Bioanalytical Assays of AAV Therapeutics: Recent Advances and Considerations

Bio distribution The evaluation of vector distribution profile following gene therapeutic administration is one of the key components to preclinical and clinical programs [3]. The biodistribution data serves to investigate the presence of vectors in both desired and undesired body fluids, tissues and organs. Q-PCR/ dd-PCR is the popular approaches for characterizing the viral vector, including the presence, persistence, and clearance [4]. During the development of qPCR based bioanalytical assays, scientists must optimize the extraction, amplification and detection of the nucleic acid analyte. Method validation is expected in supporting GxP level studies, and the validation should cover assay accuracy, assay precision, limit of quantification (LOQ), assay range, linearity and robustness [5]. As there is no bioanalytical guidance from regulatory agencies, the assay criteria are often defined according to the purpose of the studies. Despite the prevalence of qPCR in bioanalysis of gene therapeutics, it has several limitations. During the qPCR analysis, a large number of cells are required for extracting sufficient amount of DNA. During the extraction of nucleic acid, the cross-contamination across the experimental procedures limits the application of qPCR in some laboratories [5]. Liquid chromatography mass spectrometry (LC-MS): is one of the most popular platforms for the qualitative and quantitative bioanalysis of various therapeutics in laboratories [6]. Due to the advance of ion-paring reversed phase, LC-MS is also favoured in analyzing nucleic acids. The sample preparation is critical during the analysis and Liquid-liquid extraction (LLE) or/and anion exchange solid phase extraction (SPE) are recommended in this step [6]. Beyond GTs, LC-MS is used to measure the change of proteins regulated by gene therapeutics because it offers attractive sensitivity, broad range and high specificity [7]. Ligand binding assay: Such as ‘cutting ELISA’ (oligonucleotide probe hybridization/S1 nuclease protection) and fluorescent oligo probe hybridization is another approach that can be used to support the bio analysis of gene therapeutics. Hybridization ELISA often offers improved assay sensitivity in comparison with LC-MS. There are FDA and ICH guidance with LBAs that support the assay development and validation. In addition, LBA is comparable