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.

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.

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.

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 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.

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 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.

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.

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 investigate the physicochemical properties of aluminum phosphate adjuvants subjected to different numbers of autoclaving cycles and to evaluate the effect of aluminum phosphate adjuvant with varying autoclaving cycles on immunogenicity of type 6A pneumococcal polysaccharide-tetanus toxoid (TT) conjugate (6A-TT).Methods Self-prepared aluminum phosphate adjuvants were autoclaved 1, 2, and 3 times, respectively. The particle size and distribution, pH, isoelectric point, free phosphate content, phosphorus-to-aluminum molar ratio, and protein adsorption rate were examined. Additionally, the effect of aluminum phosphate adjuvants treated with different autoclaving cycles, when combined with 6A-TT, on the specific antibody titers in mice was assessed.Results After autoclaving 1, 2, and 3 times, the 50% particle distribution diameters (Dv50s) of aluminum phosphate adjuvant were 5.04, 5.22, and 5.12 μm, while Dv90s were 9.20, 9.87, and 10.60 μm, respectively. The pH values were 4.6, 4.3, and 4.3, while the isoelectric points were 5.48, 5.36, and 5.29, respectively. The free phosphate content were 4.12%, 4.79%, and 4.86%, and the phosphorus-to-aluminum molar ratios were 1.012, 0.972, and 0.944, respectively. The adsorption rates of aluminum adjuvant to TT were 92.7%, 87.8%, and 85.7%, and to 6A-TT were 93.8%, 91.6%, and 90.1%, respectively. The aluminum phosphate adjuvant, after autoclaving 1, 2, and 3 times, was able to adsorb 6A-TT and induce antibody production in mice, with geometric mean titers of 1 213, 985, and 1 056, respectively.Conclusions Increasing the number of autoclaving cycles leads to decrease in the isoelectric point and pH of the aluminum phosphate adjuvant, increase in free phosphate content, and reduction in the phosphorus-to-aluminum molar ratios and protein adsorption rate. It may also have an effect on the immunogenicity of 6A-TT pneumococcal polysaccharide antigen in mice.

Objective To investigate the epidemiological characteristics and breakthrough cases of varicella in Xuancheng City, and provide a basis for epidemic prevention and control.Methods The information of varicella cases in Xuancheng City from 2021 to 2023 was collected through the varicella epidemic report card of China Information System for Disease Control and Prevention, and the epidemiological characteristics were analyzed by descriptive epidemiological method.Results A total of 6 979 varicella cases were reported in Xuancheng City from 2021 to 2023, with an average annual reported incidence rate of 93.44 per 100 000 (χ²=122.80, P＜0.001). The incidence of varicella had significant seasonality, with a small peak from May to July and the highest peak from October to January of the following year. The three counties (cities) with highest average annual incidence rates were Jixi County, Ningguo City, and Jingxian County.The number of male cases was slightly higher than that of female cases, the age of onset was mainly under 14, and occupations were mainly students and kindergarteners.There were 2 652 breakthrough cases, accounting for 38.00% of the total cases. There were more breakthrough cases in males than in females, and the proportion of breakthrough cases in autumn was the largest. In 11 years after vaccination, longer time since vaccination was associated with more breakthrough cases.Breakthrough cases in clustered epidemic accounted for 68.45% of related cases, significantly higher than that in sporadic cases. Breakthrough cases were mainly students aged 7-13.Conclusions The incidence of varicella in Xuancheng City is on the rise, and schools and kindergartens are the key places for the prevention and control of varicella. It is nessesary to prevent the occurrence of cluster epidemic, increase 2-dose varicella vaccination, and reduce the occurrence of breakthrough cases. CDC should strengthen epidemic monitoring, review epidemic report, carry out targeted training and health education, and establish effective immune barriers to reduce the incidence of varicella.

Objective To analyze the incidence and distribution characteristics of adverse events following immunization (AEFIs) associated with diphtheria-tetanus-acellular pertussis vaccine (DTaP) before and after the implementation of new immunization strategy under the National Immunization Program in Anhui Province, and to provide evidence for evaluating the safety of the new strategy.Methods AEFI surveillance data in Anhui Province during 2024（before strategy adjustment） to 2025（after the new strategy implemention on January 1, 2025） were collected through the Immunization Program Information Management System of the Chinese Center for Disease Control and Prevention, and concurrent vaccination data were obtained from Anhui Immunization Program Information Management System. Descriptive epidemiological methods were used to analyze the distribution and incidence of AEFI. The Chi-square test or Fisher’s precision probability test were used to compare the data before and after the policy adjustment.Results From 2024 to 2025, a total of 2 375 028 doses of DTaP were administered in Anhui Province, with 4 958 AEFI cases reported. The overall incidence was 208.76/100 000 doses. Following the strategy adjustment, the incidence of general reactions increased from 186.69/100 000 doses to 221.37/100 000 doses (χ²=32.50, P<0.001)，while the incidence of adverse reactions (rare/serious) decreased from 0.66/100 000 doses to 0.20/100 000 doses, representing a reduction of approximately 69.70%. After advancing the first dose to 2 months of age, the AEFI incidence decreased from 170.57/100 000 doses to 119.65/100 000 doses (χ²=24.76, P<0.001). For the 6-year-old dose switched from the diphtheria and tetanus vaccine to DTaP, no adverse reactions were observed, with incidence of 0.00/100 000 doses (95% confidence interval: 0.00-0.83/100 000 doses ).Conclusions DTaP AEFIs in Anhui Province are predominantly general reactions, with extremely low adverse reaction rates. Advancing the first dose to 2 months of age significantly reduces the AEFI incidence, facilitating the early establishment of immune barrier. With no new safety risks monitored during this surveillance period after switching to DTaP for the 6-year-old booster, core safety indicators perform well during the study period. The new 5-dose DTaP strategy (at 2, 4, 6, 18 months and 6 years of age) demonstrates a good safety profile within the observation period.

Objective To screen differences in abnormal toxicity test (ATT) for same biological product using animals from different suppliers and to explore the scientific and rational nature of ATT by additional blood routine analysis.Methods With reference to Chinese pharmacopoeia 2025 edition (volume Ⅲ) general rule 1141 “Abnormal toxicity test method”, the differences between animals from different suppliers were compared by analyzing the weights and blood routine results of animals after immunization with different vaccines.Results The body weight growth trend of NIH mice from supplier B was statistically significantly lower than that of the current supplier (supplier A) in both control and vaccine groups (t=2.64-7.07, all P<0.05), and the difference between suppliers C and A was not significant. Using the blood routine results of NIH mice from supplier A as a reference, blood routine results of mice from supplier B had significantly more red blood cells in both vaccine groups (t=2.44-4.07, all P<0.05) and showed significant differences in many white blood cell indicators (t=2.31-5.96, all P<0.05), while that of supplier C only showed significant differences in a few indicators. The body weight growth trends of Hartley guinea pigs from 3 suppliers (B, D, E) remained the same in both control and vaccine groups, and there was no significant difference in blood routine results.Conclusions NIH mice from supplier B have significantly different quality from those from suppliers A and C, while the quality of Hartley guinea pigs from suppliers B, D, and E is similar. The difference in animal quality directly affects the determination of ATT results, and the rationality of using ATT as a testing method for vaccine batch release is worth exploring.

Objective To analyze adverse events associated with rabies vaccine for human use (RVFHU) based on data from the US Vaccine Adverse Event Reporting System (VAERS) and to evaluate the safety profile of RVFHU.Methods RVFHU-related reports were retrieved from the VAERS database for the period from January 1, 2019 to September 15, 2023. Descriptive statistical analysis was employed to characterize the distribution of adverse events, and disproportionality analysis was applied to detect vaccine safety signals.Results A total of 794 adverse event reports (comprising 2 606 clinical symptoms) were included. Product use issues were the most frequently reported clinical symptoms. In terms of System Organ Class, symptoms related to general disorders and administration site conditions accounted for the highest proportion. Regarding the composition ratios of general adverse events and serious adverse events by vaccination dose, the difference was statistically significant (χ²=10.25, P<0.05), whereas no statistically significant differences were observed for gender, age, or time of occurrence (χ²=0.32, 2.58, 2.02, respectively; P>0.05). Disproportionality analysis identified 59 positive safety signals, involving product use issues, immune system disorders, psychiatric disorders, and other aspects.Conclusions Adverse events associated with RVFHU are predominantly product use issues or transient and mild reactions. The positive safety signals detected are limited in number and require further investigation and validation.

Immunometabolism is an emerging field that explores the interactions between immune cells and metabolism. In recent years, the role of immunometabolism in autoimmunity and autoimmune diseases has become a growing area of research, focusing on how cellular metabolism influences immune cell function and responses. It is currently understood that immune cell signaling and differentiation processes can determine immune cell behavior, control numerous metabolic pathways and thereby affect immune cell function and responses. This article primarily discusses the metabolic alterations in immune cells in systemic lupus erythematosus, type 1 diabetes and rheumatoid arthritis, elucidating how immunometabolism influences disease onset, exacerbation, or remission, and summarizes drugs that modulate immunometabolism to potentially control immune responses in autoimmune diseases and their research progress.

Seasonal influenza poses a persistent threat to global public health, imposing a significant disease burden particularly on high-risk population such as the elderly, children, pregnant women, chronic diseases patients, and immunocompromised people. Although the effectiveness of existing influenza vaccines is contingent on their match with circulating strains, their protective benefits for high-risk population are well-supported by epidemiological evidence. Currently, influenza vaccination coverage in China remains considerably lower than developed countries. There is an urgent need to enhance vaccination rates among high-risk population through policy support and technological innovation to reduce the disease burden of influenza. This article systematically reviews the protective evidence of influenza vaccines in high-risk population, focusing on the effectiveness and applicability of different vaccine types (inactivated, live attenuated, recombinant protein, and adjuvanted vaccines) in the high-risk population. It also examines the effects of repeated vaccination, the role of herd immunity, and the optimization of vaccination strategies, providing references to improve protection in high-risk population.

Objective To analyze the characteristics of adverse events following immunization (AEFIs) with varicella live attenuated vaccine (VarV) in Anhui Province from 2016 to 2025.Methods Data on VarV AEFI cases in Anhui Province from 2016 to 2025 were collected through the China Disease Control and Prevention Information System, and the number of VarV vaccination doses were collected through the Anhui Immunization Program Information System. Descriptive epidemiological methods were used to analyze the incidence and distribution characteristics of AEFIs.Results A total of 2 408 VarV AEFIs were reported in Anhui Province from 2016 to 2025, with an average annual incidence of 329.46 per million doses. The incidence of common reactions and rare adverse reactions were 308.46 per million doses and 17.80 per million doses, respectively. Among all cases, 53.74% were male, 64.70% were ≤1 year old, 61.17% were from northern Anhui Province, 78.15% occurred within 24 h, and 77.24% were from the first dose. Among rare adverse reactions, anaphylactic rash accounted for 90.77%, with an average annual reported incidence of 16.16 per million doses.Conclusion VarV AEFIs are mainly common reactions in Anhui Province, with extremely rare adverse reactions, which shows an overall good safety profile of VarV.

Objective To evaluate the application effects of 3 types of sheet carriers for the production of Sabin strain inactivated poliovirus vaccine (sIPV) in order to provide basis for carrier selection in sIPV production process.Methods A 15 L basket bioreactor system was adopted to carry out Vero cell culture and prepare Sabin strain poliovirus liquid with 3 sheet carriers A, B, and C, respectively. The cell attachment rate of Vero cells inoculated on the sheet carriers within 0-3 h, the glucose metabolism characteristics during the 8-day culture cycle, the virus titer and D-antigen content of the virus harvest were investigated and compared.Results Cell attachment rates of carriers A, B, and C were all >90% at 3 h after inoculation. Cells in all 3 groups presented a typical S-shaped glucose consumption curve. The cumulative glucose consumptions of A and B were (191.1±7.6) g and (192.9±5.3) g, with no statistically significant difference (F=43.23, P=0.060), while both were statistically significantly higher than that of C at (153.1±4.4) g (F=43.23, P<0.001). D-antigen contents of A and B were (773.3±10.8) and (777.0±12.5) D-antigen unit (DU)/mL, and virus titers were (8.1±0.2), (8.2±0.1) lgCCID₅₀/mL,respectively, with no statistically significant difference (F=209.00, 27.30; P=0.946, 0.708). In contrast, the two indicators of group C at (598.3±14.2) DU/mL and (7.2±0.2) lgCCID₅₀/mL,were lower than those of group B with statistically significant differences (F=209.00,27.30; P<0.001).Conclusion Carriers A and B both exhibit clear advantages in maintaining the metabolic activity of Vero cells and promoting poliovirus replication.

Objective To identify the key binding sites and preliminarily evaluate the biological functions of fully humanized monoclonal antibody (mAb) 23F4 targeting sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15).Methods Alanine scanning mutagenesis was performed on Siglec-15 to identify key binding sites of 23F4. In vitro biological activity was assessed via ELISA and flow cytometry. In vivo antitumor efficacy was evaluated using 2 murine tumor models.Results The critical binding sites of 23F4 were identified as residue R71/H72. In vitro, 23F4 exhibited superior binding activity with median effective concentration of 68.42 ng/mL, and outperformed control antibodies in binding Siglec-15-positive cells particularly at medium-to-low concentrations. 23F4 blocked Siglec-15-receptor interaction, reversed T-cell proliferation suppression, and suppressed tumor growth in both murine tumor models.Conclusion As a Siglec-15-targeting mAb with novel binding sites, 23F4 effectively alleviates Siglec-15-mediated immunosuppression.

Objective To evaluate the transportation stability of adalimumab injection.Methods The worst-case transport conditions were simulated by conducting a round-trip road transport from Wuhan to Harbin, to assess drug stability under non-temperature factors. Destructive tests, including extreme temperature exposure and temperature cycling, were employed to simulate post-cold-chain-break scenarios. Stability under temperature excursions was evaluated by testing routine quality attributes of adalimumab injection.Results The primary packaging of all 3 batches remained intact post-transportation, with 0% breakage rate. All tested attributes complied with the company’s registered specifications, showing no statistically significant differences compared to pre-transport samples (t=﹣3.23-4.00,P=0.057-0.840). After storage at (40±2) °C or (﹣20±5) °C for 7 d, all attributes for 3 batches met the company’s registered specifications. However, monomer content (determined by size exclusion chromatography) and purity (determined by ion chromatography) exhibited statistically significant differences (t=﹣142.00-97.00，P=0.000-0.020) between samples stored at (40 ± 2) °C and those stored at 2-8 °C or (﹣20±5) °C. No statistically significant differences (t=0.70-3.46，P=0.074-0.556) were observed in any attributes between samples stored at (﹣20±5) °C for 7 days and those stored at 2-8 °C. All attributes met the company’s registered specifications after 3 freeze-thaw cycles, with no statistically significant differences compared to samples not subjected to freeze-thaw testing (t=﹣4.06-1.73，P=0.056-0.478).Conclusion Under the experimental conditions, non-temperature factors during long-distance transportation have no impact on drug quality, and short-term temperature excursions have minimal effect on the quality of adalimumab injection.

Objective To establish and validate a cation exchange chromatography detection method for adalimumab charge variants.Methods An exchange column (4 mm×250 mm) with weak cations of carboxylic functional groups was used as the stationary phase. 0.01 mol/L disodium hydrogen phosphate solution, pH 7.5 was used as mobile phase A, and 0.01 mol/L disodium hydrogen phosphate + 0.5 mol/L sodium chloride solution, pH 5.5 was used as mobile phase B for gradient elution. The content of lysine variants and acidic components were calculated by area normalization method. Specificity, repeatability, intermediate precision, linearity, accuracy, and robustness of the method were validated.Results Adalimumab target peak and solvent peak were observed. For 6 repeated tests, relative standard deviations (RSDs) of all components were all ＜2%. With different experimenters and test equipment, RSDs of each component were all ＜ 2%. The quantification limit (signal-to-noise ratio＞10) and detection limit (signal-to-noise ratio＞3) of peaks between lysine variants were 100 μg/mL and 30 μg/mL, respectively. Within the quality standard limits of Wuhan Institute of Biological Products Co., Ltd., the linearities of all components at different concentrations were good, with coefficients of determination all＞0.99. Recovery rates were within 80%-115% for the first acidic region peak, the intermediate peak between lysine variants, and lysine variant 2 peak, and 85%-110% for the second acidic region peak, main peak, and lysine variant 1 peak. Analytical results met the specified requirements and exhibited acceptable consistency under different mobile phase pH and NaCl concentration, flow rate, column temperature, and chromatographic column lot numbers.Conclusion The method established has good specificity, repeatability, intermediate precision, linearity, accuracy, and robustness, and is suitable for the determination of the purity of adalimumab charge variants.

With the increasing frequency of emerging infectious diseases globally in recent years, the response practices to previous public health crises indicate that a rapid vaccine response is crucial to effective threats containing from pathogens with large-scale epidemic potential. In 2021, the Coalition for Epidemic Preparedness Innovations (CEPI) proposed the strategic plan CEPI 2.0, also known as the 100 Days Mission. The six pillars of the plan, namely establishing a prototype vaccine library, building a global clinical trial network, identifying early immune biomarkers, developing rapid manufacturing and validation capabilities, deploying early outbreak surveillance systems, and strengthening biosafety and biosecurity capacity, aim to achieve preliminary authorization and mass production readiness for target vaccines within 100 days after confirmation of a pandemic pathogen.This paper provides a systematic review and interpretation of the scientific framework of the plan, seeking to offer reference and insights for China to better respond to potential epidemic risks and pandemic occurrences.

Objective To validate the method of cell species identification (multiplex PCR) and apply it to the identification of cells from various species.Methods Cell genomic DNA was extracted by cell species identification detection kit (multiplex PCR), and the target gene was amplified by mixed primers. Multiplex PCR combined with agarose gel electrophoresis was used to identify species and detect cross-contamination according to the band size and number of amplified products, and the detection limit, specificity and durability to 5 different sources of cells were investigated.Results The detection limits of multiplex PCR method were 500 cells for Hep-2 cells (human), canine kidney cells, Vero cells (African green monkey) and L929 cells (murine), and 5 000 cells for Chinese hamster ovary cells. When each cell type was designed with cells from different sources for cross-contamination, both main cells and contaminant cells as low as 1‰ level were detected. When the genomic DNA extracts of 5 kinds of cells were stored at ﹣18 ℃ for 1, 3 and 7 d, the amplification results were consistent.Conclusion Multiplex PCR method has high sensitivity, strong specificity, rapidity and convenience, which is helpful to improve the quality control level of cell substrates for the production of biological products.

Objective To develop and validate the isoelectric point (pI) detection method for recombinant human interferon α1b (rhIFNα1b) by imaging capillary isoelectric focusing electrophoresis (iCIEF).Methods Based on The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q2 (R2) requirement, iCIEF was used to detect the pI of rhIFNα1b, and the concentration of urea added to sample and isoelectric focus time were determined.The developed method was verified for specificity, accuracy, repeatability, intermediate precision and durability.Results In iCIEF, the focusing was performed at 3 000 V for 3 min, and 1 mol/L urea was added to treat samples. Blank control (acetate buffer) showed no interference to the determination of pI of rhIFNα1b. Isoelectric focusing of rhIFNα1b generated 2 peaks. The pIs of peak 1 and 2 were approximately 5.4 and 5.5, respectively. The method had good specificity, accuracy, repeatability, intermediate precision, and durability.Conclusion This established iCIEF method can be used for the detection of the pI of rhIFNα1b, providing a reference for the quality control of this product.

With the rapid development of microneedle technology, microneedle has gradually become a new vaccine delivery method, which has advantages such as minimally invasive, painless, self-administration, dose-saving, and enhanced immune response. The evaluation of immunogenicity is crucial for vaccine research and development. However, there is still no systematic summary of immunogenicity evaluation method of microneedle vaccines. This article summarizes the characteristics of immune responses to microneedle vaccines, systematically reviews and discusses various methods and key indicators for evaluating the immunogenicity of microneedle vaccines, as well as explores and prospects emerging immunogenicity evaluation methods in microneedle vaccines.

Objective To establish and validate an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of nonyl phenol leachable in human growth hormone (hGH) injection.Methods Referring to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines related to impurities, the safety limit of nonyl phenol was calculated. The UPLC-MS/MS method was used to determine the content of nonyl phenol leachate in hGH injection, and the linearity, specificity, limit of quantitation (LOQ), accuracy, precision, stability and robustness of the method were validated. The established method was used to detect 3 batches of hGH injection.Results The safety limit of nonyl phenol was 4.5 μg/vial. The method had a good linear relationship between peak area and concentration in the nonyl phenol concentration range of 8.2-65.5 μg/L.No nonyl phenol was detected in the blank solution.The LOQ was 1.2 μg/vial.The average recoveries of spiked samples at low, medium and high levels were all between 70%-125%, with relative standard deviations (RSDs) all ＜15%.The RSDs of repeatability and intermediate precision were both ＜15%.Nonyl phenol in the test solution was stable at room temperature for 20 h; when the flow rate in the chromatographic conditions was set to 0.27 and 0.33 mL/min, the RSD of nonyl phenol peak area in 6 detections was ＜10%.The detection results of 3 batches of samples were much lower than the limit of 4.5 μg/vial.Conclusion UPLC-MS/MS method for the determination of nonyl phenol leachate in hGH injection is successfully established, and the linearity, specificity, LOQ, accuracy, precision, stability and robustness of this method are good.

Objective To establish and validate a high performance liquid chromatography (HPLC) method coupled with an evaporative light-scattering detector (ELSD) for the determination of residual polysorbate 20 (PS20) in the monoclonal antibody SIBP-A01.Methods The HPLC-ELSD method was used to detect the residual PS20 in SIBP-A01. The method was validated in terms of specificity, precision, linearity, accuracy, limit of quantitation (LOQ), and robustness.Results No interfering peaks were observed in the diluent or the formulation buffer without PS20. For the three batches of SIBP-A01 test samples, the relative standard deviations (RSD) of repeated detection results, detection results obtained by different operators, and detection results obtained by the same operator at different time were all less than 5%. When the concentration of PS20 ranged from 12.5 to 100.0 μg/mL, a good linear relationship was observed between the logarithm of the peak area and the logarithm of the concentration, with a coefficient of determination of 0.996 8. The spiked recovery rates were in the range of 103.5% to 109.2%. The LOQ was 5.0 μg/mL. When the column temperature was set at (30±3) ℃, the samples were stored at 10 ℃ for 24 h, or different brands of PS20 were used, the difference of the detection results was less than 5%.Conclusions An HPLC-ELSD method for the determination of residual PS20 in SIBP-A01 has been successfully established. This method shows good specificity, precision, linearity, accuracy, and robustness.

Objective To develop a glucagon-like-peptide-1 receptor (GLP-1R)/gastric inhibitory polypeptide receptor (GIPR)/glucagon receptor (GCGR) triagonist peptide with balanced activity and strong resistance to dipeptidyl peptidase Ⅳ(DPP-Ⅳ) degradation using deep learning and structural modeling.Methods Based on clinical candidate Peptide 20 (MAR423), virtual mutagenesis was performed on the first 3 residues at the N-terminus， trireceptor complex stability was scored using Protein Message-passing Neural Network, and 800 sequences were screened based on the score and DDP-Ⅳ sensitivity predicted by neural network model. Top candidates were evaluated by 5 ns molecular dynamics simulations to calculate binding energy, and the median effective concentration (EC₅₀) was measured with HEK-293 reporter gene assay. DPP-Ⅳ resistance was assessed by measuring residual activity after enzyme treatment.Results Peptide A, modified with γGlu-C16, had stable conformation, with binding energy values to GLP-1R, GIPR, GCGR at (﹣151.49 ± 15.87), (﹣122.79 ± 23.42), (﹣139.25 ± 19.81) kcal·mol^﹣¹，respectively, and showed strong and balanced activity with EC₅₀ of 0.069, 0.089, 2.730 nmol/L, respectively. It retained (104.30±3.23)% activity after 24 h of DPP-Ⅳ treatment.Conclusion A rapid and effective strategy to discover triagonist peptide with improved stability and balanced potency is presented, supporting future development of multi-target peptide drugs for metabolic diseases.

Objective To validate a size-exclusion high-performance liquid chromatography (SEC-HPLC) method for determining the purity of recombinant human coagulation factor Ⅶa (rFⅦa) for injection, and apply it to stability analysis of the purity of rFⅦa finished products.Methods The SEC-HPLC method was validated for the system suitability and specificity, sample loading robustness, accuracy, repeatability, intermediate precision, and column durability using rFⅦa standard， bulk and finished product. The stability of rFⅦa stored at 2-8 °C and 40 °C for 6 months was analyzed using this method.Results The peak-to-valley ratio of dimer in the rFⅦa international standard was 1.43, and the tailing factor of monomer was 1.08. An excipient peak accounting for up to 0.4% at the monomer position in the formulation buffer. Within the protein loading range of 10-50 μg, the relative standard deviations (RSDs) of the monomer peak area percentage for rFⅦa bulk and finished product were 0.03% and 0.07%, respectively. Accuracy was evaluated through forced thermal degradation testing, which demonstrated recovery rates of 97.5%- 100.0% for percentage changes in the peak areas of polymers, dimers/oligomers, and monomers, and 98.5%-100.6% for the total peak area. The RSDs for the monomer peak area percentages in the repeatability verification of rFⅦa bulk and finished product were 0.03% and 0.02%, respectively. Intermediate precision verification showed no statistically significant differences across operators, analytical equipments, or testing dates (F=0.13-2.09,P all＞0.05), with RSDs of 0.2% for bulk and 0.9% for finished products. Column durability results showed no statistically significant differences among column batches (F=0.01, P＞0.05). After 6 months of storage at 2-8 °C, the molecular size distribution of rFⅦa finished product remained stable. Following 6 months of accelerated stability conditions at 40 °C, the monomer decreased by 3.1%-4.0%, dimer/oligomer increased by 1.9%-2.3%, and polymer increased by 1.2%-1.7%.Conclusions The validated SEC-HPLC method is efficient, accurate, and reproducible for purity determination of rFⅦa for injection. The finished product of rFⅦa for injection exhibits good stability when stored at 2–8 °C or 40 °C for 6 months.

The large-scale animal cell culture technology for production of influenza virus vaccines offers advantages such as high efficiency, stability, scalability, and safety, making it a promising alternative to traditional embryonated egg-based method. This approach is expected to become the prevailing method for manufacturing influenza virus vaccines. Gaining an in-depth understanding of the viral amplification and cellular stress responses of influenza viruses in vaccine production cell lines is pivotal for the development of production cell strains and the rational design and optimization of virus production processes. This review provides an overview of the production workflow for cell-based influenza vaccines, summarizes recent advancements in research on the viral amplification process and cellular stress responses following influenza virus infection in production cell lines, and highlights the application of mathematical modeling in influenza virus production. The aim is to offer insights and references to guide the rational optimization of cell-based influenza vaccine production processes.

The treatment of infectious bone defects faces great challenges due to the “infection-repair” vicious cycle. Antibacterial-osteogenic bifunctional hydrogels provide an innovative strategy to solve this problem by synergistically integrating antibacterial and osteogenic functions. This article reviews the latest research progress on the synergistic effect of this type of hydrogel in bone repair of bone marrow mesenchymal stem cells (BMSCs), focuses on design strategy giving the material intelligent function, and analyzes its core synergy mechanism including temporal synergy, microenvironment remodeling and spatial collaboration. The function of BMSCs is coordinated through multi-dimensional mechanisms such as physical antibacterial, immunomodulation, and intelligent drug release, effectively breaking the vicious circle of infection inhibiting bone regeneration. Although preclinical studies have shown significant efficacy, its clinical translation still faces challenges such as insufficient microenvironment response, function synergy contradiction, and biosafety. Future research needs to focus on the development of intelligent carriers and the construction of spatiotemporal sequential release systems, so as to promote bone regeneration strategies to be more intelligent and precise.

Objective To analyze the distribution of adverse events following immunization (AEFIs) in Zhaoxiang Town, Qingpu District, Shanghai from 2019 to 2023, and provide a basis for evaluating the safety of vaccination and improving the quality of vaccination services.Methods AEFI and vaccination data in Zhaoxiang Town, Qingpu District, Shanghai from 2019 to 2023 were collected through China Disease Control and Prevention Information System and Shanghai Integrated Vaccine Management and Vaccination Service Information System, respectively, and analyzed using descriptive epidemiological methods.Results From 2019 to 2023, a total of 504 AEFI cases were reported in Zhaoxiang Town, Qingpu District, Shanghai, with an average annual reported incidence of 355.18/100 000 doses. General reactions (99.01%) were mainly fever (45.24%) and local reactions (42.86%). Abnormal reactions (0.99%) were all allergic rash. There were slightly more males than females (1.20∶1.00), and children < 1 year old accounted for the highest proportion (53.77%). Among all AEFIs，83.73% occurred within 1 d after vaccination. Herpes zoster vaccine accounted for the highest reported incidence, followed by diphtheria, tetanus and acellular pertussis combined vaccine (DTaP), and DTaP-Haemophilus influenzae type b conjugate vaccine (quadruple vaccine) and 13-valent pneumococcal vaccine (conjugate) were also reported at a higher incidence.Conclusions AEFI in Zhaoxiang Town is dominated by general reactions, with a low incidence of abnormal reactions， which shows good safety of vaccination.It is recommended to focus on strengthening AEFI monitoring in children under 1 year old and within 24 h after vaccination.

Objective To evaluate the safety of combination vaccines containing diphtheria, tetanus, and acellular pertussis (DTaP) components in Nan’an District, Chongqing.Methods Data on adverse events following immunization (AEFI) reported for DTaP-containing combination vaccines and corresponding vaccination data in Nan’an District, Chongqing from 2020 to 2024 were collected through the Immunization Program Information Management System of the Chinese Center for Disease Control and Prevention and the Chongqing Immunization Program Information Management System.Results From 2020 to 2024, a total of 191 299 doses of DTaP-containing combination vaccines were administered in Nan’an District, Chongqing, with 296 AEFI cases reported. The reported AEFI rate was 154.73 per 100 000 doses. Among these, 291 cases were general reactions (152.12 per 100 000 doses) and 5 cases were abnormal reactions (2.61 per 100 000 doses). The male-to-female ratio was 1.33∶1.00. Children aged 18-24 months accounted for 70.95% of cases. Cases were reported throughout the year, with the majority (78.38%) occurring from April to September. The reported AEFI incidence rate for booster doses was higher than that for primary immunization, and the difference was statistically significant (χ²_trend = 266.21, P < 0.01).Conclusion From 2020 to 2024, AEFI reports for DTaP-containing vaccines in Nan’an District, Chongqing are predominantly general reactions, with rare abnormal reactions, indicating that these vaccines have a favorable safety profile.

T cell-mediated antitumor immune responses play a central role in the treatment of solid tumors. In recent years, multiple T cell-based therapeutic strategies, involving the expansion and adoptive transfer of tumor-reactive T cells, the engineering of antigen recognition in transferred T cells, and the in vivo redirection of endogenous T cells, have achieved important clinical advances and regulatory milestones in solid tumors. This review summarizes the major technological approaches and representative evidence for T cell-based therapies for solid tumors, focusing on tumor-infiltrating lymphocyte therapy, T-cell receptor (TCR)-engineered T-cell therapy, TCR-based CD3 T-cell engagers, chimeric antigen receptor T-cell therapy, and antibody-based CD3 T-cell engagers. Based on products approved, re-approved, or under pivotal development, this review compares the clinical translation patterns of different modalities and highlights structural constraints in target population coverage, safety management, manufacturability, access, and evidence maturity. Overall, precise patient selection, manageable immune-related toxicities, and mature manufacturing and supply systems will be key determinants of sustainable clinical translation.

Objective To analyze the epidemiological characteristics of varicella in Qingpu District, Shanghai, from 2020 to 2024, and to provide a basis for improving varicella prevention and control measures.Methods Varicella cases in Qingpu District, Shanghai, from 2020 to 2024 were collected from the Immunization Program Information Management System of the Chinese Center for Disease Prevention and Control. Descriptive research methods were used to analyze the temporal, regional, and population distribution of varicella cases. Data were organized and analyzed using SPSS 26.0 software, and the chi-square test was used to compare rates among multiple groups.Results A total of 1 225 varicella cases were reported in Qingpu District from 2020 to 2024, showing an overall downward trend in incidence (χ² _trend = 110.73, P < 0.001). The 2 high-incidence periods for varicella were from May to July and from November to January of the following year, respectively. Cases were reported in all subdistricts and towns, with a concentration in central urban areas including Xiayang Subdistrict and Xujing Town. Cases were distributed across multiple age groups, with the highest number of cases occurring in the < 5-year-old group, although its proportion showed a declining trend with constituent ratio among all cases decreasing from 22.34% in 2020 to 13.46% in 2024. Among all occupations, students accounted for the majority of cases (38.04%), followed by scattered children (15.10%).Conclusions The incidence of varicella in Qingpu District has declined during 2020-2024, but attention should be paid to key institutions and populations, and standardized outbreak response measures should be implemented promptly. Meanwhile, the coverage rate of varicella vaccine should be steadily increased.

Objective To explore the feasibility of detecting bevacizumab products from 3 manufacturers using the HEK293 reporter gene assay (RGA).Methods The biological activities of bevacizumab products from 3 manufacturers were detected by RGA respectively. The linearity, accuracy, and precision of this method were verified, and the effects of fetal bovine serum (FBS) concentration, cell seeding density, and cell culture passage number on RGA detection results were investigated.Results The four-parameter curves plotted for the RGA detection of bevacizumab products from 3 manufacturers showed that manufacturer 2 had a high signal-to-noise ratio, and manufacturer 2 and 3 exhibited high goodness of fit. When the theoretical relative potency ranged from 50% to 150%, the theoretical logarithm of relative potency showed a good linear relationship with the measured potency value. The recovery rates of the products from 3 manufacturers all fell within the range of 70% to 130%, and the relative standard deviations were all less than 20%. When the FBS concentration was 5% or 10%, the cell seeding density was 0.6×10⁵, 0.9×10⁵, or 1.2×10⁵ cells per well, and the cell passage numbers were 8, 16, or 23, the fitting curves of activity determination were all good. Conclusions RGA can be used to detect the biological activities of bevacizumab products from 3 manufacturers. This method has good linearity, accuracy, and precision, but the FBS concentration, cell seeding density, and cell passage number have certain effects on the detection results of RGA.

Objective To express the truncated glycoprotein E (gE) of varicella zoster virus (VZV) in eukaryotic cells using Chinese hamster ovary (CHO) cell expression system and evaluate its immunogenicity.Methods The VZV-gE 1-546 amino acid gene fragment was cloned into the vector pCHO1.0. The recombinant expression plasmid pCHO1.0-VZV-gE 1-546 was constructed and was transiently transfected into CHO cells, amplified and cultured, and the supernatant was harvested. Highly purified recombinant truncated gE, obtained through ion exchange chromatography, was mixed with a complex adjuvant (aluminum adjuvant + CpG 1018 adjuvant) in a certain proportion. Mice were immunized twice by intraperitoneal injection, and blood was collected to isolate serum for IgG antibody level detection. Three weeks after the last immunization, mice were euthanized and spleen lymphocytes were collected for enzyme-linked immunospot assay.Results Gene sequencing results showed that the recombinant expression plasmid pCHO1.0-VZV-gE 1-546 was constructed correctly, and the purity of the purified recombinant truncated gE reached over 95%. The IgG antibody level of anti-VZV and the geometric mean number of spots of IFN-γ and IL-2 cytokines in the serum of the experimental group mice after immunization were higher than those in the control group mice, and the complex adjuvant group was higher than the non-adjuvant group.Conclusion Recombinant truncated VZV gE is successfully constructed and expressed, which can induce a good immune response in mice.

Objective To analyze the epidemiological and spatio-temporal characteristics of varicella in Susong County from 2019 to 2024, in order to provide scientific basis for varicella surveillance, prevention and control.Methods Case data of varicella reported in Susong County from January 1, 2019 to December 31, 2024 were obtained from the China Disease Control and Prevention Information System . Descriptive epidemiological methods were used to analyze the epidemiological characteristics (temporal, population, and geographical distribution). SaTScan software (version 10.1) was employed to perform retrospective space-time scan statistics, with township as geographical unit and month as time unit, to identify spatio-temporal clusters of varicella incidence.Results A total of 1 091 varicella cases were reported in Susong County from 2019 to 2024. The annual reported incidence rate fluctuated between 14.23/100 000 and 35.39/100 000, with average annual incidence rate at 21.06/100 000. Based on chi-squared test for trend, a significant decreasing trend in the annual reported incidence was observed (χ² = 28.76, P < 0.01). The incidence exhibited a bimodal seasonal pattern, with primary peak occurring from October to January (autumn and winter) and secondary peak from May to July (late spring and early summer). Spatio-temporal scan analysis identified 4 statistically significant clusters (P<0.05), covering 10 townships. High-risk areas were mainly concentrated in the central and northern parts of the county, particularly in townships surrounding the central Fuyu Town. More cases were male (n=615, 56.37%) than female (n=476, 43.63%). Cases were predominantly observed in children and adolescents (<15 years old, 64.54%), with the highest incidence in the 5-9 age group (30.26%), followed by the 10-14 age group (23.46%). By occupation, students constituted the majority of cases (n=723，66.27%), followed by preschool children. A significant portion of the cases (78.00%) had no history of varicella vaccination.Conclusions From 2019 to 2024, the reported incidence of varicella in Susong County shows a significant declining trend. Students are the most affected population, and the disease distribution exhibits spatio-temporal clustering. To further control varicella outbreaks, it is recommended to continuously strengthen varicella surveillance, with a particular focus on cluster outbreaks in schools and childcare institutions, and to implement and improve the coverage of the 2-dose varicella vaccine schedule, especially for age-appropriate children and individuals with no prior immunization history.

Objective To observe the toxic reaction of aluminum hydroxide adjuvant intramuscular injection in mice.Methods In 50 specific pathogen free grade KM mice, preliminary experiments were performed with 10 female mice, and the remaining 40 mice (half males and half females) were randomly divided into treatment group and saline control group. High concentration (13 mg/mL) aluminum hydroxide adjuvant was administered by single intramuscular injection at dosage of 260 mg/kg. Mortality and toxic reactions were observed within 14 d after administration, and gross anatomy of systemic organs and histopathological examination of lesions were performed at the end of the study.Results The 20 mice in treatment group moved normally after intramuscular injection in the leg, and there was no death within 14 d.The body weight of female mice in treatment group was statistically significantly lower than that of control group on the 2nd and 3rd day after administration (t values were 2.23 and 2.61, respectively, P < 0.05), and the body weight of male mice in treatment group was statistically significantly lower than that of control group on the 2nd,3rd,7th and 14th day after administration (t values were 4.04, 3.93, 2.25 and 3.04, respectively, P < 0.05). The food intake of mice in treatment group was statistically significantly lower than that of control group on the 1st day after administration (t = 4.21, P < 0.01), and the water intake of mice in treatment group was statistically significantly lower than that of control group on the 1st and 3rd day after administration (t values were 3.29 and 3.49, respectively, P < 0.01). A white nodule with ~2 mm diameter was observed at the administration site of mice in treatment group, and no obvious pathological changes were observed in other tissues. Pathological examination showed that mice in treatment group presented partial degeneration and necrosis of muscle fibers, infiltration of inflammatory cells,partial proliferation of connective tissues and formation of a small number of megakaryocytes， as well as a small amount of bleeding at the injection site. The mice in control group showed no abnormality during the experiment.Conclusion A single intramuscular injection of high-dose aluminum hydroxide adjuvant in mice can cause muscle irritation at the injection site to a certain extent, food intake and water consumption reduction, and body weight loss, but no other abnormalities.

Objective To establish and validate a double-antibody sandwich ELISA for the detection of rabbit milk protein residues in recombinant protein drugs expressed by transgenic rabbit mammary gland bioreactor.Methods Polyclonal antibodies were obtained by immunizing sheep with non-transgenic rabbit milk, and a double-antibody sandwich ELISA for detecting rabbit milk protein residues was established. The method was validated for specificity, accuracy, precision and ruggedness, and its limit of quantitation, linearity and range were determined. The established method was applied to detect the residual amount of rabbit milk protein in recombinant human C1 esterase inhibitor expressed by transgenic rabbit mammary gland bioreactor.Results The average absorbance value difference of specificity test between 450 and 630 nm was ≤0.1, with good linearity at the concentration range of 5-800 ng/mL and limit of quantitation of 5 ng/mL. The recoveries of low, medium and high spiked samples were 111.0%, 117.5% and 110.4%, with relative standard deviations (RSDs) of 5.0%, 4.7% and 8.9%, and RSDs for intermediate precision of 7.9%, 8.5% and 7.6%, respectively. For the ruggedness test, RSDs of the determined values of low, medium and high spiked samples under 3 different experimental conditions were 8.0%, 4.7% and 7.6%, respectively.Conclusion A double-antibody sandwich ELISA for the detection of rabbit milk protein residues in products expressed by the transgenic rabbit mammary gland bioreactor is successfully established, which has good specificity, accuracy, precision, ruggedness and sensitivity.