Digital PCR: Quantitative quality control for plasmid and viral vector development

 

Quality control is essential at every stage of the development process to create reliable cell and gene therapies.  With the ability to provide an absolute count of nucleic acids, digital PCR (dPCR) is used for many applications, including:


Vector copy number—assess the number of integrated vector genomes during characterization studies and monitor product stability and identity
Virus quantification—precisely quantitate genomic titer, viral particle titer, and full/empty capsid ratios during purification and lot release to accurately asses dose
Purity & safety testing—identify product and process-related impurities such as residual DNA or microbial contamination for in-process and release testing

 

Benefits of digital PCR for cell and gene therapy development
 

Precise absolute quantification across a range of nucleic acid concentrations

For cell and gene therapy workflow steps that require absolute quantification, such as viral titer and vector copy number, dPCR provides accurate measurements without the use of a standard curve. This removes a source of variability, which makes the process more robust and efficient.

Application Note: Automated high-throughput digital PCR for accurate and consistent AAV quantification

 

Data plot showing expected vs. observed AAV concentrations across a dilution series
Figure 1. Expected vs. observed AAV concentrations across a dilution series, obtained using the Absolute Q AAV Viral Titer dPCR Assay on the QuantStudio Absolute Q AutoRun Suite.

Simple and scalable automated dPCR workflows

Reclaim valuable time and resources while helping to maximize throughput with an automated system that enables flexible configurations and walk-away workflows for high throughput digital PCR.

The Applied Biosystems QuantStudio Absolute Q AutoRun Digital PCR  Suite is an integrated system solution that scales precise nucleic acid quantification while keeping it simple.

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Vector copy number
 

Vector copy number (VCN) refers to the number of vector genomes that have been incorporated into the genome of  target cells. Because viral transduction can be variable, precise measurement of vector copy number assumes pivotal significance, serving as a vital metric for monitoring sustained transgene expression and for determining therapeutic dose.

dPCR enables robust absolute quantification for precise copy number calculation

Digital PCR, which utilizes simple counting of target molecules to achieve absolute quantification, enables higher precision and accuracy to detect and quantify copy number variations, even if the changes are small.

Application Note: Lentiviral vector characterization using the QuantStudio Absolute Q Digital PCR System

Learn more in the education center ›

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Virus quantification
 

Gene and cell therapies utilize a variety of viral vectors for gene transfer, including adeno-associated viruses (AAV), lentiviruses, retroviruses, herpes viruses, adenoviruses, and others. Controlling the concentration of a potential therapeutic is difficult since viral particles are produced in living cells and then purified. The viral titer must be measured accurately, precisely, and consistently to ensure correct formulation..

dPCR provides high precision for genome titer, viral particle titer and full/empty capsid ratio calculation

Applied Biosystems Absolute Q Viral Titer dPCR assays enable easy and accurate quantification of viral vectors. The assays can be run individually or multiplexed using a custom assay with your target gene of interest.

 Poster: Measuring rAAV Genomic Titer, Viral Particle Titer and Full/Empty Capsid Ratio on the QuantStudio Absolute Q dPCR System

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dPCR fluorescent plots measuring proviral copies in cells transduced with lentivirus and table with proviral concentration calculations
Figure 4: Representative dPCR fluorescent plots measuring proviral copies in cells transduced with lentivirus at the specified MOI and subsequent proviral conentration calculations, obtained with the ViralSEQ Lentivirus Proviral DNA Titer Kit on the QuantStudio Absolute Q System

 


Purity and safety testing: residual DNA & microbial contamination
 

The process of generating and purifying viral vectors and cell therapies is also susceptible to contamination by host cell DNA, mycoplasma, and other contaminants. Such product and process-related impurities must be detected and screened out with the highest sensitivity to maintain the highest safety standards and meet regulatory requirements. 

dPCR enables quantitative detection residual host DNA or plasmid contaminants and microbial agents

Applied Biosystems Absolute Q Viral Titer dPCR assays and SEQ-technology kitted solutions provide a simple and accurate way to test for contamination in a broad range of sample types, from raw materials and in-process samples to purified final products.

Tech Note: Residual plasmid quantification in gene therapy manufacturing workflows using the QuantStudio Absolute Q Digital PCR System

Application Note: Assessment of using the QuantStudio Absolute Q Digital PCR System to quantify residual E. coli DNA

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Graph depicting the concentration of plasmid DNA determined by KanR target quantification
Figure 5: Concentration of plasmid DNA determined by KanR target quantification, run on the Absolute Q dPCR System using the resDNASEQ Quantitative Plasmid DNA - Kanamycin Resistance Gene (pDNA-KanR) Kit.


Assays and kits

Absolute Q dPCR assays for viral titer

Applied Biosystems Absolute Q Viral Titer dPCR assays enable easy and accurate quantification of viral vectors with the following features:

  • Simple—streamlined workflow for ease-of-use with your digital PCR instrument
  • Fast—minimal hands-on time; results in 90 minutes when used with the QuantStudio Absolute Q Digital PCR System
  • Dependable—analyze your data with confidence using verified assays backed by a performance guarantee*
     

Ordering information

To order an Absolute Q Viral Titer Assay please add it to cart by entering the Catalog Number and Assay ID (if applicable) provided below into ThermoFisher.com’s Quick Order tool.

 

Target

Dye

Catalog No.

Assay ID

Absolute Q Viral Titer Assay

eGFP

VIC-MGB/20X

A53736

DT2W7KW

Absolute Q Viral Titer Assay

GFP

VIC-MGB/20X

A53736

DTFVKJZ

Absolute Q Viral Titer Assay

SV40 polyA signal

VIC-MGB/20X

A53736

DTZTDZY

Absolute Q Viral Titer Assay

AAV ITR-2

VIC-MGB/20X

A53736

DTH49PU

Absolute Q Viral Titer Assay

CMV promoter region

VIC-MGB/20X

A53736

DTYMJF2

Go to Quick Order tool

Other assays and kits for digital PCR analysis

Assay-design-hub

Custom dPCR assays

Can’t find the assay you’re looking for? Design a digital PCR assay with a self service tool or get help with our design services

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Lentiviral kits

Lentivirus characterization solutions

  • ViralSEQ Lentivirus Physical Titer Kit—quantitate the total number of genome-containing lentiviral particles by targeting the conserved long terminal repeat (LTR) region of the genome
  • ViralSEQ Lentivirus Proviral DNA Titer Kit—measure vector integration in transduced cells by targeting LTR in the provirus

Learn more ›

A51970 resDNASEQ

Residual DNA testing solutions

  • resDNASEQ dPCR E1A DNA Fragment Length Kit—quantify residual DNA fragment sizes in HEK293 production systems and other cell lines transformed with the E1A gene
  • resDNASEQ dPCR E. coli. DNA Kit—quantify residual DNA in E. coli production systems, commonly used for production of recombinant proteins and plasmids
  • resDNASEQ Quantitative Plasmid DNA - Kanamycin Resistance Gene Kits—detect residual plasmid containting kanamycin-resistant genes

Learn more ›

 

taqman-doc-files

TaqMan Assays

Adapt an existing TaqMan Assay for use on the QuantStudio Absolute Q Digital PCR System.

Download quick reference guide ›

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Resources

dPCR and qPCR workflow tool

dPCR and qPCR workflow tool

Explore workflows, view product recommendations, and find regulatory documentation for cell and gene therapy.

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Customer testimonial

Customer testimonial

See why Senti Biosciences is using Absolute Q dPCR System to characterize their gene circuit technology.

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Talk to a Digital PCR specialist

Talk to a digital PCR specialist

Have questions about implementing dPCR in your lab? Let us help you get started.

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*Terms and conditions apply. For complete details of the guarantee, go to thermofisher.com/absoluteqassayguarantee

For Research Use Only. Not for use in diagnostic procedures.

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