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RESIDUAL DNA ANALYSIS IN INFLUENZA VACCINE PROCESSING

机译：流感疫苗加工中的残留DNA分析

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In cell-based influenza vaccine production, the European Pharmacopoeia demands a host cell residual DNA concentration of less than 10 ng per dose. To reliably measure residual DNA in both process samples and final vaccine using quantitative PCR, DNA preparation prior to analysis is a necessity. Samples from the vaccine purification process contain different buffers, salts, and cell-based compounds, and vary 3-4 logs in DNA concentration from harvest to the final product, which all put strain on the DNA preparation. For accurate determination of DNA concentration, recovery is of high importance. There are many commercially available DNA preparation kits that use different techniques to bind DNA, from spin columns with a DNA-binding membrane or medium (resin) to magnetic beads. However, these kits are mainly developed to purify DNA fragments from gel electrophoresis or genomic DNA from tissues such as blood or cultured cells, and do not have recovery as a priority. Few kits are intended for residual DNA determination in samples with high concentration of a protein or virus product. In this study, prototype media for DNA preparation, in bind-elute and batch mode, were evaluated for recovery, hands-on time, and throughput. In batch mode, recoveries of > 80% were achieved, but the technique exhibited matrix effects on real process samples. In bind-elute mode, recoveries of 40%-60% were achieved after elution. However, recovery could be improved by determination of DNA concentration, while keeping DNA bound to the medium.
机译：在基于细胞的流感疫苗生产中，《欧洲药典》要求宿主细胞残余DNA的浓度应低于每剂10 ng。为了使用定量PCR可靠地测量过程样品和最终疫苗中的残留DNA，必须在分析前进行DNA制备。疫苗纯化过程中的样品包含不同的缓冲液，盐和基于细胞的化合物，从收获到最终产物的DNA浓度变化为3-4个对数，所有这些都对DNA制备造成了压力。为了准确测定DNA浓度，回收非常重要。有许多可商购的DNA制备试剂盒，它们使用不同的技术来结合DNA，从带有DNA结合膜或介质（树脂）的旋转柱到磁珠。但是，这些试剂盒主要用于从凝胶电泳中纯化DNA片段或从组织（如血液或培养细胞）中纯化基因组DNA，并不优先考虑回收。很少有试剂盒可用于高浓度蛋白质或病毒产物样品中残留DNA的测定。在这项研究中，评估了结合洗脱和分批模式下用于DNA制备的原型培养基的回收率，动手时间和通量。在批处理模式下，回收率> 80％，但该技术对实际过程样品表现出基质效应。在结合洗脱模式下，洗脱后回收率达到40％-60％。但是，通过确定DNA浓度，同时保持DNA与介质的结合可以提高回收率。

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