Establishment of the 1st WHO Establishment of the 1st WHO

International Standard for Detection of International Standard for Detection of

Antibodies to Hepatitis B Virus Core Antibodies to Hepatitis B Virus Core

Antigen (anti-HBc)Antigen (anti-HBc)

SoGAT XXISoGAT XXI29 May 200929 May 2009

Dr. Heiner ScheiblauerDr. Heiner ScheiblauerPaul-Ehrlich-Institut, Langen/GermanyPaul-Ehrlich-Institut, Langen/Germany

PEI-IVDPEI-IVD

2

Why Anti-HBc?Why Anti-HBc?

Anti-HBc are the first antibodies after Hepatitis B virus infection and persist trough out life

Anti-HBc can detect a persistent HBV viraemia, that is not easy detectable by HBsAg or HBV DNA

Anti-HBc can detect HBV infections with HBV escape mutants or genotypes which are not detectable by certain HBsAg or HBV DNA test kits

Anti-HBc screening can prevent transfusion-transmitted HBV infections

Anti-HBc has a regulatory impact: requirement for obligatory screening in some countries worldwide

3

WHO Anti-HBc StandardWHO Anti-HBc StandardDemandDemand

Estimation of the analytical sensitivity for anti-HBc tests

Calibration of anti-HBc test systems by manufacturers

Accuracy and reproducibility verification in batch testing

Current national PEI anti-HBc standard (no 82, serum, 100 PEI

U/ml) has been used widely

Sensitivity of assays and anti-HBc content of samples expressed as

PEI-U/ml

PEI anti-HBc standard stocks declined, replacement preparation

needed

4

WHO Anti-HBc Collaborative StudyWHO Anti-HBc Collaborative StudySamples TestedSamples Tested

Institute NIBSC PEI PEI CBER

Designation 95/522 No. 82 108166 Panel #11

Code Sample A Sample B Sample C Sample D

Sample nature plasma pool serum poolplasma pool

(4 units from one donor)

dilutions of plasma from individual

donors

Anti-HBc Candidate standardPEI anti-HBc

standard(100 PEI-U/ml)

anti-HBc low positive(isolated anti-HBc)

8 reactive, 2 negative members

Anti-HBc IgM negative negative negative negative

Anti-HBs >1000 IU/ml negative negative negative

Anti-HBe/HBeAg negative negative negative negative

HBsAg negative positive negative positive

HBV-DNA negative negative (<12 IU/ml) positive (<12 IU/ml) n.a.

HIV 1/2 and HCV negative negative negative negative

Aggregate status dry lyophilized liquid liquid liquid

Preservative no no no thiomersal

Storage temp. -20°C -70°C -70°C +2-8°C

Reconstitution 1 ml dest. H2O N/A N/A N/A

Filling volume (ml) 1 0.5 0.5 2

Ampoules available 2700 500 1000 n.a.

N/A = not applicablen.a. = not available

Candidate Material

5

WHO Anti-HBc Collaborative StudyWHO Anti-HBc Collaborative Study Study designStudy design

Analytical sensitivity of Samples A (NIBSC 95/522) and B (PEI 82) at the intercept with the assay´s cut-off (endpoint titer, detection limit in U/ml)

Potency of NIBSC 95/522 relative to PEI 82 (U/ml) GMV ratio of the detection limit Parallel line model

Correlation between analytical sensitivity for NIBSC 95/522 and anti-HBc detection in Samples C (PEI 108166) and D (CBER Panel #11)

Variability in results between assay methods (repeatability), and between laboratories (reproducibility) Geometric coefficient of variation (GCV%)

Statistical significance of differences between methods and laboratories

Stability

6

WHO Anti-HBc Collaborative StudyWHO Anti-HBc Collaborative StudyParticipantsParticipants

1. Hemocentro de Sao Paulo, Brazil

2. CBER, USA

3. INTS, France

4. Abbott Laboratories, USA

5. KFDA, Seoul, Republic of Korea

6. American Red Cross, Gaithersburg, USA

7. National Institute of Infectious Diseases, Tokyo, Japan

8. NIBSC, Hertsfordshire, UK

9. AFSSAPS, France

10. National Institute for the Control of Pharmaceutical and Biological Products Beijing Zhongyuan, China

11. NRL, Australia

12. VQC Laboratory Sanquin-CLB Diagnostics, Netherlands

13. Paul-Ehrlich-Institut, Germany

7

WHO Anti-HBc Collaborative StudyWHO Anti-HBc Collaborative StudyTest kitsTest kits

1. ADVIA Centaur HBcT

2. Architect Anti-HBc

3. AxSYM Core

4. Bioelisa anti-HBc

5. Corzyme

6. Elecsys Anti-HBc

7. Enzygnost Anti-HBc monoclonal

8. Genedia Anti-HBc ELISA Plus

9. Hepanostika anti-HBc Uniform

10. Immulite 2000 anti-HBc

11. Immuncomb II HBc IgG

12. IMx Core

13. Anti-HBc EIA Lumipuls

14. Presto HBc Ab-N

15. Monolisa anti-HBc Plus

16. Murex anti-HBc

17. Ortho anti-HBc

18. PRISM HBCore

19. ST AIA-Pack HBcAb

20. Architect Anti-HBc II (ex-US) /

Architect Core (US)

8

WHO Anti-HBc Collaborative StudyWHO Anti-HBc Collaborative StudySummary of test kits characteristicsSummary of test kits characteristics

Assay Code Product name Manufacturer.

Assay procedure Test format Antigen Conjugate Ig-class

Measu-ring

Pretreat-ment

1. 1 ADVIA Centaur HBcT Siemens Med. Solutions 1) Centaur Sandwich rHBcAg rHBcAg IgG/IgM ECL No 2. 3 Architect Anti-HBc Abbott GmbH Co. KG Architect Indirect rHBcAg Anti-human

IgG/IgM IgG/IgM CMIA No

3. 5 AxSYM Core 2.0 Abbott GmbH Co. KG AxSYM Competitive rHBcAg MAb rHBcAg IgG/IgM MEIA DTT 4. 6 Bioelisa anti-HBc Biokit S.A. Manually Competitive rHBcAg PAb rHBcAg IgG/IgM ELISA No 5. 7 Corzyme Abbott Diagnostics Manually Competitive rHBcAg PAb rHBcAg IgG/IgM ELISA No 6. 8 Elecsys Anti-HBc Roche Diagnostics GmbH Elecsys Competitive rHBcAg MAb rHBcAg IgG/IgM ECL DTT 7. 9 Enzygnost Anti-HBc

monoclonal Siemens Med. Solutions 2) BEPII/III

BEP2000 Competitive rHBcAg MAb rHBcAg IgG/IgM ELISA No

8. 1 Genedia Anti-HBc ELISA Plus Green Cross Corp. Manually Competitive rHBcAg Human anti-HBc IgG/IgM ELISA No 9. 1 Hepanostika anti-HBc Uniform bioMérieux SA Manually Competitive rHBcAg MAb rHBcAg IgG/IgM ELISA No 10. 1 Immulite 2000 anti-HBc Siemens Med. Solutions 3) Immulite Competitive rHBcAg MAb rHBcAg IgG/IgM CLEIA No 11. 1 Immuncomb II HBc IgG Orgenics Ltd. Rapid Indirect rHBcAg Anti-human IgG IgG EIA No 12. 1 IMx Core Abbott GmbH Co. KG IMx Competitive rHBcAg MAb rHBcAg IgG/IgM MEIA DTT 13. 2 Anti-HBc EIA Shanghai Kehua Co. Ltd. Manually Competitive rHBcAg MAb rHBcAg IgG/IgM EIA No 14. 1 Lumipuls Presto HBc Ab-N Fujirebio Diagnostics Inc. Lumipuls Presto Competitive rHBcAg MAb rHBcAg IgG/IgM CLEIA No 15. 1 Monolisa anti-HBc Plus Bio-Rad Laboratories Manually Indirect rHBcAg MAb anti-human

IgG+IgM IgG/IgM ELISA No

16. 1 Murex anti-HBc Abbott/Murex Biotech Ltd. Manually Competitive rHBcAg MAb rHBcAg IgG/IgM ELISA No 17. 1 Ortho anti-HBc Ortho Clinical Diagnostics Manually Indirect rHBcAg MAb anti-human

IgG+IgM IgG/IgM ELISA No

18. 1 PRISM HBCore Abbott GmbH Co. KG PRISM Competitive rHBcAg MAb rHBcAg IgG/IgM ChLIA Cysteine 19. 2 ST AIA-Pack HBcAb Tosoh Bioscience Tosoh Competitive rHBcAg MAb rHBcAg IgG/IgM EIA No 20. 4 Architect Anti-HBc II (ex-

US)/Core (US) 4) Abbott Diagnostics Architect Indirect rHBcAg Mab anti-human

IgG/IgM IgG/IgM CMIA Reductant

9

Collaborative Study - Suitability for Collaborative Study - Suitability for variety of tests, mfcts, labs, geography variety of tests, mfcts, labs, geography

20 anti-HBc test kits included in the study from 15 different

manufacturers and from 7 different countries

Tested in 10 different countries (Australia, Brazil, China, France, Japan,

Germany, Korea, Netherlands, UK, USA)

Including the current available anti-HBc test technologies

Competitive test format (n=14)

Indirect test format (n=5)

Sandwich test format (n=1)

Reductant pre-treatment (e.g. DTT) with (n=5) or without (n=15)

Manually conducted (n=9)

Automated (n=10)

Rapid assay (n=1)

All anti-HBc test kits coated with recombinant HBc antigen

10

WHO Anti-HBc Collaborative StudyWHO Anti-HBc Collaborative StudyPotency of NIBSC 95/522 relative to PEI 82Potency of NIBSC 95/522 relative to PEI 82

Dilution ranges for NIBSC 95/522 (sample A) was in the dynamic measuring range of assays

Within an individual assay, Sample A and B (PEI 82) gave comparable dose responses

0

1

2

3

4

5

6

7

8

9

10

0.03 0.06 0.13 0.25 0.50 1.00 2.00

PEI-U/ml - PEI 82

S/C

o

0

1

2

3

4

5

6

7

8

9

0.08 0.16 0.31 0.63 1.25 2.50 5.00

PEI-U/ml - NIBSC 95/522

S/C

o

PEI-82 NIBSC 95/522

11

WHO Anti-HBc Collaborative Study WHO Anti-HBc Collaborative Study Endpoint titersEndpoint titers

Dilution equivalent to cut-off – NIBSC 95/522

Endpoint dilution

1/32

1/64

1/128

1/256

1/512

1/1024

Laboratory

Assay 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

7 12 10 12 13 1 7 12 12 1 4 7 12 9 12 7 5 12 2 6 12 10 8 2 5 7 11 12 5 9 12 4 12 1 3 4 12 8 1

12

WHO Anti-HBc Collaborative Study WHO Anti-HBc Collaborative Study Overall PotencyOverall Potency

Overall potency was 49.8 U/ml (95%-CI 44.2 - 56.1 U/m)

Assay 1 had a potency of 22.8 U/ml (mean of 2 labs)

Assay 8 had a potency of 73.3 U/ml

Overall potency excluding assays 1 and 8 was 51.1 U/ml (95%-CI 45.3 - 57.5 U/ml)

Distribution of Potency of NIBSC 95/522

GMV ratios (PEI 82 - 100 U/ml)

Potency (IU/ml)

Lab

ora

tori

es

17.5 22.5 27.5 37.5 42.5 47.5 52.5 57.5 62.5 72.5 77.5

05

1015

L 7 A 1 L 12 A 1 L 10 A 2

L 12 A 2

L 13 A 2L 1 A 3L 7 A 3

L 12 A 3 L 12 A 4

L 1 A 5

L 4 A 5

L 7 A 6

L 12 A 6

L 9 A 7

L 12 A 7

L 7 A 8

L 5 A 9

L 12 A 10

L 2 A 11

L 6 A 12

L 12 A 12

L 10 A 13

L 8 A 14

L 2 A 15

L 5 A 15

L 7 A 15

L 11 A 15

L 12 A 15

L 5 A 16

L 9 A 16

L 12 A 16L 4 A 17

L 12 A 17L 1 A 18

L 3 A 18

L 4 A 18

L 12 A 18

L 8 A 19

L 1 A 20

Distribution of Potency of NIBSC 95/522

GMV ratios (PEI 82 - 100 U/ml)

Potency (IU/ml)

Lab

ora

tori

es

17.5 22.5 27.5 37.5 42.5 47.5 52.5 57.5 62.5 72.5 77.5

05

1015

L 7 A 1 L 12 A 1 L 10 A 2

L 12 A 2

L 13 A 2L 1 A 3L 7 A 3

L 12 A 3 L 12 A 4

L 1 A 5

L 4 A 5

L 7 A 6

L 12 A 6

L 9 A 7

L 12 A 7

L 7 A 8

L 5 A 9

L 12 A 10

L 2 A 11

L 6 A 12

L 12 A 12

L 10 A 13

L 8 A 14

L 2 A 15

L 5 A 15

L 7 A 15

L 11 A 15

L 12 A 15

L 5 A 16

L 9 A 16

L 12 A 16L 4 A 17

L 12 A 17L 1 A 18

L 3 A 18

L 4 A 18

L 12 A 18

L 8 A 19

L 1 A 20

13

WHO Anti-HBc Collaborative Study WHO Anti-HBc Collaborative Study CommutabilityCommutability

Analytical sensitivity (low detection limits) in Sample A (NIBSC 95/522) correlated with positive score for anti-HBc in Samples C (PEI 108166) and D (CBER panel #11) One assay of the study did not follow this correlation

Cand. Std. PEI 82 Sample

Assay A B D10 D4 D9 D5 D8 C D7 D1 D2

code Detection limit Results (s/co, co/s or reading)

11 0.16 0.10 ind pos pos pos pos pos pos pos pos

16 0.27 0.15 0.57 0.76 1.25 2.00 2.28 6.21 6.32 7.78 8.80

3 0.44 0.20 0.57 0.68 1.13 1.41 2.17 7.87 7.35 10.72 14.82

12 0.79 0.38 0.54 0.63 0.85 1.12 1.47 5.67 5.04 11.61 21.88

6 0.82 0.42 0.64 0.64 0.90 1.28 2.74 87.94 124.81 55.16 160.58

20 0.88 0.43 0.15 0.22 0.56 0.92 2.00 4.71 6.56 3.41 7.05

15 0.9 0.45 0.11 0.22 0.26 0.96 1.10 3.09 3.31 3.50 5.70

18 0.91 0.41 0.65 0.65 0.85 0.90 1.24 2.32 1.93 2.81 4.66

17 1.02 0.6 0.12 0.13 0.31 0.91 1.55 5.40 6.40 4.66 6.62

4 1.15 0.55 0.45 0.47 0.52 0.61 1.28 1.13 2.38 1.81 5.62

9 1.19 0.55 0.28 0.30 0.37 0.52 1.60 2.90 2.16 2.74 4.45

2 1.23 0.66 0.18 0.21 0.47 0.82 1.80 3.15 6.25 2.95 7.43

13 1.38 0.8 0.26 0.25 0.35 0.35 1.61 8.77 4.30 1.38 4.09

7 2.12 1.06 0.17 0.18 0.22 0.24 0.97 1.17 1.38 1.52 3.60

10 2.18 1.06 0.33 0.32 0.37 0.40 1.59 1.69 1.44 1.88 6.03

19 2.4 1.24 0.00 0.00 0.34 0.36 1.70 1.19 1.94 1.42 1.91

5 2.57 1.39 0.42 0.42 0.56 0.59 2.35 2.77 4.55 1.70 5.68

14 2.91 1.41 0.00 0.10 0.20 0.30 0.46 1.57 4.43 1.27 3.73

8 3.03 2.22 0.52 0.50 0.60 0.57 1.66 1.19 7.53 1.06 4.79

1 0.62 0.15 0.22 0.31 0.22 0.55 1.19 1.51 0.82 5.91 13.64

14

WHO Anti-HBc Collaborative StudyWHO Anti-HBc Collaborative Study StabilityStability

The proposed anti-HBc standard NIBSC 95/522 is likely to be highly stable

when stored at the recommended temperature of -20°C

NIBSC 95/522 Stability after 4 years tested with Architect anti-HBc

0

10

20

30

40

50

60

70

80

90

0 1 2 3 4 5 6 7 8 9 10

Replicates

En

dp

oin

t ti

ter

fresh +4°C +20°C 37°C 45°C

15

1st International Anti-HBc Standard 1st International Anti-HBc Standard ConclusionsConclusions

NIBSC 95/522 1st International Standard (IS) for detection of anti-HBc

Assigned unitage of 50 IU per ampoule (ml)

The IU provides continuity with the currently used PEI units/ml

Analytical sensitivity for anti-HBc was effective for estimation of

sensitivity performance the lower the detection limit with the IS,

the higher the positive score for anti-HBc concentration in other

samples

The proposed International anti-HBc Standard was found suitable for

estimation of analytical sensitivity for anti-HBc detection

calibration of anti-HBc test kit sensitivity by manufacturers

to calibrate secondary standards

for quality control procedures, e.g. in batch release testing

16

1st International Anti-HBc Standard 1st International Anti-HBc Standard AcknowledgementsAcknowledgements

NIBSC: Dr. Morag Ferguson

CBER: Dr. Robin Biswas

PEI: Dr. Peter Volkers

Dr. Sigrid Nick

Dr. Micha Nübling

WHO: Dr. Ana Padilla

17

Thank you for your attention!