As rAAV vectors more frequently head toward the clinic for gene therapy trials, there is an increasing need to share pharmacokinetic, toxicologic, and efficacy data. This need is currently confounded by the lack of standardization of critical vector parameters such as vector strength and potency. The standardization issues arise because different assays or different protocols for the same assay are often used by individual investigators to measure an identical vector property. The introduction of a widely accepted rAAV reference standard would allow laboratories to characterize AAV vectors in terms of common units, therefore facilitating comparison of doses determined by disparate assays and permitting safe and effective dosage at equivalent levels. Furthermore, efficacy and toxicology data reported in the literature could be used as a guide for initial dosing in animals and humans.
Here we have described the characterization of the first rAAV reference standard, an AAV serotype 2 vector. The goal of the AAV2RSWG was to provide a stable, high-quality, highly characterized RSM that would be both accepted and easily accessed by the AAV research community. As pointed out by FDA officials at the beginning of the effort, a reference standard material does not need to be pure or the “best,” it just needs to be well characterized. Furthermore, there are many examples of viral reference standard materials from the World Health Organization (WHO, Geneva, Switzerland) and the National Institute for Biological Standards and Control (NIBSC, Potters Bar, UK) that are not pure (e.g., poliovirus and hepatitis B virus references). Although the rAAV2 RSM was made in a research vector core and not at a current Good Manufacturing Procedure (cGMP) facility, it was extensively tested for adventitious agents and contaminants. The final rAAV2 RSM product was negative for adventitious agents in all tests to which it was subjected, although the harvest material was exposed to mycoplasma that was cleared and/or inactivated in the purification process, because the purified bulk tested negative for viable mycoplasma and mycoplasma DNA (Potter et al.
). Because the rAAV2 RSM is a reference standard to be used in research and quality control (QC) laboratories and is not intended for use in humans, the AAV2RSWG recommended that filling, banking, and characterization proceed. A summary of the mycoplasma testing will be included on the product information sheet supplied with each shipment of the rAAV2 RSM, stating that the reference standard has been exposed to mycoplasma, but is mycoplasma-free. Thus, institutions and companies requesting the rAAV2 RSM will be fully informed and can decide if they want to bring it into their QC laboratories. The reference material is intended to be restricted to QC laboratories, isolated from production suites. In addition, it is envisioned that internal reference standards will be calibrated against the AAV2 RSM one time and then used on a routine basis for product-specific testing.
The short-term stability testing performed on the surrogate AAV2-GFP vector as well as on the final vialed rAAV2 RSM material suggested that some loss of vector potency was occurring on storage. Initially this loss was assumed to be due to absorption to the surfaces of the vial as was seen in the previous study using vials that were not siliconized (Potter et al.
); however, when vector genomes were assayed no corresponding loss was seen when using siliconized vials (). One explanation for the loss of potency observed may be the omission of a stabilizing excipient in the final formulation (Croyle et al.
; Wright et al.
). The beta test stability results influenced the way the rAAV2 RSM was handled during the testing phase; aliquots were thawed only once and transduction and infectivity assays were performed within 1
hr of this thaw. Regarding future use of the reference material for potency assays, it would seem essential that a similar protocol be followed when normalizing internal reference standards against the rAAV2 RSM. For physical titer assays such as particle and vector genome assays, storage and refreezing are permissible. Plans for assessing the long-term stability of the rAAV2 RSM by yearly testing for capsid protein integrity, infectious titer, transducing titer, and vector genome titer are in place. Data will be reported by the AAV2RSWG through the Reference Standards section of the International Society for BioProcess Technology website (www.ISBioTech.org
The characterization phase of the rAAV2 RSM project successfully fulfilled the goals of the AAV2RSWG by obtaining mean titers and 95% confidence intervals from a large number of representative assays performed by numerous test centers. The tightest confidence intervals were obtained for the nonbiological assays (particle titer and vector genome titer) whereas the biological assays (infectious titer and transduction titer) gave wider intervals (). This pattern might be expected because the biological assays are inherently more variable. The tight confidence interval observed for the vector genome titer is relevant because this titer has been used exclusively in dosing regimens and a high degree of precision is important for the use of the rAAV2 RSM in dose standardization.
One obvious trend in the quantitative assay data was the degree of variation between institutions for each assay ( and ) despite the relatively tight correlation of assay results within an institution (). This poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. The variation may be explained by the use of different reagents (i.e., other than those provided, such as tissue culture media, PCR primers, and PCR mixes), equipment, and/or operator technique. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. This need is especially apparent when it is considered that fundamentally dissimilar tests are often used to measure the same parameter (e.g., qPCR and dot–blot for vector genome titer) and that even when different laboratories use the same assay, different protocols are usually followed. For some assays the variation is not large, with the most important measure, vector genome titer (qPCR), which is almost exclusively used for dosing in preclinical and clinical studies, having low variation (confidence interval of less than 0.5 log). Despite the spread of infectious titers, the mean value represents the best titer based on multiple replicates conducted at the different sites on different test dates.
Because the rAAV2 RSM supply is limited, it is not intended that it be used routinely, but rather for the calibration of laboratory-specific internal reference standards, which can then be run concurrently with test samples in subsequent assays that have been validated. The initial calibration would involve titering the RSM alongside the internal standard in the same assay; the difference between the titer determined in this assay and the accepted titer of the RSM would act as a conversion factor for calculating the titer of the internal standard in reference standard units (RSU). Once the internal standard titer is known in reference standard units per milliliter, the titer of test samples can be calculated similarly in the same units, during subsequent assays. It is envisaged that the RSM will be used in this way for standardizing the genome titer, particle titer, and infectious titer of AAV2 vectors. A prerequisite for qPCR or hybridization-based vector genome/infectivity titering methods would be that the internal AAV standard share enough genome sequence with the rAAV2 RSM for oligonucleotide or labeled probe annealing. Several common transcriptional elements are included in the rAAV2 RSM genome for this purpose and many existing internal reference standards will therefore be candidates for calibration. If this is not the case, new internal standards will need to be produced that harbor DNA elements in common with the AAV2 RSM. For transducing titers, the encoded transgene provides the basis of detection and, therefore, with the exception of GFP-expressing vectors for preclinical studies, these titers will generally not be amenable to standardization using the rAAV2 RSM.
Although the primary intent of the rAAV2 RSM was to provide a reference point for AAV2 serotype vectors it is possible that for nonbiological assays such as vector genome titration, the rAAV2 RSM could be used for the calibration of other AAV serotypes. Because the vector capsid is not directly involved in these types of assays, it might be argued that there is no capsid specificity and that the capsid serotype would not have an impact. As an example, in the vector genome titer assay it might be assumed that different capsids are equally susceptible to PCR heat treatment for liberation of the vector genome. However, conditions would need to be optimized because equal susceptibility of AAV serotypes to heat has not been definitively demonstrated. In addition, proteolysis is often used to liberate the vector genome and it is known that different capsid serotypes have different susceptibilities to protease treatments (Van Vliet et al.
). Similarly, serotype-independent methods of determining particle titer (e.g., high-performance liquid chromatography, spectrophotometry) could be calibrated, using the rAAV2 RSM, but the same assumption of capsid independence would apply, and for spectrophotometric measurements the proper extinction coefficient would need to be incorporated (Sommer et al.
). If data are available to demonstrate that an assay is indeed capsid independent, then the use of the rAAV2 RSM for other serotypes may well be acceptable, but thorough review with the appropriate regulatory agency is recommended. For biological assays such as infectious titer, the paramount roles of the capsid, the requisite target cell line, and the helper virus preclude the use of the rAAV2 RSM to calibrate other serotypes. For these assays, investigators must await the development of further reference standard materials such as the AAV8 material currently under production (Moullier and Snyder, 2008
The rAAV2 RSM carries a single-stranded DNA vector genome. Self-complementary AAV vector genomes, generated with a mutation within the terminal repeat (McCarty et al.
), have become popular for gene transfer because they bypass the rate-limiting genome conversion of single-stranded to double-stranded DNA during transduction of target cells. The rAAV2 RSM can be used to normalize “in-house” reference standards for both the classic single-stranded vectors and self-complementary vectors. Because self-complementary vectors carry double the genome complement of single-stranded vectors, a simple conversion is necessary when calculating vector genome titers for these two vector types.
In the United States, the FDA Center for Biologics Evaluation and Research (CBER), Office of Cellular, Tissue, and Gene Therapies (OCTGT), Division of Cellular and Gene Therapies (DCGT) recommends reference materials as benchmarking tools for qualifying and validating “in-house” reference standards and assays by comparison with the collective data. It should be noted that it is not the intent of the FDA to standardize assay methods across the field or to require that the values assigned to the rAAV2RSM be duplicated during validation studies. Furthermore, there is no requirement in the United States to follow rAAV2 RSM procedures when assaying particle concentration, genome copy number, or infectious titer. Sponsors of adeno-associated virus-related investigational new drugs (INDs) should consult with the FDA/CBER or appropriate national agency for further guidance. The rAAV2 RSM fulfills many of the requirements of a reference standard material in that it (1) is sufficiently homogeneous and stable with respect to specified properties, (2) is established to be fit for its intended use in measurement, (3) is accompanied by documentation, (4) provides relevant property values that are based on multiple measurements conducted at different locations, and (5) is accompanied with associated measurement uncertainty.
From the outset, the vision of the AAV2RSWG for the rAAV2 RSM was that it would represent the first step toward standardization of AAV-based gene therapy dosing and provide a blueprint for the development of reference standards for other AAV serotypes. This vision is becoming reality through the successful production and characterization reported here, and with the effort to develop the AAV8 reference standard material underway. The requirement that the reference materials be universally accepted by the AAV community has dictated the need for a voluntary communal effort in the production and characterization phases. Despite the numerous drawbacks, difficulties, and delays inherent in this type of approach, the AAV gene therapy community has responded selflessly and with enthusiasm. It is hoped that the ultimate success of this collaboration will inspire future reference standard efforts and contribute to the development and commercialization of AAV-based gene therapeutics.