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 57081 Medical Device Manufacturing Owner/Operator Number: 9049048

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 QuantificationPhosphate Assay• Set up standard curve (concentration vs.

OD600), 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 QuantificationPhosphate Assay

Inorganic phosphate standard curve used to determine copy number (4.6 copies) of HIV RNA in each Armored RNA particle

y = 1.7539x - 0.0127

R2 = 0.9981

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.1 0.2 0.3 0.4 0.5

Phosphorus (ng/ul)

A60

0

y = 0.0342x + 0.0274

R2 = 0.9801

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15

AR (ng/ul)A

600

Armored RNA Quant

In vitro Armored RNA QuantificationHPLC 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 QuantificationHPLC Assay

Determination of RNA concentration by HPLC

Armored RNA Quant

In vitro Armored RNA QuantificationHPLC Assay

C: y = 41657x + 4375.5

R2 = 0.9976

G: y = 93552x + 7806.9

R2 = 0.9987

U: y = 61456x + 9250.4

R2 = 0.9957

A: y = 159672x + 18137

R2 = 0.9973

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

0 1 2 3 4 5 6 7 8

Armored RNA (ug)

Pea

k A

rea

CUAGLinear (C)Linear (G)Linear (U)Linear (A)

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 QuantificationComparison

PhosphateAssay HPLC Assay Roche Monitor®

Assay

Batch 1 4.6 4.1 3.7

Batch 2 3.9 3.5 3.4

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

SummaryAn 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)

AcknowledgementsJun 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.