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 construct a granulocyte-macrophage colony-stimulating factor (GM-CSF) Fc and DXd conjugate (GM-DXd), and explore its therapeutic effect on targeted killing of acute myeloid leukemia (AML) cells. Methods The GM-CSF receptor (GMR) targeting GM-DXd was prepared, and identified by reducing SDS-PAGE. The biological activity of GM-DXd was detected by Western blot, and the internalization of GM-DXd by AML cell lines MV4-11 and THP-1 was detected by flow cytometry. The viability inhibition and apoptosis induction in GMR-positive AML cells by GM-DXd were assessed by cell proliferation-cytotoxicity detection and flow cytometry, respectively, and the expression of apoptotic proteins in GMR-positive AML cells was detected by Western blot. Results The GM-DXd prepared had stable biological activity and was successfully endocytosed by AML cells. Compared to control group, GM-DXd inhibited AML cell viability and the median inhibitory concentrations to MV4-11 and THP-1 cells were 15.91 and 37.64 nmol/L, respectively. GM-DXd (20 nmol/L) greatly promoted apoptosis in AML cells with ~90% and ~60% apoptotic cells in MV4-11 and THP-1 cells, respectively, and the AML cell killing ability was stronger than DXd only. Conclusion The small molecule conjugate drug GM-DXd can effectively kill AML cells by targeting GMR and can be a potential drug for the treatment of AML.

Objective To optimize the culture medium for Chinese hamster ovary (CHO) cells expressing anti-human IL-17A/F monoclonal antibody (anti-hIL-17A/F), and screen domestic culture media with high expression and comparable quality of antibody. Methods A CHO cell line expressing anti-hIL-17A/F was used to optimize the culture medium. The optimal basic medium and feed medium combination was selected, and the feed strategy optimization and 2 L bioreactor screening were carried out. Then the antibody expression and quality were measured in bioreactor final confirmation.Results The combination of basic medium BM-01, feed medium FM-01 and FM-02 was the optimal medium combination for the expression of anti-hIL-17A/F in recombinant CHO cells. Among them, the feed medium FM-01 was supplemented with a feed strategy of day3/6/8/10/12 (3%-7%), and the ratio of FM-01 to FM-02 was 10∶1. In 2 L bioreactor， the protein expression exceeded 6.5 g/L, more than twice of that of the original process, and the product quality was consistent.Conclusion High expression and comparable quality of anti-hIL-17A/F in CHO cells are achieved by using the combination of basic medium BM-01, feed medium FM-01 and FM-02, which lays a foundation for large-scale production of anti-hIL-17A/F using domestic culture media.

Objective To investigate the effect of replacing analytically pure phosphate with pharmaceutical grade phosphate on critical quality attributes of diluent for meningococcal polysaccharide vaccine. Methods The quality attributes of analytically pure and pharmaceutical grade dibasic sodium phosphate and potassium dihydrogen phosphate, including pH, insoluble matter in water and drying weight loss, were compared and analyzed according to Chinese pharmacopoeia 2020 edition volume 4. The key quality attributes of vaccine diluents prepared with phosphates of different grades were compared and their stability was investigated. Results There was no statistically significant difference in the quality of two phosphates (t=-1.38-1.67，P=0.126-0.954) and diluents produced with two phosphates (t=0.09-0.26，P=0.376-0.524). The quality of both diluents complied with the requirements of Chinese pharmacopocia and standard of enterprise.Both diluents were stable within 36 months storage under 25 ℃±2 ℃ and 60%±5% relative humidity. Conclusion The quality of pharmaceutical grade phosphate meets the regulatory requirements, which can ensure the stable and controllable quality of meningococcal vaccine diluent, and analytically pure phosphate can be replaced.

Objective To evaluate the sterilization effectiveness of sterilization and filtration process of live attenuated Japanese encephalitis vaccine (JEV) final bulk. Methods Filter performance and sterilization effectiveness were analyzed by testing filter integrity, extractables, bacterial viability, bacterial challenge levels, and chemical compatibility in the sterilization and filtration process of JEV final bulk. Results Under the conditions of production process, the maximum diffusion flow of JEV final bulk (simulated solution) was 11.6 mL/min, the minimum foaming point was 3.200×10⁵ Pa, and the solution tested was not bactericidal at 2-8 ℃ and room temperature. Under the worst conditions, defective short-wave Aeromonas with a challenge level of 10⁷ CFU/cm² effective filtration area was retained, virus titers and pH adsorption measurements after filtration of JEV final bulk were within acceptable range, and there was no interaction between final bulk and filters. Conclusion The filters are structurally intact after contact with final bulk with good bacterial viability, bacterial challenge and chemical compatibility, and are suitable for the production process of JEV.

Objective To establish a double antibody sandwich ELISA for quantitative detection of recombinant porcine trypsin (RPT), and to perform methodological validation and preliminary application. Methods A double antibody sandwich ELISA for quantitative detection of RPT content was established.Paired mouse monoclonal antibodies against RPT were selected through antibody screening, optimal working concentrations of coating and enzyme-labeled antibodies as well as type and concentration of blocking agent were determined. The linear range, sensitivity, accuracy, precision and specificity of the method were determined. This established method was used to detect the content of RPT in viral background liquid during the vaccine production, and to verify the detection efficiency of this method for different brands of natural extracted porcine trypsin. Results The coating antibody was determined to be RPT-5C2D12 with optimal working concentration of 6 000 μg/mL， the enzyme-labled antibody was RPT-1E10A5 with optimal dilution of 1∶5 000, and the blocking solution was 1% bovine serum albumin. A four-parameter logistic fitting standard curve achieved correlation coefficient ≥0.990 and detection range of 0.156-40.000 ng/mL. Limits of quantification was 0.156 ng/mL. Spike recovery rate for accuracy ranged from 92.3% to 106.7%. Precision validation showed inter-assay coefficient of variation (CV) ≤6.7% and intra-assay CV ≤5.9%. Except for RPT, there was no cross-reactivity with other control proteins. The spike recovery rate for three batches of viral background fluid samples ranged from 87.4% to 98.2%. The relative detection efficiency for two brands of natural extracted porcine trypsin was 59% and 68% compared to RPT. Conclusion The established double antibody sandwich ELISA for quantitative detection of RPT has good linearity, sensitivity, accuracy, precision, and specificity, and can be used for assessing trypsin residues in vaccine type biological products.

Objective To validate the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of Berol 185 residues in diphtheria, tetanus and pertussis (acellular, component) vaccine (DTacP) . Methods LC-MS/MS was used to screen quantitative transitions, external standard method was used to quantify Berol 185 in the samples, and the methodology was verified. Results The quantitative transition of 630.05→89.10 was screened for final quantification. Linearity range was 5-120 μg/L (coefficient of determination>0.990 0, and the recovery rate was 80%-120%). The relative standard deviations (RSDs) of reproducibility were 1.8%-4.2%, the RSD of intermediate precision were 3.0%-3.6%, and the recoveries were 96.1%-111.9%. The results were stable at different temperatures (35, 37 and 40 ℃) or different detection times (0, 4, 8, 12, 16, 20, 24 h). Conclusion The method has good linearity, precision, accuracy and durability, and can be used for quantitative detection and process monitoring of Berol 185 in DTacP.

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.

The drug qualified persons（QPs） engage in the quality management activities of pharaceutical enterprises, take responsibilities for the product release，and ensure that the production and inspection of each batch of released products meet the relevant laws and regulations, drug registration requirements and quality standards.The professional ethics and legal consciousness of the drug QPs directly affect the quality and safety of drugs. This paper states the basic situation and existing problems of the implementation of the drug QP system in China, as well as puts forward professional ethics requirements for the drug QP, and sorts out the legal responsibilities and risks that the drug QP needs to bear, aiming to improve the professional quality and legal awareness of drug QPs and further ensure the ability of drug QPs to perform their duties.

Legionella pneumophila is the pathogen of Legionella pneumonia. With the prevalence of Legionella pneumonia, the research and development of vaccines is increasingly urgent. This paper summarizes the status and achievements of vaccine development since the initial outbreak of Legionella pneumonia in 1976. This paper highlights three major vaccines with greater application prospects, including the recombinant protein vaccine, nucleic acid vaccine and epitope vaccine. Meanwhile, the advantages and shortcomings of various Legionella pneumophila vaccines are briefly reviewed, and some new ideas for the research of Legionella pneumophila vaccine are proposed.

Pertussis is a highly contagious respiratory disease. With the increase in pertussis cases in recent years, it has become more and more concerned. Currently, vaccination is the most effective prevention method for pertussis, and developing new vaccines is a major priority in pertussis prevention and treatment. This paper summarizes the current pertussis epidemiological trends, analyzes the causes of pertussis resurgence, and tracks the latest progress in pertussis vaccine development, in order to provide a reference for developing novel pertussis vaccines.

Adenovirus vectors, renowned for their efficacy as gene delivery tools, have garnered significant attention in vaccine research over the past decades. Currently, adenovirus vectors have been successfully applied to the vaccine development of Ebola virus and severe acute respiratory syndrome coronavirus 2, and have shown good protection and safety. However, the application of adenovirus vectors in vaccines still faces challenges, such as interference of pre-existing antibodies and poor targeting. This article provides a review of the application, existing problems, and improvement directions of adenovirus vectors in vaccine research.

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.