Description
Host cell DNA contamination in products such as vaccines, drugs and other biopharmaceuticals imposes great health risks. Therefore, its safe limits are tightly regulated by agencies such as the FDA and WHO. Clear guidelines for the upper limits of residual DNA are established based on the nature of drug administration, infectivity and oncogenicity of the contaminating cell DNA. For instance, parenteral administration of non-tumorigenic cell DNA should be limited to 10 ng/dose and a maximum length of 200 bp, whereas less than 100 µg/dose of residual DNA is recommended by WHO for orally administered vaccines. Detection of such contaminations requires highly sensitive and accurate measurements of the extremely low amounts of specific host cell DNA present in the products. Digital PCR is the preferred method for residual DNA (resDNA) quantification. This is due to its unmatched sensitivity and accuracy, especially at trace levels of contamination compared to other detection methods such as qPCR. QIAcuity Residual DNA Quantification Kits provide the accurate and precise detection of Escherichia coli (E. coli), Chinese Hamster Ovary (CHO) and Human Embryonic Kidney 293 (HEK293) host cell DNA. The QIAcuity HEK293 resDNA Sizing Kit allows quantification and fragment size determination to meet guidelines for residual DNA size (<200 bp). This kit also allows for multiplexing with oncogene targets like E1A (Geneglobe ID DMA00938), E1B (GeneGlobe ID DMA01000) or SV40 (GeneGlobe ID DMA00936) for added safety testing flexibility. The QIAcuity resDNA Quant Standard Kits are dPCR-verified absolute quantification standards that can be used in combination with resDNA Quant and Sizing Kits for validation of quantitation accuracy or bridging studies.
The QIAcuity Residual DNA Quantification Kits provide accurate CHO, E. coli and HEK293 residual DNA quantification results with or without extraction, even in the presence of PCR contaminants or other inhibitory reagents. The assays are multicopy target assays that accurately determine even very low amounts highly fragmented residual host cell DNA. QIAcuity HEK293 resDNA Sizing Kit targets a proprietary multicopy locus, enabling sensitive detection and sizing of HEK293 or human residual DNA via two amplicons (short with less than 200 bp, and long with more than 200 bp). This allows for precise quantification and assessment of DNA size distribution in a single reaction. Assay Target Copy Number Amplicon Size Conversion Factor cp/µL to fg/µL QIAcuity E. coli resDNA Quant Kit 7 <200 0.35 QIAcuity CHO resDNA Quant Kit ~ 1 million, undefined (repeated element) <100 0.28 QIAcuity HEK293 resDNA Quant Kit ~ 1 million, undefined (repeated element) <100 1.54 QIAcuity HEK293 resDNA Sizing Kit > 20 copies per genome Short Amplicon (~83 bp) 0.129 Long Amplicon (~345 bp) 0.143 Digital PCR provides higher sensitivity of detection at a lower template input range in comparison to qPCR and therefore enables a more robust application. QIAcuity E. coli resDNA Quant Kit detects as low as 5 fg of residual DNA in a single reaction Loading amount per reaction (fg/rxn) E. coli Standard (copies/µL) Internal Control (copies/µL)* 50000 2949.7 99.9 5000 291.6 91.7 500 27.6 91.4 50 2.8 93.2 25 1.46 92.5 5 0.45 90.6 NTC 0 98.9 *100 copies/µL expected for Internal Control QIAcuity CHO resDNA Quant Kit detects as low as 5 fg of residual DNA in a single reaction Loading amount per reaction (fg/rxn) CHO Standard (copies/µL) Internal Control (copies/µL)* 50000 4939.3 108.7 5000 508.3 104.9 500 50.2 97.6 50 4.7 99.5 25 2.6 101.3 5 0.6 100.4 NTC 0 104.7 *100 copies/µL expected for Internal Control QIAcuity HEK293 resDNA Quant Kit detects as low as 50 fg of residual DNA in a single reaction Loading amount per reaction (fg/rxn) HEK293 Standard (copies/µL) Internal Control (copies/µL)* 50000 1104.9 96.6 5000 104.5 92.5 500 8.83 92.4 50 0.99 94.4 NTC 0 96.2 *100 copies/µL expected for Internal Control The kits work in conjunction with the QIAcuity Digital PCR System and QIAcuity Nanoplates, offering an end-to-end fast dPCR workflow comparable to qPCR, but delivering absolute quantification of resDNA in your sample without the need for standard curves. The kits have been designed according to the requirements of bioprocess manufacturing and QC.
The principle of the dPCR reaction in the nanoplates is described here. Using QIAcuity digital PCR for host cell DNA monitoring improves the LOD/LOQ through partitioning of the bulk sample with the help of QIAcuity Nanoplate 26k. Partitioning increases the effective concentration of the target, allowing a small amount of host cell DNA to be captured and measured with higher accuracy and precision. The ability to load high template input amounts in the QIAcuity Nanoplate 26k combined with an increased number of partitions is one of the reasons for the higher accuracy precision and reproducibility residual host cell DNA detection.
The sample or extracted template DNA is added to the reaction mix together with the internal amplification control in a standard PCR plate. The reactions are transferred to a Nanoplate and target DNA concentration (copies/µL) is measured by absolute quantification on the QIAcuity System. Copies/µL are converted to fg/µL using the provided conversion factor.
