Researchers as the US National Institute of Standards and Technology and the US Pharmacopeia have found that one of the most popular measurement techniques used in gene therapy is "problematic".1
Results of the study revealed that PCR-ELISA — out of the four measurement methods analysed — was the least accurate and precise assay.
This is notable as PCR-ELISA is widely used by producers of adeno-associated viruses (AAVs) to test their safety and efficacy.
From these results, the NIST has determined that this assay type needs further development and standardisation to ensure its accuracy and reliability.
Methodology
To determine the accuracy of the four different measurement assays, researchers were asked to quantify the concentration of genetic material and viral particles in the sample vectors.
The assays tested in this study included PCR-ELISA, SEC-MALS, SV-AUC and A260/A280 dual wavelength ultraviolet spectrophotometry, endeavouring to identify their strengths and limitations.
Researchers quantified the "accuracy" of each assay by how close each measurement was to the known correct value, while "precision" refers to whether the method produced consistent results.
PCR-ELISA's limitations uncovered
During testing, it was revealed that PCR-ELISA's lack of precision meant that the method had "poor reproducibility", meaning that results were unlikely to be replicated reliably between labs.
Researchers also advise gene therapy developers to avoid the use of PCR-ELISA for measuring AAV vectors quantitatively until the method is further developed and standardised.
PCR-ELISA combines two measurement methods together, and has been in use within the pharmaceutical industry for decades.
However, since there are many iterations of each technique currently available on the market, there are subtle differences in how PCR-ELISA is utilised, meaning that the results obtained may vary significantly from lab to lab.
“It’s like a recipe for the same chocolate cake,” said Vreeland, who was the principal investigator on the study.
“You can give someone the same ingredients, but when you use different equipment to make them or bake them in different ovens, the cakes don’t turn out the same.”
The other methods assessed
The team at the NIST also assessed SEC-MALS (size exclusion chromatography with multi-angle light scattering and tandem UV/Vis and/or refractive index) which was revealed to be the most accurate and precise of all the methods tested.
SV-AUC (sedimentation velocity-analytical ultracentrifugation), which is often considered the 'gold standard' for AAV vector measurement, was found to be better than SEC-MALS in creating a detailed distribution map of the genetic and viral particles within the AAV vector, though it was less accurate and precise than SEC-MALS overall.
Therefore, scientists recommend that “SEC-MALS could be implemented as a general method with [SV-AUC] being used for more complete analysis as necessary.”
A260/A280 dual wavelength ultraviolet spectrophotometry, however, was plagued with significant limitations compared with other methods, as it cannot distinguish between partially filled or overfilled AAV vectors.
It was also determined that AAV protein particles are too big for the equipment to process without error.
“All the different methods we tested have their limitations and uncertainties,” said Vreeland. “What’s important is that you understand what your measurement technique can and cannot tell you.”
Reference
1 https://pubmed.ncbi.nlm.nih.gov/39723438/
[Image credit: Thomas Cleveland (IBBR/NIST)]