Objective　To establish a rapid method for the detection of mycoplasma using real-time quantitative PCR (qPCR) technology.Methods　Eight mycoplasma species available for validation under European Pharmacopoeia（10.0 edition） 2.6.7 were selected. Specific primers were designed based on mycoplasma 16S RNA sequence. A qPCR method for the detection of mycoplasma was established and validated for specificity, sensitivity and reproducibility.Results　Suitable primers for the detection of mycoplasma by qPCR were successfully screened. Eight mycoplasma species showed a cycle threshold (CT) between 20-30 at a concentration of 10⁵-10⁶ CFU/mL, and there was no CT for the bacterial control at same concentration. Except for Mycoplasma sperminum, which could be detected at a concentration of 10-100 CFU/mL, the other 7 mycoplasma species could be detected at a concentration of 10 CFU/mL. CTs were between 35-40 at 10 CFU/mL. In specificity experiment that repeated 3 times, the control bacteria were all undetected, CTs of 8 species within 10⁵-10⁶ CFU/mL were between 20-30, and for each species, the coefficient of variation (CV) for 3 times was less than 10%. In sensitivity experiment that repeated 3 times, the CTs of each species with different dilution concentrations were between 20-40 in each experiment, and the CVs were less than 10%.Conclusion　The established method has good specificity, sensitivity and reproducibility, and can support timely control of possible mycoplasma contamination risks in biologics.

Objective　To evaluate the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus combined vaccine formulated with 4 adjuvants Al(OH)₃, MF59, AS03 and QS21 in mice.Methods　Adjuvants Al(OH)₃, MF59, AS03 and QS21 were used respectively to prepare SARS-CoV-2 (inactivated) and tetravalent influenza virus (split) combined vaccine. BALB/c mice were immunized by intraperitoneal injection on day 0 (D0) and D14, respectively. Blood samples were collected on D14 and D28 to detect antibody titers against SARS-CoV-2 and hemagglutination inhibition titers of influenza. Spleen lymphocytes were isolated on D28 to detect cellular immune responses to both SARS-CoV-2 and influenza virus.Results　SARS-CoV-2 and influenza virus combined vaccine with different adjuvants induced both antigen-specific antibody responses and cellular immune responses in mice. At D28 post-initial immunization, MF59 adjuvant group induced high levels of SARS-CoV-2 binding antibodies and neutralizing antibodies, with geometric mean titers (GMTs) of 89 144 and 5 418, respectively, and MF59 group also induced high levels of hemagglutination-inhibiting antibodies against the quadrivalent influenza virus strains (H1N1, H3N2, BV, BY), with GMTs of 4 457, 5 120, 1 470 and 5 881, respectively. Both the MF59 and AS03 groups induced robust Th1-type (IFN-γ, IL-2) cellular immune responses against SARS-CoV-2, with spot forming units (SFUs) statistically significantly higher than those of Mock group （IFN-γ： H=16.69，P＜0.01； IL-2： H=15.21， P＜0.05）. The AS03 group induced a strong Th1-type (IFN-γ, IL-2) cellular immune response against the quadrivalent influenza virus strains (H1N1, H3N2, BV, BY), with SFUs statistically significantly higher than those of Mock group （IFN-γ： H=12.93, 12.17, 11.82, 13.61, P＜0.05； IL-2： H=12.24, 12.42, 11.72, 12.43， P＜0.05）.Conclusion　The immunogenicity as well as specific antibody and cellular immune responses of SARS-CoV-2 and influenza virus combined vaccine with different adjuvant formulations are different, indicating the importance of adjuvants in the development of combined vaccine formulations.

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.

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.

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 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.

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.

值此中华医学会成立110周年之际，作为《国际生物制品学杂志》的编委与忠实读者，回顾这本杂志与我科研生涯的交织轨迹，恰似一部中国生物医药发展的微观史。从1984年踏入上海生物制品所实验室的青涩学子，到如今带领团队实现抗体药物产业化的研究者，这本杂志始终是我获取专业知识、开拓学术视野的重要伙伴，也见证了中国生物制品学界的发展进步。在此，我很高兴同大家分享我的成长故事和点滴体会。

1984年大学毕业后，我便被分配到上海生物制品所（后文简称为“上生所”）细胞生物实验室工作，从事正在兴起的利用DNA重组技术开发蛋白重组疫苗的研发工作。作为刚走出校门的年轻科技工作者，虽热情有余，但经验及方法严重不足。首先面对的就是如何开展科研工作的问题，其中如何快速有效查找并获取科研信息是重要一关。幸运的是，当时上生所拥有非常齐全的当代有影响力的生命科学领域杂志，并拥有许多外文书籍。鉴于当时国内外交流信息比较迟缓，上生所还主办了颇具特色的《国外医学•生物制品分册》。杂志及时刊登最新的科技信息，并翻译、摘录最新的国外生物制品研究领域的科研动态，是我们科研人员了解世界科研动态的重要媒介。每期新杂志一到，同事们争相阅读，个个沉浸在获取前沿资讯的喜悦之中。 

当时编辑部旁边的图书馆，是年轻科研工作者的殿堂。为了帮助大家在浩瀚的文献海洋中快速准确找到需要的资料，德高望重的向建之教授还亲自向年轻科研人员讲解检索文献的方法。记得我读的第一本英文书是关于启动子如何调节蛋白表达的专著。书不算太厚，大概300页，由于当时对许多专业名词不熟悉，我花费了整整3个月的业余时间才将这本书读完。但知识的果实是甘甜的。对那本书的潜心研读不仅使我对重组DNA技术有关启动子调节蛋白表达的理论知识有了比较全面的了解，还让我累积了大量分子生物学英文专业词汇，为我后来的职业生涯插上了飞翔的翅膀。自此，工作之余，我常躲在图书馆内阅读，既是自我提升，也是一种精神享受。阅读最多的，还是我们生物所自己编译的那本杂志（1985年起更名为《国外医学•预防、诊断、治疗用生物制品分册》）。记得有一次读到有关用昆虫病毒在家蚕中表达蛋白的信息，既兴奋又好奇。幸运的是，不久后我所在的科室就与加拿大某研究所就利用家蝉昆虫病毒表达乙型肝炎病毒表面蛋白开展了合作课题，我也有幸代表科室赴加拿大进行合作研究，并圆满完成了任务，将研究成果在国际病毒学会议上口头发表。

在之后的研究生涯里，最初累积的知识及阅读前沿资讯的习惯使我受益良多。在做任何工作之前，我都力求对相关知识及原理有全面、深刻的认知。在国外工作时，面对表达蛋白包涵体的复性问题，我花费大量时间和精力查阅有关蛋白质变性、复性的专著，并将掌握的相关理论应用于实践中，开发了一套行之有效的方法，使n个蛋白的复性率达到90%以上，并将该成果分享给全系同仁。

2005年我回到上生所工作。鉴于当时信息全球化趋势以及科研工作者外语水平的显著提高，研究人员对专业文献及行业资讯获取的便捷性有了天翻地覆的提升。我国期刊行业也进行了科学整合，上生所主办的《国外医学•预防、诊断、治疗用生物制品分册》几经改革，转由与中华医学会联合主办，并于2006年正式更名为《国际生物制品学杂志》，主要刊登生物制品领域最新研究进展及相关综述，为实验室的年轻同事及研究生提供学术交流平台。而我作为杂志的忠实读者，也有幸被邀请担任编委，一晃已有近20年。

结合在国外的学习研究情况及对生物制品领域未来发展方向的研判，从国外回来后，我成立了新的研发团队，主研方向为治疗性单抗药物开发，但重组蛋白技术仍然是抗体研发的重要基石之一，分子生物学及免疫学也仍然是药物开发最基本的知识。回国后的近20年，我们从无到有，搭建了以人源化技术开发单抗的新药平台、抗体偶联小分子药物平台、双抗平台及抗体产业化平台等，目前已有2款单抗药物获批上市、1款单抗药物申报上市、多个1类新药获得临床试验通知书，其中4个抗体偶联药物产品正在进行临床试验并初步取得了较好的安全性及疗效结果。这些成绩的取得，近在身边的《国际生物制品学杂志》也有一份功劳。回顾过去，杂志给予我最初的科学营养，为我开启了了解科技前沿的一扇心窗，为我后期的个人提升奠定了基础。

在此，预祝《国际生物制品学杂志》越办越好！

Hand, foot and mouth disease (HFMD) is an acute infectious disease caused by enterovirus infection. It has strong infectivity and multiple transmission routes, and is a long-term epidemic in the world. In the past decade, the pathogen spectrum of HFMD has undergone great changes worldwide. The dominant pathogenic agents transfer from enterovirus A71 (EV-A71) and coxsackievirus A(CV-A)16 to other enteroviruses, among which CV-A6 has the most significant growth trend. Although the monovalent inactivated EV-A71 vaccine has effectively controlled the infection and decreased the number of severe cases caused by EV-A71, it offers no cross-protection against enteroviruses of different serotypes, thus can not fully meet the prevention and control requirements of HFMD. It is urgent to develop multivalent vaccines that can simultaneously protect against multiple epidemic enteroviruses. This paper reviews the pathogen spectrum changes and vaccine research of HFMD in home and abroad.

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.

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 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 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.

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 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.

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 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 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 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.

Liposomes are artificially prepared nanometer-scale spherical vesicular structures composed of phospholipids, cholesterol, surfactants, etc. Liposomes exhibit a variety of potential applications in drug delivery and vaccine development due to advantages such as flexible charge, particle size regulation and surface modification properties. In cancer therapy, liposomes have become an important tool for improving therapeutic efficacy and reducing side effects by enhancing drug targeting, improving drug biocompatibility and stability and controlling drug release. Liposomes are the key technology for improving drug delivery efficiency and enhancing the efficacy of anti-tumor treatments. This article reviews the current state of the application of liposomes in pharmaceuticals to provide reference for developing novel liposomes.

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.

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.

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.

Objective To establish and compare 3 tetanus antibody detection methods in vitro. Methods The indirect method, double antigen assay and toxin binding inhibition (ToBI) test were established. The developed methods were validated for linearity and range, intermediate precision, accuracy and detection limit. Results The linear range of indirect method was 0.007 8-1.000 0 mili-international unit (mIU)/mL [coefficient of determination (R²) = 0.998 6], of double antigen assay was 0.078-10.000 mIU/mL (R²=0.998 7) and of ToBI test was 1.95-31.25 mIU/mL (correlation coefficient > 0.98). The validation results indicated that three methods meet the guiding principles of analytical method validation in terms of precision, accuracy, and specificity. Comparing the positive detection rates of these methods, there was no statistically significant difference (χ²=1.34，P=0.513). Conclusion Three detection methods for tetanus antibody in vitro, namely indirect method, double antigen assay and ToBI test, are successfully established, laying research foundation for in vitro method to replace in vivo neutralizing assay.

Immunoglobulin A nephropathy (IgAN) tends to occur in young people, and some patients may develop end-stage kidney disease, causing serious family and social burden, but for current clinical treatment, IgAN-specific drugs are scarce. With the development of disease cognition, drugs discovery and drug development technology in recent years, it is hopeful that IgAN could be overcome in the near future. At present, the recognized pathogenesis of IgAN is the "four-fold strike" theory, in which the complement system plays an important biological role in the occurrence and development of IgAN. In this paper, the basic concept, components, activation pathway, function and related drug discovery and development progress of the complement system are briefly introduced. Then, the pathogenesis of IgAN and the role of the complement system in IgAN are analyzed in detail, as well as the strategies for treating IgAN based on the complement system, and the discovery and development of new drugs are discussed. Finally, the prospects and challenges of the complement system drugs in the treatment of IgAN are prospected.

Objective　To establish a chromogenic substrate method for the determination of residual heparin in human coagulation factor Ⅷ(FⅧ ).Methods　The chromogenic substrate method (including kinetic method and endpoint method) was established using fully automated coagulation analyzer to determine the residual heparin in FⅧ. The method was verified for specificity, linearity, accuracy, intermediate precision, quantification limit and detection range.Heparin residues in three batches of FⅧ samples were measured using the above method.Results　The specificities of the kinetic method and endpoint method were good. In the range of 0.000-0.064 international unit (IU)/mL, the concentration of heparin standard solution for 2 methods showed a good linear relationship with the logarithm of absorbance value or its change rate. The mean values of spiked recoveries were all in the range of 75%-120%. The reproducibility RSDs were all≤8%, and the intermediate precision RSDs were all≤8%. The quantification limit of kinetic method was 0.007 IU/mL, and the detection range was 0.007-0.048 IU/mL.The quantification limit of endpoint method was 0.014 IU/mL, and the detection range was 0.014-0.048 IU/mL.The heparin residues of the 3 batches of samples tested by the 2 methods were below the limit of quantification.Conclusion　The determination method of heparin residue in FⅧ is successfully developed, which has good specificity, linearity, accuracy, repeatability and intermediate precision.

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.

According to difference of principle, virus detection methods can be divided into electron microscope method, cell culture method, immunoassay, nucleic acid testing, etc. Electron microscope method with a high resolution can show the structure of the viruses directly, which is suitable for the study of unknown virus. Cell culture methods can obtain the virus activity and detect virus titers. Immunoassay has strong specificity, low cost, and high speed, which is suitable for large-scale population screening. Nucleic acid detection methods allow simultaneous detection of multiple viruses with high sensitivity and strong specificity. Researchers should make a trade-off in the choice of method. This article discusses advantages and disadvantages of virus detection methods and applications, to provide reference for development of more accurate and efficient virus detection, prevention and control strategies.

Objective　To investigate the relationship between specific activity and isoelectric point distribution of equine tetanus immunoglobulin F（ab'）₂, so as to establish a better quality characterization for optimizing the process.Methods　The isoelectric points of equine tetanus immunoglobulin F（ab'）₂ before and after immunoaffinity chromatography purification were measured by capillary isoelectric focusing（CIEF） electrophoresis, and the experimental results were statistically analyzed.Results　The isoelectric point distribution ranges of the samples before and after purification by immunoaffinity chromatography were both 4.97-9.88. The isoelectric point distribution of the purified samples was more concentrated. After purification, the propartions of peak area in the isoelectric point ranges of 6.01-7.00 and 8.01-9.00 increased compared with those before purification, with an average increase of 70% and 37%, respectively. In the isoelectric point ranges of 7.01-8.00 and 9.01-10.00, the peak area proportion decreased by 25% and 26%, respectively.Conclusion　According to the results of this study, it is speculated that the isoelectric points of the antibody with strong neutralizing activity against tetanus toxin in equine tetanus immunoglobulin F(ab')₂ may mainly be distributed in the ranges of 6.01-7.00 and 8.01-9.00.

Objective　To compare the effects of different signal peptide sequences on secretory expression of influenza virus hemagglutinin (HA) in Sf9 insect cells.Methods　The HA of H1N1 subtype influenza virus A/Victoria/2570/2019 (IVR-215) was used as a target.The full-length HA gene sequence was selected and naïve signal peptide, honeybee melittin signal peptide, glycoprotein 67 signal peptide（Gp67sp）, chitinase signal peptide, and human azurocidin gene signal peptide were connected to the 5´terminal of the gene， respectively. The genes were cloned into the genome of the pFastBac1 transfer plasmid， and five recombinant baculoviruses were constructed by the Bac-to-Bac system. The viral titers were detected by BacPAK^TM qPCR titration kit. The recombinant baculoviruses were used to infect Sf9 cells at different multiplicities of infection, and the cell supernatants were harvested at different time points after infection, and the protein expression levels were detected by immunoblotting to optimize the conditions for HA secretory expression.Results　The titers of the five recombinant baculoviruses harvested were all ＞10⁶ PFU/ml, and the Sf9 cells were able to correctly express and secrete HA, with a relative molecular mass of about 80 000. The amount of HA expressed was the highest when mediated by Gp67sp.The highest expression of HA was achieved when the recombinant baculovirus AcMNPV-Gp67sp-HA infected Sf9 cells for 96 h with multiplicity of infection 5.0 or 10.0, and the hemagglutination efficiency was 256 hemaqqlutinetion units/50 μl.Conclusion　All signal peptides tested effectively guide the secretory expression of influenza virus HA protein in Sf9 cells, among which Gp67sp is particularly advantageous for the efficient secretory expression of HA.

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 validate a quantitative ELISA method for the detection of residual host cell protein (HCP) in recombinant hepatitis B vaccine (Saccharomyces cerevisiae).Methods　The S. cerevisiae HCP ELISA Kit was used to detect HCP residues in recombinant hepatitis B vaccine (Saccharomyces cerevisiae), and the linearity, accuracy, repeatability, limit of quantification, specificity and intermediate precision of the method were verified.Results　The ELISA standard curve had good linearity (coefficient of determination ≥ 0.97), with the lowest absorbance (A_405/492) value <0.300, and the highest A_405/492 value >1.000. With same experimenter, samples added with low, medium and high concentration standards were detected 3 times, and the recovery rates were 80%-120%. Medium concentration standard-added sample was detected 6 times, and the relative standard deviation (RSD) was <10%, indicating good accuracy and repeatability of the method. The A_405/492 values of the sample background solution and the sample diluent were both smaller than the lowest point of the curve, indicating that the method was highly specific. The medium concentration standard-added sample was detected by different experimenter groups at different time for 6 times, and the RSD was <10%, indicating good intermediate precision of the method.Conclusion　The ELISA method for the detection of HCP residues in recombinant hepatitis B vaccine (Saccharomyces cerevisiae) has good linearity, accuracy, repeatability, limit of quantification, specificity and intermediate precision, thus can be used for the process control of hepatitis B vaccine production.

Objective To investigate the mechanism of pyroptosis production in colorectal cancer cells by Newcastle disease virus (NDV) via natural killer (NK) cell activation through granzyme A (GZMA) secretion, with specific focus on the role of gasdermin B (GSDMB) expression level in this process. Methods NK cells were co-cultured with NDV at different concentrations [NDV1, 2, 3: 0.001, 0.010, 0.100 hemagglutinin/mL] for 16 h, and blank control (culture medium only) as well as non-stimulated NK cell control were set. All groups were subsequently incubated with high (SW837) or low (SW480) GSDMB expression colorectal cancer cells. Quantitative PCR and ELISA were used to detect the gene expression and protein secretion levels, respectively, of effector molecules in NK cells. Cell proliferation, cytotoxicity and lactate dehydrogenase (LDH) release were measured to assess the cell viability and pyroptosis in 2 tumor cell lines. Cell morphology observation and Western blot of GSDMB cleavage were applied to verify NK cell-mediated pyroptosis effect. Results Quantitative PCR and ELISA results indicated that NDV stimulation upregulated the gene expression and protein secretion levels of GZMA, IFN-γ, and perforin in NK cells. The highest protein secretion levels of GZMA (t = 6.70, P = 0.003) and IFN-γ (t = 13.66, P < 0.001) were reached in NDV3 stimulation, and were statistically significantly higher than non-stimulated NK control. Comparing with non-stimulated NK cell control treated, cell viability inhibition [NDV3-NK at effector-to-target ratio (E/T) =1, 2: t = -10.17, -25.94, both P < 0.001], LDH release (NDV3-NK at E/T=1, 2: t = 13.70, 9.75, both P < 0.001), and N-terminal GSDMB (NDV2-NK at E/T=1, 2: t = 6.80, 3.07, both P < 0.05) of with stimulated NK cell treated SW837 all increased statistically significantly. Pyroptosis-related features such as plasma membrane blebbing and swelling were observed in SW837, but not in SW480 cells, even though viability inhibition and cell damage were seen in SW480. Conclusion NDV-activated NK cells induce pyroptosis in colorectal cancer cells via GZMA secretion, particularly in cells with high GSDMB expression.

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 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.