Objective  To establish investigation and analysis for microbial data deviation (MDD) in the laboratory and apply it to practice. Methods  By consulting literature, a laboratory MDD investigation process was established. The investigation and analysis were conducted on a case of MDD exceeding the standard in a sterility test. Results  From the 12th day, there was bacterial growth in the sample in culture medium, and the identification result was Achromobacter denitrificans. The investigation results indicated that the bacteria detected in this case were not caused by contamination during laboratory testing, but from the sample itself. Conclusion  A preliminary laboratory MDD investigation and analysis scheme is established.

Objective  To investigate the feasibility and influencing factors of the two mycoplasma detection methods in viral-vectored products.  Methods  Two different mycoplasma nucleic acid amplification detection methods based on PCR technology were used to detect mycoplasma in two recombinant Sendai virus vector vaccines respectively expressing type II herpes simplex virus antigen gD and ICP27 (SeV-dF/HSV-2 gD、SeV-dF/HSV-2 ICP27). The specificity and minimum detection limit of the mycoplasma detection in harvest and purified liquids from vaccine products were studied, and the mycoplasma contamination in multiple batches of harvest liquid and purified liquid was detected.  Results  There was no PCR inhibition to detect mycoplasma with normal PCR method in virus harvest liquid, but the inhibition was obvious in purified liquid, and M.orale and M.pneumoniae at 100 CFU/ml were not detected. The real-time quantitative PCR (qPCR) method had no such inhibition in the detection of viral harvest and purified liquid. Moreover, the sensitivity of qPCR for M.orale, M.pneumoniae and other mycoplasma reached 10 CFU/ml, and the sensitivity of the nucleic acid copy number of M.orale and M.pneumoniae reached 50 genome copies per ml.  Conclusion  The qPCR method has higher specificity and sensitivity, and it can be used as a quality internal control method for rapid detection of viral vector products and timely detection of mycoplasma contamination to ensure product quality.

Viral biological products include viral vaccines and various viral vector drugs for gene therapy and oncolytic virotherapy. In the process of research and production，accurate and rapid assay methods should be established to evaluate the process and final products. Titration is essential for the detection of the viability and potency of viral biological products，and is one of the key indicators to evaluate the quality of biological products. This article mainly reviews the principles，characteristics and research progress of virus titration methods，including virus plaque formation assay，half-infection dose measurement method，quantitative polymerase chain reaction，plaque focus immunochemical method，and reporter gene assays.

Objective  To develop and optimize the perfusion culture process for shear stress sensitive mammalian cell line to obtain high protein production. Methods  High-density seed cells were obtained by N-1 seed stage perfusion process for N stage intensified fed-batch process development. The perfusion medium suitable for the cell line was screened out and the process was further optimized at TPP tube scale. The scale-up and process optimization in benchtop bioreactors was then executed. Results  The unit volume capacity of the hybrid process of "seed perfusion process+intensified fed-batch process" was increased by 46% compared to original fed-batch process. Using the established medium rating system, one mixed medium (basal+feed medium) and one commercial perfusion medium were screened out and were scaled up to 2 L benchtop bioreactors. The mixed medium process increased the unit volume capacity by 335%, and the commercial perfusion medium process increased it by 460%. Conclusion  The perfusion culture processes for shear stress sensitive mammalian cell line at N-1 and N stages are established respectively, and designed for best production capacity at N stage.

Vaccination is a common method for the prevention of infectious diseases. Because live attenuated vaccines have the possibility of infection, identifying wild type and vaccine type viruess is the basis for identifying pathogen and performing diagnosis. Traditional identification methods include gene sequencing，PCR-restriction fragment length polymorphism analysis，etc. These identification methods with high accuracy are commonly used，but they also have the disadvantages of long time and high requirements. Real-time quantitative PCR technology reflects the amplification process through the interaction of fluorescent substances and nucleic acid fragments in the PCR reaction system. Real-time quantitative PCR technology has the advantages of high specificity，high sensitivity, and short time consuming besides the ability to identify wild type and vaccine type viruses with comparable detection accuracy， which can help with rapid identification and clinical diagnosis.

Objective  To establish and culture primary hamster kidney cells (PHK cells) by using 40-layer cell factory（CF40) process, the production process of live attenuated Japanese encephalitis vaccine was optimized. Methods  Cultured PHK cells of different inoculation densities with different concentrations of newborn calf serum.Compared the number of PHK cells in each group, and determined the PHK cell inoculation density and newborn calf serum concentration of the CF40 process. By comparing the PHK cell culture situations with different newborn calf sera in CF40 and 15 L rolling bottle, the selectivity of the two processes on newborn calf serum was compared. A one-way ANOVA was performed on the number of PHK cells cultured in the cell factories of 4 manufacturers to determine the selectivity to the cell factories. Results The optimal inoculation density of PHK cell in the CF40 process was 6.1×10⁵ ml^-1, and the optimal concentration of newborn calf serum was 6%. The CF40 process was less selective for newborn calf serum, and cultured more PHK cells more stably than the rolling bottle process. There was no statistically significant difference in the number of PHK cells cultured in the cell factories of the 4 manufacturers (F=0.23,P=0.873)，and the selectivity to cell factories was low.  Conclusion  CF40 can be used to prepare PHK cells that meet the requirements for the production of live attenuated Japanese encephalitis vaccines.

Objecive To investigate potential influencing factors on the mRNA content detection in mRNA vaccines by RiboGreen method. Methods The mRNA contents of mRNA vaccines were detected and analyzed using the RiboGreen fluorescent dye under different lysis buffer concentrations, lysis time, sample dilutions and reference standards. Results There was no statistically significant difference in the results of mRNA content when samples were diluted to 0.5-1.5 μg/ml and lysed in 0.1%-2.5% Triton X-100 lysis buffer for 5-30 min. There was statistically significant difference in the results of mRNA content with different reference standards. Conclusion Based on the recovery of mRNA contents, the conditions are determined initiatively for mRNA content detection in mRNA vaccines.

Objective  To evaluate the feasibility of preparing bacterium suspension by recovering the standard bacterium in equal volume and culturing 18-24 h for microbial verification. Methods  Standard bacteria were prepared in equal volume. Then the recovered strains were counted after storing for 0, 3, 6, 9, 12, 13 months. The colony counting results were analyzed by single-factor analysis of variance. Results  Compared with 0 month, the counts of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans after storing for 3, 6, 9, 12, 13 months were stable and of no statistically significant difference, with F values of 2.24, 2.21, 0.58 and 1.59, respectively, and all P values >0.05. Conclusion  Using recovered standard bacterium in equal volume to prepare the bacterium suspension for microbial verification of biological products is feasible,and with bacterium concentration stable .

Virus detection plays an increasingly important role in viral infection prevention, diagnosis, treatment and quality control of viral vaccines. Through detecting the fluorescent signals related to the amplified quantity of the target gene in real time, real-time fluorescent quantitative PCR (qPCR) is the most commonly used virus detection technology with high sensitivity, rapid detection and low pollution. This article describes the application of qPCR in the rapid diagnosis of virus infectious diseases, the quantification of viral nucleic acid and the identification of virus types.  

As a new substitute for antibodies, affibody not only binds to target protein specifically with high affinity, but also has many characteristics such as relatively small molecular mass (approximately 6 500), strong penetration, weak immunogenicity and simple preparation process. Therefore, affibody has more prominent advantages and feasibility in tumor targeting therapy, in vivo imaging diagnosis and biotechnology application. The characteristics of affibody and research progress of application are reviewed in this paper.

Objective  To investigate the data fitting model of ELISA for determination of gentamicin residues in attenuated Japanese encephalitis vaccine (JEV), so as to validate and optimize the quantitative detection system. Methods  The JEVs were tested for gentamicin residue using commercial gentamicin residue ELISA kit, and the test data were fitted respectively through quadratic polynomial model, log-linear model and four-parameter logistic model. Then the indicators including the coefficient of determination (R²), experimental point distribution and recovery rate were analyzed and compared. Results  The variables tests of quadratic polynomial model, log-linear model and four-parameter logistic model were all of statistical significance (P<0.05), and the mean values of R² were 0.997 0, 0.994 4 and 0.999 3, respectively, among which the quadratic polynomial model and four-parameter logistic model presented the best fitting. The distribution of standard points on both sides of the standard curve was uniform for all models, and the four-parameter logistic model was the best, especially in the range of medium and low concentrations (0.1-0.9 ng/ml). The recoveries of standard substances in the concentration range of 0.1-8.1 ng/ml of each fitting model met the requirements of Chinese pharmacopoeia, and the differences were not statistically significant. Conclusions  Quadratic polynomial model, log-linear model and four-parameter logistic model are all suitable for the data analysis of ELISA for gentamicin residues, but the quadratic polynomial model and four-parameter logistic model fit well, which has important guiding significance for continuous optimization of this quantitative determination.

 Objective  To explore the disinfection method of disinfectant in clean area and ensure effective disinfection of clean area. Methods   Bromogeramine and peracetic acid were used to disinfect the clean area, the microbial limit was detected, and disinfectant residue was tested. The influence of disinfected area, materials of contact surface, and disinfection time on disinfection effect was examined. Results  With either disinfectant, replacing the cleanroom wipers fulfilled with  disinfectant every 4 m² and disinfectant contact with the clean interface for 5 min obtained the best microbial elimination effect. After the second wipe with detergent, the residual amount of disinfectant did not affect human health and product quality. Conclusion  A suitable method for disinfection in clean areas was established.

The clustered regularly interspaced short palindromic repeat and associated protein (CRISPR-Cas) system, derived from the adaptive immune mechanism of prokaryotes, quickly stands out from many competitive gene-editing technologies by its high specificity, high exploitability, simplicity, and efficiency. Not only has it brought revolutionary breakthroughs to in vivo gene-editing technology, but it has also opened up a new world in the field of in vitro diagnostics. In recent years, many fast, portable, economical, and efficient in vitro diagnostic technologies based on the CRISPR-Cas system have emerged. These techniques show extraordinary performance in pathogen detection as well as having unlimited potential in tumor gene diagnosis, genetic disease screening, transplant rejection detection, etc. This article only briefly describes the working principle of the CRISPR-Cas system, the detection methods based on the CRISPR-Cas system, and the application progress in the field of pathogen nucleic acid detection.

Objective  To establish an accurate and rapid analytical method to detect the content of β-propiolactone (BPL) in inactivated vaccines. Methods  The content of BPL was determined by gas chromatography and calculated by external standard method. The specificity, system adaptability, linearity and range, accuracy, precision, detection limit, quantitative limit and durability of the analytical method were verified. Results  The peak area of BPL in BPL solution and sample solution was observed during the corresponding retention time, and there was no BPL peak observed in reference solution. The relative standard deviation (RSD) of BPL peak was less than 2% and the degree of separation from other peaks was greater than 1.5. The concentration of BPL in the range of 1.11-35.52 μg/ml was linearly related to the peak area. The recovery rates of 2.22, 4.44, and 8.88 μg/ml solution samples were between 97.3% and 106.9% , and the RSDs were all less than 3%. The detection limit was 0.30 μg/ml, and the quantitative limit was 0.49 μg/ml. Conclusion  The established method has good specificity, system adaptability, linearity and range, accuracy, precision and durability, and is accord with requirements of quantitative measurement.

Objective To improve the sterility test of biological products with mercuride. Methods Sodium thiosulfate was added to 0.1% peptone aqueous solution and used as flushing buffer to eliminate the bacteriostasis of biological products with mercuride. Validation of microbial recovery was used to identify the toxicity of sodium thiosulfate flushing buffer at different concentrations. The optimal concentration was selected by two vaccine samples. The effectiveness of the optimal concentration was confirmed through the sterility test applicability verification by membrane filtration method. Results  Sodium thiosulfate-contained flushing solution at concentration of 0.05-0.80 mol/L was non-toxic to the tested micro-organisms. Flushing buffer with 0.10 mol/L sodium thiosulfate could be used in sterility test of biological products with mercuride. Introduction of sodium thiosulfate greatly reduced the amount of flushing solution and the time for test. Conclusion  The sterility test of biological products with mercuride is successfully improved by adding sodium thiosulfate.

Objective  To optimize the chromatography colorimetry method and verify by analytic methodology, to compare optimized chromatography colorimetry with chromatography integration, and to evaluat the applicability of the two methods to determine the molecular size of typhoid Vi refined polysaccharide. Methods  The concentration range of O-acetyl standard curve of colorimetry was optimized, and its accuracy, repeatability, intermediate precision, linearity and durability were verified. The molecular size of multiple batches typhoid Vi was tested using chromatography colorimetry and chromatography integration, and the results were statistically analyzed. Results  The accuracy recovery rates of the optimized chromatography colorimetry were all 100%. The relative standard deviations（RSDs) of corresponding repeatability of samples were both＜5.0%, the RSDs of intermediate precision were both 3.4%. The standard curve was analyzed for 5 time, and coefficients of determination were all greater than 0.9990. The RSD of durability was 4.6%. There was no statistically significant difference between optimized chromatography colorimetry and chromatography integration in the detection of molecular size of typhoid Vi refined polysaccharide (t=-1.372, P > 0.05). Conclusions  The optimized chromatography colorimetry has good accuracy, repeatability, intermediate precision, linearity and durability. Both methods are applicable in detecting the molecular size of typhoid Vi refined polysaccharide.