Characterization in process development
The percentage of full capsids is an important quality attribute for this viral vector used for gene therapy delivery. During process development optimization there’s a need to quickly assess changes in % full capsids ― between different process steps and peak fractions and from starting material to purified full capsids.
Conventional characterization methods for empty full analysis
Traditionally, adeno-associated virus (AAV samples, of any purity level) are analyzed using qPCR/ddPCR and ELISA to determine the titer of the genome-containing capsids and total capsids, respectively. To determine the % full capsids the ratio between PCR:ELISA is commonly used. However, the drawback of this method is that each assay has its variation, and using the ratio between them may enhance the variation.
Typically, the % full capsids from qPCR/ddPCR:ELISA needs to be confirmed by using an orthogonal or additional method. Many methods with varying performance and cost are available to choose from; most require high purity and often a relatively high amount to give accurate results.
The need for a quicker, robust method
Our teams at Cytiva successfully developed an anion exchange (AEX)-based chromatography process to achieve baseline separation of empty and full capsids at scale, and we showed robust results for several serotypes. Here we show how AEX with Capto™ Q columns (previously reported/published) can be used analytically to determine % full AAV capsids in 5 to 15 min per run.
The method can be run on ÄKTA pure™ systems, with bypassed mixer and using a sensitive 10 mm path length UV detector and (fluorescence detector (FLD)). Or it can be run on a high-performance liquid chromatography (HPLC) system with FLD detector. Capto™ Q packed into Tricorn™ 5/20 0.5 mL or prepacked HiTrap™ columns (1 mL) can be used. The buffers were 20 mM Tris or Bis-tris propane (BTP), pH 9 with constant 2 mM MgCl2 (A buffer) and either NaCl or NaAc salt (250 mM) for elution (B buffer). Sample loads as low as 1 × 1010 viral particles/run and flow rates 1 to 5 mL/min can be used.
The optimal conductivity (% B buffer) for empty capsid elution is determined by a prescreening with small conductivity steps; one prescreening is needed for each capsid serotype or variant. A final two-step protocol elutes empty capsids first in step 1 followed by full capsid elution in step 2. Peak UV260:280 ratio confirms empty and full capsid purity, and then UV280 or FLD signal area in the peaks is used to calculate % full capsid in the sample.
The reproducibility was good between triplicate runs, and the UV260:280 ratios in the peaks were consistent between runs.
Detailed protocol for our empty full analysis using AEX
Table 1. Methods used for prescreening (needed once for each AAV capsid serotype or variant to determine % B buffer for step 1 eluting the empty capsids), analytical two-step protocol for AAV8 and AAV9, and recommended CIP protocol (to avoid precipitation of MgCl2 inside column)
CIP is cleaning in place. VP is viral particle. CV is column volume.
Empty full analytical results
Fig 1. Analysis of AAV8 and AAV9 using a Capto™ Q resin (HiTrap™ column, 1 mL) connected to an ÄKTA pure™ 25 system, comparing using % peak 2 area (UV280) or qPCR:ELISA ratio to determine the % full capsids in the samples. Typical UV260:280 (orange: blue lines) for empty peak 1 and full peak 2 are indicated in the chromatograms.
Figure 2 legend: blue line - measured percentage of full AAV capsids based on peak area; orange dotted line - theoretical percentage of full AAV capsids from 0% to 100%.
Fig 2. AAV8 was preparatively purified into full and empty fractions (purity confirmed by qPCR:ELISA and UV260:280 ratios) and mixed in different ratios with 10% intervals from 3% up to 89% full capsids. The AAV8 sample mixes were run on a Capto™ Q 0.5 mL Tricorn 5/20 column connected to an HPLC system (1260 Infinity LC system, Agilent Technologies) fluorescence detector (FLD, 280 nm excitation/350 nm emission). The % intrinsic tryptophan fluorescence signal in peak 1 and 2 of the empty and full AAV capsids was used to calculate the % full capsids in the mixed samples.