目的:利用数字PCR(dPCR)技术检测重组腺相关病毒(recombinant adeno-associated virus, rAAV)产品中质粒DNA残留。方法:针对质粒抗性基因KanR序列的4个不同区段设计引物探针,建立单重和双重dPCR检测KanR残留量;在双重dPCR中,双阳性孔代表2组引物对之间的序列完整,通过测试单、双阳性孔的比例,评价KanR序列的分布情况;利用DNase Ⅰ消化游离的质粒DNA,评价病毒衣壳内错包装的质粒DNA情况。结果:4组单重dPCR实测KanR残留量之间基本一致(1.054×108~1.467×108 copies·μL-1);双重dPCR与单重dPCR实测结果间无明显差异;双重dPCR在6~6 000 copies·μL-1浓度范围内显示出良好的线性关系(r＞0.999)和重复性(RSD<20%);1/2,2/3和3/43种组合的双重PCR的双阳性孔占比基本一致,表明KanR基因的断裂可能更趋向于随机断裂;DNase Ⅰ处理前后结果提示,错包装的大片段质粒DNA(1/4双阳性)比游离的占比更高。结论:dPCR技术可用于检测rAAV产品中质粒DNA残留量,多重dPCR还可评估质粒DNA片段大小及分布情况。
Objective: To detect plasmid DNA residues in recombinant adeno-associated virus (rAAV) products by digital PCR (dPCR). Methods: Primer probes were designed for four different segments of the plasmid resistance gene KanR sequence, and singleplex and duplex dPCR were established to detect KanR residues. In duplex dPCR, double positive wells represent the sequence integrity between two sets of primer pairs, and the distribution of KanR sequences was assessed by testing the proportion of single and double positive wells. The presence of incorrectly packaged plasmid DNA within the viral capsid was evaluated by digesting free plasmid DNA with DNase Ⅰ. Results: The KanR residues measured in four groups by single dPCR were basically consistent (1.054×108-1.467×108) copies·μL-1. There was no significant difference between the measured results of duplex dPCR and singleplex dPCR in each channel. It showed good linearity (r＞0.999) and repeatability (RSD<20%) in 6-6 000 copies·μL-1 concentration range for the established duplex dPCR. The proportion of double positive wells in the dual PCR of 1/2, 2/3, and 3/43 combinations was basically consistent, indicating that the breakage of the KanR gene may tend to be more random. The results before and after DNase Ⅰ treatment suggested that the proportion of large fragments of plasmid DNA (1/4 double positive) that are wrongly packaged was higher than that of free plasmid DNA. Conclusion: The dPCR technology can be used to detect plasmid DNA residues in rAAV products, and multiple dPCR can also evaluate the size and distribution of plasmid DNA fragments.
[1] 王兰, 王军志. 关于生物制品残余DNA质量控制问题[J]. 中国新药杂志, 2011, 20(8):678
WANG L,WANG JZ. Issue on quality control of residual DNA in biological products[J]. Chin New Drugs J, 2011, 20(8):678
[2] 陈平, 张利娟, 李娜. 腺相关病毒载体的研究进展[J]. 中国生物制品学杂志, 2022, 35(4):500
CHEN P, ZHANG LJ, LI N. Progress in research on adeno-associated virus vectors[J]. Chin J Biol,2022, 35(4):5007
[3] 杜梦潭, 刘兴健, 胡小元, 等. 腺相关病毒的生产方式及其在基因治疗中的应用[J]. 生物技术进展, 2019, 9(4):326
DU MT, LIU XJ, HU XY, et al. The production method of adeno-associated virus and its application in gene therapy[J]. Curr Biotechnol, 2019, 9(4):326
[4] 陈静斌. 重组腺相关病毒制备方法的建立[D]. 陕西:陕西师范大学, 2016
CHEN JB. Establishment of a Production Method for Recombinant Adeno-Associated Virus[D]. Shanxi:Shanxi Normal University, 2016
[5] OLIVIER V, ERIC S, VALéRIE D, et al. Comparative analysis of the performance of residual host cell DNA assays for viral vaccines produced in Vero cells[J]. J Virol Methods, 2019,268: 9
[6] 唐月明, 伊洁. 数字聚合酶链反应(dPCR)技术在病原体基因检测应用中的研究进展[J]. 现代检验医学杂志, 2021,36(5):174
TANG YM, YI J. Recent advances in research on digital polymerase chain reaction(dPCR) in pathogen gene detection[J]. J Mod Lab Med, 2021, 36(5):174
[7] HUSSAIN M. A droplet digital PCR method for CHO host residual DNA quantification in biologic drugs[J]. J Anal Pharm Res, 2017, 4(3):107
[8] HUSSAIN M. Isothermal droplet digital PCR method for quantification of CHO residual DNA[J]. J Pharm Biomed Anal, 2022,211:114564
[9] WANG Y, COOPER R, BERGELSON S, et al. Quantification of residual BHK DNA by a novel droplet digital PCR technology[J]. J Pharm Biomed Anal, 2018,159: 477
[10] HAYDEN RT, GU Z, INGERSOLL J, et al. Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus[J]. J Clin Microbiol, 2013, 51(2):540
[11] HUSSAIN M, FANTUZZO R, MERCORELLI S, et al. AS direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells[J]. J Pharm Biomed Anal, 2016,123:128
[12] HUSSAIN M. A direct qPCR method for residual DNA quantification in monoclonal antibody drugs produced in CHO cells[J]. J Pharm Biomed Anal, 2015,115: 603
[13] PLOTKA M, WOZNIAK M, KACZOROWSKI T. Quantification of plasmid copy number with single colour droplet digital PCR[J]. PLoS One, 2017,12(1):e169846
[14] PINHEIRO LB, COLEMAN VA, HINDSON CM, et al. Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification[J]. Anal Chem, 2012,84(2):1003
