QPCR Services
In house validated assays available for both standard CHO and E. Coli Residual DNA Analysis
Avacta Analytical can provide polymerase chain reaction (PCR), Quantitative PCR (QPCR) and reverse transcriptase QPCR) services to support clinical studies, biodistribution studies, viral screening, residual DNA analysis studies, detection of replication competent adenovirus, gene expression studies and for mycoplasma detection studies.
The polymerase chain reaction (PCR) is a molecular biology technique which allows a small amount of DNA to be amplified exponentially. QPCR has many advantages over the standard polymerase chain reaction:
-
Higher throughput method
-
Quantitative rather than qualitaive
-
Higher sensitivity
-
Data is recorded in real time and so removes the need for gel electrophoresis to view the results
It is recommended that QPCR assays are validated in accordance with the appropriate ICH topic Q2 (R1) guidelines to ensure the assay has a specificity and accuracy capable of detecting only the target sequence and its quantification limit or sensitivity is such that accurate detection is achieved at low levels. Other important elements to consider are the precision (in terms of repeatability and reproducibility) and the robustness of the assay taking into consideration any variations in reagents and operators.
Cross contamination controls
DNA/RNA is added into the QPCR in a specific order to avoid the possibility of cross contamination (negative controls, sample, spiked samples and positive controls). All work is carried out in separate laboratories dedicated to the respective sample types to provide segregation both spatially and temporally. Dedicated equipment and clothing are also used for each separate laboratory with a single direction of workflow to provide additional cross contamination protection.
QPCR controls are used to ensure validity of the final results:
- Internal positive control to indicate correct cycling conditions have been achieved and any negative results are valid.
- Sentinel controls, where water is added to wells containing reaction mix instead of the DNA template and left uncapped to monitor airborne contamination.
- Water controls, where water is added to wells containing reaction mix instead of the DNA template and capped immediately to monitor reagent contamination.
- Inhibition control, where the test sample is spiked with positive control DNA, to evaluate any possible inhibitory effects of the test material on the QPCR reaction
Residual DNA Analysis
The amount of host cell residual DNA in protein, plasmid and viral products requires determination for regulatory purposes. The most recent guidance issued by WHO (1998), conclude that up to 10 ng of DNA/purified dose is acceptable in the product. Specific assays can be developed and validated according to our clients requirements or standard ‘off the shelf’ assays can be used.
We currently have in house validated assays for both standard CHO and E. Coli RDA analysis.
Clinical studies
QPCR assays can be designed and validated to detect and quantify the client’s gene therapy vector. This assay can then be used to determine the presence, distribution and excretion of the vector in patients undergoing clinical trials. QPCR assays can also be used to determine the viral load of patients undergoing an anti-viral therapy to assess its efficacy. QPCR assays can be designed and developed according to our clients requirements or standard ‘off the shelf’ assays can be used.
Biodistribution studies
Regulatory requirements for gene therapy vectors are that pre-clinical safety data is compiled. Biodistribution studies can play an important part in pre-clinical safety evaluation as they can be used to determine into which tissues the vector has been integrated, specifically whether the vector is detected in the target tissues and germ line cells and the clearance of the vector from the tissues. Biodistribution of gene therapy vectors can be assessed by extraction of the DNA from a selection of tissues or bodily fluids (blood, urine etc) followed by QPCR analysis. The location of the vector and the quantity per microgram of tissue can then be determined. Furthermore, gene expression of the vector can be assessed in the tissues by extracting the mRNA and performing RT-QPCR analysis.
Replication Competent Adenovirus testing (RCA)
Adenoviral gene therapy vectors are made replication incompetent by the deletion of the E1 gene. It is possible for a replication defective adenovirus to become replication competent by recombination with a wild type replication competent adenovirus. Regulations suggest that a small amount of RCA in the lot is allowed and is dependent upon the application of the vector. Recent guidance by the FDA (2008) sets a maximum level of RCA contamination of less than 1 RCA in 3x1010 virus particles. QPCR is a useful tool for screening gene therapy vectors lots for the presence of RCAs.
Viral screening for Adventitious agents
Due to the possibility of contamination with an adventitious virus, it is necessary to perform studies to demonstrate their absence from a product. Avacta Analytical can provide QPCR based assays for the detection of species specific viruses. We have experience of developing assays to detect a wide range of viruses and are happy to develop and validate new assays to meet our client’s requirements.
To get a quote please e-mail: