Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors

Abstract

Adeno-associated virus (AAV) vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. An inherent characteristic of the manufacturing process is production of empty capsids that lack the transgene and are therefore unable to provide the intended therapeutic benefit. The effect of empty capsids on clinical outcomes is not well understood, but there are immunogenicity and efficacy concerns, and these empty capsids are considered a product-related impurity. Therefore, empty capsids should be controlled during the manufacturing process and monitored through analytical testing, but there are limited techniques available that are capable of quantifying capsid content and even fewer that are amenable to validation and implementation as registered release tests in a regulated environment. In addition, there is currently not a widely accepted gold standard technique for quantifying capsid content, and the understanding of how the results compare between different orthogonal technologies is limited. The current study utilizes a comprehensive assessment to evaluate diverse analytical techniques for their ability to quantitate capsid content.

Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer

“…While CDMS was not used to quantitate particle content for the mixtures in this study, the data included represent a proof of concept that the technology could be used for such purposes. A similar study was performed on AAV capsids, which demonstrated that CDMS was highly complementary to AUC ( Werle et al, 2021 ).…”

“…Capsid protein analysis via reversed-phase high performance liquid chromatography (RP-HPLC) can also parse out particle maturity via analysis of viral protein content and processing ( Lehmberg et al, 1999 ; Vellekamp et al, 2001 ; Chelius et al, 2002 ; Takahashi et al, 2006 ). In addition to the more routine techniques, additional-state-of-the-art techniques, including charge detection mass spectrometry (CDMS) ( Contino and Jarrold, 2013 ; Pierson et al, 2016 ; Draper et al, 2018 ; Draper and Jarrold, 2019 ) are available for direct mass determination of intact viral particles ( Kondylis et al, 2019 ; Werle et al, 2021 ).…”

Applications of Charge Detection Mass Spectrometry in Molecular Biology and Biotechnology

“…Along with capsids that have packaged the full genome of interest, empty capsids are generated, and some capsids package a partial genome. − The encapsidation of DNA impurities, such as heterogeneous fragments of host cellular, helper, or plasmid DNAs, also occurs. , Finally, aggregation can reduce bioavailability and influence biodistribution and may also cause an immune response . CDMS has emerged as a valuable tool to analyze the DNA content of rAAV vectors. , It can go beyond characterization of the empty/full ratios and provide information about particle heterogeneity including packaging of heterogeneous DNA Figure a shows a CDMS mass distribution measured for empty AAV8 particles separated by ultracentrifugation with a CsCl gradient.…”

“…225 CDMS has emerged as a valuable tool to analyze the DNA content of rAAV vectors. 222,226 It can go beyond characterization of the empty/full ratios and provide information about particle heterogeneity including packaging of heterogeneous DNA. 227 Figure 10a shows a CDMS mass distribution measured for empty AAV8 particles separated by ultracentrifugation with a CsCl gradient.…”

Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM

“…Table 1 provides an overview of methods commonly used for detecting full and empty capsid particles. A recent comparison of these techniques demonstrated good correlation between the listed methods [ 40 ]. Within this set of orthogonal methods, AUC is a popular technique due to its capability of discriminating the population of partially filled capsids, a population that is gaining more and more interest in the industry.…”