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Column on Quality Evaluation Techniques and Methods for Gene Therapy Products(1)
Editor: Invited Column Editor-in-chief： ZHOU Yong

    Gene therapy in a broad sense refers to the introduction of nucleic acid fragments into cells in an appropriate manner, enabling the genetic information of nucleic acid sequences to perform corresponding functions and achieve the goal of treating diseases.

    Since the 1990s, gene therapy has developed rapidly. In this century, a number of gene therapy products at home and abroad have been approved for marketing, of which seven varieties of recombinant adeno-associated virus (rAAV) vector gene therapy products have been approved for marketing worldwide. In recent three years, 26 rAAV products have been approved for clinical trials, with indications including eye diseases, blood system diseases, nervous system diseases and infectious diseases, serotypes involving type 2, 5, 8, 9, and 10.

    As more and more gene therapy products enter clinical practice, potential safety risks are gradually exposed. Therefore, regulatory authorities in various countries are also accelerating the development of relevant regulations and technical guidelines to guide the research and evaluation of gene therapy products. The 2020 edition of the Pharmacopoeia of the People’s Republic of China, Part Three “General Introduction to Human Gene Therapy Products”, and the relevant guiding principles for rAAV gene therapy products issued by the Drug Evaluation Center (CDE) of the National Drug Administration have put forward principled requirements for quality control. However, as rAAV is a “live” virus, its production and quality control require higher requirements, and the current testing methods still need to be continuously improved, the durability of the method and the repeatability of the results also need to be improved. Therefore, in order to promote the early application of new concepts, technologies, tools, and standards in the quality control of gene therapy products, a “Column on Quality Evaluation Techniques and Methods for Gene Therapy Products” has been specially established to showcase the relevant research progress of quality analysis technology. The column includes 9 articles, which are divided into 2 issues due to their long length. The topics and focus of this issue includes: ①Critical considerations for quality control testing technology of recombinant adeno-associated virus（rAAV）gene therapy products, which introduces the exclusive testing project settings and method specific inspection points for rAAV products. ②Biological activity detection method for gene transcription level of rAAV2-ND4 injection, focuses on introducing a biological activity detection method with dual calibration of internal reference genes and reference substances. ③Study on digital PCR methods for rAAV5 genomic titer determination, which focuses on the comparison between micro droplets and chip digital PCR. ④Analysis of the proportion content of recombinant adeno-associated virus (rAAV) capsid protein by CGE-LIF, which describes the detection method for relative quantification of capsid protein subunits using CE-LIF technology. ⑤Analysis of particle titer and full rate of a recombinant adeno-associated virus (rAAV) by SEC-UV-MALS-RI technique, which shares the application of SEC-MALS combined technology in quality control of rAAV products.

    This column is established to raise the attention of the industry and research institutions to the quality of rAAV products, continuously improve the understanding of practitioners in rAAV product quality control, improve inspection and testing capabilities, and explore and establish corresponding experimental plans to promote the resolution of quality control problems, thereby promoting the construction of the quality control system and technical specifications for rAAV gene therapy products in China, and accelerating the high-quality development of gene therapy products.

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  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products (1)
    Critical considerations for quality control testing technology of recombinant adeno-associated virus(rAAV) gene therapy products*
    QIN Xi, YU Lei, TAO Lei, BI Hua, WANG Guang-yu, SHI Xin-chang, ZHOU Yong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(11): 1813-1819. https://doi.org/10.16155/j.0254-1793.2023.11.01
    Abstract (55) PDF (35)   Knowledge map   Save
    In recent years, the domestic gene therapy industry has developed rapidly, and innovative gene therapy products are emerging constantly. How to effectively control the quality of such products has become a highly concerned issue in the industry. This article draws inspiration from relatively mature, effective, and practical methods in the field of quality control of gene therapy products. Taking recombinant adeno-associated virus gene therapy products as an example, starting from the selection of testing items and methods, precautions, and formulation of quality standards, it elaborates on their quality control inspection items and related technical requirements. We hope this article can provide reference for practitioners to conduct quality control research on such products, and through subsequent communication and discussion, a certain consensus can be reached, thereby accelerating the development of gene therapy industry in China.
  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products (1)
    Biological activity detection method for gene transcription level of rAAV2-ND4 injection*
    QIN Xi, CHEN Long, SHI Xin-chang, YANG Jing-qing, PAN Yan-qun, YU Lei, BI Hua, PEI De-ning, AN Yi-fang, PAN Yue, LI Xiang, ZHOU Yong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(11): 1820-1825. https://doi.org/10.16155/j.0254-1793.2023.11.02
    Abstract (36) PDF (14)   Knowledge map   Save
    Objective: To establish and validate a biological activity detection method for detecting the transcription level of rAAV2-ND4 injection. Methods: rAAV2-ND4 sample (3.0×1010 vg·mL-1 at 360 μL per well) was infected with HEK293T cells (3.0×105 cells·mL-1, 0.8 mL per well, pre-cultured at 37 ℃ with 5% carbon dioxide for 24 h), followed by 2 h of culture. An appropriate amount of culture medium was added and continued to culture for 24 h, then the cells were collected. Cell total RNA was extracted according to the operating instructions of the cell total RNA extraction kit (cell lysis, adsorption, deproteinization, impurity removal, and elution). Using the extracted total RNA of cells as a template, the Ct values of mRNA and β-actin internal reference gene of ND4 were detected using qPCR method. By simultaneously setting up rAAV2-ND4 reference samples for parallel testing,the relative biological activity of rAAV2-ND4 was detected using double calibration detection with β-actin internal reference genes and the rAAV2-ND4 reference substance. The methodological validation included accuracy, precision, linearity, and specificity. The inter batch consistency of the three products was also investigated. Results: The results of qPCR detection of ND4 mRNA transcription and reference correction method indicated a dose-response relationship between ND4 mRNA and rAAV2 ND4 infect titers. According to the verification of methodology, the deviation rate between the detection value and the expected value of the method was between 100% and 130%. The RSD of 9 results for different personnel at different times was 13.75%. The method was linearity from 1.0×1010to 5.0×1010 vg·mL-1 of rAAV2-ND4 titer, r>0.97. The method had good specificity. The RSD of the three batches of rAAV-ND4 detection values was 9.3%. Conclusion: A preliminary biological activity detection method for rAAV2 ND4 transcription level has been established.
  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products (1)
    Study on digital PCR methods for rAAV5 genomic titer determination*
    ZHENG Hong-mei, QIN Xi, LI Yong-hong, YU Lei, BI Hua, RAO Chun-ming, ZHU Liu-qiang, YANG Jing-qing, SHI Xin-chang, ZHOU Yong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(11): 1826-1832. https://doi.org/10.16155/j.0254-1793.2023.11.03
    Abstract (34) PDF (17)   Knowledge map   Save
    Objective: To establish and verify a digital PCR method for the determination of recombinant adeno-associated virus type 5(rAAV5) genome titer, and to compare the performance of chip and droplet digital PCR methods. Methods: By examine the different lysing outcome from various concentrations of SDS or EDTA, the adeno-associated virus lysis method was optimized. To ensure other experimental conditions being consistent, the rAAV5 sample was amplified with WPRE primers in the droplet digital PCR method, and the annealing temperature was set to 60, 58, 54, 52 ℃. With amplification effect compared, the annealing temperature was optimized. Both chip and droplet digital PCR were tested to determine the genomic titer of rAAV5, and the consistency of the results of the two methods was compared. Results: The final concentration of 1% SDS and 62.5 mmol·L-1 EDTA was mixed 1∶1(v/v) and then rAAV5 was lysed. The measured genome copy number was high, the lysing outcome was good. At annealing temperature 54 ℃, the positive droplets in the droplet digital PCR and the separation effect of negative droplets was ideal, and the amplification specificity is the strongest. Comparing the two methods, it was found that the laboratory precision and recovery rates of the two determination results are similar, and the results of determining the same pyrolysis sample were relatively close (on the same order of magnitude). Conclusion: A preliminary digital PCR method has been established and validated, laying the foundation for further validation in the future.
  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products (1)
    Analysis of the proportion content of recombinant adeno-associated virus (rAAV) capsid protein by CGE-LIF*
    LI Xiang, GAO Tie, SHI Xing-chang, CHEN Hong-xu, TANG Hong-mei, QIN Xi, ZHOU Yong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(11): 1833-1839. https://doi.org/10.16155/j.0254-1793.2023.11.04
    Abstract (35) PDF (15)   Knowledge map   Save
    Objective: To established a capillary gel electrophoresis tandem laser induced fluorescence detector (CGE-LIF) method to analyze the proportion content of recombinant adeno-associated virus (rAAV) capsid proteins VP1, VP2 and VP3, and to verify the methodology. Methods: The CGE-LIF analysis method for rAAV capsid protein was as follows: rAAV samples were pre denatured with a solution of 4% SDS-150 mmol·L-1 NEM (70 ℃, 5 min); labeled with P503 dye (70 ℃, 10 min); denatured with 1% SDS (70 ℃, 5 min), then the processed rAAV samples were mixed well and tested. The method validation included accuracy, repeatability, linearity, detection limit, quantification limit, durability, etc. In addition, the conditions of P503, TAMRA, and FQ dyes, as well as the consistency between different serotypes and between some batches of rAAV were investigated. Results: CGE LIF analysis of different serotype of rAAV (including types 2, 5, 8, 9, and 10) could separate their capsid proteins VP1, VP2, and VP3 at baseline and had sharp peaks. The accuracy showed that the correlation coefficient r was >0.99 between the measured results of capsid proteins VP1, VP2, and VP3 and the expected results. The repeatability results showed that the migration time RSD of each capsid protein VP1, VP2, and VP3 was less than 1.5%, and the peak area percentage RSD was less than 5%. The intermediate precision results showed that the migration time RSD of capsid proteins VP1, VP2, and VP3 were all less than 1.5%, and the peak area percentage RSD were all less than 5%. The linear result was displayed from 0.6×1012 vg·mL-1 to 3.0×1012 vg·mL-1, within the range, there was a good linear relationship with the peak area of capsid protein. The detection limit and quantification limit of VP3 were 7.2×1010 vg·mL-1 and 2.2×1011 vg·mL-1. Three different dyes (P503, FQ, and TAMRA) were used for CGE-LIF detection, and the results were good. The inter batch stability results of this method were relatively ideal. Conclusion: A CGE-LIF analysis method for rAAV capsid protein has been established, which can separate and quantify the proportion of three capsid proteins in rAAV products.
  • Column on Quality Evaluation Techniques and Methods for Gene Therapy Products (1)
    Analysis of particle titer and full ratio of a recombinant adeno-associated virus (rAAV) by SEC-UV-MALS-RI technique*
    LI Xiang, SHI Xin-chang, WANG heng, HAN Chun-mei, QIN Xi, ZHOU Yong
    Chinese Journal of Pharmaceutical Analysis. 2023, 43(11): 1840-1846. https://doi.org/10.16155/j.0254-1793.2023.11.05
    Abstract (56) PDF (18)   Knowledge map   Save
    Objective: To analyze the particle titer and full rate of a recombinant adeno-associated virus (rAAV) by using size exclusion chromatography (SEC) with tandem ultraviolet detector (UV), multi angle laser scattering detector (MALS) and differential detector (RI). Methods: SEC-UV-MALS-RI series analysis was performed on an rAAV, its empty shell reference substance, and a mixture of the two samples. The mobile phase was 50 mmol·L-1 phosphate buffer, with a flow rate of 0.3 mL·min-1, and a sample loading volume of 50 μL. The temperature is room temperature and the collection time was 30 min. The UV detector selected dual channels of 260 nm and 280 nm. 18 angle laser scattering detection and differential detection adopted the system default settings. The results were analyzed using WYATT software. Results: The particle titer of the sample was 4.816×1012 vp·mL-1, with an RSD of 0.37%. The particle titer of the empty shell reference substance was 3.199×1012 vp·mL-1, with an RSD of 0.75%. The particle titer RSD of the 5 samples mixed according to the preset different ratios were all within 3%, and the RSD of full ratio was all within 4%. The measurement results of particle titer and full ratio of the mixed sample were basically consistent with the expected values. Conclusion: The SEC-UV-MALS-RI method can be used to analyze the particle titer, full ratio, and other items of rAAV products, which is suitable for quality control of particle titer and full ratio of rAAV products.
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