10 October 2025, Volume 48 Issue 5
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International Journal of Biologicals. 2025, 48(5): 301-308. https://doi.org/10.3760/cma.j.cn311962-20250211-00011Objective 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.
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International Journal of Biologicals. 2025, 48(5): 309-314. https://doi.org/10.3760/cma.j.cn311962-20250119-00008Objective 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.
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International Journal of Biologicals. 2025, 48(5): 315-320. https://doi.org/10.3760/cma.j.cn311962-20250107-00001Objective 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.
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International Journal of Biologicals. 2025, 48(5): 321-325. https://doi.org/10.3760/cma.j.cn311962-20250117-00006Objective 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.
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International Journal of Biologicals. 2025, 48(5): 326-330. https://doi.org/10.3760/cma.j.cn311962-20250117-00007Objective 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.
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International Journal of Biologicals. 2025, 48(5): 331-336. https://doi.org/10.3760/cma.j.cn311962-20241217-00089Objective 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.
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International Journal of Biologicals. 2025, 48(5): 337-342. https://doi.org/10.3760/cma.j.cn311962-20241215-00087Objective 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.
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International Journal of Biologicals. 2025, 48(5): 343-349. https://doi.org/10.3760/cma.j.cn311962-20241219-00090Objective 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.
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International Journal of Biologicals. 2025, 48(5): 350-356. https://doi.org/10.3760/cma.j.cn311962-20241114-00078Objective 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.
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International Journal of Biologicals. 2025, 48(5): 357-360. https://doi.org/10.3760/cma.j.cn311962-20241224-00094Objective 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.
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International Journal of Biologicals. 2025, 48(5): 361-369. https://doi.org/10.3760/cma.j.cn311962-20250801-00056Chikungunya 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.
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International Journal of Biologicals. 2025, 48(5): 370-377. https://doi.org/10.3760/cma.j.cn311962-20250728-00055Chikungunya 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.
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International Journal of Biologicals. 2025, 48(5): 378-385. https://doi.org/10.3760/cma.j.cn311962-20241112-00075Bone 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.
创刊：1978年10月
主管：中华人民共和国国家卫生健康委员会
主办：中华医学会
上海生物制品研究所有限责任公司
编辑：《国际生物制品学杂志》编辑委员会
出版：《中华医学杂志》社有限责任公司
ISSN 1673-4211
CN 31-1962/R
主管：中华人民共和国国家卫生健康委员会
主办：中华医学会
上海生物制品研究所有限责任公司
编辑：《国际生物制品学杂志》编辑委员会
出版：《中华医学杂志》社有限责任公司
ISSN 1673-4211
CN 31-1962/R
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