Scientists have examined various methods used to assess modified viruses in gene therapy, revealing that one method, SEC-MALS, stands out for its precision and accuracy, while another method has significant flaws.
In a study significant for advanced medical treatments, researchers assessed several measurement techniques that are typically used in gene therapy. Their findings indicated that one of the most widely used techniques is “problematic” and needs further refinement and standardization.
Gene therapy involves replacing or altering a person’s defective genes to either treat or prevent diseases. With approximately two dozen products available and hundreds of clinical trials underway or in planning, this therapy is recognized as a groundbreaking approach to addressing the genetic origins of various diseases, from sickle cell disease to cancer.
Certain gene therapy treatments utilize modified adeno-associated viruses (AAVs) to carry therapeutic genetic material into a patient’s cells. These AAVs are engineered to target specific cell types, with their harmful genetic material replaced by therapeutic genes before being administered to the patient.
Accurate measurement of AAV vectors is crucial for ensuring their safety and effectiveness.
A recent study involved researchers from the National Institute of Standards and Technology, the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL), and the U.S. Pharmacopeia (USP), who enlisted six industry laboratories across the U.S. and Europe to measure sample AAV vectors.
The labs were tasked with quantifying the amount of genetic material and viral particles in the vectors, employing four different measurement techniques and reporting their findings to the study’s authors.
Among the four methods utilized, polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) demonstrated the least accuracy and precision. Accuracy refers to how close a measurement is to the true value, while precision indicates whether a method consistently yields similar results.
The lack of precision in PCR-ELISA led the researchers to determine that this method exhibits “poor reproducibility.” Its results could not be reliably replicated both within individual laboratories and between different labs.
Consequently, they warned that this method “should not be used for quantitative [measurements of AAV vectors] without further method enhancement and standardization.”
PCR-ELISA consists of two distinct tools combined into one: PCR quantifies genetic material, and ELISA assesses the proteins making up the viral shell. Both techniques are well-established, with numerous versions available on the market today.
Because there are subtle yet significant variations in how PCR-ELISA is manufactured and applied, NIST chemical engineer Wyatt N. Vreeland pointed out that many may believe they are performing the same test, but they often are not.
“It’s akin to following a recipe for the same chocolate cake,” explained Vreeland, who led the study. “You might use the same ingredients, but differing equipment or oven conditions can lead to distinct outcomes.”
The study also examined the other three measurement methods, with the following findings:
- SEC-MALS (size exclusion chromatography with multi-angle light scattering and tandem UV/Vis and/or refractive index) was determined to be the most accurate and precise of the methods tested.
- SV-AUC (sedimentation velocity-analytical ultracentrifugation) that uses UV/Vis and/or Rayleigh interference optics was found to be less accurate and precise than SEC-MALS, which was unexpected since SV-AUC has typically been regarded as the “gold standard” for measuring AAV vectors. However, SV-AUC excels in creating detailed maps of the distribution of genetic and viral particles within the AAV vector. The study proposed that “SEC-MALS could serve as a general method, while [SV-AUC] could be utilized for more thorough analyses as required.”
- A260/A280 dual wavelength ultraviolet spectrophotometry, which assesses the absorption of ultraviolet light by samples, presented considerable limitations compared to the other methods. It is unable to differentiate between partially filled or overfilled AAV vectors. Moreover, AAV protein particles are too large for this technique, leading to potential errors. These shortcomings in spectrophotometry are well recognized, and the method is generally not trusted for highly accurate measurements.
The study did not draw conclusions concerning previous, ongoing, or future gene therapy research that utilized these methods for measuring AAV vectors, nor did it offer any policy or regulatory advice.
In future endeavors, researchers at NIST, USP, and NIIMBL—an initiative supported by NIST to promote biopharmaceutical manufacturing—plan to create standard operating procedures (SOPs) for the SV-AUC method. SOPs provide a clear, written set of instructions for executing specific technologies or methodologies. The scientists believe that developing widely accepted SOPs for SV-AUC will enhance its reproducibility performance to match that of SEC-MALS, which already has established SOPs.
“All the various methods we assessed come with their own limitations and uncertainties,” Vreeland noted. “What’s crucial is understanding what your measurement technique can reliably convey.”
