SynTura: A New Ribonuclease Resistant

Viral RNA Control Material

Ralf Schönbrunner, Ph.D.SoGAT XXI

Brussels, BelgiumMay 28, 2009

SynTura Project

• The SynTura project was created to provide RNA Internal Controls and Quantification Standards which behave like the target they intend to control

• Synthetic & Natural = SynTura

SynTura Project Objectives

• Develop technology platform which can provide control material close to real analyte

• HCV was used as model system• Also functional as QS and IC

The Best Theoretical Alternative for Real HCV

• Similar virus to HCV

• Easy to culture

• Grows to high titers

• Non infectious to humans

• Detectable by HCV assays

CONFIDENTIAL

Viruses Related to HCV

Flaviviridae

Flavivirus

Yellow fever virus,

West Nile virus

Dengue virus

St. Louis encephalitis virus

Tick-borne encephalitis virus

Japanese encephalitis virus

Pestivirus

• Classical swine fever virus (CSFV) • Border disease virus (BDV)• Bovine Viral Diarrhea Virus 1 & 2

(BVDV)

Hepacivirus HCV

CONFIDENTIAL

BVDV, HCV and MS2

BVDV HCV MS2

Mammalian virus Mammalian virus E. Coli Bacteriophage

Flaviviridae Flaviviridae Leviviridae

Genome 12 kb Genome 9.5 kb Genome 3.57 kB

Enveloped (lipid bilayer) Enveloped (lipid bilayer) Protein coat

Detergent sensitive Detergent sensitive Detergent resistant

HCV & BVDV Genome Comparison

BVDV genome is similar to HCV but can be easily cultured and manipulated

Target Region of NAT HCV assays

Flaviviridae TranslationHCV & BVDV

5’UTR Secondary Structure

• Critical 5’UTR secondary structure– Transcripts often do not have correct

secondary structure– Might give misleading results for certain

HCV genotypes

• Is the Flaviviridae 5’-3’ Ring formation important?

5’ & 3’ NTR Strategies

SynTura

Position of HCV 5’NTR in BVDV

5’HCV NTRNFAR

Generation of a BVDV-Hybrid

BVDV Plasmid(BVDV in blue)

Insert desired Sequence (red)

Make RNA Transcripts

Transfect Cell Line

Grow transfected Cell in culture

Viral Particles

Infected Cells produce Virus

DNA RNA

Development Timeline

Milestones Time Receive and confirm synthetic DNA Sequence for cloning 2 - 4 weeks

Cloning into 3’NTR (DNA into 20 kb Plasmid) 4 – 5 weeks or longer Transfection 1 week

Grow first passage – initial titer (used as seed stock for subsequent lots)

2 weeks

Grow material for first lot (100 mL – 300 mL) 3 weeks Inactivation and confirmation 2 weeks

Total time: 4 – 6 month

SynTura HCV Growth Curve

growth curve

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1

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4

5

6

7

8

9

10

10 20 30 40 50 60 70

time (h)

titr

e/ m

L (

log

10)

NCP7

B5UH

Inactivation of BVDV

• Inactivated with ß-Propiolactone

• Protein-modifying agent – reacts with amides of Lys or Arg

• Commonly used for BVDV vaccines

• Alternative: Heat – 90% loss

Initial Titer Determination

Samples Ct QS Ct Dilution Factor log IU/ml Final Conc IU/ml

HCV-BVDV K8 20.3 31.4 10 7.46 2.9.E+08

HCV-BVDV K4 20.2 31.4 10 7.49 3.1.E+08

HCV control 26.6 31.3 1 5.30 2.0.E+05

Effect of Inactivation on TMA

SAMPLE Invalid/valid (IC)

Interpretation Avg S/CO

(n=5) Active 1E2 Valid reactive 5.65 (n=5) Inactive 1E2 Valid reactive 5.83 (n=5) Active 1E3 valid reactive 9.07 (n=5) Inactive 1E3 valid reactive 8.73

Results with ProCleix Ultrio TMA Assay

• No impact of ß-PL inactivation on TMA

Sequence Stability

• 3 passages 5 clones partially sequenced– No mutations found in 5 clones

• 7 passages 5 clones partially sequenced – 1 clone no mutation – 2 clones had 1 point mutation in BVDV sequence– 2 clones had 1 point mutation in HCV insert

• Rate of mutation in insert similar to virus

SynTura Thermal Stability

Thermal Stability of β-Propiolactone inactivated SynTura HCV

0

20

40

60

80

100

0 20 40 60 80 100

Temp. (ºC)

% R

eco

very

BVDV β-PL

37ºC Accelerated Stability

37ºC

-6

-4

-2

0

2

4

6

0 5 10 15 20 25

Days

Ref C

t-Sa

mpl

e Ct

BVDV

HCV

SynTura HCV as Calibrator

HCV vs.BVDV-HCV as Calibrators

BVDVy = -3.8043x + 43.98

R2 = 0.9772

HCVy = -3.8196x + 44.319

R2 = 0.9911

15

20

25

30

35

40

45

0.00 2.00 4.00 6.00 8.00

Log conc (IU/mL)

Ct

HCV

BVDV

Linear (BVDV)

Linear (HCV)

OptiQuant-S HCV RNA Quantification Panel

• Based on SynTura Technology

• 100, 500, 5e3, 5e4, 5e5, 5e6 & 2.5e7 IU/mL

• Plasma Matrix

Multicenter Study

0.00

2.00

4.00

6.00

8.00

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Expected Titer (log10 IU/ml)

Ob

serv

ed

Tite

r (l

og

10 IU

/ml)

Tricore

NYPH

CCPHL

ACL

BayCare

Covance

Overall Results from all Testing Sites

Roche CAP/CTM HCV IVD

BayCare Laboratories

y = 0.9462x + 0.3496

R2 = 0.9984

0.00

2.00

4.00

6.00

8.00

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

Expected Titer (log10 IU/ml)

Ob

serv

ed

Tite

r (l

og

10 IU

/ml)

Comparison of two Abbott realTime HCV ASR Assays

OptiQuant-S HCV Quantification Panel inAbbott Systems

Abbott Lab 2y = 1.0004xR2 = 0.9977 Abbott Lab 1

y = 0.9892xR2 = 0.9992

1

2

3

4

5

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7

8

1 2 3 4 5 6 7 8

Expected Titer log10 (IU/ml)

Ob

serv

ed T

iter

log

1

0

(IU

/ml)

Abbott Lab 1

Abbott Lab 2

Key Results

• SynTura HCV titer 2.0E+08 IU/mL

• Concentrated to 1.5eE+10 IU/mL

• Stable over 7 passages– Insert remains intact– No mutation after 3 passages – Only 1 point mutation after 7 passages

Summary

• SynTura has properties very similar to HCV and probably other enveloped mammalian viruses

• SynTura technology allows integration of defined RNA sequences

• SynTura technology can be used for RNA assays as:– QS and IC– Positive Control– Calibrator

Thank You

• Martin Luther Universität Halle/Saale– Sven Behrens – Martina Behrens

• Acrometrix– Mona Shahbazian– Jerry Boonyaratanakornkit– Reina Karunaratne

• Steve Young, Jessie Kilgore –Tricore

• Hanna Rennert, John Sipley, - NYPH, Cornell Medical Center

• Melody Hung-Fan, Kara Lee, -Contra Costa Public Health Lab

• Linda Sabatini, Lech Mazur, -ACL Central Lab

• Maura Pieretti, Carolyn Dowell, -BayCare Lab

• Ted Schutzbank- Covance