumococcal polysaccharide conjugate vaccine and the optimal mouse gender and immune dose by comparing the immune response in BALB/c, KM, and CD1 mice.Methods The 13-valent pneumococcal polysaccharide conjugate vaccine was used to immunize 3 strains of mice with doses of 0.05, 0.10, 0.20 and 0.40 μg, respectively. ELISA was used to analyze the immune responses to evaluate the optimal mouse model. Response of male and female mice of the optimal model immunized with 4 different doses was compared to evaluate the optimal mouse gender. Response of optimal mouse model of optimal gender immunized with 4 different doses was compared to evaluate the optimal immune dose.Results The immune response values of most serotypes were the highest in CD1 mice, and the weakest in KM mice. There was a significant dose-response relationship in CD1 mice. Immune responses of both male and female mice were low at 0.05 μg dose. At doses of 0.10, 0.20, and 0.40 μg, almost all serotypes showed higher immune responses in female mice than in male mice. The immune response of CD1 female mice immunized with 0.20 μg was the highset in most serotypes except 3, 9V, 19F, and 23F.Conclusion CD1 female mouse is the optimal immunogenicity evaluation model for 13-valent pneumococcal polysaccharide conjugate vaccine, and 0.20 μg is the optimal immunization dose.

Objective To provide basis for the quality control of impurities in oral hexavalent reassortant live attenuated rotavirus vaccine (Vero cell) by analyzing the residual levels of porcine trypsin, bovine serum albumin（BSA）, Vero cell DNA，and the DNA fragment size in monovalent virus bulk.Methods The residual levels of porcine trypsin and BSA in 30 batches of monovalent virus bulk were detected by ELISA. The residual Vero cell DNA in 30 batches of monovalent virus bulk was purified by magnetic bead extraction method and then quantified by fluorescent staining method, and the size and distribution of Vero cell DNA fragment were detected by capillary gel electrophoresis.Results In 30 batches of monovalent virus bulk tested, the residual levels of porcine trypsin was 3.8-7.2 μg/mL, BSA was 85-268 ng/mL, and Vero cell DNA was 48-115 μg/mL. The DNA fragments of Vero cells in the range of 201- 2 000 bp counted for 73.4%-91.9% of fragments.Conclusion The residual impurities in the monovalent virus bulk of oral hexavalent reassortant live attenuated rotavirus vaccine (Vero cell) are controllable.

Objective To establish and validate a method for the simultaneous determination of residual Triton X-100 and polysorbate 80 (PS80) in influenza virus split vaccine (MDCK cell).Methods Triton X-100 and PS80 residues in influenza virus split vaccine were detected by high performance liquid chromatography (HPLC)-evaporative light-scattering detector (ELSD) method. The specificity, accuracy, precision, linearity, limit of quantitation and robustness of this method were verified.Results In the HPLC-ELSD analysis, no chromatographic peaks were observed in blank solvent. Two target chromatographic peaks were visible in both mixed standard solution and test sample, with the resolution from adjacent peaks greater than 1.5. The spiked recovery rates of Triton X-100 and PS80 at various concentrations were 90.0%-105.0% and 80.0%-105.0%, respectively. When the same experimenter performed repeated injections six times, the relative standard deviations (RSDs) of Triton X-100 and PS80 contents were 0.7% and 1.4%, respectively. When two inspectors determined the residual amounts of Triton X-100 and PS80 on different working days, the RSDs were both ≤ 5.0%. When Triton X-100 mass fraction was in the range of 0.010%-0.100%, a good linear relationship was observed between the concentration and peak area, with limit of quantitation at 0.010%. When PS80 mass fraction was in the range of 0.006%-0.060%, a linear relationship was found between the concentration and peak area, and the limit of quantitation was 0.003%.Conclusions HPLC-ELSD detection method for Triton X-100 and PS80 residues in influenza virus split vaccine is successfully established. The specificity, accuracy, precision, linearity and robustness are good.

Objective To develop a purification process for anti-receptor activator of NF-κB ligand (RANKL) monoclonal antibodies.Methods The upstream cell culture supernatant was clarified by depth filtration to remove cells. The clarified harvest was then subjected to affinity chromatography (AC) capture, low-pH viral inactivation, anion-exchange chromatography (AEX), cation-exchange chromatography (CEX), viral filtration (VF), ultrafiltration/diafiltration (UF/DF), and sterile filtration to obtain the antibody bulk. AC purification efficiency was evaluated based on the yield and removal of product-related impurities. The dynamic binding capacity (DBC) of resin was calculated by monitoring the breakthrough point. Low-pH viral inactivation conditions were determined by evaluating the impact of pH and temperature conditions on sample purity and activity.The optimal AEX buffer system was determined by assessing the stability of target protein in different buffer conditions and the removal of process-related impurities. Purification efficiency and DBC were also evaluated. CEX process parameters were optimized using linear gradient elution and fraction collection to analyze impurity clearance. The VF filter type was selected based on throughput, flux, and flow decay.Membrane pore size, flow rate, and transmembrane pressure were determined based on UF/DF and sterile filtration manufacturer specifications and platform technology. Concentration and dialysis buffer exchange were performed accordingly. Finally, the bulk was obtained through a 0.2 μm membrane sterile filtration, and the purity and impurity were accessed.Results AC used MabSelect SuRe resin (DBC: 45.3 mg/mL resin) with 25 mmol/L Tris-HCl (pH7.5) for equilibration and 150 mmol/L HAc (pH2.8) for elution. Viral inactivation was accomplished with pH3.5 for 1 h. AEX used Sepharose Q FF resin (max DBC: 60 mg/mL resin) with 50 mmol/L Tris-HAc (pH7.5) for equilibration. CEX used SP HP resin of 21.9 mg/mL loading capacity with linear elution from 100% buffer A (10 mmol/L citrate， pH5.5) to 100% buffer B (10 mmol/L citrate + 1 mol/L NaCl， pH5.5) over 20 column volumes. Viresolve Pro was used for VF and PLCTK 30 kDa ultrafiltration cassette (C-channel) was used in UF/DF to concentrate to (40.00 ± 5.00) mg/mL, followed by 10× diafiltration and sterile filtration to yield bulk. Both the purity and impurity levels met predefined standards.Conclusion A robust process for anti-RANKL monoclonal antibody purification is successfully developed, yielding drug substance with acceptable qualities.

Objective To establish a capillary zone electrophoresis (CZE) method to detect the polysaccharide content and molecular size distribution of group A, C, Y, and W135 meningococcal polysaccharide vaccine.Methods CZE method was developed to detect all 4 polysaccharides, and the optimal separation voltage and temperature were investigated. Four polysaccharides were characterized in a single experiment, and their contents and molecular size distributions were determined. The method’s linearity, repeatability, accuracy, and specificity were validated.Results When contents of all 4 polysaccharides ranged from 0.081 3 to 0.487 5 μg/μL, the CZE method demonstrated good linearity with coefficient of determination > 0.98. For low, medium, and high concentration samples, the recovery rates were between 95% and 110%, and the relative standard deviation for 6 experiments was < 2.0%. No impurity peaks were observed at the target peak positions.Conclusion The CZE method shows good linearity, accuracy, repeatability, and specificity for detecting the four meningococcal polysaccharide, and is suitable for determining the polysaccharide content and molecular size distribution of group A, C, Y, and W135 meningococcal polysaccharide vaccine.

Genital herpes, a widespread sexually transmitted disease, is caused by herpes simplex virus (HSV) infection.HSV can establish lifelong latent infection in the host and cause severe and recurrent diseases. In addition, HSV infection significantly increases the risk of HIV infection. Although anti-HSV drugs have some effects, they are not capable of preventing reactivation of latent HSV. Therefore, it is urgent to develop effective vaccines to control HSV infection, and limit spread and relapse of the disease. In this paper, the etiology and epidemiology of genital herpes and development progress of genital herpes vaccines are reviewed.

WHO Technical Report Series (TRS) provides important guidances for the drug industry, covering technical specifications and standards for the entire life cycle ranging from product research and development, clinical trials to commercial production, distribution and transportation, and discontinuation. It guides enterprises to establish a comprehensive knowledge system to ensure their products meet international standards. This paper introduces the source and numbering principle of WHO TRS, its guiding significance for the medical product industry, and the challenges faced by TRS. The WHO TRS is listed and introduced, which is divided into quality management, premises and facilities, quality control, production management, storage and distribution, and inspection. Based on years of practical experience and insights in quality management and WHO prequalification by the authors, this article shows how to effectively use WHO TRS to guide enterprises through comprehensive quality management work and acceleration of WHO prequalification projects.

Objective To efficiently express the self-constructed recombinant human IFNα2b (rhIFNα2b)-TNFα stimulated gene 6 (TSG6) fusion protein in Escherichia coli BL21 using principles and methods of synthetic biology, and to assess its anti-inflammatory and anti-viral activities.Methods The tertiary structure of the fusion protein was predicted using Swiss-model software online. The codon-optimized human IFNα2b gene was linked to a TSG6 gene fragment via a linker peptide. The pET-32a recombinant plasmid containing rhIFNα2b-TSG6 fusion gene was constructed and transformed into E. coli BL21(DE3) strain. Fermentation, inclusion body denaturation-renaturation, and purification processes were undertaken to obtain the fusion protein. Purity assessment along with protein content analysis were conducted through SDS-PAGE while Western blotting was applied to confirm specificity. The antiviral activity of rhIFNα2b-TSG6 was determined by WISH/vesicular stomatitis virus activity detection system with micro-cytopathic effect inhibition assay. Protective effects on hepatocytes as well as IL-β mRNA expression suppression were observed in mouse hepatitis models infected with murine cytomegalovirus (MCMV). The rhIFNα2b-TSG6 in vivo pharmacokinetic study was performed in rabbits.Results The results of bioinformatics analysis showed that rhIFNα2b-TSG6 met the design expectation. The target protein was successfully expressed in the form of inclusion bodies. The apparent relative molecular weight of rhIFNα2b-TSG6 was ~68 000, and the expression rate was 30%-40%. The purity of purified rhIFNα2b-TSG6 was up to 90%. The fusion protein could specifically bind to anti-IFNα and anti-TSG6 monoclonal antibodies. The specific antiviral activity of rhIFNα2b-TSG6 was (1.80±0.16)×10⁶ international unit/mg, 3.5 times of rhIFNα2b alone. It also protected the liver and inhibited inflammation in the MCMV mouse infected hepatitis model.The blood drug concentration reached peak at 24 h in rabbits.Conclusion Efficient prokaryotic expression of rhIFNα2b-TSG6 is achieved, accompanied by pronounced antiviral activities, laying foundational ground work for large-scale production aimed at clinical applications addressing acute chronic viral diseases.

Objective To analyze the blocking effect of measures for preventing maternal-infant transmission of hepatitis B virus (HBV) in Qingdao and to explore related influencing factors.Methods A total of 869 HBV infected parturients giving birth in Qingdao in 2021 and their children who completed hepatitis B serological marker detection (HBV-exposed children) were selected as research objects. Through follow-up results and questionnaire surveys, the maternal-infant transmission rate of HBV and parents’ awareness of knowledge related to HBV maternal-infant transmission were obtained. Univariate and multivariate logistic regression methods were used to analyze the influencing factors of blocking failure.Results Of 869 HBV-exposed children surveyed, the maternal-infant transmission rate of HBV was 0.69%. Uncompleted full-course hepatitis B vaccination in HBV-exposed children [odds ratio (OR) = 0.030, 95% confidence interval (CI) = 0.004-0.232] and maternal premature rupture of fetal membrane(OR = 9.570, 95% CI: 1.343-68.201) might be independent risk factors for maternal-infant transmission of HBV. There were differences between parents of children in HBV surface antigen study group and control group in the awareness regarding necessary medication during pregnancy reducing the risk of maternal-infant transmission, the need for testing after HBV-exposed children receiving full-course hepatitis B vaccination, hepatitis B vaccination procedure, the number of vaccinations, and the intervention and blocking measures for HBV-exposed children after birth.Conclusions Enhancing prenatal care, reducing the occurrence of premature rupture of fetal membrane, and timely implementation of combined immunization strategies and full-course hepatitis B vaccination procedures for HBV-exposed children after birth can significantly improve the efficacy of maternal-infant blocking. Strengthening health education for key populations and improving the awareness rate of knowledge related to hepatitis B prevention are effective ways to reduce maternal-infant transmission of HBV.

Objective To evaluate the pharmacokinetic parameters of human coagulation factor Ⅸ (FⅨ) after single intravenous injection in patients with hemophilia B, and to preliminary analyze the clinical implications. Methods In a single-arm, open-label, multicenter clinical trial， 13 male patients aged 25-64 years with hemophilia B received a single intravenous injection of FⅨ at 50 international unit (IU)/kg under non hemorrhagic conditions. Blood samples were collected at 2 h before injection, 15 min, 30 min, and 1, 3, 9, 24, 48, 72, 96 h post-injection to measure FⅨ activity. The FⅨ pharmacokinetic parameters were calculated by non-compartment model. In addition, FⅨ inhibitors in patients were measured 30,90 d after injection.Results The plasma FⅨ concentrations peaked at 51.5-69.3 IU/dL in all patients after single intravenous injection of FⅨ for 15 min-1 h. The baseline-corrected pharmacokinetic parameters （±s） of FⅨ were below： peak time (0.37±0.22) h, elimination half-life (29.732±4.576) h, peak concentration (58.3±5.3) IU/dL, clearance (0.028±0.006) dL/kgh, area under the plasma drug concentration time curve (zero time to last time point) (1 626.189±285.365) %h, and incremental recovery rate (1.166±0.106) IUdL^-1/IUkg^-1. At 1 h post-injection, FⅨ activities were 40%-60% in all 13 patients, which maintained 40%-60% in 12 patients 3 h post-injection, and were still 40%-60% in 8 patients 9 h post-injection. No FⅨ inhibitors were detected in all 13 patients. Conclusion After single intravenous injection of FⅨ at 50 IU/kg in patients with hemophilia B, the major pharmacokinetic parameters, such as elimination half-life and clearance, indicate effective maintainence of blood concentrations in vivo, which can be used for further clinical studies on the efficacy of replacement therapy.

Objective To evaluate the non-clinical safety of live attenuated Japanese encephalitis vaccine with changed stabilizer by long term toxicity test in SD rats.Methods A total of 160 SD rats were subcutaneously injected with Japanese encephalitis vaccine with changed or unchanged stabilizer or NaCl(negative control) for 3 times. Animals were clinically observed, monitored for body weight, food intake, blood cell count, blood biochemistry, urinalysis, body temperature, clinical pathology, T lymphocyte subsets, and specific IgG antibody as well as performed urinalysis and ophthalmic examination.Results During the test, no significant abnormal reactions related to administration was observed. The body weight gain, food intake, blood cell count, and blood biochemistry showed statistically significant differences between certain vaccine groups and negative control （t=2.03-4.26，P=0.011-0.042），but no abnormal changes related to the stabilizer change were seen. There were no statistically significant changes in body temperature, coagulation function,and T lymphocyte subsets (t=1.35-1.98，P =0.052-0.186). By the end of recovery period, antibodies were detected in all animals in all vaccination groups, and there was no significant decrease in antibody titers. At 3 days after the last dose of administration and at the end of recovery period, no significant abnormal change was observed in the gross anatomy and histopathological examination of euthanized animals in all vaccination groups and negative control groups. There was no significant difference in toxicity, local irritation, and immunogenicity between vaccine groups with changed or unchanged stabilizer.Conclusion The long-term toxicity test results of live attenuated Japanese encephalitis vaccine with changed stabilizer in rats meet the requirements of animal safety evaluation.

Bone injury is a common clinical disease, and existing clinical treatments are ineffective in treating some difficult conditions. The research results of bone tissue engineering provide a new solution to this problem. One mature bone tissue engineering injury repair method currently in use is bone marrow mesenchymal stem cell transplantation. However, there are several problems in its clinical application, such as the difficulty of seed cell sources, the lack of allogeneic stem cell seed cells or universal seed cells, etc. Recently, peripheral blood monocytes are found to have not only the potential for multidirectional differentiation, but also a strong proliferative capacity and availablility, thus can be used as an alternative material for bone injury repair. This paper reviews monocytes and their roles in bone injury repair and their mechanism, aiming at improving the life quality of patients with bone injuries and providing more effective treatment without side effects.

Objective  To evaluate hemolysis and vascular irritation of human coagulation factor Ⅷ (hFⅧ).  Methods  Using rabbits as experimental animals, hemolysis and vascular irritation of hFⅧ were studied by in vitro and in vivo stets.  Results   Hemolysis and agglutination of rabbit erythrocytes were not observed during the in vitro test within 3 h. The vascular irritation reaction  of ear vein was not observed with intravenous administration of hFⅧat 40 IU/kg.  Conclusion  There is no hemolysis and vascular irritation when intravenous administration of hFⅧ at recommended dosage.

Objective To establish and validate a method for determination of total protein content after desorption in 13-valent pneumococcal conjugate vaccine.Methods The total protein content of the 13-valent pneumococcal conjugate vaccine was determined by the Lowry method after desorption. The linearity, accuracy, repeatability and durability of the established method were verified.Results The optimum desorption condition was to add 10% volume of 0.3 mol/L NaOH. Within the range of 40-200 μg/mL of protein standard, the linearity of the standard curve was good, coefficient of determination＞0.995. In the accuracy test, the recovery rates of protein standards at concentrations of 20,40,60,80 and 120 μg/mL ranged from 95.25% to 104.77%,demonstrating good accuracy. In the repeatability test,the relative standard deviation (RSD) of 6 test results of the sample at different time points was 3.82%, indicating good repeatability.In the durability test, no statistically significant difference (t = 0.059 and 0.238, P > 0.05) was observed in test results after samples were stored at room temperature for 15 or 30 min post-desorption, with RSD values of 4.82% and 5.14%，respectively, indicating good durability.Conclusion The established method can effectively, accurately and stably detect the total protein content of 13-valent pneumococcal conjugate vaccine.

Objective To explore the application of parallelism test in the data analysis of four-parameter model and parallel line model ELISAs for non-physiologically active organisms.Methods In 2 ELISA methods without parallelism test, the four-parameter model of enterovirus 71 (EV71) in vitro relative efficacy measured by interpolation method, and the parallel line model of EV71 antigen content detection, test data of different levels were selected. The results obtained without parallelism test, and results with non-significant or significant deviation from parallel after parallelism test, were compared.Results Different data analysis modes did not cause result fluctuation, with coefficients of variation at 2.9% to 17.9%. The paired t-test analysis was carried out between the interpolation results of four-parameter model and results with non-significant or significant deviation from parallel, and showed no statistically significant difference except the comparison with significant deviation parallel results at 2.0 level (t=0.11， P<0.001). There was no statistically significant difference between the original calculation results of parallel line model and results with non-significant or significant deviation from parallel.Conclusion  Since the calculation results are consistent with original calculation results whether or not deviation from parallel in parallelism test is significant, ELISA can selectively use parallelism test after data analysis model screening and complete methodology validation.

Diabetes mellitus type 2 is a systemic endocrine metabolic disease characterized by insulin resistance and impaired function of pancreatic β cells. At present, most patients with diabetes mellitus type 2 are obese patients with metabolic disorders. Semaglutide is a glucagon-like peptide-1 receptor agonist， which can achieve blood glucose control and weight loss through multiple mechanisms, such as suppressing appetite, delaying gastric emptying, and improving pancreatic function. Semaglutide is widely used in the treatment of diabetes mellitus type 2. This article reviews the mechanism of action, research progress, clinical application issues and related suggestions of semaglutide in the treatment of diabetes mellitus type 2, aiming to provide a reference for the clinical application of semaglutide in the treatment of diabetes mellitus type 2 patients.

Objective To analyze pathogens, severe cases and population distribution characteristics of hand foot mouth disease (HFMD) after the launch of inactivated enterovirus A71 (EV-A71) vaccine in China, and to provide references for the prevention and control of HFMD in China.Methods The literature published openly from 2016 to 2025, explicitly involving the application effect evaluation of EV-A71 vaccine and containing basic data such as the changes of pathogens before and after the application of vaccine, severe cases or incidence rates, and population distribution characteristics, was collected from China National Knowledge Infrastructure and comprehensively analyzed by chi-square test.Results The population distribution of HFMD cases before and after the use of EV-A71 inactivated vaccine showed that scattered children accounted for the highest proportion, followed by children in kindergartens and nurseries, and students and other groups accounted for the lowest proportion. However, after the application of EV-A71 vaccine, the composition of scattered children in HFMD cases decreased by 11.35% compared with that before the vaccine application, the proportion of children in kindergartens and nurseries increased by 25.62%, and the proportion of students and others increased by 61.38%. The differences in population distribution before and after the vaccine application were all statistically significant (χ²=15 659.45, 11 028.25, 3 587.36; P<0.001). After the application of EV-A71 vaccine, the average number of HFMD cases caused by EV-A71 decreased by 56.17%, while the average number of cases caused by coxsackie virus A16 (CVA16) and other enteroviruses increased by 8.08% and 62.37%, respectively, and the differences were all statistically significant (χ²=41.94, 310.80, 39.37; P<0.001). Non-EV-A71 enteroviruses (such as CVA16, CVA6 and CVA10, etc.) became dominant pathogens. After the application of vaccine, the average number of severe cases decreased by 42.70%, and the proportion of those caused by EV-A71 in severe cases dropped by 17.89%. The differences were statistically significant (χ²=4 087.32, 4 215.92；P< 0.001). However, approximately 59% of severe cases were still caused by EV-A71 infection after the use of the vaccine, suggesting that EV-A71 remained the main pathogen leading to critical HFMD.Conclusion EV-A71 vaccine has significantly reduced the incidence of severe HFMD cases, but EV-A71 remains the primary pathogen causing severe cases, while non-EV-A71 enteroviruses have become dominant pathogens.

Objective To establish an ELISA method for the quantitative detection of coxsackievirus A16 (CV-A16) antigen in CV-A16 vaccine.Methods A double antibody sandwich ELISA method for quantitative detection of CV-A16 antigen was established using rabbit polyclonal antibody against CV-A16 as coating antibody and mouse monoclonal antibody against CV-A16 labeled with horseradish peroxidase as detection antibody.The linear range of the method was determined, and the accuracy, precision, specificity and durability were verified. The content of CV-A16 antigen in the stock solution of CV-A16 vaccine and intermediate samples in the production process were detected to examine the applicability of this method.Results The linear range of established ELISA was 3.125-400.000 U/mL, and coefficient of determination was 0.983 9-0.991 3, indicating good linearity. The recovery rates of CV-A16 national antigen standards with different concentrations were 80%-120%, and the relative standard deviation（RSD）was ≤11%, indicating good accuracy. The RSDs for reproducibility and intermediate precision with different antigen concentrations were all ≤ 12%, indicating good precision. There was no cross reaction with enterovirus A71, CV-A10, CV-A6 antigen and other components during CV-A16 vaccine process, indicating good specificity. The durability test for different influencing factors results showed that the recovery rates were 80%-120% and the RSD was ≤7%, indicating good durability. The antigen content of CV-A16 bulk and intermediate products in the preparation process had good parallelism and linearity, indicating good applicability.Conclusion A double-antibody sandwich ELISA method for quantitative detection of CV-A16 inactivated vaccine (Vero cells) antigen is established, and this method can be used for quantitative detection of CV-A16 antigen.

Five years after the implementation of vaccine resident inspection system, it has achieved remarkable results in ensuring the bottom line of vaccine safety production and improving the GMP compliance level of enterprises.With China’s deep participation in the international drug regulatory system, such as International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use and Pharmaceutical Inspection Co-operation Scheme, the current inspection mechanism needs further consideration and improvement in terms of international integration and professional capacity building. Based on the practice and development of the “four-in-one” collaborative supervision mode of vaccine resident inspection in Shanghai, this paper discusses the internationalization of benchmarking, careful organization and implementation of resident inspection, highlighting the key points of resident inspection and optimizing resident inspection strategies, aiming at building a more scientific and efficient vaccine quality supervision system and continuously improving the supervision efficiency.

 Folate receptor α (FRα) is a single-chain glycophosphatidylinositol- anchored protein on the cell surface, which binds folate and transports it into cells. Folate is a basic component of cell metabolism as well as DNA synthesis and repair. FRα is overexpressed in specific malignant tumors of epithelial origin while restrictively expressed in normal cells, and its expression level increases with tumor progression. Thus, FRα has been selected as a target and marker for cancer therapy. Various therapeutic agents linked to folate can then enter cancer cells via FRα-mediated endocytosis. Monoclonal antibodies targeting folate receptors have also shown potential in cancer diagnosis and therapy. The tissue-specific expression of FRα enables selective delivery of cytotoxic agents by monoclonal antibodies into malignant cells rather than normal tissues for killing tumor cells or preventing tumor progression.

Objective To explore the inactivation effect of 84 disinfectant on Japanese encephalitis virus (JEV) and establish a detection method.Methods According to "Technical Specification for Disinfection" issued by National Health Commission in 2002, the suspension quantitative method was adopted to carry out the interaction between disinfectant and virus on the surfaces of representative laboratory materials (glass, colored steel, polyvinyl chloride, and stainless steel). Appropriate neutralizer was determined by neutralization identification tests. Detection method was established to detect virus activity, and the detection results were evaluated.Results At room temperature, neutralizers lecithin (10 g/L) and Tween 80 (10 g/L) , along with their neutralization byproducts, resulted in the death of host cells (primary golden hamster kidney cells). However, neutralizer sodium thiosulfate (1 mg/L) and its neutralization byproducts demonstrated no toxicity to the host cells, no impact on cellular proliferation, and no effect on the virulence of JEV. So sodium thiosulfate (1 g/L) was selected as neutralizer. Following the interaction between 84 disinfectant and the virus, and neutralization with sodium thiosulfate (1 g/L), plaque assay detection of the resultant mixture failed to identify any JEV particles.Conclusion The 84 disinfectant with available chlorine content of 800 mg/L can effectively inactivate JEV after reacting for 5 min, and the inactivation effect meets the expected goal.

Objective To investigate the hemolysis and vascular irritation of human coagulation factor Ⅸ (FⅨ) products.Methods According to the provisions of the Technical Guidelines for Studies on Irritation, Sensitization, and Hemolysis of Drugs issued by the Center for Drug Evaluation of the National Medical Products Administration, for FⅨ products, the in vitro test tube method was used to evaluate the hemolytic potential of the drug, and the self-control method with left-right side of the same animal was adopted to conduct the rabbit irritation test by auricular vein injection of 1.6 mL/kg FⅨ or saline for 7 d.Results At a concentration of 50 international unit（IU）/mL, FⅨ did not induce hemolysis or erythrocyte agglutination. During the drug administration period and the recovery period after drug withdrawal in rabbits, no significant abnormalities were observed in general condition, and the vascular irritation scores were all 0. Fourteen days after drug withdrawal, the vascular irritation scores of rabbits remained 0. At 96 h after the last administration and at the end of the 14 d recovery period, the mean pathological scores of rabbits were both 0.Conclusion FⅨ at a concentration of 50 IU/mL does not exhibit hemolytic activity or vascular irritation in rabbits.

In recent years， bispecific antibodies （BsAbs） have presented unique therapeutic potential in tumor immunotherapy by improving the targeted killing capacity of immune cells and blocking multiple signaling pathways to produce synergistic effects. Over 200 BsAbs for antitumor therapy are currently in clinical development worldwide， with 15 approved for use in hematologic malignancies or solid tumors. BsAbs are effective in treating malignant hematologic tumors. However， many challenges remain in solid tumor therapy， including target selection， drug delivery， safety and drug resistance. This article summarizes the clinical applications and research progress of licensed BsAbs in cancer treatment， providing a reference for BsAbs development and clinical utilization.

Objective To establish and validate the ELISA method for the determination of porcine trypsin residues in attenuated Japanese encephalitis vaccine (JEV).Methods Double-antibody sandwich ELISA was used to establish a method for the determination of porcine trypsin residues in JEV. The accuracy, repeatability, intermediate precision, durability, linearity, specificity, and detection and quantification limits of the method were verified.Results The recovery rates of test samples ranged from 92.23% to 118.16%. Coefficients of variation (CVs) of the same batch were 6.6%-12.1%. CVs of the same batch was 8.1%-11.5%, 6.7%-8.5%, and 8.4%-9.6%, respectively, for different test personnel, different test dates, and different batch kits. Within the temperature range of （37±1） ℃, the recovery rates of samples showed no statistically significant difference (F=0.43, P=0.662). Coefficients of determination were ＞ 0.98 in the range of 3.12-200.00 ng/mL. The method showed no cross-reaction with the substrate. The limits of detection and quantitation were 1.25 ng/mL and 3.12 ng/mL, respectively.Conclusion The method has good accuracy, repeatability, intermediate precision, durability, linearity and specificity, thus is suitable for the determination of porcine trypsin residues in JEV.

Ankylosing spondylitis (AS) is a chronic autoimmune disease that primarily affects the sacroiliac joints, spine and adjacent tendons, soft tissues. The typical pathological progression of AS involves processes such as immune inflammation-driven responses, bone destruction, and new bone formation. These processes recur at same sites, ultimately leading to gradual joint stiffness and deformation. This article discusses the pathogenic mechanism， regulatory role, and current research status of IL-17 in AS, aiming to clarify the advantages of IL-17 as target in AS treatment and lay foundation for the development of IL-17-related drugs and the clinical management of AS.

Objective To establish, verify and preliminarily apply a real time fluorescent quantitative reverse transcription PCR (RT-qPCR) detection method for xenotropic murine leukemia virus (X-MuLV). Methods Specific primers were designed according to gene sequence of X-MuLV provided by American Type Culture Collection. Recombinant plasmid and RNA standard containing target sequence were constructed, quantified through digital PCR and serially diluted to establish RT-qPCR method. Sensitivity, specificity, accuracy, precision, and robustness of RT-qPCR were investigated. The method was applied to validate virus clearance procedure by nanofiltration and chromatography, and compared with cytopathic effect assay.Results The established RT-qPCR detection method for X-MuLV showed a linear range of 2.5×10¹-2.5×10⁸ copies/μL，and the amplification efficiency was 98%. The sensitivity of detection was 2.5×10¹ copies /μL. No cross-reaction with other commonly used viruses in the lab was detected. The coefficients of variation of repeatability and intermediate precision validation were all less than 1%. Protein and buffer had no matrix effect on virus detection of samples. When the method was employed to access the X-MuLV clearance of nanofiltration process, >4 lg copies reduction was shown in virus load between pre- and post-treatment, consistent with the result of cytopathic effect assay. When applied to evaluate composite cation-exchange chromatography, the RT-qPCR method detection results reflected the distribution of X-MuLV particles throughout the process.Conclusion The RT-qPCR method established for X-MuLV has good sensitivity, specificity, accuracy, precision and robustness, applicable for virus clearance processes validation of biological products.

As an important nucleic acid quantification technology in molecular biology, digital PCR has been widely used in the fields of pathogen detection, gene editing detection, and disease diagnosis in recent years due to its advantages of high sensitivity, accuracy, and specificity. This article reviews the basic concepts and principles of digital PCR, as well as the application progress of digital PCR in the field of biological detection, in order to provide a reference for researchers to choose appropriate detection methods.

Objective  To construct a plasmid vector expressing recombinant fusion protein Exendin-4-GLP-1/IgG4(Fc), and study activity of the fusion protein.  Methods   The gene encoding recombinant fusion protein Exendin-4-GLP-1/IgG4 (Fc) was inserted into expression vector pOptiVEC™-TOPO^® to construct recombination plasmid Exendin-4-GLP-1/IgG4(Fc)-pOptiVEC™-TOPO^®. The constructed recombination plasmid was transfected into CHO/DG44 cells. The expression product was prified by affinity chromatography and detected by Western blotting. The effect of the recombinant fusion protein on insulin secreting of INS-1 cells was determined by insulin releasing test. The regulation of the recombinant fusion protein on blood glucose was studied in CD1 mice.  Results  CHO/DG44 cells transfected with the constructed recombination plasmid successfully expressed Exendin-4-GLP-1/IgG4(Fc). Western blotting showed that relative molecular masses of the expression product were consistent with expectations. Relative molecular masses of the monomer and dime of the recombinant fusion protein were about 35 000 and 70 000, respectively. Insulin releasing test showed that amounts of insulin secreted by INS-1 cells increased with Exendin-4-GLP-1/IgG4(Fc) concentration under the same glucose concentration. CD1 mice study showed the recombinant fusion protein had regulating function on blood glucose in mice with streptozotocin-induced diabetes. Blood glucose in mice with streptozotocin-induced diabetes treated with Exendin-4-GLP-1/IgG4(Fc) was significantly lower than that in control mice with streptozotocin-induced diabetes.  Conclusion   Exendin-4-GLP-1/IgG4(Fc) has activity of  native GLP-1 and can be as a GLP-1 receptor agonist for treatment of type 2 diabetes.

Objective To evaluate the non-clinical safety comparability of Reassortant Rotavirus Vaccine, Live, Oral, Hexavalent (Vero Cell) produced in pilot and commercial facilities by assessments of single- and repeated-dose toxicity.Methods SD rats were administered by gavage with vaccines produced in pilot and commercial facilities, respectively, at 6 mL dosage, while the proposed clinical dosage was 2.0 mL. The long-term toxicity of the vaccine was assessed through 4 repeated intragastric administrations at doses of 2 or 6 mL per rat on day 1, 15, 29, and 43, with a 4-week recovery period. Results In the single-dose toxicity test, maximum tolerated dose was 6 mL per rat for vaccines produced in both facilities, which was 50-200 times the intended human dose converted by body weight. The body weight gain from day 0—14 and mean daily food intake of rats that received vaccines produced in pilot and commercial facilities were simliar to control groups， which were 33-44 g and 18-20 g, respectively, for female rats and 109-123 g and 25-29 g, respectively, for male rats. In the repeated-dose toxicity test, the no-observed-adverse-effect level was 6 mL per rat. The trends of body weight, mean daily food intake and body temperature in low- and high-dose groups with vaccines produced in pilot and commercial facilities were all simliar to control groups. The blood and biochemical indicators of animals did not show abnormal changes related to different facilities.In equivalent dose vaccine satellite groups, there were no statistically significant differences in seroconversion rate (P=0.474-1.000) and geometric mean titer (GMT) (t=0.05-0.85,P=0.442-0.965) of serum IgG antibodies against rotavirus, and the change trend was consistent. The peak of IgG antibody seroconversion rates and GMTs (1∶lgx) in low- and high-dose vaccine (produced in pilots facilities) satellite groups were 80.0%, 80.0% and 1.61,1.81, while the corresponding vaccine （produced in commercial facilities） satellite groups were 90.0%,100.0% and 1.79,1.91,respectively. The organs and histopathological sections of animals did not show abnormal changes related to different facilities.Conclusion Reassortant Rotavirus Vaccines, Live, Oral, Hexavalent (Vero Cell) produced in pilot and commercial facilities both show good safety in SD rats and are comparable in single-and repeated-dose toxicity studies.

Objective To compare the digestive effects of trypsin and trypsin-like enzyme TrypLE on Vero cells, 2BS cells, and MDCK cells, in order to determine their suitability for cell culture and digestion processes.Methods After trypsin solution and TrypLE were used to digest Vero cells, 2BS cells, and MDCK cells, key parameters such as digestion time, dispersion effect, residue after digestion, cell count, and cell viability were analyzed to compare the effects of different digestive enzymes. Digested cells were then inoculated into cell factories and basket bioreactors for cultivation. During the cultivation period, cell morphology and confluence in cell factory were observed daily, and the adhesion effect of cells and glucose consumption in basket bioreactor were measured.Results There were no statistically significant differences in digestion time, cell count, or cell viability of Vero cells and 2BS cells between trypsin and TrypLE digestions (t=﹣0.46-3.46, P=0.074-0.885), and dispersion effect and residue after digestion were similar. However, the digestion time for MDCK cells using TrypLE was statistically significantly longer than using trypsin (t=13.28, P＜0.01), and the digestion effect of TrypLE was inferior to that of trypsin. When these 3 types of cells were re-cultured in the cell factory after digestion, they all had regular shapes and good confluence. When re-cultured in the basket bioreactor, the adhesion rates of 3 types of cells within 3 h were all above 80%, and there was no statistically significant difference in the total glucose metabolism (t=﹣1.05-1.01，P=0.405-0.783).Conclusion TrypLE can replace trypsin for the digestion process of Vero cells and 2BS cells, while trypsin is more effective for digesting MDCK cells.