Collaborative Study for Establishment of a Ph. Eur. BRP for

B19 Virus DNA Testing of Plasma Pools by NAT

K.H. Buchheit, A. Daas, C.M. Nübling, J.M. Spieser

3 July 2003

SoGAT XVI

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B19 positive plasma (ca 109 IU/ml), from window phase

Liquid bulk material (106 IU/ml)– Diluent: Human plasma negative for HCV,

HBV, HIV1 (NAT & serology)

Lyophilisation: – ca 5400 vials, 0.5 ml– residual water: 0.98 ± 0.65%

Candidate BRP

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Collaborative study

• 16 labs (OMCLs, manuf., NAT test labs, kit manuf.)

• Calibration vs 1st IS (500 000 IU/vial)• B19 negative plasma as dilution matrix• Qualitative assay (13 labs)

– 4 independent tests– dilution series proposed – ≥ 3 half-log dil. around dilutional end point

• Quantitative assay (5 labs)– Measure in parallel, diff. conc., linear range

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Results (1)(Values as log10 IU/ml)

• Qualitative assay (n = 12)5.64 ± 0.17 (C.L.: 67 - 150%)

• Quantitative assay (n = 5)5.83 ± 0.05 (C.L.: 88 - 113%)

• Combined5.80 ± 0.05 (C.L.: 86 - 117%)

630 957 IU/ml

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Results (2)

Grey cells: qualitative assays Black cells: quantitative assays

6 2 1 16 14 15 13 16 5 11 5 9 10

12 7 4 8 3 14.9 5.1 5.3 5.5 5.7 5.9 6.1 6.3 6.5 6.7

Potency (log10 IU B19-DNA/ml)

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cBRP suitable for intended purpose• Assigned potency: 105.8 IU/ml• Dilution for threshold control: 1/101.8 = 1/63

No. positive results/ no. assays for each dilutionDilution Laboratory codes

1 3 4 5 7 8 9 10 11 12 13 14 16100.0 1/110-1.0 1/1 1/1 1/1 1/110-1.5 3/310-1.7 3/3

10-2.0 1/1 1/1 1/1 1/1 1/1 4/4 1/1 20/20 5/5 1/1 4/4 1/110-2.3 1/110-2.5 40/40 3/3 3/310-2.7 3/410-3.0 1/1 4/4 4/4 1/1 4/4 4/4 1/1 54/60 5/5 3/3 1/1 4/4 1/110-3.3 3/310-3.5 3/3 3/3 3/3 17/40 4/4 3/3 3/3 3/3

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Next Steps

• OMCL guideline for B19 NAT validation (M. Nübling)

• Validation panel

• PTS studies for OMCLs & manufactures (if requested)