Residual DNA Quantitation
To ensure product quality, the amount of residual DNA in a drug's final dosage form must meet regulatory guidelines established by the World Health Organization (WHO), the European Pharmacopoeia, the US Food and Drug Administration (FDA), and other regulatory agencies.
A host cell quantitation system should be designed with the sensitivity to meet or exceed regulatory requirements, while enabling high throughput and cost-effectiveness to enable robust process clearance study design.
Features and Benefits:
- PCR sensitivity: accurate quantitation down to 1.5 pg/mL of host cell DNA
- Wide dynamic range: up to 6 logs of dynamic range
- Robustness: dilution linearity and efficiency in complex matrices
- Specificity: highly target DNA-specific with no cross reactivity to unrelated DNA
- An oligonucleotide probe is constructed containing a fluorescent reporter dye on the 5' end and a quencher dye on the 3' end. While the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET).
- If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5' nuclease activity of Taq DNA polymerase as this primer is extended.
- This cleavage of the probe:
- Separates the reporter dye from the quencher dye, increasing the reporter dye signal.
- Removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process.
- Additional reporter dye molecules are cleaved from their respective probes with each cycle, resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced.
The software solution for mycoplasma detection, protein analyses and residual DNA
The Applied Biosystems™ AccuSEQ™ Real-Time PCR Detection Software supports the unique needs of analytical testing of contaminants and impurities during the biopharmaceutical manufacturing process, as well as routine qPCR assays.
AccuSEQ software is part of the integrated workflow for Mycoplasma detection, residual Protein A quantitation, and residual DNA quantitation. Automated analysis tools enable one-click processing of complex Ct, amplitude (DV), and melting temperature for the MycoSEQ™ Mycoplasma detection assay according to user specifications. Nonlinear curve-fitting enables rapid analysis of ProteinSEQ Protein Quantitation System data. Next-generation algorithms deliver accurate quantitation data for the resDNASEQ™ Residual Host Cell DNA Quantitation.
- Interfaces with the Applied Biosystems™ 7500 Fast Real-Time PCR instrument
- Accurate quantitation of host cell residual DNA
- Single software platform for multiple SEQ real-time PCR assays
- Automated Purification of Residual DNA Using the PrepSEQ™ Residual DNA Sample PreparationKit and the MagMAX™ Express 96 Instrument
- resDNASEQ™ Quantitative NS0 DNA Kit
- resDNASEQ™ Quantitative MDCK DNA Kit
- resDNASEQ™ Quantitative E. coli DNA Kit
- resDNASEQ™ Quantitative Pichia pastoris DNA Kit
- resDNASEQ™ Quantitative Vero DNA Kit
Protocols and quick reference cards
- Host cell protein and residual DNA quantification-part 1 - Protein PCR: the new ELISA of protein impurities
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