Adeno-Associated Virus Serotype 2 Reference Standard Material
Supported by the NGVL

(Updated October 27, 2010)
Donors

Bid Submission Forms
Bids Submitted Awarded Bids Meeting Minutes Presentations Articles
Container
Stability Study
Production
Schedule
Protocols and Test Records
for AAV Reference Standard Material
AAV2 Working Group
Member List

Working Cell Bank - Certificate of Analysis


The AAV2 reference material (an AAV2-GFP viral vector) is now available from ATCC

The AAV2 reference material (RSM) ATCC VR-1616 is available from ATCC at a nominal cost. Each vial contains 0.5 mL of AAV2 RM at 3.28 x 10E10 vg/mL, or 4.37 x 10E9 IU/mL with TCID50 analysis. The pTR-UF-11 vector plasmid ATCC MBA-331 used to produce the reference material is also available from ATCC.  However, the HEK-293 production cells are not available for sale, and you should plan to use your own cell bank to develop and test your internal reference material.  Other relevant materials available from ATCC include the HeLa RC32 cell line (ATCC CRL-2972) and the adenovirus type 5 helper virus (ATCC VR-1516) that are used to conduct the infectious titer assay.


The AAV2 RSM is intended for use in calibrating internal (product-specific) reference materials and assays for recombinant AAV viral gene transfer products, with the purpose of making data from different pre-clinical and clinical studies more comparable.

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 material as benchmarking tools for qualifying and validating “in house” reference material and assays by comparison to the collective AAV2 RSM 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 rAAV2 RSM 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 INDs should consult with the FDA/CBER or appropriate national agency for further guidance.

AAV2 Working Group Member List
For more information, please contact the
Chair of the AAV Reference Material Working Group:
Richard O. Snyder, PhD
PO Box 110585, University of Florida, Gainesville, FL 32611-0585
Phone: 386-418-1642 • Fax: 386-462-7365Email: rsnyder@cerhb.ufl.edu

Working Cell Bank - Certificate of Analysis

Bid Submission Forms:

RFP 1.0 - Production
RFP 2.0 - Purification
RFP 3.0 - Formulation
RFP 4.0 - Vialing and Freezing
RFP 5.0 - Repository Services
RFP 6.0 - Plasmid Production  
RFP 7.0 - Plastic Ware Donation
RFP 8.0 - Medium Donation
RFP 9.0 - Sterility and Long-Term Stability Study
 

Donors:

Bids Submitted:

As of October 26, 2010, all submitted bids have been awarded

Awarded Bids:

RFP 1.0 - Production and Production Addendum
RFP 2.0/3.0 - Purification/Formulation
RFP 4.0 - Vialing and Freezing
RFP 5.0 - Repository Services
RFP 6.0 - Plasmid Production
RFP 7.0 - Plastic Ware Donation
RFP 8.0 - Medium Donation
RFP 9.0 - Sterility and Long-Term Stability Study

Meeting Minutes:

WG Meeting on 6/5/2003
WG Meeting on 6/3/2004
Attendee List - WG Meeting 6/3/2004

Presentations:

Overview
Manufacturing
Characterization
ASGT Presentation by Richard Snyder

Articles:

AAV2 Production
Utility of a Recombinant Adeno-Associated Viral Vector Reference Standard.
Flotte, TR, Burd, P and Snyder, RO.
BioProcessing J. 1: 75, 2002.

Manufacture and Stability Study of the Recombinant Adeno-associated Virus Serotype 2 Vector Reference Standard.
Potter, M, Phillipsberg, G, Phillipsberg, T, Pettersen, M, Sanders, D, Korytov, I, Fife, J., Zolotukhin, S, Byrne, BJ, Muzyczka, N et al.
BioProcessing J. 7: 8-14, 2008.
 
AAV2 Characterization
Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.
Lock M, McGorray S, Auricchio A, Ayuso E, Beecham EJ, Blouin-Tavel V, Bosch F, Bose M, Byrne BJ, Caton T, Chiorini JA, Chtarto A, Clark KR, Conlon T, Darmon C, Doria M, Douar A, Flotte TR, Francis JD, Francois A, Giacca M, Korn MT, Korytov I, Leon X, Leuchs B, Lux G, Melas C, Mizukami H, Moullier P, Müller M, Ozawa K, Philipsberg T, Poulard K, Raupp C, Rivière C, Roosendaal SD, Samulski RJ, Soltys SM, Surosky R, Tenenbaum L, Thomas DL, van Montfort B, Veres G, Wright JF, Xu Y, Zelenaia O, Zentilin L, Snyder RO.
Human Gene Ther.  Oct 21(10):1273-1285, 2010.

Container Stability Study:

This is an on-going study to look at the short-term stability (1 hour, 1 day, 3 days, 7 days) of the vector at room-temperature (the temperature for the vial filling) and long-term -80°C storage temperature (1 hour, 1 day, 14 days, 35 days, 124 days) in these containers. We conducted this comparative study with glass vials since we only had stability data in polypropylene cryovials.

The vector was dispensed into both glass vials and cryovials, and stored: A) frozen; and B) room temp for the indicated time. The infectious titer (green cells) was tested at each timepoint in triplicate. (48) vials (cryo and glass) were filled with 0.5 mL at 2 x 10E11 vg/mL each (= 24 mL @ 2 x 10E11 vg/mL = 4.8 x 10E12 vg total used), as specified for the Reference Material.

Please see the attached stability data for the AAV2-GFP vector filled into cryovials (standard container for UF) and glass vials.

Production Schedule:

With an NIH NCRR grant and donations from industry and numerous academic organizations, production and purification of the AAV2 RM began in March 2006 at the University of Florida’s Powell Gene Therapy Center. Approximately 150 mL of AAV2-GFP = 5.69 x 10E14 vg (= 3.79 x 10E12 vg/mL) was made.

The Reference Material was diluted to the proper concentration (2 x 10E11 vg/mL), filtered and divided into two portions. In March 2008, ATCC filled 0.5 mL into each vial for a total of 2,087 vials, and the vials were frozen and transferred to the repository at ATCC. Characterization of the AAV2 RM was performed by 16 laboratories worldwide starting Q2 of 2008, followed by statistical analysis.

Sterility Testing and Associated Data

Protocols and Test Records for AAV2 Reference Standard Material:

 
Testing Protocols
Packet to Report Data
Progen_AAV2_ELISA_kit_PRATV
Preparation_of_Reference Material_for_qPCR
AAV2_RSM_Infectious_titer_assays_V2
AAV2_RSM_Identity-Purity_assay
AAV2_RSM_genome_copy_titration_QPCR
AAV2_capsid_titer_assay_V2
AAV2_Reference_Material_Handling_Instructions
(form rev'd Sept. 28, 2010)
AAV-sterility-test-302.01
(form added Sept. 28, 2010)
 
AAV2 RSM Characterization Results
Test Results Summary
AAV2 RSM Mycoplasma Testing
 
AAV2 RSM Stability Study
AAV2 RSM Stability Study
2010 Data
2011 Data
 

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