RESUMO
Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.
RESUMO
An analytical method using the imaged CE (iCE) technology has been developed and validated for measuring contents of charge variants for mAb molecules. The method could generate similar information that require both conventional IEF electrophoresis and ion exchange liquid chromatography to generate as an iCE analysis would produce both the pI of the molecules and quantitative contents of charge variants. Thus, it offers unique advantages over the IEF and ion exchange methods in terms of identification, separation and quantitation of charge variants. The data presented in this study demonstrated that the iCE method is suitable not only for research purposes but also for quality control purpose of mAb clinical or commercial manufacture as the technique can be validated and possesses sufficient robustness. The developed generic iCE method has been tested for a wide range of therapeutic mAbs and proved its suitability for multiple mAb molecules. The tested mAbs are a group of molecules with a wide range of charge compositions (acidic species ranging from 10 to 70%) and a diverse range of pI of 6.9-9.6. Developing platform analytical technologies is an efficient way to meet the demand of rapid growth of therapeutic mAb candidates under clinical and preclinical development. The iCE technology is a good candidate to become a platform method as it could cover a broad range of pH gradient. The experiences learned during the developmental process would provide important and valuable information to the biotech industry for the evaluation of charge heterogeneity of mAbs in terms of release testing, characterization, stability study, process development support and comparability study.
