Gene therapies involving recombinant adeno-associated viral (rAAV) vectors have been approved by FDA, EMA, and used in well over 100 clinical trials, demonstrating efficacy against many genetic diseases.
Improvements to AAV production and purification processes have focused on improving gene delivery safety profiles by removing process impurities, fulfilling current good manufacturing practices (CGMP). Commonly observed impurities in rAAV include defective particles ("empty" AAV capsids and particles lacking infectivity), illegitimate DNA, process-related chemicals, and residual proteins from the biological production systems employed. Careful analysis of listed impurities is recommended when designing an AAV manufacturing process.
Residual proteins in purified AAV vector products can originate from the producer cell line, the helper viruses employed, or potential adventitious viruses. We use both traditional enzyme-linked immunoassay methods (ELISA) and by liquid chromatography mass spectrometry (LC-MS) to detect residual host cell protein (HCP) impurities in rAAV.
Due to ease-of-use and adaptability in quality control, ELISAs developed at our site in Erkrath, Germany have been used to detect HCPs in support of process development and product monitoring. Recently, LC-MS detection of HCPs has become an orthogonal approach expected by regulatory authorities, providing identification of the specific HCPs potentially causing immunogenicity or impacting product stability.
Identification and quantification of AAV HCPs using LC-MS:
|Host Cell Lines||Helper Viruses|
|Insect Cells (sF9)||Baculovirus|
|HeLa||Herpes Simplex Virus (HSV)|
Using untargeted LC-MS, specific proteins are detectable at or below 100 parts per million (ppm), as routinely achieved by data dependent acquisition (DDA) and label-free quantification (LFQ) methods. Targeted analysis of specific HCPs to further aid process development and product safety can achieve lower detection limits (~1-10 ppm).
Additional LC-MS approaches include data independent acquisition (DIA), or absolute quantitation (AQUA) employing synthetic peptide standards supporting multiple reaction (MRM) or parallel reaction monitoring (PRM).
Our Mass Spectrometry laboratory in Shrewsbury, Massachusetts has developed LC-MS methods that are now available to support your diverse AAV production programs. These LC-MS strategies complimenting ELISA may include mass spectrometry-based evaluation of the ELISA reagent and MS-based ELISA coverage determination.
If you are developing an AAV product and are ready to perform residual protein determination, our complete packages can be customized to streamline the process and accommodate your unique needs.
For more information on our host cell protein assays and mass spectrometry offering or to speak with one of our experts, contact us at [email protected].