ACUVRA: Anion-Exchange Chromatography UV-Ratio Analysis—A QC-Friendly Method for Monitoring Adeno-Associated Virus Empty Capsid Content To Support Process Development and GMP Release Testing

Anion-Exchange Chromatography UV-Ratio Analysis

Anion-exchange chromatography utilizes the difference in surface charge, resulting in species separation. Theoretically, empty capsids lacking the encapsulated DNA have a less negative charge than full capsids, causing them to bind less tightly to the anion-exchange column and elute first in shallow salt gradient conditions. There are many examples in the literature of IEC separations developed and optimized to exploit this net charge difference to achieve resolution between full and empty capsids for a range of serotypes (21, 22). However, each of these published methods is only applicable to the serotype it was developed for, and often required extensive method development and gradient optimization to achieve adequate resolution between empty and full capsids. Moreover, none of the reported applications have shown that the final optimized separation conditions were robust to product charge heterogeneity changes that may arise from batch-to-batch process variability and other product-related related variants that are often encountered in gene therapy early phase process development and scale-up.

We took a novel approach, where the goal was to develop a method that could become a platform, which would enable shortened method development timelines without sacrificing the method performance that is necessary for successful deployment and validation in a GMP QC setting. We use a shallow salt gradient to first elute DNA and other process-related impurities, followed by AAV capsids. Some empty/full separation may occur, but is not the objective and does not pose challenges to data interpretation, as integration of all capsid-related peaks is used to determine the A260/A280 ratio (Fig. 1).

Fig. 1figure 1

Overlays of 260 nm (solid) and 280 nm (dashed) ACUVRA chromatograms for a buffer blank, b rAAV2 5% Empty capsid calibration standard, and c rAAV2 51% Empty capsid calibration standard. Peaks in (a) labeled with * are buffer-related or system peaks observed in all standards and samples. Integration window for the capsid peak is denoted in inset in (b) and (c). Representative TEM images taken at 39,000 × magnification for d 5% Empty calibration standard and e 51% Empty calibration standard. A representative empty capsid (red circle) and full capsid (green circle) are shown in (d)

Calibration Model for Accurate Determination of %Empty Capsids

The A260/A280 ratio has previously been used for estimating vector genome titer in purified samples (17). It is noted that the correlation between this ratio and %Empty capsids in a sample is not linear due to changing DNA and protein contribution to UV absorbance as the %Empty capsids changes. The ACUVRA calibration curve (Fig. 2) was created by creating mixtures from mostly full and mostly empty rAAV2 material, as determined by TEM. While AUC is considered the “gold standard,” the large volumes required precluded its use in this instance. The small sample volumes required for TEM allowed for analysis of multiple replicates on multiple days, thus reducing the uncertainty in the final reported %Empty results. In this study, we evaluated two calibration models: a quadratic model, and a more complex alternative model, derived from physical principles (see Supplementary Information), which we call the “r3” model. As demonstrated, both quadratic and r3 models performed similarly within the range of the calibration curve. The decision to use the quadratic model (Fig. 2) for the final method was based on it being the simplest to implement for routine QC testing with the ability to perform all calculations within the validated Chromatography Data System (CDS) software. However, the r3 model does have advantages in certain situations, which is discussed further in the Supplementary Information.

Fig. 2figure 2

Calibration curve with quadratic fit. Calibration standards were prepared by co-mixing of rAAV2 materials to final levels of 5%, 17%, 31%, and 51% Empty capsids, as determined from TEM analysis. The A260/A280 response is the ratio of the integrated peak areas for the rAAV2 capsid peaks in the 260- and 280-nm channels. The mean of n = 2 measurements is shown with each error bar constructed from the min and max of the data

Proteins have an absorbance maximum at 280 nm, but DNA also contributes to absorbance at this wavelength. This relationship is complicated by the effect of changing the empty/full sample composition, making the conversion of the A260/A280 ratio to a %Empty capsid result less straightforward. Recently, SEC-UV-RI-MALS has been applied for analysis of AAV, combining the signals from each detector to calculate the Empty and Full content, along with capsid and genome titer (16). This approach also uses the A260/A280 ratio, but avoids the need for external calibration standards by using the RI signal to calculate the sample mass in absolute terms. However, this calculation relies on several assumptions: dn/dc of protein, dn/dc of DNA, extinction coefficient of protein, extinction coefficient of DNA, 1:1:10 ratio of VP proteins, and that AAV particles are isotropic scatterers. While any one of these assumptions is derived from established theory, the compound error of this many assumptions could be sizable. The approach we take here makes no assumptions except that TEM can measure %Empty accurately. An additional drawback of SEC is its being an isocratic separation, and there is an upper limit on the volume of sample that can be injected while maintaining good peak shape (29, 30). IEC has advantages when analyzing low concentration samples, as injection volume does not impact the efficiency of the separation, and the sample is effectively concentrated at the top of the column prior to the gradient elution. Unfortunately, the increasing salt gradient poses a challenge for coupling with RI detection, thus precluding UV-RI-MALS quantitation approach with this mode of chromatography.

Our %Empty standard calibration curve approach eliminates the necessity for assumptions surrounding extinction coefficient calculations or for the resolution of empty and full capsid peaks to characterize the %Empty capsids in a sample. This also helps control for any instrument-to-instrument differences that could impact the absorbance response, such as UV detector lamp age and flow-cell dimensions.

It should be noted that the A260/A280 ratio range which we measured was small, with the 51% Empty calibrant measuring ~ 1.2 and 5% Empty calibrant measuring ~ 1.45. This narrow range requires that assay precision be given due consideration, as small changes in these values can have significant impact on results. Therefore, we took steps to uniformly integrate all capsid peaks in the chromatograms at each wavelength and found that application of a smoothing factor greatly improved the consistency of the baseline integrations. Precision was further controlled by performing n = 2 injections of the calibrants and samples and setting sample acceptance criteria around the relative difference of the A260/A280 ratio between replicates. Calibrants were not created for sample with more than 51% Empty capsids because this is outside of the expected range for this rAAV2’s process intermediates, drug substance, and drug product samples. However, a wider range could be advantageous, especially in research and academic settings, to allow for analysis of a broader range of samples. As noted in previous studies, samples with compositions of predominantly empty capsids have been modeled to show that precision of the absorbance ratio decreases as the percentage of empty capsids increases (17). In our work, we generated four calibration levels. While additional levels may improve the fitting of the calibration model, we demonstrate that four levels are sufficient to achieve acceptable accuracy and precision for quantitation of %Empty capsids in the range relevant for our rAAV2 samples.

Analysis of rAAV2 Process Intermediates and Drug Substance

Two rAAV2 process intermediates and a drug substance sample were analyzed using the above-described ACUVRA method. Process intermediate 1 and process intermediate 2 represent different fractions recovered following the iodixanol density gradient ultracentrifugation step, which is the first purification step in the downstream process. The presence of iodixanol in the process intermediates caused significant interference in detection of the capsid peaks at both 260 and 280 nm, as shown in Fig. 3. However, we found that buffer-exchanging six times was effective in removing this interference and enabled successful detection and quantitation of the capsid peaks in these process intermediate samples. Drug substance samples with salt concentrations > 100 mM NaCl required only an initial dilution in mobile phase A buffer prior to analysis to enable binding to the anion-exchange resin.

Fig. 3figure 3

a 260-nm and b 280-nm chromatograms representative of rAAV2 process intermediates from the post-iodixanol density gradient ultracentrifugation step showing overlaid profiles before (red) and after (blue) performing six buffer exchanges into drug substance formulation buffer. Enhanced view of capsid peak at ~ 7 min is shown in inset panels, showing effective removal of iodixanol by buffer exchanging to enable detection and integration of the capsid peak at both 260 and 280 nm. Buffer-related peaks are denoted with *

Representative chromatograms and TEM images for rAAV2 process intermediate 1, process intermediate 2, and drug substance are shown in Fig. 4. A comparison of the %Empty capsid results by ACUVRA to orthogonal TEM analysis (Table I) demonstrates the good agreement between the two methods and confirms that the calibration curve quantitation approach is suitable for both process intermediates and purified drug substance. While the accuracy of 143% for process intermediate 1 is outside the typical 70–130% acceptable range per ICH Q2(R1) Validation of Analytical Procedures guidance (31), the relative accuracy requirements for our purposes are less stringent for samples from early process steps than for drug substance. The ACUVRA results for process intermediates are suitable for characterizing trends across the multiple downstream process steps.

Fig. 4figure 4

Overlays of 260 nm (solid) and 280 nm (dashed) ACUVRA chromatograms for rAAV2: a process intermediate 1, b process intermediate 2, and c drug substance. Blue-shaded region on the chromatograms indicates the integration window for the capsid peaks, and buffer-related peaks are labeled with *. Representative TEM images for each sample are shown to the right of each chromatogram, with a representative empty (red circle) and full (green circle) capsid labeled in each image. TEM images in panels (a) and (b) were acquired at 21,000 × magnification, while (c) was acquired at 39,000 × magnification

Table I %Empty Capsid Results for rAAV2 Process Intermediates (from Iodixanol Density Gradient Ultracentrifugation Step, Post-Buffer-Exchange) and Drug Substance Analyzed by Anion-Exchange Chromatography UV-Ratio Analysis (ACUVRA) in Comparison to Orthogonal TEM AnalysisMethod Qualification and Validation for rAAV2

The goal of this work was to develop a QC-friendly method to measure %Empty capsids in the rAAV2 drug substance and drug product for batch release and stability studies. Qualification experiments were conducted in accordance with ICH Q2(R1) to assess Repeatability, Intermediate Precision, Accuracy, and Range. Method performance attributes are summarized in Table II, and show repeatability of 7.9% RSD, intermediate precision of 7.4% RSD, and accuracy (expressed as recovery to TEM results) of 112.9%. The range of the method is defined by the low and high level calibration standards, which in this instance is 5–51% Empty capsids. The qualification results were used to set the acceptance criteria for method validation in a quality control laboratory. Method validation results (Table II) show consistent performance between the development and QC laboratories, meeting all validation acceptance criteria, thus demonstrating this as a viable QC-friendly approach for %Empty capsid determination.

Table II ACUVRA Method Performance Results from Method Qualification for rAAV2 Drug Substance Performed in the Analytical Development Laboratory, and Method Validation for rAAV2 Drug Product Performed in the Quality Control LaboratoryAdditional Considerations

It should be mentioned that the established ACUVRA approach is not able to distinguish between partially filled and empty capsids. This is also a limitation of IEC methods that do separate the empty from full capsids, as well as the SEC-UV-RI-MALS (16) and SEC-UV (19) approaches. Currently, AUC is the only broadly available technique that has the resolving power to separate the partially filled from empty and full capsids (7, 23), although more recently icIEF (15) and mass photometry (32) have been shown to resolve partially filled capsids in certain circumstances, while CDMS has been used to characterize AAV genome packaging (10). None of these techniques, including ACUVRA, can identify if the capsid is packed with DNA from host cells or other exogenous debris; however, this quality and safety concern can be addressed with specifically designed residual impurity assays. From a Chemistry, Manufacturing, and Controls (CMC) and patient-safety perspective, we classify as product-related impurities any capsids that do not contain the complete target genome. In our method, we report “%Empty capsids” and treat this as an impurity method, but one could also report results as “%Full capsids” and consider it as an expression of product purity.

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