Armored RNA Quant ™ Development of Armored RNA ® as a Primary Standard June 5, 2003 Cindy WalkerPeach, Ph.D. Director of Research and cGMP Operations Ambion Diagnostics, Austin, Texas USA BSI EN ISO9001:2000 Certified Quality System Regulations (21CFR§820) BSI Registration Number: FM Medical Device Manufacturing Owner/Operator Number: SoGAT 2003

Armored RNA Quant  RNA Controls and Standards Stable – ribonuclease resistant Homogenous – single, sequence-defined transcript Quantifiable – known copy number Compatible – performs broadly with RNA-base viral assay technologies Flexible – allows for addition to human matrix of choice Non-infectious – ease in handling and shipping

Armored RNA Quant  In vivo Armored RNA Packaging System

Armored RNA Quant  RNA Controls and Standards Stable – ribonuclease resistant Homogenous – single, sequence-defined transcript Directly Quantifiable – known copy number Compatible – performs broadly with RNA-base viral assay technologies Flexible – allows for addition to human matrix of choice Non-infectious – ease in handling and shipping

Armored RNA Quant  In vitro Armored RNA Packaging System

Armored RNA Quant  In vitro Armored RNA Quantification Phosphate Assay Set up standard curve (concentration vs. OD 600 ), using a phosphate standard from NIST Digest Armored RNA by phosphodiesterase and alkaline phosphatase to nucleosides and inorganic phosphate Measure the phosphate concentration of the digested Armored RNA and calculate the value using the standard curve

Armored RNA Quant  In vitro Armored RNA Quantification Phosphate Assay Inorganic phosphate standard curve used to determine copy number (4.6 copies) of HIV RNA in each Armored RNA particle

Armored RNA Quant  In vitro Armored RNA Quantification HPLC Assay Set up standard curve (concentration vs. peak area) for standard ribonucleosides (A, U, C, G) by HPLC Digest Armored RNA, using phosphodiesterase and alkaline phosphate to nucleosides and inorganic phosphate Measure the nucleoside peak areas by HPLC Based on the Armored RNA sequence, calculate the concentration of the RNA in the Armored RNA preparation

Armored RNA Quant  In vitro Armored RNA Quantification HPLC Assay Determination of RNA concentration by HPLC

Armored RNA Quant  In vitro Armored RNA Quantification HPLC Assay HPLC assay standard curves used to determine copy number (4.1 copies) of HIV RNA in each Armored RNA particle

Armored RNA Quant  In vitro Armored RNA Quantification Comparison Phosphate Assay HPLC Assay Roche Monitor ® Assay Batch Batch Determination of RNA copy number per Armored RNA particle Roche Amplicor Monitor Assay results consistent with those of NIST-traceable phosphate assay and standardized HPLC assay in the quantification of in vitro Armored RNA, within a 20% error range

Armored RNA Quant  Summary An in vitro Armored RNA ® packaging system has been developed to produce Armored RNA that is comprised entirely of a single, defined transcript (target sequence), allowing for direct quantification by two independent, direct, physical methods (NIST- traceable phosphate assay and standardized HPLC assay) Acknowledgements Jun Wang Ginny Lai Marty Badgett Britt Pasloske The project described was supported by grant number AI40529 from the NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH awarding component. Armored RNA ® technology was jointly developed by Ambion, Inc. and Cenetron Diagnostics, LLC. Armored RNA ® is a registered trademark of Ambion and Cenetron.