Preparation, characterization and establishment of a WHO International

Biological Reference PreparationDr Sjoerd Rijpkema

Division of BacteriologyNIBSC

How are International Standards How are International Standards prepared?prepared? Experts select material that is suitable to serve as a

standard for diagnostic assays Filled and freeze dried Long term stability Suitability for use is established in a collaborative study Statistical analysis of raw assay data Arbitrary unitage is assigned

Reference panel for Chagas disease

Chagas disease is a global threat to public health

Antibody panel oSuitable for all diagnostic testsoSpecific for Tc I and Tc IIoCross-reactive antibodiesoAntibody titre range

Source material

Represent geographic origins of disease Informed consent/local ethical approval Serum/Plasma/Recalcified plasma Pooled samples or individual sample Certified free of HIV1/2, HepA/B/C Certified free of T. cruzi parasites Source material ‘fit for purpose’ pre & post freeze drying Pilot study FD conditions ‘fit for purpose’ post freeze drying 1000 – 5000 ampoules

Aim of the collaborative study

Establish suitability of the candidate standard in a range of assays including the ‘gold’ standard

Collect international data set Statistical analysis of raw data Assign a unitage Evaluate differences in assay methods Evaluate inter-laboratory differences (GCV)

Collaborative study design

Create project team Select international participantsoRepresent reference and specialist diagnostic

laboratories Select samples for analysis pack Select test or range of testsoSamples tested as part of routine oNo re-imbursement for testing

Project Team

InternaloProject leader oStatisticianoFormulation scientist oFill & freeze dry/packaging & distribution

External oExpert(s) oSpecialist diagnostic laboratoriesoParticipants of the collaborative study

Collaborative study (1)

Participants oGlobal distribution

Duplicate sample packs distributedoBlinded samplesoCandidate standard duplicatedoPositive + Negative control samples

Tests oSelection agreed in the CS designoCarried out in duplicate on at least two

separate days = 4 tests per sample

Sample pack for the collaborative study of 05/122 & 05/132

Study Code

NIBSC Code

Sample RPR TPPA EIA Ig IgM

HS HS 1st International Standard 24 IU/ml

64 >1280 pos pos

G, E 05/132 Pooled candidate standard

8 1280 pos pos

C 05/122 Pooled candidate standard

neg 1280 pos neg

F 97/682 Normal serum neg neg neg neg A 05/142 Positive serum neg 1280 pos pos B 83/571 Sera with high level

of cortisol

neg neg neg neg D 82/585 neg neg neg neg

Collaborative study (2)

Collate raw test data Calculate mean test value, CI and GCV Accelerated degradation studiesoTest stability of candidate standards stored at

+37oC to –20oC Draft report and propose unitage Final report endorsed by all members of CS Report send to WHO-ECBS for approval Publish study

Collection of raw data

Titration methods: yield endpoint titreoEach sample tested 4x on two separate dayso8 data points/sample

EIA Methods: produce a numerical valueoEach sample tested in 4 sequential dilutions on two

separate days o8 data points/sample

In-house methods: standard procedures Commercially available tests: manufacturer’s protocol

Stability of biological standards

The stability of a biological standard is predicted by an accelerated thermal degradation test.

Samples of the standard are stored at elevated temperatures at 4oC to 56oC.

Storage time ranges from 6 months to 2 years. The activity of these samples is then calculated

relative to those samples stored at -20oC (T0).

The method

Biological activity is assumed to degrade exponentially with time. This is a “first-order” process.

An estimate of k(T0) is required to predict the stability of the standard, but k(T) can only be estimated for T>T0.

Arrhenius equation

log{k(T)} is:o Linear with 1/To log{k(T)} = + /T o Independent of time

Log

{ k(

T) }

1 / T37oC 20oC 4oC -20oC

Accelerated degradation Predicts degradation process & rate

Samples removed from storage site at

Sample removed from-20oC 4oC 20oC 37oC 45oC 56oC

1 month √ √ √ √2 months √ √ √ √3 months √ √ √ √ (√)6 months √ √ √ (√)12 months √ √ √ √Subsequent times √ √ √ √

Effect of accelerated degradation on potency of 05/122 and 05/132

Storage Temp. for one year

TPPA endpoint Potency relative to -20oC#1 #2 #3 #4

05/122 -20oC 640 640 640 640 -+ 4oC 640 640 640 640 1.00+20oC 640 640 640 640 1.00+37oC 320 320 320 640 0.59

05/132

-20oC 5120 5120 5120 5120 -+ 4oC 5120 5120 5120 5120 1.00+20oC 5120 5120 5120 5120 1.00+37oC 5120 5120 2560 2560 0.71

Timeline of a collaborative study

• MTA agreed 1 mo • Source samples shipped +1 mo 2 mo • Pilot study freeze dry +2 mo 4 mo• Test FD samples +2 mo 6 mo• Fill and freeze dry +2 mo 8 mo• Ship sample packs +3 mo 11 mo• Collect results +3 mo 14 mo • Analyse data +3 mo 17 mo • Submit report • Approval by WHO ECBS 2011? • Publication

Biological standards produced by NIBSC meet WHO criteria

ISO 9001 and ISO 13485 All containers in the batch are identical Potency is unchanged after processing Stable – indefinite shelf life Low moisture content Oxygen level (<1%) Sterility testing: pre & post filling & freeze drying Fill weight variation control Full production records

References

For a detailed description of the standard specification see:o WHO technical bulletin series 932. ISBN 92-4-120932-1 o http://www.who.int/biologicals/expert_committee/TRS932CVR%

20with%20full%20Texts.pdf

For accelerated degradation see:o Kirkwood TBL. Predicting the stability of biological standards and

products. Biometrics 1977; 33: 736-742.o Shin J, Nam J. Validation of a computer software program for

statistical analysis of accelerated stability studies on biological standards. Biologicals 2007; 35: 27-30.

Acknowledgements

Peter Rigsby, Statistical Division Paul Matejtschuk, Centre for Biological

Reference Materials