PROTEIN A MIX-N-GO Unnatural Recombinant （F610-ELISA Kit）
F400-ELISA Kit
Protein A ELISA Kit
Protein A ELISA Kit
Cygnus Protein A检测试剂盒
仓鼠细胞宿主蛋白酶联检测试剂盒（ELISA）（专属定制）
仓鼠细胞宿主蛋白酶联检测试剂盒（ELISA）
仓鼠细胞宿主蛋白酶联检测试剂盒（BAT1806 HCP 专属试剂盒）
仓鼠细胞宿主蛋白酶联检测试剂盒（ELISA）（BAT1706专属定制）
Cygnus CHO Host Cell Proteins检测试剂盒
宿主细胞残留DNA样本前处理磁珠法I
宿主细胞残留DNA样本前处理磁珠法II
宿主细胞残留DNA样本前处理磁珠法III
CHO残留DNA检测试剂盒PCR荧光探针
2×qPCR SHENmix(荧光探针法)
CHO-F Primer
CHO-R Primer
CHO-Probe
Nuclease-Free Water
支原体DNA提取纯化试剂盒（磁珠法）I
支原体DNA提取纯化试剂盒（磁珠法）II
支原体DNA提取纯化试剂盒（磁珠法）III
支原体DNA检测试剂盒（PCR-荧光探针法）
肺炎支原体
口腔支原体
VEGF165贮存液
IL-6R贮存液
TNF-α贮存液
P40-Fc抗原贮存液
CTLA-4-ECD-Fc抗原贮存液
PD-1抗原贮存液
IL17A贮存液. IL-17A抗原贮存液
HRP标记的山羊抗人IgG-Fc抗体（J
HRP标记的山羊抗人IgG-Fc抗体贮存液（Sigma）
酶标二抗（kappa）. Anti-Human Kappa Light Chains（Bound andFree）
放线菌素D贮存液
DMEM basic（1X）. DMEM培养基
Celltiter-Glo LuminescentCell Viability Assay
0.25%Trypsin-EDTA 1x
DMEM basic（1X）
Cell Counting Kit-8
RPMI Medium 1640（1X）
稳定型萤火虫荧光素酶报告基因检测液
0.2%台盼蓝染色液
FBS
One-Glo Luciferase Assay System
基础培养基
IL-12贮存液
脱脂奶粉
Human IFN-γ ELISA Set
TMB Substrate Reagent Set
Bio-Glo Luciferase Assay System

The basic principles of nucleic acid amplication technologies (NAT) and denitions of the various techniques are covered in Nucleic Acid-Based Techniques—General 〈1125〉. This chapter covers the analytical procedures used to quantify residual DNA in biopharmaceuticals.
Process characterization and the theoretical safety concerns associated with process-related impurities highlight the need for residual DNA testing in biopharmaceutical products. The ability of a manufacturing process to remove residual DNA from a biopharmaceutical product is an indicator of the quality and consistency of the process and that the process is under control. Additionally, the cells used to produce a biopharmaceutical can be sources of a range of complex, heterogeneous, and potentially unsafe impurities, with host cell DNA among these impurities. For continuous cell lines, the potential risk of residual DNA arises from both of its biological activities, namely infectivity and oncogenicity. Infectivity could be due to the presence of an infectious viral genome in the cellular DNA of the cell substrate. The oncogenicity activity of residual DNA could arise through its capacity to induce a normal cell to become transformed, which may lead to tumorigenicity. Although animal testing has shown that extraneous DNA can cause tumors or infections, no reports to date have demonstrated this risk in humans. Residual DNA content, up to 10 ng of residual DNA per parenteral dose, may be considered for DNA originating from mammalian cell cultures, but the acceptable residual DNA content may vary depending on the source of the residual DNA and the route of administration of the product. One can address residual DNA in biopharmaceutical processes in two ways: 1) by validating clearance during process validation; and/or 2) by
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OFFICIAL
2021/8/25 USP-NF 〈1130〉 Nucleic Acid-Based Techniques—Approaches for Detecting Trace Nucleic Acids (Residual Dna Testing)
monitoring residual DNA levels through routine testing of the drug substance. Generally, 10 ng per dose is the accepted limit, by health authorities, of host residual DNA derived from mammalian cell cultures. The level of concern regarding residual DNA can be tied to its source and the route of administration, so the residual DNA specication and procedure for monitoring DNA clearance for a given product should be developed in consultation with regulatory agencies. Regardless of whether routine testing of a drug substance is used to determine residual DNA content or whether DNA clearance is demonstrated by process validation, analytical procedures for the quantication of residual DNA are required. DNA amplication techniques, such as quantitative PCR (qPCR), are used most often for residual DNA testing because of their superior sensitivity and unique advantages (e.g., high specicity). The expectation is that the analytical procedure used to quantify residual DNA in biopharmaceuticals has a detection limit well below the DNA level allowed by regulators for biopharmaceuticals (often 10 ng/dose). Assays based on hybridization, DNA-binding protein, and qPCR are typically the techniques of choice because they can meet the sensitivity expectation.
SAMPLE PRETREATMENT
Analysis of residual DNA requires accurate quantication of picogram levels of DNA in mg (or larger) quan

e because they can meet the sensitivity expectation.
SAMPLE PRETREATMENT
Analysis of residual DNA requires accurate quantication of picogram levels of DNA in mg (or larger) quantities of product, which may be in a variety of matrices. In certain circumstances, the sample can be analyzed neat in the analytical procedure with acceptable recovery and precision. When the product or other sample components interfere with the assay sample, dilution may be all that is required to overcome the interference, so long as the specied DNA content of the sample remains within the useful range of the analytical procedure. When sample dilution is not effective in reducing assay interference, it may be necessary to use more extensive sample pretreatment procedures, such as proteolytic digestion, chemical dissociation, or extractions. It may be necessary to use a combination of different pretreatment steps to remove interference to an acceptable level. Extensive sample manipulation can lead to losses of DNA or introduction of environmental DNA, and should be a consideration when using one or more sample pretreatment steps. Contamination with environmental DNA may only be a concern when using a residual DNA procedure that is not sequence specic.
Protein samples may only require digestion with proteinase (e.g., Proteinase K, Pronase) to allow the analytical method to quantitatively recover the residual DNA. It may also be possible that the DNA is bound to the sample components, and chemical dissociation (e.g., detergents) may disrupt the binding, allowing sucient recovery in the residual DNA assay. Residual DNA test procedures often use protein reagents and the use of a chemical dissociation reagent. These materials must be used at a suciently low level or removed so that the analysis is not compromised.
It may be necessary to extract the DNA from the sample to remove the inhibitory substances that are causing the reduced DNA recovery. Extraction procedures are typically based on precipitating the DNA from the sample or DNA-specic binding to a matrix
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2021/8/25 USP-NF 〈1130〉 Nucleic Acid-Based Techniques—Approaches for Detecting Trace Nucleic Acids (Residual Dna Testing)
(e.g., magnetic beads). Historically, extraction methods based on phenol and chloroform, followed by ethanol precipitation, have been applied to the purication of DNA in molecular biology research. The phenol/chloroform extraction technique may be a useful pretreatment for residual DNA samples before analysis, but the phenol/chloroform extraction technique might not be the best choice for the low levels of DNA typically found in biopharmaceutical samples. Because of these low levels, quantitative DNA recovery with ethanol precipitation may be dicult. For this reason, a carrier molecule (e.g., glycogen) may be necessary to aid in DNA recovery if the phenol/chloroform extraction technique is used. Commercial kits are available and have been used successfully for pretreatment of residual DNA samples for improved recovery in the residual DNA assay. For example, some kits use a chaotrope (sodium iodide) and a detergent (sodium N-lauroyl sarcosinate) to disrupt the association of the DNA with the sample. The DNA is then co- precipitated using glycogen as the carrier molecule in the presence of isopropanol. Extraction of DNA from the sample, based on binding to a solid matrix, can be found in various formats. One of the most popular formats uses magnetic beads, where the beads are added to a sample with a binding solution to capture the sample DNA on the beads. The beads are then captured and held in the sample tube using a magnetic stand while the supernatant containing the interfering substances is removed and discarded. The beads are washed repeatedly using a magnetic stand and a wash solution. Finally, the DNA is eluted from the beads for the assay using an elution buffer, with the beads being removed from the sample preparation using the magnetic stand.
The sample manipulation involved with pretreatment may reduce the recovery of the residual DNA or introduce environmental DNA into the sample. Great care must be taken during any sample manipulations to avoid DNA losses or contamination. The addition of target DNA-spiked samples in the residual DNA assay is a common practice. The target DNA-spiked sample should not be confused with the internal positive control (IPC), which is typically a nontarget DNA added after the sample pretreatment step to detect the presence of PCR inhibitors and to evaluate DNA amplication during the analysis. The IPC may also be introduced before the extraction to improve the control of this step. A recovery of 50%–150% of the spiked target DNA is often applied to residual DNA assays to ensure that the assay yields acceptable results. When sample characteristics (e.g., matrix effects or sample preparation method) make achieving a recovery acceptance criterion of 50%–150% impractical, then correcting the observed DNA concentration by using the load recovery percentage is also an acceptable approach.
HYBRIDIZATION-BASED RESIDUAL DNA ASSAY
The rst residual DNA assays were based on DNA hybridization, wherein a DNA probe created from host cell DNA detects and quanties the amount of complementary DNA present in the product under assay. Double-stranded host cell DNA consists of two complementary strands of DNA that are held together by hydrogen bonding. The double-stranded DNA in the test sample is denatured to single strands and immobilized to a membrane, typically a nitrocellulose or nylon membrane. The sample is probed using host cell
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仪器：酶标仪：D4-C87，2022.06.23；微孔板孵育器：D4-B91，2022.04.26；20~200μL12通道移液器：4K440FI102，2022.09.13。
试剂：Pierce BCA Protein Assay Kit: RL-0005585 TI-0032859；BAT0606STD：B220171002RSTD-135、-134；0.05%PBST：P211105001。