1. Practical aspects of standardization for a global control manufacturer Example: Enterovirus SoGAT XX Warsaw, Poland June 12, 2007 Ralf Schönbrunner, AcroMetrix

2. AcroMetrix’ Control Development AcroMetrix Quality System –ISO 13485:2003 certified –FDA 21CFR Part 820 Quality System Regulations (QSR) –cGMP –Calibrators and Controls values assigned following ISO 17511:2003 In vitro diagnostic medical devices —Measurement of quantities in biological samples — Metrological traceability of values assigned to calibrators and control materials

3. ISO 17511:2003 Five Metrological Levels Upper end of the metrological traceability chain –Level 1: Traceable to SI –Levels 2 – 5: Not traceable to SI See ISO 17511:2003 Introduction p. vii

4. Metrological Levels See ISO 17511:2003 Introduction p. vii

5. Metrological Level: 5 Requires In-house measurement procedure(s) In-house calibrator(s) –Example: Enterovirus, majority of viruses

6. Level 4: Calibration Hierarchy with International Standard WHO Standard

7. Level 5: Calibration Hierarchy without International Standard

8. Enteroviruses Metrological Level 5 Group: Group IV (+)ssRNA Family: Picornaviridae Genus: Enterovirus –No WHO Standard available

9. Genus enterovirus Human enterovirus A, B, C, D Bovine enterovirus Porcine enterovirus A, B Simian enterovirus Human rhinovirus A,B almost 100 different serotypes –Source: Fields Virology, 5 th edition, 2007 –Coxsackie viruses –Echovirus –Poliovirus

10. First EV Quantitation Zhang, HY, Yousef GE, Bowles NE, Archard LC, Mann GF, Mowbray JF J Med Virol. 1988 Dec;26(4):375-86. Detection of enterovirus RNA in experimentally infected mice by molecular hybridisation: specificity of subgenomic probes in quantitative slot blot and in situ hybridisation. Department of Experimental Pathology, St. Mary's Hospital Medical School, London, England In situ-hybridization reportedly detected 10 to 100 enteroviral genomes

11. First EV PCR Quantitation T A Martino, M J Sole, L Z Penn, C C Liew, and P Liu J Clin Microbiol. 1993 October; 31(10): 2634–2640. Quantitation of enteroviral RNA by competitive polymerase chain reaction. Center for Cardiovascular Research, Toronto Hospital, Ontario, Canada. Used competitive internal control standard Concentration measured at 260 nm Based on gels and radioactive [α- 32 P]dCTP quantitation Quantitation possible from 15 – 6000 TCID 50 Calibration Curve: [Standard] (pg) against CPM Std / CPM Virus

12. Cepheid Xpert EV assay FDA approval March 16, 2007 Reverse transcription polymerase chain reaction (RT-PCR) Qualitative detection of enterovirus (EV) RNA Cerebrospinal fluid (CSF) specimens Individuals with signs and symptoms of meningitis

13. EV Serotypes tested with Xpert EV Assay

14. Xpert EV LOD for 4 EV Genera representatives The LOD of the five (5) serotypes, one from each of the enterovirus species are shown in the table below. CVA6 (A), CVA9 (B), CVA17 (C), EV70 (D) and PV1 (C - poliovirus)

15. Challenges for Development of a Low PC useful for Xpert EV No international EV standard Which enterovirus should be used? Which unit should be used? What is correct level for control? –(about 3 - 5 x LOD)

16. EV in-House working calibrator Step 1in ISO17511 to generate EV Low PC Which EV strain should be used? –Meningitis causing – see assay intended use –Defined LOD –CVA6 or CVA9 Which unit should be used? –Copies –Genome equivalents –IU –TCID 50 –pg RNA What is best level for in-house working calibrator? –Significantly higher than Low PC

17. Objective for in-house working calibrator Requirements: –Proxy for non existing International Standard –Same strain as Low Positive Control –Easily available stock material Not necessary to know: –Number of viruses in calibrator –Number of Signal Generating Units (SGU)

18. EV strain for in-House working calibrator Human Enterovirus Species B Type: Coxsackie A9 (CVA9) Strain: P.B. (Bozek) Geographic Origin: New York Illness yielding prototype virus: Meningitis Accession Number: D00627 Heat inactivated, 56ºC, 30 min –K.E. van Vliet et al. J.Clin.Microbiol. (2001) 39, p.3390

19. in-House working calibrator unit Copies – No. Avoid confusion with other people’s copies SGU – No. Dependent on assay sensitivity Genome equivalents – Ideally yes, but no general accepted reference measurement procedure available IU – Requires international protocol for value assignment by international scientific organization TCID 50 – Heat inactivated: TCID 50 = ∞ pg RNA – No accepted int. reference method Generated independent in-house unit –according ISO 17511:2003 clause 4.1.1 d

20. Enterovirus Unit (EVU) Basis to deliver consistent control Not based on SI unit Arbitrarily defined unit –similar to WHO IU No claim about number of viruses Traceable or commutable to other units –Qiagen copies –Cepheid TCID 50 –Future WHO Standard?

21. In-house working calibrator level 500 times higher than Low Pos Control Low Pos Control about 3 - 5 x LOD of Xpert EV –Target level of EV low PC: 100 EVU/mL In-house calibrator: 50 000 EVU/mL –Named: EV APS (AcroMetrix Primary Standard) Product calibrator: 100 EVU/mL –Named: EV LPC AQS (AcroMetrix QC Standard)

22. EV in-house working calibrator & EV product calibrator development Obtained high titer stock CVA9 Determined dilution factor to 4xLOD Diluted 500 fold less to generate EV in-house working calibrator (EV APS) Defined EV APS at 50 000 EVU/mL Diluted EV APS 1:500 to generate EV product calibrator (EV LPC AQS) Defined EV LPC AQS at 100 EVU/mL

23. OptiQual EV Low PC Heat inactivated CVA9 Level: 100 ± 30 EVU/mL Fill volume: 300 µL Storage: -70ºC –Goal: switch to -20ºC

24. Results with OptiQual EV LPC 2 independent labs

25. Possible Downsides Meaning of EVU? –No SI unit –Metrological based –Driven by ISO 17511 What’s an IU? Many different enteroviruses –CVA9 one of many enteroviruses –Similar to HIV subtypes and HCV genotypes –Consider naming it CVA9 unit?

26. Conclusions OptiQual EV control very reproducible results with geneXpert EV assay Level probably higher than 3 – 5 LOD –CV very low for 3 – 5 xLOD –labs use low CV of control to asses variability –evaluations ongoing –some other EV assays show high CV Adjustment to different level easily possible, due to availability of in-house calibrators

27. Proposal Quickly & proactively create “WHO Standards” for “orphan pathogens”* and *: Pathogens currently without International Standard Provide protocol for value assignment Act before commercial assays are available Unit: IU