Anion-exchange HPLC assay for separation and quantification of empty and full capsids in multiple adeno-associated virus serotypes

Abstract

Gene therapy has entered a new era where numerous therapies for severe and rare diseases are generating robust and compelling clinical results. The rapid improvements in gene therapies over the past few years can be attributed to better scientific understanding of the critical quality attributes that contribute to a safe and efficacious product, as well as a better understanding of the manufacturing processes that are required to yield consistent products, which routinely meet the quality standards required for clinical studies. Of particular concern is the need for an effective, quality control (QC)-compatible, and versatile test method for the quantification of empty and full capsids in recombinant adeno-associated virus (rAAV) samples from multiple serotypes. In that regard, we describe the development of a QC-compatible anion-exchange chromatography method consisting of a modular discontinuous gradient to achieve full baseline peak separation and quantification of empty and full AAV capsids. Using an rAAV6 vector, our assay was shown to be precise, linear, robust, and accurate-correlating well with orthogonal methods such as analytical ultracentrifugation (AUC) and cryogenic transmission electron microscopy (Cryo-TEM). Additionally, we demonstrate the versatility of our approach by adapting the method to separate and quantify empty/full capsids in samples from several rAAV serotypes.

Clinical enrollment assay to detect preexisting neutralizing antibodies to AAV6 with demonstrated transgene expression in gene therapy trials

“…AAV titers were determined by quantitative polymerase chain reaction (qPCR). Full and empty capsid ratio was determined for applicable lot using high-performance liquid chromatography [12].…”

Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids

“… 26 − 28 Although this approach remains time-consuming and each serotype requires condition optimization, it must be noted that recently several groups have been working on the optimization of the technique across different serotypes. 29 , 30 OD, or UV absorbance spectroscopy, utilizes the extinction coefficient (an indicator of light absorbance at a given wavelength) of capsid proteins and DNA, and it estimates the ratio of capsid particle to vector genome based upon the absorbance ratio. 31 OD analysis time can be as low as 15 min/sample; however, it has very high purification requirements to prevent interference with the UV absorbance.…”

Surface Modified Nano-Electrospray Needles Improve Sensitivity for Native Mass Spectrometry

“…Native mass spectrometry (MS), which uses nondenaturing ionization prior to mass analysis, has become a useful tool for characterizing protein and macromolecular complexes. − For applications like intact adeno-associated viral (AAV) capsids that yield unresolvable charge states, native MS is coupled with charge detection–mass spectrometry (CD-MS) to simultaneously measure the charge and m / z , enabling mass analysis for highly complex systems. ,, AAVs are used for gene therapy, vaccines, and drug delivery, − and CD-MS provides direct ratios of empty versus filled capsids. , However, CD-MS analysis of AAVs often requires relatively high concentrations of sample in the range of 5–100 × 10 12 capsids per milliter, which is roughly 10–300 nM. − More broadly, native MS is also limited in sensitivity and usually requires approximately micromolar concentrations of analyte . A comparable method, mass photometry, has lower nanomolar levels of detection for protein complexes but with lower resolution than native MS. , …”