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 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 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.

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.

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.

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.

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.

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.

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.

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.

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.