Wako 残留DNA提取试剂盒 DNA with DNA Extractor® Kit

This article was written by Hiroki Fukuchi, Life Science Research Laboratories of FUJIFILM Wako Pure Chemical Corporation, Japan.

导言
因为许多生物药物，包括疫苗，都是由培养的细胞或微生物(如大肠杆菌指出宿主细胞来源的DNA可能存在于合成的药物物质和产物中.鉴于残留DNA可能转移宿主细胞或病毒衍生的癌基因，病毒DNA可能导致感染事件，定量检测残留DNA是制造业的重要测试，也是生物制药过程验证等测试的一部分。正如一份报告所指出的那样，每一剂量的残余DNA最多可达100 pg，这对于生物制药来说是可以接受的，1)不仅在美国和欧洲国家，而且在其他国家，越来越需要对宿主细胞中的残留DNA进行定量检测。为了检测残留DNA的痕迹，必须从样品中提取出高收率的残留DNA。本文旨在介绍DNA提取器的用途。®用于检测残留DNA作为DNA纯化试剂的试剂盒。

DNA提取器®试剂盒(#295-50201，Fujifilm Wako纯化学公司)
在对生物制品中的总DNA进行检测和定量之前，有必要从蛋白质等其他生物成分中分离纯化生物制品中的DNA。一般来说，DNA的分离是通过蛋白酶介导的样品消化，然后用苯酚和/或氯仿提取的。然而，这种方法的缺点是，必须使用有害的苯酚和氯仿，需要时间和人力的提取，虽然可以获得相当高纯度的DNA。此外，二氧化硅载体固相萃取，由于载体吸附导致DNA丢失，对DNA的提取效果不理想。有机溶剂的提取也不利于DNA的有效回收，这是DNA提取试剂需要解决的关键问题之一。

DNA提取器®试剂盒(产品代码：295-50201)，我们于1992年推出，解决了上述限制，使用钠碘提取高纯度的DNA，通过简单的程序。

DNA提取原理®试剂盒
工具包的组成部分*含有碘化钠和表面活性剂，当2-丙醇加入时，它们通过溶解蛋白质和其他生物成分，选择性地沉淀(共沉淀)核酸(主要是DNA)和糖原，作为蛋白质增溶剂(潮致离子)。2)与上述方法不同，纯化步骤被简化为在没有载体或有机溶剂的情况下产生沉淀物，从而能够以较高的收率提取微量DNA。

*工具包的组成部分：
碘化钠溶液，N-月桂酸盐溶液，洗涤液(A)，
洗涤液(B)，糖原溶液

DNA提取器提取DNA的实例®试剂盒和总DNA定量
本文介绍了一份关于在稀释剂或辅料存在的情况下DNA产量的报告，该报告最初发表在瓦科纯化学杂志第60卷第3期(1992年)第28页。本实验通过在磷酸盐缓冲液中溶解常用作稀释剂或辅料(如精氨酸、尿素)或蛋白质(BSA和人γ-球蛋白)的常用或过量的物质制成模型溶液，并在每个模型溶液中加入一定量的小牛胸腺脱氧核糖核酸(PG)。然后，根据dna提取器标签上的说明，从模型溶液的400μL中提取dna。®基特。所得沉淀物溶于500μL磷酸盐缓冲液中，用阈值法测定总dna。®总DNA分析系统，*3,4)来测量DNA的产量。测量结果见表1。

Introduction

Since many biopharmaceuticals, including vaccines, are manufactured from cultured cells or microorganisms such as Escherichia coli, it is pointed out that host cell-derived DNA may remain in resultant drug substances and products. Given the possibility that the residual DNA may transfer host cell- or virus-derived oncogenes and that viral DNA may cause infectious events, quantitative detection of residual DNA is important testing for manufacturing and as part of testing such as process validation for biopharmaceuticals. As indicated by a report recommending that up to 100 pg of residual DNA per dose is acceptable for biopharmaceuticals,1) there is increasing need for quantitative detection of residual DNA from host cells as a quality test not only in the US and European countries but also in other countries. To detect traces of residual DNA, it is necessary to extract traces of residual DNA from a sample at a high yield. This article is intended to introduce the usefulness of DNA Extractor® Kit in detecting traces of residual DNA as a DNA purification reagent.

DNA Extractor® Kit (#295-50201, FUJIFILM Wako Pure Chemical Corporation)

Prior to detection and quantification of total DNA in a biologics, it is necessary to separate and purify DNA in the biologics from other biological components such as proteins. In general, DNA is isolated through protease-mediated digestion of specimen followed by extraction with phenol and/or chloroform. However, this method has disadvantages that deleterious phenol and chloroform have to be used and that time- and labor-consuming extraction is required, although quite highly pure DNA can be obtained. In addition, solid-phase extraction via silica carriers, which results in loss of DNA due to adsorption on carriers, is not ideal to recover traces of DNA. Extraction with organic solvents is also not favorable with inefficient recovery of traces of DNA, which is a key issue to be addressed with DNA extraction reagents.

DNA Extractor® Kit (Product code: 295-50201), which we launched in 1992, resolves the aforementioned limitations by using sodium iodide to extract highly pure DNA at high yields through simple procedures.

Principle of DNA Extractor® Kit

The components of the kit* contain sodium iodide and surfactants, which act as protein solubilizers (chaotropic ions) by solubilizing proteins and other biological components in specimens to precipitate (coprecipitate) nucleic acids (mainly DNA) and glycogen selectively when 2-propanol is added.2) Purification steps are simplified to produce precipitates without carriers or organic solvents, unlike the aforementioned methods, enabling the extraction of traces of DNA at high yields.

* Components of the kit:
Sodium Iodide Solution, Sodium N-Lauryl Sarcosinate Solution, Washing Solution (A),
Washing Solution (B), Glycogen Solution

Example of DNA extraction with DNA Extractor® Kit and total DNA quantification

A report on yields of DNA in the presence of diluents or excipients with the use of the kit, which was originally published on page 28 in Wako Pure Chemical Jiho Vol. 60 No. 3 (1992), is introduced here. In this experiment, model solutions were prepared by dissolving usual or excessive doses of substances often used as diluents or excipients (e.g., arginine, urea) or proteins (BSA and human γ-globulin) in phosphate-buffered saline, and a given amount (pg) of calf thymus DNA was added to each model solution. Subsequently, DNA was extracted from 400 μL of model solution according to the instructions on the label of DNA Extractor® Kit. The obtained precipitates were dissolved in 500 μL of phosphate-buffered saline, and total DNA was quantified using Threshold® Total DNA assay system,*3,4) to measure the yield of DNA. Measurement results are presented in Table 1.