Demonstration that host cell and process-based impurities are removed during the purification of biopharmaceutical products is required so that these impurities are reduced to levels below those guided by regulatory agencies globally. The resDNASEQ kit is a quantitative PCR based assay that was designed and developed specifically to enable sensitive and accurate quantitation of residual host cell DNA. When combined with PrepSEQ sample preparation, we provide a complete solution for extraction and quantitation of host cell DNA from sample matrices typical of biopharmaceutical purification processes. The demonstrated high percentage, consistent DNA recovery provided by PrepSEQ combined with the sensitivity accuracy and range of the assay enables design and execution of robust purification process validation studies to demonstrate clearance of host cell DNA to levels that meet or exceed to most rigorous regulatory expectations.
Learn more about residual plasmid DNA quantitation
The resDNASEQ system is a fully integrated assay leveraging real-time PCR technology for quantitation of residual host cell DNA with a qualified DNA reference standard. The resDNASEQ solution, on the Applied Biosystems QuantStudio 5 Real-Time PCR System or the Applied Biosystems 7500 Fast Real-Time PCR System, features a complete residual host cell DNA testing workflow from sample preparation to results in less than 5 hours. This established GMP compliant solution is widely adopted for routine use in major global biopharma companies.
resDNASEQ residual host cell quantitation is now available for our Applied Biosystems QuantStudio Absolute Q Digital PCR System. We are optimizing our trusted resDNASEQ qPCR kits for digital PCR to enable our customers to choose the platform that best suits their needs.
USP <509> Residual DNA Testing, details the application of quantitative PCR for measurement of residual host cell DNA levels during manufacturing of biopharmaceuticals. ResDNASEQ combined with PrepSEQ sample preparation is a fully integrated qPCR-based solution for extraction and quantitation of host cell DNA that is manufactured and QC tested specifically for use in biopharmaceutical development and manufacturing. The overall performance of the resDNASEQ method, including sensitivity, accuracy, precision, range and linearity meets or exceeds the example described in USP <509>. Implementation of a fully integrated testing solution reduces the expense and effort in procurement of assay components from multiple vendors and qualification of critical reagents that is required for in-house developed assays.
Figure 1: The resDNASEQ system provides a sample-to-results solution, including the proprietary genomic DNA standard and leverages automated sample prep and reporting software compliant with 21 CFR Part 11. These kits are designed to detect fragmented and unfragmented DNA resulting in confident quantitation.
Our newest kits provide the same quality and performance that has established the resDNASEQ system as an industry-leading solution with wide adoption for routine use at major biopharma companies. The fully integrated real-time qPCR system for quantitation of residual host cell DNA helps provide a high degree of confidence in quantitation data obtained from a wide range of sample types—from in-process samples with different sample matrices to purified final product.
Applied Biosystems resDNASEQ Quantitative Sf9 and Baculovirus DNA Kit
The first commercially available duplex quantitative qPCR-based system for the detection of residual DNA from the Sf9 baculovirus platforms used in the development of gene therapies, cell-based vaccines, and similar biotherapeutics.
Applied Biosystems resDNASEQ Quantitative HEK293 DNA Kit
A qPCR-based system for the detection of host-cell DNA from HEK293 cell lines used in the development of gene therapies, cell-based vaccines, and similar biotherapeutics.
Applied Biosystems resDNASEQ Quantitative E1a DNA Fragment Length Kit
Accurately quantitate residual HEK293 fragments with this E1a fragment sizing assay. Sensitive analysis is achieved using three single-plex assays to quantitate residual DNA fragments of varying length. This kit is a perfect complement to the HEK293 DNA kit for gene therapy customers and cell-based vaccine manufacturers or those researchers using host cells that contain E1a.
To verify product quality, the amount of residual DNA in a drug's final dosage form must meet regulatory guidelines established by the US Food and Drug Administration (FDA), the European Pharmacopoeia, World Health Organization (WHO), and other regulatory agencies. Each regulatory agency has specific guidelines for acceptable limits range from 10 pg to 10 ng DNA per therapeutic dose depending on the product and therapeutic modality. The Applied Biosystems resDNASEQ Quantitative DNA solution was designed to help you meet these regulatory guidelines.
Understand the components of a method validation per ICH Q2(R1) in the context of a quantitative polymerase chain reaction (PCR) assay for sensitive, accurate and rapid quantitation of residual DNA from a variety of host-cell systems, including Chinese hamster ovary (CHO), E. coli, Vero and human/human embryonic kidney 293 (HEK293).
Biopharmaceutical companies that produce biopharmaceutical products must verify the quantity of host cell DNA impurities carried over through the purification process and in the drug’s final dosage to meet or exceed regulatory guidelines. Validation of residual host cell DNA assays may vary by regulatory agency, but general expectation is to meet validation requirements outlined in ICH Harmonised Tripartite Guideline Validation of Analytical Procedures Q2(R1) (ICHQ2R1). Here we’ll share some validation data from our resDNASEQ host cell DNA quantitation kits, focusing on some of the most challenging validation parameters including limit of quantitation (LOQ), specificity, and accuracy.
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of assays for low levels of compounds in sample matrices and is used particularly for the determination of impurities and/or degradation products. For resDNASEQ kits, the LOQ is 1.5 pg/mL for mammalian DNA; and LOQ to 15 pg/mL for bacterial/yeast DNA.
Assay accuracy is measured by spiking replicate samples with a known concentration and calculating recovery from a standard curve. Assay precision is defined and measured by percent coefficient of variation (%CV), which is calculated by dividing the standard deviation by the average Ct of the number of replicates. The resDNASEQ assay was evaluated by running 100 pg of spiked control DNA in a simulated sample matrix with PrepSEQ and the corresponding resDNASEQ kit. Results are shown in the table below for four of the resDNASEQ assays. Typical %CVs for resDNASEQ assays are in the low single‐digit range and recoveries are between 78–92%, depending on the cell line. In addition, there was consistent high-level DNA recovery and quantitation across all spike concentrations tested including 10 pg and 1 pg.
Spike recovery for CHO cell line DNA
|100 pg||10 pg||1 pg|
Figure 2: Triplicate samples spiked with 100 picograms of standard DNA.
For accurate quantitation, the assay must detect only the target DNA and not other DNA present in the sample. An example is shown below where the resDNASEQ Pichia Pastoris assay is unaffected by the presence of other DNA.
|S. cerevisiae||Und||10 ng|
|E. coli||Und||10 ng|
|Pichia pastoris (KM71)||31.7||10 pg|
Figure 3: Amplification is only seen for Pichia pastoris, and clearly denotes the specificity of the target DNA
For the most accurate quantitation of residual host cell DNA, the assay results must be unaffected by the size of the DNA molecules present in the test sample. Shown below are results generated from analysis of a dilution series of high molecular weight and fragmented CHO standard DNA in several different matrices representing typical monoclonal antibody purification scenarios. The resDNASEQ assay maintained consistent performance even with fragmented DNA across the entire concentration range of the analysis.
Figure 4: Spiked CHO resDNASEQ recoveries at 5 concentration levels, showing high molecular weight DNA (uncut) and low molecular weight DNA (Sau961). Matrices are M1: ion exchange, pH 7.5; M2: hydrophobic Interaction; M3: ion exchange, pH 6; M4: protein A eluate; M5: purified drug substance.
Applied Biosystems products offer a complete residual host cell quantitation workflow that includes sample preparation with automation (manual preparation also available), residual host cell analysis kit, qPCR detection and analysis software. A streamlined workflow enables validation, minimize hands-on time, and improve overall productivity.
Our Applied Biosystems PrepSEQ kits for residual host cell DNA sample preparation are a magnetic bead based nucleic extraction and purification workflow, with best-in-class recovery efficiency for applications where consistent absolute quantitation and highest sensitivity detection are required. Easy to follow protocols for both manual and automated workflows are available based on your process needs.
The KingFisher Apex magnetic particle processor instrument offers an easy workflow with high throughput so you can process up to 96 samples with 1 hours of total hands-on time using PrepSEQ sample preparation kits.
Figure 5: Standard curve for CHO resDNASEQ showing 6 orders of magnitude linearity and curve fitting parameters using our AccuSEQ software.
The Applied Biosystems AccuSEQ Real-Time PCR Detection Software supports the unique needs of analytical testing of residual host cell DNA during the biopharmaceutical manufacturing process, as well as routine qPCR assays. The software is designed to work with the Applied Biosystems QuantStudio 5 (QS5) Real-Time PCR Instrument with 0.1 mL, 96-well plates for either Fast or Standard cycling and the Applied Biosystems 7500 FAST Real-Time PCR instrument. Next- generation algorithms deliver accurate quantitation data for resDNASEQ Residual Host Cell DNA analysis and allow for easy sample concentration determinations with relevant statistics.
The Applied Biosystems QuantStudio 5 (QS5) system is designed for both new and experienced users who need simple and affordable real-time PCR systems without compromising performance and quality. The QuantStudio 5 Real-Time PCR Systems detects differences in target quantity as small as 1.5-fold in single plex reactions, and provides 10 logs of linear dynamic range.
The Applied Biosystems 7500 Real-Time PCR System is a powerful platform for labs requiring superior performance and maximum dye versatility. It is a sophisticated platform for users who require extended capabilities and maximum versatility. The third generation platform features an innovative optical system that enhances sensitivity.
The Applied Biosystems resDNASEQ host cell residual DNA quantitation systems offer a portfolio of kits to measure residual DNA levels from common host cell lines used in the production of biopharmaceutical products.
*Customers working with HEK293 cell lines in gene therapy or cell-based vaccine manufacturing may want to consider the Applied Biosystems resDNASEQ Quantitative E1A DNA Fragment Length Kit to accurately quantitate residual HEK293 fragments. This E1A fragment sizing assay is also applicable for manufacturing processes using other host cells that are transformed by the E1A gene.
Custom services for in-process and lot-release testing
If you can’t find the resDNASEQ assay specific to your cell line, we can help. Our Custom Services Team can work with you to develop a custom TaqMan assay to meet your in-process and lot release testing needs. We can also support the development and validation of a newly developed custom resDNASEQ assay process.
Validation consulting services provide technical project management to help verify that the assay is tested for required parameters.
Timely, cost-effective, audit-ready documentation managed by a compliance specialist so that your instrument is installed, operating, and performing to manufacturer’s specifications.
Application and instrument training programs available at our training centers located throughout the world, your lab, or through web-based instruction.
Scheduled planned maintenance and guaranteed response times to help avoid unnecessary downtime, reduce strain on laboratory staff, and help extend the life of your instrument.
For Research Use Only. Not for use in diagnostic procedures.