In a finding with implications for one of the most promising cutting-edge medical treatments, the researchers evaluated several measurement techniques commonly used in gene therapy. The study determined that one of the most popular techniques was “problematic” and requires further development and standardization.
In gene therapy, a person’s defective genes are replaced or modified to treat or prevent diseases. With approximately two dozen products on the market and hundreds of clinical trials underway or planned, the treatment is hailed as a revolutionary approach to attacking the fundamental genetic causes of diseases ranging from sickle cell anemia to cancer.
Some gene therapy treatments use modified adeno-associated viruses (AAV) to deliver therapeutic genetic material to the patient’s cells. These AAVs are designed to target specific cell types. Their infectious genetic material is then replaced with therapeutic genetic material and administered to the patient.
Correctly measuring AAV vectors, as they are called, is critical to their safety and effectiveness.
In a recently published study, researchers from the National Institute of Standards and Technology, the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), and the US Pharmacopeia (USP) recruited six industrial laboratories in the United States and Europe to measure sample AAV vectors. .
Laboratories were asked to quantify the concentration of genetic material and viral particles in the vectors. They used four measurement techniques and reported their results to the study authors.
Of the four measurement methods used, the polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) had the lowest accuracy and precision. By precision, researchers mean how close the measurement is to the correct value. Precision refers to whether the method produces consistently similar results.
The imprecision of PCR-ELISA led the study authors to conclude that the method had “poor reproducibility.” Their results could not be reliably replicated either within the same laboratory or in different laboratories.
The method “should not be used for quantitative analysis.” [measurements of AAV vectors] without further development and harmonization of methods,” they added.
PCR-ELISA are actually two independent tools combined into one. PCR is used to quantify genetic material and ELISA measures the proteins that make up the viral envelope. Both tests have been around for decades, and there are dozens of versions of each sold on the market today.
As a result, there are subtle but important differences in the way PCR-ELISA is manufactured and used, said NIST chemical engineer Wyatt N. Vreeland. Everyone may think they’re doing the same thing when they take a PCR-ELISA test, he said, but that’s often not the case.
“It’s like a recipe for the same chocolate cake,” said Vreeland, who was the study’s principal investigator. “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.”
Here’s what the study found about the other three methods:
- SEC-MALS (size exclusion chromatography with multi-angle light scattering and tandem UV/Vis and/or refractive index) was the most accurate and precise of the methods tested.
- SV-AUC (Sedimentation Velocity Analytical Ultracentrifugation) with UV/Vis and/or Rayleigh interference optics was less accurate and precise than SEC-MALS, which surprised researchers since SV-AUC is considered the “gold standard.” ” for the measurement of AAV vectors. However, SV-AUC was better than SEC-MALS at creating a detailed “map” of the distribution of genetic and viral particles in the AAV vector. The study suggested 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, which measures the absorption of ultraviolet light by a sample, has significant limitations compared to other methods. It cannot distinguish between partially filled or overfilled AAV vectors. Additionally, the AAV protein particles are too large for the team to handle without errors. These problems with spectrophotometry are well documented, and this technique is generally not relied upon for high-precision measurements.
The study did not draw conclusions about past, ongoing, or future gene therapy research that relied on these methods for measuring AAV vectors. Nor did it make any policy or regulatory recommendations.
In future work, researchers from NIST, USP, and NIIMBL, a public-private partnership supported by NIST to support biopharmaceutical manufacturing, plan to develop standard operating procedures (SOPs) for the SV-AUC. SOPs are a detailed, written set of instructions for a specific technology or methodology. Scientists believe that having widely accepted SOPs for SV-AUC will improve its reproducibility performance to the same level as that of SEC-MALS, for which SOPs already exist.
“All the different methods we tested have their limitations and uncertainties,” Vreeland said. “The important thing is that you understand what your measurement technique can and cannot tell you.”