residual host cell dna testing

Demonstration that host cell and process-based impurities are removed during the purification of biopharmaceutical products is required to ensure 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.

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Easy-to-use solution for residual host cell DNA testing

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 DNA quantitation system

All-in-one kit from a trusted source

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 eliminates 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.

DNASystem

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.

Introducing our newest resDNASEQ Quantitative DNA kits

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 ensure 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.

Designed to meet the rigors of regulatory guidance

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.

Helpful webinars
Webinar-Picture

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).

Validation guidance for residual host cell DNA testing of biologics

  • Considerations when qualifying a residual host cell DNA assay and establishing acceptance criteria for validation
  • Overview of an example validation study design for a quantitative test for impurities per ICH Q2 (R1), Validation of Analytical Procedures
  • Review of data generated during execution of the example validation study

Watch webinar ›

Development & validation of quantitative real-time PCR for the detection of residual HEK-293 host cell DNA

  • Overview of current regulatory requirements for residual host DNA clearance for biological products in gene therapy applications
  • A streamlined, automated workflow for quick quantification of residual HEK-293 host cell DNA
  • Performance specifications for the resDNASEQ Quantitative HEK-293 DNA system

Watch webinar ›

Designed for purpose, confirmed in validation, implemented globally

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.

Sensitivity—limit of quantitation (LOQ)

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.

Accuracy—spiked sample recovery

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

  100pg 10pg 1pg
CHO Recovery% CV% Recovery% CV% Recovery% CV%
1 97.9 5.9 91 2.8 82.1 7.9
2 95.4 4.5 89.8 2.2 80.1 4.9
3 107.5 3.7 93.6 3.3 100.3 6.4
4 109.6 5.2 94.6 4.8 97.1 3.8
Avg 102.6   92.3   89.9  
SD 7   2.2   10.3  
CV% 6.8   2.4   11.4  

Figure 2: Triplicate samples spiked with 100 picograms of standard DNA.

Specificity—confidence in detection of target 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.

Spike Ct Input
CHO Und 10 ng
Vero Und 10 ng
Hamster Und 10 ng
Chicken Und 10 ng
Pig Und 10 ng
S. cerevisiae Und 10 ng
Bovine Und 10 ng
Mouse Und 10 ng
Rat Und 10 ng
Dog Und 10 ng
Human 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

Confidence in results regardless of molecular weight fragments size

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.

DNA added M1 M2 M3 M4 M5
Un-cut Sau961 Un-cut Sau961 Un-cut Sau961 Un-cut Sau961 Un-cut Sau961
1 ng 122% 91% 122% 100% 95% 88% 103% 93% 85% 105%
0.1 ng 95% 94% 95% 108% 109% 86% 99% 107% 82% 110%
10 ng 91% 84% 91% 98% 96% 89% 84% 98% 86% 101%
1 ng 88% 82% 88% 87% 91% 79% 85% 82% 66% 101%
0.1 ng 91% 87% 91% 80% 100% 87% 82% 86% 76% 115%

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.

Get results in less than 5 hours with a streamlined workflow

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.

Get consistent performance regardless of sample matrix complexity

nucleic acid sample extraction, prepseq, sample prep, residual host cell DNA

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.

Increase sample processing throughput using automated sample preparation

The KingFisher Flex 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.

  • Consistent, high-quality nucleic acid recovery from a wide variety of sample types
  • Validated nucleic acid purification method for the resDNASEQ workflow to enable consistent, reproducible results
  • Cross-contamination control leveraging the bead transfer technology
KingFisher Flex magnetic particle processor instrument for automated sample preparation

Simplify data analysis with integrated instrument and analysis 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.

Key features:

  • Interfaces with the 7500 and QS5 Fast Real-Time PCR instruments
  • Intuitive interface to set up sample runs and interpret impurity data
  • Security, audit, and e-signature capabilities to help enable 21 CFR Pt 11 compliance
  • Windows 10 compatible

AccuSEQ assay results report for CHO samples

AccuSEQ data table
Figure 6: Table showing quantitation results for spiked CHO samples at two levels, each one run in triplicate.

Powerful platform. Regulatory preferred.
QuantStudio 5 qPCR for residual host cell DNA quantitation

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.

Learn more about our QS5

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 3rd generation platform features an innovative optical system that enhances sensitivity.

Learn more about our 7500 system ›

7500 qPCR for residual host cell DNA quantitation

Shop resDNASEQ quantitation kits by cell line 

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.


Custom Solutions

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 experts 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.

Learn more about our custom solutions ›

Worldwide service and support to ensure your scientific success

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Validation & implementation guidance

Validation consulting services provide technical project management to help verify that the assay is tested for required parameters

Compliance services

Timely, cost-effective, audit-ready documentation managed by a compliance specialist to ensure your instrument is installed, operating, and performing to manufacturer’s specifications

Training services

Application and instrument training programs available at our training centers located throughout the world, your lab, or through web-based instruction.

Service plans

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.

Bioprocessing resources

Access a targeted collection of scientific application notes, case studies, posters, white papers and more for bioprocessing: