Successful Validation Of Capsid Titer And Host-Cell Derived DNA Impurity Assays Extends Our rAAV Batch Release QC Portfolio

By F. Thoennissen, M. Magerl, C. Zach, M. Gora, T. Kloetzler, P. Zanotti, S. Beer, R. Kober, A. Schoberth, S. Schermann, S. Geiger

Quantifying capsid titer and packaged host-cell-derived (HCD) impurities are critical quality attributes for recombinant adeno-associated virus (rAAV) batch release. This study established AAV9 capsid titer determination using the automated immunoassay system Gyrolab xPlore, employing commercially available AAV9 empty capsids from two manufacturers as standard and trending controls. Robustness studies examined the impact of using different lots of empty capsids for standard and control kit components.

For HCD determination, a droplet digital PCR (ddPCR) method was developed, targeting the 18S ribosomal RNA gene locus as a surrogate marker for packaged DNA impurities in rAAV. The method underwent extensive qualification and robustness testing, including assessments of droplet lifetime and sample dilution storage, in compliance with ICH Guideline Q14 for analytical procedure development. Analytical validation of both methods, performed under GMP conditions, adhered to ICH Guideline Q2(R2), evaluating parameters such as specificity, working range, calibration model suitability, precision, and accuracy.

These methods, adaptable for various serotypes and production cell lines with minor protocol modifications, present significant potential for expanding rAAV batch release testing capabilities.