Recombinant adeno-associated virus (AAV) products for the treatment of various conditions such as cancer and central nervous system diseases continue to gain importance in the realm of gene therapies. In consideration of the specialized testing packages AAV products require for regulatory submission, we have developed a number of new methods for vector characterization and quality control testing to move your program forward and make it to market within your timeline. In addition to AAV products, these tests are also applicable to lentiviral and cellular therapy products.
Specialized Methods and Their Advantages
Vector Capsid Characterization by Analytical Ultracentrifugation (AUC)
Many protein analysis assays do not accurately distinguish empty from full capsids due to the similarity in the diameters of each, as well as the lack of access to the full capsids’ nucleic acids which are fully shielded from the bulk solution by the outer capsid. Proven to be reliable in determining the ratio of empty and full viral capsids in AAV preparations, analytical ultracentrifugation (AUC) is gaining widespread acceptance as an optimal method for the characterization of gene delivery vectors. A development platform that lacks this crucial result will likely face questioning by regulatory bodies and ultimately impede progress to market; partnering with a provider with demonstrated expertise in performing this test is essential.
Vector Aggregation Characterization by Size-Exclusion Chromatography (SEC)
Our size-exclusion chromatography (SEC) method is proven to be indicative of stability for an AAV product and can be used to quantify overall and relative amounts of intact, aggregated, and fragment capsids. Profiling of the A280 vs. A260 protein ratio of each chromatographic peak enables nucleic acid- and protein-related peaks to be differentiated. Helpful in demonstrating product integrity for regulatory submissions, this beneficial method both monitors product stability with respect to aggregation and provides important lot vs. lot characterization data.
Vector Protein Characterization by Capillary Electrophoresis Sodium Dodecyl Sulfate (CE-SDS)
The protein components of a vector capsid can be resolved and profiled by capillary electrophoresis sodium dodecyl sulfate (CE-SDS). A typical profile can be achieved using our optimized sample preparation approach. The method provides the benefit of a relatively rapid and well-resolved product characterization/release method to confirm the ratio of the three AAV viral proteins, VP1, VP2 and VP3, within a product. It is also an important safeguard to demonstrate that no other protein contaminants are present within the material.
Quantitation of Residual Polyethyleneimine by Reverse-Phase Chromatography
Considering regulatory agencies’ concern around failure to demonstrate the removal and control of polyethyleneimine (PEI) – a widely used transfection agent – we recently validated our reverse-phase chromatographic method for the quantitation of residual PEI. If required, more sensitive methods which utilize an alternative chromatographic approach are available. PEI is a challenging analyte to measure, and our method provides reliable specificity and recovery of PEI in the presence of either or both protein and nucleic acid contaminants.
Quantitation of Plasmid Supercoiling Levels by High-performance Liquid Chromatography (HPLC)
Regulatory bodies are also concerned with evidence of acceptable levels of plasma supercoiling; thus, we have implemented a plasmid supercoiling chromatographic method using a NaCl salt gradient and a TSKgel DNA-NPR HPLC column, which enables us to analyze the ratio of supercoiled vs. non-supercoiled plasmid. This method can be easily modified to apply to a wide range of plasmid species.
Vector Protein Sequence by Liquid Chromatography
We have developed a novel method to predict protein function from sequence. The three AAV viral proteins, VP1, VP2 and VP3, may be identified in their intact state using liquid chromatography and mass spectrometry (LC-MS) by matching against the corresponding theoretical amino acid sequence. Protein mapping analysis may also be conducted to detect and monitor single amino acid changes, modification, and degradation products. In addition to providing valuable data on amino acid sequence, this method also provides indications of peptides with post-translational modifications often found in AAV samples.
Detection and Characterization of Host Cell Protein Contaminants by Nano-Liquid Chromatography & Mass Spectrometry (nano LC-MS)
Residual host cell protein (HCP) contaminants are identified using a combination of high resolution nano-liquid chromatography and mass spectrometry (nano LC-MS) proteomics methods. The samples are prepared and analyzed in triplicate at different protein injection amounts using a label-free quantitation (LFQ) workflow, which provides a higher dynamic range of measurement than more standard approaches. Once residual host cell proteins are identified, the MS/MS spectra corresponding to peptides comprising HCP contaminants are stored in spectral libraries which may be used for ongoing batch-to-batch quantification of those HCPs.