WHO/BS/2013.2219

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 21 to 25 October 2013

Report on a Collaborative study for proposed 3rd

International

standard for Tumor Necrosis Factor -alpha (TNF-)

Meenu Wadhwa

1, Chris Bird, Paula Dilger, Jason Hockley, Peter Rigsby

National Institute for Biological Standards and Control

Blanche Lane, South Mimms, Potters Bar, Herts, EN6 3QG, UK

1

Email address: Meenu.Wadhwa@nibsc.org

Note:

This document has been prepared for the purpose of inviting comments and suggestions on the

proposals contained therein, which will then be considered by the Expert Committee on

Biological Standardization (ECBS). Comments MUST be received by 4 October 2013 and

should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:

Quality Safety and Standards (QSS). Comments may also be submitted electronically to the

Responsible Officer: Dr Jongwon Kim at email: kimjon@who.int.

© World Health Organization 2013

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WHO/BS/2013.2219

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Summary

The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS)

in 2012 recognized the need for a replacement International Standard for human sequence

recombinant Tumor Necrosis Factor - alpha (TNF-) for the assignment of potency to

preparations of human TNF- used therapeutically and those which serve as critical reagents for

potency evaluation of several TNF- antagonists.

We report here the characterization of two candidate standards for human TNF- in comparison

with the existing International Standard coded 88/786 by bioassay and immunoassay in an

International Collaborative Study carried out by 18 laboratories in 10 countries.

The mean estimate of the TNF- bioactivity of the candidate standard, coded 12/154, is 42,882

IU per ampoule. It is proposed that it is established as the third International Standard for human

TNF- with an assigned bioactivity of 43,000 IU per ampoule.

The results of this study also indicate that the candidate standard appears sufficiently stable, on the

basis of a thermally accelerated degradation study, to serve as an international standard.

Responses from study participants

Responses were received from twelve of the 18 participants. Minor comments were received

relating to typographical errors or omissions in the description of methodologies (Table 3) or the

names of participants (Appendix 1) and these have been corrected. All responses received were

in agreement with the proposal that the preparation coded 12/154 is suitable as the WHO 3rd IS

for TNF- with an assigned bioactivity of 43,000 IU per ampoule.

Introduction

Human Tumor Necrosis Factor - alpha (TNF-), a non-glycosylated protein of 17 kDa (157

amino acids) involved in the regulation of immune cells is produced mainly by macrophages.

Based on its ability to induce cytotoxic activity and inhibit tumorigenesis, it is used

therapeutically as an adjunct to surgery for soft tissue sarcoma of the limbs. However, since

TNF- promotes inflammatory responses which, in turn, cause many of the clinical problems

associated with autoimmune disorders such as rheumatoid arthritis and psoriasis, much interest is

in developing inhibitors of TNF-activity. Consequently, several TNF-antagonists are

approved for clinical use in various indications e.g., rheumatoid arthritis, psoriasis and Crohn’s

disease.

The second International Standard (IS) for human TNF- (88/786) consisting of a highly purified

preparation of TNF- derived from BALL cells (Fukuda et al; 1988) was established by the

WHO Expert Committee on Biological Standardisation (ECBS) in 2003 (WHO Technical Report

Series 927, 2005). The IS has proved suitable for the potency labelling of approved TNF-

products (INN tasonermin) and is widely used for the calibration of preparations of human TNF-

which serve as critical reagents for potency evaluation of TNF-antagonists. The global

requirement for such a standard is evidenced by the high sustained demand for the current

standard and the continued expansion in the number of TNF-antagonist products available or

in development worldwide.

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Stocks of the current IS, 88/786, are nearly exhausted and a replacement is required. In 2012, the

WHO ECBS recognized the need for a replacement international standard for human TNF- and

agreed that lyophilized candidate preparations should be evaluated in a study and, subject to their

suitability, be considered to serve as a potential replacement standard. Therefore, two candidate

preparations were evaluated in an international collaborative study organized with expert

laboratories to facilitate the value assignment of the proposed 3rd

International Standard relative

to the current 2nd IS as per WHO procedures (WHO Technical Report Series 932, 2006).

Aims of the Study

To characterize a candidate WHO 3rd

IS for the bioassay of human TNF-and assign a unitage for

activity, the study sought

1. To assess the suitability of ampouled preparations of human TNF- to serve as 3rd

IS for

the bioassay of human TNF- by assaying their biological activity in a range of routine,

'in-house' bioassays.

2. To assess the activity of the ampouled preparations in different assays (e.g., bioassays,

immunoassays etc) in current use for these materials and to calibrate the candidate IS against

the 2nd IS (88/786).

3. To compare the ampouled preparations with characterised 'in-house' laboratory standards

where these are available.

Materials and Methods

Two pure preparations of recombinant human sequence TNF-expressed in E coli kindly donated

to WHO were evaluated in the study. One of these candidate preparations (88/784) was derived

from the previous collaborative study for establishment of 1st IS for TNF-(Meager and Gaines

Das, 1994) lyophilized as per the previous procedures used for International Biological Standards

(WHO Technical Report Series 800, 1990; Annex 4). The other preparation, 12/154 was lyophilized

at NIBSC as per the procedures used for International Biological Standards (ECBS guidelines -

WHO Technical Report Series 932, 2006) following pilot lyophilization to confirm that the

lyophilized preparation performed appropriately relative to the bulk material in two different

bioassays for TNF-; a cytotoxicity assay and a reporter gene assay. The former exploits KD-4

clone 21 human rhabdomyosarcoma cells, which are very susceptible to the cytotoxic effect of

TNF- (Meager, 1991) while the latter employs human erythroleukemic K562 cells transfected

with the TNFα responsive NFκB regulated Firefly luciferase (FL) reporter-gene construct

together with a Renilla luciferase (RL) reporter gene under the control of a constitutive minimal

thymidine kinase promoter (Lallemand et al; 2011).

Buffers, final compositions as shown in Table 1, were prepared using nonpyrogenic water and

depyrogenated glassware. Buffer solutions were filtered using sterile nonpyrogenic filters (0.22m

Stericup filter system, Millipore, USA) where appropriate.

For the study, the two rDNA derived preparations were coded as described in Table 1. The mass

content of the preparations was determined by the manufacturers. As the protein content of the

ampoules cannot be verified by direct measurement of absolute mass, the content is assumed to

WHO/BS/2013.2219

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be the theoretical mass, calculated from the dilution of the bulk material of known protein mass

content, and the volume of formulated solution delivered to the ampoule. This mass value is

given as “predicted g”.

For the two preparations, the appropriate volume was added to the buffer to provide a solution of

TNF- at a concentration which when distributed in 1.0ml aliquots, gives the theoretical protein

content per ampoule as 1g/ml for both preparations as shown in Table 1.

For each fill, a percentage of ampoules were weighed. The mean fill weights are shown in Table

2. Each solution was lyophilized, and the ampoules were sealed under dry nitrogen by heat

fusion of the glass and stored at –20°C in the dark. Residual moisture of each preparation,

measured by the Karl-Fischer method, is shown in Table 2. Headspace oxygen content was

determined by frequency modulated spectroscopy using the Lighthouse FMS-760 Instrument

(Lighthouse Instruments, LLC). Testing for microbial contamination using total viable count

method did not show any evidence of microbial contamination.

Participants

Samples were despatched in October 2012 to 19 laboratories in 10 countries. The participants

comprised 2 control laboratories, 1 academic laboratory, 1 contract research organization, 12

manufacturers’ laboratories and 2 regulators; 18 participants submitted data and are listed in

Appendix 1.

Assay Methods and Study Design

A summary of the assay methods, bioassays and immunoassays used in the study is given in

Table 3A and B. A majority of laboratories used bioassays which measured the cytotoxic effect

of TNF- in the murine fibroblast cell-line L929 although murine or human fibrosarcoma or

rhabdomyosarcoma cell-lines were also used (Meager and Gaines Das, 1994) as in Table 3A.

These assays however employ different readouts for assessing the cytotoxic effect. In rare

instances, apoptosis assays using the human histiocytic lymphoma cell-line, U937 or reporter

gene assays were used (Lallemand et al; 2011). While all laboratories conducted bioassays, two

laboratories also performed immunoassays. One laboratory performed ELISAs using different

commercially available reagents/kits while another laboratory used in-house assays using either

an in-house monoclonal antibody against TNF- (Findlay et al 2010) or the approved TNF-

inhibitors, adalimumab, infliximab or etanercept as capture reagents (Table 3B).

Participants were asked to assay all samples including the current IS (88/786) concurrently on a

minimum of three separate occasions using their own routine bioassay methods within a

specified layout which allocated the samples across 3 plates and allowed testing of replicates as

per the study protocol (Appendix 2). It was requested that participants perform eight dilutions of

each preparation using freshly reconstituted ampoules for each assay. Where available they were

asked to include their own in-house reference material.

Participating laboratories were sent five sets of four study samples coded A-C along with the

current IS (88/786) as detailed in Table 1. Samples A and C were coded duplicate samples of the

same material (candidate replacement standard 12/154).

Participants were requested to return their raw assay data, using spreadsheet templates provided.

WHO/BS/2013.2219

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All laboratories are referred to by a code number, allocated at random and not representing the

order of listing in Appendix 1, to retain confidentiality in the report. Where a laboratory returned

data from more than one method, the different assay methods were analysed and reported

separately and coded, for example, laboratories 1a and 1b.

Statistical Analysis An independent statistical analysis of all bioassay data was performed at NIBSC using the

EDQM CombiStats software. Where possible, assays were analysed by fitting a sigmoid model

comparing assay response to log concentration, using the full range of responses. In some

instances the analysis did not converge to determine asymptotic limits and the data for these

plates were analysed with a parallel-line model based on a linear portion of the dose response

curve (Finney, 1978), using a log transformation of the assay response. Assay validity was

assessed by calculation of the ratio of slopes for the two test samples under consideration. The

samples were concluded to be non-parallel when the slope ratio was outside of the range 0.80 to

1.25 and no potency estimates were calculated.

Laboratory means were calculated as unweighted geometric means. Overall means were

calculated as the unweighted geometric mean of laboratory means. Variability within and

between laboratories has been expressed using geometric coefficients of variation (GCV = {10s-

1}×100% where s is the standard deviation of the log10-transformed potency estimates). Analysis

of variance with Duncan’s multiple range test (Duncan, 1975) using the log transformed potency

estimates was used to compare laboratories and samples (p<0.05 used to conclude significance).

The agreement between duplicate samples was assessed by calculating the difference in log

potency estimates (relative to 88/786) of samples A and C for each assay, calculating the mean

of the squared difference for each laboratory, taking the square root to give a root mean square

(RMS) value, and expressing this as an average percentage difference.

Stability Analyses

Accelerated thermal degradation study

Samples of the candidate standard 12/154 were stored at elevated temperatures (4°C, 20°C, 37°C

and 45°C) for up to 10 months and assayed using the KLJ Reporter Gene bioassay at NIBSC. A

total of six independent assays were performed. Samples were tested concurrently with those

stored at the recommended storage temperature of -20°C, and baseline samples stored at -70°C.

The assays were analysed as described for the main collaborative study, except using analysis of

variance to assess linearity and parallelism using a 5% level of significance (p<0.05), and the

potencies of the samples stored at different temperatures were calculated relative to the

appropriate -70°C baseline samples.

Stability after reconstitution

Samples of the candidate standard 12/154 were reconstituted and stored at 4°C and 20°C for

periods of 1 day and 1 week. The reconstitutions were timed to allow all samples to be assayed

concurrently with a freshly reconstituted sample. The assays were analysed as described for the

main collaborative study, except using analysis of variance to assess linearity and parallelism

using a 5% level of significance (p<0.05). Three independent assays were performed, with each

WHO/BS/2013.2219

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sample replicated across three plates within each assay. The potencies of all samples were

calculated relative to fresh samples.

Stability on freeze-thaw

Samples of the candidate standard 12/154 were reconstituted and subjected to a series of freeze-

thaw cycles (1 up to 4). They were then assayed concurrently with a freshly reconstituted

ampoule. Two independent assays were performed, with each sample replicated across 2 plates

within each assay. The assays were analysed as described for the main collaborative study,

except using analysis of variance to assess linearity and parallelism using a 5% level of

significance (p<0.05). The potencies of each freeze-thaw cycle were calculated relative to fresh

samples.

Results

Data Received

Results were received from 18 laboratories. Laboratories 1 and 3 returned two sets of data from

two different bioassay methods (Table 3A), which have been analysed separately as if from

different laboratories, and are referred to as 1a, 1b, 3a and 3b. Laboratories 1 and 5 also

submitted data from immunoassays as briefly stated in Table 3B.

The majority of laboratories returned data from three plates in each of three assays. Some

laboratories included data from an additional pilot assay, but these were not used in the analysis.

Laboratory 5 returned data from a single assay with 3 plates. Laboratories 9, 11, 14, 15 and 18

returned data from 3 assays each with a single plate. Laboratory 1a returned data from 3 assays

each with 4 plates. Laboratories 3, 12 and 13 returned data from 6, 5 and 13 assays respectively,

with each assay containing data from 3 plates.

In some assays, data points at the upper end of the response range were removed where the

responses dropped sharply after reaching an upper limit. This is a reasonably well characterised

occurrence in dose-response curves and removing them improved the fit of the sigmoid model.

Three laboratories also submitted raw bioassay data from 48 plates using three TNF antagonists

(etanercept, infliximab, adalimumab) and two different assay types (reporter gene, cytotoxicity).

Of the 48 plates, only one showed any problems, with CombiStats unable to estimate asymptotes

for the model.

Parallelism of dose-response curves

Slope ratios from individual plates of samples A, B and C relative to IS 88/786 are shown in

Figures 1, 2 and 3. Assay results where the ratio of slopes was outside of the range of 0.80 to

1.25 were excluded from further calculations. Of the 180 plates analysed, this consisted of

excluding 19, 22 and 19 plates for samples A, B and C respectively, corresponding to 10.6%,

12.2% and 10.6% of cases. If a range of 0.67 to 1.50 was used to conclude parallelism then 2, 2

and 3 plates (1.1%, 1.1% and 1.7%) would be excluded for samples A, B and C respectively.

Samples A and C were coded duplicates of the same material. Slope ratios for A relative to C, as

shown in Figure 4, showed non-parallelism on 23 of the 180 plates (12.8%). If a range of 0.67 to

1.50 was used to conclude parallelism then this occurred on 6 of the 180 plates (3.3%).

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Potencies of samples A-C relative to IS 88/786

The laboratory geometric mean potencies of samples A, B and C relative to the current IS

(88/786 – assigned unitage of 46,500 IU/ml) are shown in Tables 4 - 6, along with the intra-

laboratory (within-laboratory) GCV. The overall geometric mean, the 95% confidence intervals

and the inter-laboratory (between-laboratory) GCV values are included below the laboratory

results. The laboratory mean potency estimates are also shown in histogram form in Figures 5

and 6. Each box represents a laboratory geometric mean estimate, and the boxes are labelled with

the laboratory code. The unshaded boxes represent cytotoxicity assays while the shaded boxes

identify all other assays used in the study.

The intra-laboratory variability ranges from 2% (laboratory 11, sample C) to 78% (laboratory 18,

sample C). Many laboratories achieved GCV values of 20% or less, with 9 labs achieving this

for all three samples. Some higher GCV values were observed when a wider slope ratio range of

0.67 to 1.50 was used to conclude parallelism.

Inter-laboratory variability, as measured by the between-laboratory GCV values shown in Tables

4 – 6 (17%, 16% and 11% for samples A, B and C respectively), indicates a good level of

agreement between the laboratories. The laboratory geometric mean potencies were analysed

using Duncan’s Multiple Range Test, but no laboratories were determined to be outliers, and so

all were included in the overall potency calculations.

The mean estimated potencies are 42,967 IU/ml (Sample A), 64,469 IU/ml (Sample B) and

42,796 IU/ml (Sample C), with a geometric mean of these estimates for samples A and C

(proposed standard 12/154) of 42,882 IU/ml. Using a slope ratio range of 0.67 to 1.50 to

conclude parallelism, similar mean potency estimates were obtained for all samples in all

laboratories except for laboratory 18 where the result for sample A was almost 50% lower. The

mean estimated potencies using this alternative slope ratio range and excluding laboratory 18 are

42,357 IU/ml (Sample A), 67,039 IU/ml (Sample B) and 42,854 IU/ml (Sample C) with a

geometric mean of these estimates for samples A and C (proposed standard 12/154) of 42,605

IU/ml.

Data from immunoassays was mainly derived from two laboratories using different methods

(Table 3B). In laboratory 1, data were consistent using different methods and gave a GCV of up

to 10% (Tables 9A, B and C). In laboratory 5, the potency estimates varied, a GCV of 5-58%

was seen depending on the immunoassay used and the sample tested. The potency estimates for

5B were higher for samples A and C relative to other assays. A close examination of the data

revealed that the current IS was not recognised as effectively in this assay in comparison with

other assays. However, if 5B is excluded, potency estimates are quite consistent and provide

geometric mean of 47,406 IU/ml for A and 50,745 for C with a mean value of 49,076 IU for

12/154 which is slightly higher than the estimate of 42,882 derived by bioassay. For sample B,

5C gave a much lower estimate relative to other assays, the reason for this is not clear.

Agreement between duplicates

Samples A and C were coded duplicates of the same material. The overall potency estimates

relative to 88/786 were in very close agreement (42,967 and 42,796 IU/ml). There is also good

agreement between the laboratory mean estimates for samples A and C (Tables 4 and 6) for most

laboratories. Laboratory 14 had no valid estimates for sample C and is thus not included in these

calculations.

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The agreement between the potency estimates of A and C can be assessed in two ways. Firstly,

the intra-laboratory GCV values for the potency of sample A calculated relative to sample C,

shown in Table 7, represents the variability between assays of direct comparisons of A to C.

They range from 4% (laboratory 11) to 101% (laboratory 7). Laboratory 7 showed particularly

high variability for this analysis, but also showed high variability in the potency estimates for A

and C calculated relative to 88/786.

The average differences in potency estimates of samples A and C were calculated (root mean

square difference in log potency) for each laboratory and these differences, expressed as a

percentage, are shown in Table 8. These range from 6% (laboratory 11) to 77% (laboratory 7),

and in most laboratories show similar levels of variability compared with intra-laboratory GCV

results discussed above.

Accelerated thermal degradation study

Geometric mean potency estimates of 12/154 following storage at different temperatures are

shown in Table 10A; both time points were used in the analysis to predict stability. The

Arrhenius model for accelerated degradation was applied (Kirkwood and Tydeman, 1984) to

obtain a predicted loss of potency per year of <0.001% when stored at the recommended

temperature of -20°C. The predicted loss when stored at 37°C is 0.340% per month. These

figures indicate that the material is stable for long-term storage at -20°C, and for limited

excursions at higher temperature during transportation. It is sufficiently stable to serve as an

International Standard.

Stability after reconstitution

The potency estimates, calculated relative to fresh samples, of the reconstituted ampoules of

12/154 are shown in Table 10B, along with the GCV values for between-assay variability. The

potency of 12/154 is not diminished after a week at 4°C, but shows a loss at 20°C with an

observed potency of 93% of the freshly reconstituted material. This is within the limits of assay

variability.

Stability on freeze-thaw

The potencies of the reconstituted ampoules of 12/154 are shown in Table 10C, along with the

GCV values for between-assay variability. From the results, it is clear that the potency of 12/154

does not decrease with these numbers of freeze-thaw cycles (the confidence interval after 4

cycles spans 1).

Discussion

A majority of laboratories used a bioassay which was recommended as a reference bioassay in

the previous study for the 1st IS and measured TNF mediated cytotoxicity in the presence of the

metabolic inhibitor, actinomycin D in the murine fibroblast cell-line L929 (Meager, Gaines Das,

1994). However, murine or human fibrosarcoma or rhabdomyosarcoma cell-lines were also used

as demonstrated previously (Meager and Gaines Das, 1994). These assays, however, employ

different readouts for assessing the cytotoxic effect. In rare instances, assays based on the ability

of TNF- to induce apoptosis in a human histiocytic lymphoma cell-line, U937 which exhibits

various properties typical of macrophages (Minafra et al, 2011) or reporter gene assays have also

been used (Lallemand et al; 2011).

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Results from this study showed that acceptable parallelism was achieved between the study

samples and the current IS as indicated by the slope ratios obtained in the majority of bioassays

(~89%) employed in the study (Figures 1-3). This was also confirmed for the coded duplicates,

A and C (Figure 4).

In many laboratories, there was good within laboratory repeatability, with GCVs less than 20%

for all three samples tested in the bioassays used. Although a high intra-laboratory variability,

ranging from 2% – 78% was observed in some laboratories for sample C, many laboratories

were able to achieve a GCV value of less than 20%. The high variability in data from some

laboratories was not specific for any particular bioassay type (Figures 5 and 6). Considering that

cytotoxicity assays can be extremely variable, the study data were remarkably good. Of the

eleven laboratories using the L929 cytotoxicity assay (reference bioassay – Meager and Gaines

Das 1994), a minority (n=3) showed variable results. Limited data were available from

laboratories using the apoptosis assay (n=2) and although this suggests that the assay is variable

(based on the data submitted), more data is needed to substantiate this finding.

Further analysis showed that despite the within laboratory variability, the inter-laboratory

variability was low and between 11 – 17% depending on the sample, indicating a good

agreement between laboratories. From Tables 4 – 6 and the data illustrated in Figures 5-6, the

bioassays from a majority of laboratories provided potency values for A and C that were

clustered around a range of 42,000 – 43,000 IU relative to the current IS (coded 88/786) despite

a few exceptions, for example a low value of 32,168 or a high value of 57,897 for sample A.

The mean values for samples A and C based on the laboratories performing bioassays are 42,967

and 42,796 IU/ml respectively with a geometric mean of these estimates for samples A and C of

42,882 IU/ml.

For sample B, the potency value was higher at around 64,000 IU relative to 88/786. As opposed

to the full length sequence (157 amino acids) in A and C, sample B contains a TNF-variant with 2

amino acid deletion at the N terminus. Such variants in comparison with the full length molecule

have been associated with increased cytotoxicity particularly in murine cell-lines. A higher potency

for sample B (88/784) therefore was not unexpected - it was seen previously with this sample as

it was included as a candidate preparation in the original collaborative study conducted to

establish the 1st IS for TNF-(Meager and Gaines Das, 1994). From data presented in the

previous study, the estimated potency of 88/784 was ~ 66,500 IU which is in close agreement

with the results from the current study (conducted after ~ 22 years), and providing further

evidence of the long term stability of lyophilized TNF- preparations.

Despite use of different immunoassays, the potency estimates were quite consistent with the

exception of 5B. It was apparent that the current IS (containing natural protein) was not

recognised as effectively as samples A and C in the assay of 5B in comparison with other assays

indicating that antigenic determinants in the TNF-molecule are differently recognised by the

antibodies used in this particular assay. Overall, immunoassay data provided slightly higher

potency estimates for samples A and C in comparison with bioassays.

However, since data from bioassays in this study is largely consistent between the different

laboratories and given that the potency of the current IS was derived on the basis of bioassays in

the previous study, it seems reasonable to assign the potency for the candidate preparation,

12/154 using the mean potency estimate from bioassays alone. For the candidate standard 12/154,

therefore, the mean value derived from bioassay data is 42,882 IU and is in continuity with the

WHO/BS/2013.2219

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2nd

IS for TNF- (current standard coded 88/786). The candidate standard contains rDNA

derived TNF-as opposed to the natural TNF-in the current standard but this is unlikely to have

any impact as the protein is non-glycosylated in the natural form. Moreover, rDNA derived TNF-

has been used previously as the standard sincethe 1st IS for TNF-coded 87/650) contained

rDNA derived TNF- A limited assessment of TNF antagonists (etanercept, adalimumab and

infliximab) in a few laboratories has shown that the standard is suitable to serve as a bioassay

calibrant for measuring the inhibitory activity of TNF antagonists.

Stability studies indicated that the candidate preparation (code 12/154) is stable for long term

storage at -20˚C and the potency is not diminished after 1 week of storage at either 4˚C or 20˚C

following reconstitution or after repeated freeze-thaw cycles.

These results clearly indicate that candidate preparation (code 12/154) is stable and suitable for

use as the 3rd International Standard for TNF-. It is therefore proposed that a value of 43,000

IU/ampoule is assigned to the 3rd

International Standard for TNF- in continuity with the units

assigned to the current IS for TNF-.

Conclusions and Proposal

Based on the results of this study, it is clear that the TNF- candidate (sample A coded 12/154)

is suitable to serve as the WHO 3rd IS for TNF-for assessing potency of current TNF-

products and reagents. It is proposed, therefore, that the candidate preparation 12/154 be

accepted as the WHO 3rd IS for TNF- with an assigned value of 43,000 IU/ampoule of TNF-

activity.

Acknowledgements

We are very grateful to the manufacturers (Mochida Pharmaceutical Ltd, Japan, Dainippon

Pharmaceutical Ltd, Japan and Xiamen Amoytop, China) for the supply of candidate materials

and to the participating laboratories for performing the laboratory tests. We are grateful to Paul

Matejtschuk and Kiran Malik for the pilot fill and for assessing the characteristics of the

lyophilized preparations and staff of SPD for lyophilizing and despatching the candidate

materials of the study.

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U937 cell line. Leuk Res. 35(2):226-36.

WHO/BS/2013.2219

Page 12

Table 1: Materials used in the study

Ampoule Code

Fill Date Study Code

No in Stock

TNFα (predicted

Mass-g)

Type and expression

system

Excipients

12/154 14/6/12 A, C 7828 1.0 157 amino acids, full length; E.Coli

expressed

6 salt PBS, 0.6% HSA, 0.1% Trehalose

88/784

09/3/89 B 3219 1.0 155 amino acids, 2 amino acid deletion at N-

terminus; E.Coli expressed

6 salt PBS, 0.6% HSA

88/786

16/3/89 Current IS

499 1.0 157 amino acids, full length; BALL-

1 cell derived

6 salt PBS, 0.6% HSA

Table 2: Mean fill weights and residual moisture content of candidate preparations

Ampoule Code

Study Code

Mean Fill weight(g)

CV Fill weight

%

Mean Residual

Moisture %

CV Residual Moisture

%

Mean Headspace Oxygen %

CV Headspace Oxygen %

12/154

A, C 1.006 (264) 0.1000 1.294 (12) 29.912 0.430 (11) 32.32

88/784

B 1.005 (87) 0.0951 0.191 (3) 4.231 0.217 (6) 89.13

88/786

Current IS

1.008 (75) 0.0913 0.156 (3) 11.108 0.361 (6) 33.50

The numbers in parentheses indicate the number of determinations. Residual moisture of each

preparation was measured by the coulometric Karl-Fischer method (Mitsubishi CA100).

Headspace oxygen content was determined by frequency modulated spectroscopy (Lighthouse

FMS-760)

WHO/BS/2013.2219

Page 13

Table 3: Brief details of (A) bioassays and (B) immunoassays contributed to the study

A

Laboratory

Code

Bioassay

Cell Line**

Assay

Type

Assay

Duration

(hrs)

Assay Readout

1a

KD4 Clone 21 Cytotoxicity 18-20 Colorimetric (Cell Titer 96

AQueous One, MTS)

1b KLJ Reporter-

gene 4

Luminescence (Steadylite-

plus)

2 L929

Cytotoxicity 20-24 Colorimetric (MTT)

3a WEHI-164

Cytotoxicity 24 Colorimetric (Cell Titer 96

AQueous One, MTS)

3b L929

Cytotoxicity 18-24 Colorimetric (Cell Titer 96

AQueous One, MTS)

4 KLJ Reporter-

gene 4 Luminescence (SteadyGlo)

5 L929

Cytotoxicity 18 Fluorescence (Resazurin)

6 WEHI-164

Cytotoxicity 24 Colorimetric (MTS)

7 L929

Cytotoxicity 20 Colorimetric (MTS)

8 HEK-293 Reporter-

gene 16 Luminescence (SteadyGlo)

9

L929 Cytotoxicity 20-22

Colorimetric (Cell Titer 96

AQueous One, MTS)

10

L929 Cytotoxicity 18-20

Colorimetric (Cell Titer 96

AQueous One, MTS)

11 L929 Cytotoxicity 18 Colorimetric (WST-1)

12

L929 Cytotoxicity 16-20

Colorimetric (Cell Titer 96

AQueous One, MTS)

13

L929 Cytotoxicity 18-24 Colorimetric (MTT)

14 U937

Apoptosis 2-2.5 Luminescence (Apoptosis

detection substrate)

15 L929

Cytotoxicity 18 Luminescence (Cell Titer

Glo)

16

L929 Cytotoxicity 18

Colorimetric (Cell Titer 96

AQueous One, MTS)

17

WEHI-164 Cytotoxicity 20-24

Colorimetric (Cell Titer 96

AQueous One, MTS)

18 U937

Apoptosis 2-2.5 Luminescence (Apoptosis

detection substrate)

* * L929 and WEHI-164– murine; KD4, U937, KLJ, HEK – human

WHO/BS/2013.2219

Page 14

B

Laboratory

Code Immunoassay

Commercial/In-house

1A ELISA using anti-TNF- (MAb 101-4) to capture and

polyclonal antibody to detect

In-house

1B-D ELISA using three human TNF- antagonists* to

capture and anti-TNF- (MAb 101-4) to detect

In-house

5A Fluorokine Map Cytokine Multiplex Human TNF-

Commercial 5B High Sensitivity Human TNF- Immunoassay

5C ELISA (Quantikine) Human TNF-a Immunoassay

5D ELISA (Quanti-Glo) Human TNF- Immunoassay

TNF- antagonists, etanercept, adalimumab and infliximab used in the immunoassays in 1B, 1C and 1D

WHO/BS/2013.2219

Page 15

Table 4: Potencies of sample A relative to IS 88/786 (in IU/ml)

Laboratory Laboratory GM

1 n

1 GCV

1 Laboratory GM

2 n

2 GCV

2

1a 41794 12 18% 41794 12 18%

1b 43326 11 17% 42675 12 18%

2 43356 9 6% 43356 9 6%

3a 43917 8 14% 42940 9 15%

3b 44152 9 5% 44152 9 5%

4 54548 8 28% 53651 9 27%

5 43464 3 15% 43464 3 15%

6 42507 8 10% 41847 9 11%

7 32413 7 41% 31573 9 35%

8 45997 9 23% 45997 9 23%

9 38619 3 7% 38619 3 7%

10 37465 7 41% 38869 9 75%

11 42589 3 3% 42589 3 3%

12 36985 8 18% 36985 8 18%

13 43919 33 14% 43835 38 14%

14 51702 3 55% 51702 3 55%

15 32168 2 n/a 36025 3 53%

16 51982 8 32% 50668 9 31%

17 40142 9 18% 40142 9 18%

18 57897 1 n/a 30127 3 104%

GM

42967 41641

95% C.I. (39999 – 46156) (38910 – 44565)

GCV 17% 16%

n 20 20

1

Calculations excluding cases with slope ratios outside of 0.80 to 1.25

2 Calculations excluding cases with slope ratios outside of 0.67 to 1.50

WHO/BS/2013.2219

Page 16

Table 5: Potencies of sample B relative to IS 88/786 (in IU/ml)

Laboratory Laboratory GM1

n1

GCV1

Laboratory GM2

n2

GCV2

1a 69306 12 26% 69306 12 26%

1b 72287 12 17% 72287 12 17%

2 62971 9 8% 62971 9 8%

3a 62616 8 24% 63806 9 23%

3b 54327 8 4% 54678 9 4%

4 78341 6 16% 83599 9 20%

5 55586 3 20% 55586 3 20%

6 67116 9 15% 67116 9 15%

7 50805 4 12% 51025 9 18%

8 68728 9 25% 68728 9 25%

9 62887 3 8% 62887 3 8%

10 65809 8 29% 65809 8 29%

11 68580 3 9% 68580 3 9%

12 60533 8 31% 62400 9 31%

13 68998 36 20% 68803 39 20%

14 47355 1 n/a 104374 2 n/a

15 57372 2 n/a 66732 3 39%

16 74456 7 45% 74396 9 39%

17 65974 9 12% 65974 9 12%

18 88767 1 n/a 58615 3 126%

GM

64469 66591

95% C.I. (60149 – 69098) (61970 – 71556)

GCV 16% 17%

n 20 20

1 Calculations excluding cases with slope ratios outside of 0.80 to 1.25

2 Calculations excluding cases with slope ratios outside of 0.67 to 1.50

WHO/BS/2013.2219

Page 17

Table 6: Potencies of sample C relative to IS 88/786 (in IU/ml)

Laboratory Laboratory GM

1 n

1 GCV

1 Laboratory GM

2 n

2 GCV

2

1a 41741 12 22% 41741 12 22%

1b 47617 11 15% 48301 12 15%

2 44919 9 5% 44919 9 5%

3a 42831 7 12% 42099 8 12%

3b 44996 9 8% 44996 9 8%

4 49426 8 18% 48996 9 17%

5 36205 3 17% 36205 3 17%

6 43102 9 11% 43102 9 11%

7 33145 5 66% 36578 8 63%

8 48306 9 16% 48306 9 16%

9 41258 3 17% 41258 3 17%

10 37822 8 35% 42430 9 56%

11 44506 3 2% 44506 3 2%

12 39456 8 18% 39456 8 18%

13 45172 35 13% 45236 39 13%

14 n/a 0 n/a 39968 3 229%

15 44292 2 n/a 41708 3 12%

16 49910 8 54% 46120 9 60%

17 41094 9 18% 41094 9 18%

18 41532 3 78% 41532 3 78%

GM

42796 42787

95% C.I. (40673 – 45030) (41149 – 44490)

GCV 11% 9%

n 19 20

1

Calculations excluding cases with slope ratios outside of 0.80 to 1.25 2 Calculations excluding cases with slope ratios outside of 0.67 to 1.50

WHO/BS/2013.2219

Page 18

Table 7: Potencies of Sample A calculated relative to Sample C

Laboratory

code Potency n GCV

1a 1.001 12 26%

1b 0.897 10 13%

2 0.965 9 6%

3a 1.022 7 19%

3b 0.981 9 6%

4 1.104 8 12%

5 1.200 3 8%

6 0.982 8 9%

7 0.998 3 101%

8 0.952 9 16%

9 0.936 3 13%

10 0.950 7 56%

11 0.957 3 4%

12 0.937 8 23%

13 0.979 30 14%

14 n/a n/a n/a

15 1.110 1 n/a

16 1.042 8 27%

17 0.977 9 17%

18 0.730 1 n/a

Laboratory 14 had no valid estimates for sample C

WHO/BS/2013.2219

Page 19

Table 8: Average differences between samples A and C within each lab

Laboratory code

Average %

difference

between A and C

1a 25%

1b 18%

2 7%

3a 18%

3b 6%

4 16%

5 21%

6 8%

7 77%

8 16%

9 13%

10 51%

11 6%

12 22%

13 14%

14 n/a

15 11%

16 26%

17 16%

18 37%

Laboratory 14 had no valid estimates for sample C

WHO/BS/2013.2219

Page 20

Table 9A: Potencies of sample A relative to IS 88/786 (in IU/ml)

Laboratory Laboratory GM1

n1

GCV1

1A 45226 8 2%

1B 39083 4 9%

1C 44879 4 8%

1D 40972 4 5%

5A 55021 9 11%

5B 98629 9 13%

5C 53288 8 26%

5D 56467 9 6%

GM

51953

Using all

laboratories

95% C.I. (40674 – 66358)

GCV 34%

n 8

GM

47406

Excluding

laboratory 5b

95% C.I. (41368 – 54326)

GCV 16%

n 7

Table 9B: Potencies of sample B relative to IS 88/786 (in IU/ml)

Laboratory Laboratory GM1

n1

GCV1

1a 60205 8 3%

1b 58763 4 10%

1c 62594 4 5%

1d 63790 4 9%

5a 61155 9 11%

5b 65743 8 7%

5c 32799 6 58%

5d 65477 9 5%

GM

57648

Using all

laboratories

95% C.I. (47514 – 69942)

GCV 26%

n 8

GM

56576

Excluding

laboratory 5b 95% C.I. (45185 – 70838)

GCV 28%

n 7

WHO/BS/2013.2219

Page 21

Table 9C: Potencies of sample C relative to IS 88/786 (in IU/ml)

Laboratory Laboratory GM

1 n

1 GCV

1

1A 44598 8 2%

5A 49737 9 8%

5B 97977 9 14%

5C 53163 7 36%

5D 56232 9 6%

GM

57882

Using all

laboratories

95% C.I. (39555 – 84701)

GCV 36%

n 5

GM

50745

Excluding

laboratory 5b 95% C.I. (43305 – 59464)

GCV 10%

n 4

Sample C was not assessed in 1B, 1C and 1D.

In all tables above, 1

calculations excluding cases with slope ratios outside of 0.80 to 1.25

WHO/BS/2013.2219

Page 22

Table 10A: Potency estimates of ampoules of 12/154 after storage at different

temperatures, calculated relative to ampoules stored at -70oC by weighted, semi-weighted

or unweighted mean as appropriate.

Temperature Storage Time GM Potency 95% Confidence Interval n GCV

-20°C 6-7 months 0.988 0.928 – 1.053 8 7.8%

4°C 6-7 months 0.987 0.904 – 1.079 7 10.0%

20°C 6-7 months 1.003 0.937 – 1.074 7 7.7%

37°C 6-7 months 0.902 0.793 – 1.026 7 14.9%

45°C 6-7 months 0.897 0.861 – 0.935 10 5.9%

-20°C 10 months 0.970 0.886 – 1.061 4 15.1%

+4°C 10 months 1.005 0.933 – 1.084 2 n/a

+20°C 10 months 0.981 0.930 – 1.035 4 7.5%

+37°C 10 months 1.006 0.903 – 1.121 2 n/a

+45°C 10 months 0.840 0.769 – 0.918 4 14.0%

Predicted loss of potency of < 0.001% per year when stored at -20°C

Predicted loss of potency of 0.340% per month when stored at +37°C

Table 10B: Potency estimates of ampoules of 12/154 after reconstitution assays, calculated

relative to fresh sample

Duration Temperature GM Potency 95% Confidence Interval n GCV

Day +4°C 1.032 0.924 – 1.153 7 12.7%

+20°C 1.075 0.952 – 1.214 6 12.3%

Week +4°C 1.015 0.943 – 1.092 8 9.2%

+20°C 0.927 0.873 – 0.985 4 8.5%

Table 10C: Potency estimates of ampoules of 12/154 after freeze-thaw cycles, calculated

relative to fresh sample

Cycles GM Potency 95% Confidence Interval n GCV

1 1.025 0.979 – 1.074 3 2.5%

2 1.033 0.958 – 1.113 2 n/a

3 0.975 0.918 – 1.036 3 7.9%

4 0.974 0.924 – 1.026 4 9.6%

WHO/BS/2013.2219

Page 23

Figure 1: Slope ratios of sample A relative to IS 88/786 by laboratory

Figure 2: Slope ratios of sample B relative to IS 88/786 by laboratory

1817161514131211109876543b3a21b1a

2.0

1.50

1.25

1.0

0.80

0.67

0.50

Laboratory

Ra

tio

of

Slo

pes

1817161514131211109876543b3a21b1a

2.0

1.50

1.25

1.0

0.80

0.67

0.50

Laboratory

Ra

tio

of

Slo

pes

WHO/BS/2013.2219

Page 24

Figure 3: Slope ratios of sample C relative to IS 88/786 by laboratory

Figure 4: Slope ratios of coded duplicates (A relative to C) by laboratory

1817161514131211109876543b3a21b1a

2.0

1.50

1.25

1.0

0.80

0.67

0.50

Laboratory

Ra

tio

of

Slo

pes

1817161514131211109876543b3a21b1a

2.0

1.50

1.25

1.0

0.80

0.67

0.50

Laboratory

Ra

tio

of

Slo

pe

s

WHO/BS/2013.2219

Page 25

Figure 5: Laboratory mean potencies of samples A and C calculated relative to IS 88/786

(IU/ml)

Num

ber

of Labs

0

1

2

3

4

5

6

7

8

9

10

11

12

Potency (IU/ml)

21000 42000 84000

7

7

15

5 9

10

10

12

12

17

6

9

11

17

18

1a

1a

2

5

6

11

13

15

1b

3a

3a

3b

2

8

13

3b

8

1b

4

14

16

16 4 18A

C

A

C A

A

C

A

C

A

A

C

A

C

C

A

C

A

A

C

C

A

C

A

A

C

A

C

A

C

C

C

C

C

A

C

A A A

Cytotoxicity Assays Other Assays

WHO/BS/2013.2219

Page 26

Figure 6: Laboratory mean potencies of sample B calculated relative to IS 88/786 (IU/ml)

Num

ber

of Labs

0

1

2

3

4

5

6

7

8

9

10

Potency (IU/ml)

31000 62000 124000

14 7 3b 5 15 2

9

12

3a

10

17

6

8

11

13

1a

1b 16 4 18

Cytotoxicity Assays Other Assays

WHO/BS/2013.2219

Page 27

Appendix 1 List of Participants

The following participants contributed data to the study. In this report, each laboratory has been

identified by a number from 1 to 18 that is not related to this order of listing.

Chris Bird, Paula Dilger and Isabelle Cludts, Cytokines and Growth Factors Section,

Biotherapeutics Group, NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts, EN6

3QG, UK

Zeng Yan and Meihua Yang, Xiamen Amoytop Biotech co., Ltd, No. 330, Wengjiao

Road, Haicang, Xiamen, Fujian, P.R.China

Gao Kai and Zhang Feng,Yu Chuanfei, Division of Monoclonal Antibodies, NIFDC,

No2.Tiantan Xili, Beijing, 100050, P.R.China

Michael Tovey and Christophe Lallemand, Laboratory of Biotechnology & Applied

Pharmacology (LBPA), Ecole Normale Supérieure de Cachan,61 Avenue du Président

Wilson,94235 Cachan Cedex, France

Hishani Kirby and Jan Broadbridge, UCB Celltech, 216 Bath Road, Slough, Berks, UK

Guoping Wu, Lori Ablett and John Beauchamp, R&D Systems, Inc.614 McKinley Place NE, Minneapolis, MN 55413,USA

Joon Ho Eom, Advanced Therapy Products Research Division, KFDA, 187

Osongsaengmyeong 2-ro, Osong-eup, Cheongwon-gun, Chungcheongbuk-do, 363-700,

Republic of Korea

Allison Jones, Biochemistry Section, Office of Laboratory & Scientific Services,

TGA,136 Narrabundah Lane, Symonston, ACT 2602, Australia.

MN Dixit, Manjunath Patil, Bioanalytical Laboratory, 3rd

Floor Clinigene International

Limited , Clinigene House, Electronics City, Phase 2 , Bangalore 560100, India

Cornelius Fritsch, TRD Biologics, Novartis Pharma AG, Klybeckstrasse 141, CH-4002

Basel, Switzerland

Camille Dycke and Ravi Vekaria, Covance Laboratories Ltd, Biotechnology - Building

LB6.1, Otley Road, Harrogate HG3 1PY, UK.

Venkata Ramana and Pradnya D Palande, Therapeutic proteins group, Reliance Life

Sciences Pvt Ltd, Dhirubhai Ambani Life Sciences Centre, Plot No:R282, TTC area of

MIDC, Thane Belapur Road, Rabale, Navi Mumbai-400701, India.

Renate Kron, Dominik Dorer and Julia Spanowsky, AbbVie Deutschland GmbH & Co

KG, Knollstrasse, 67061 Ludwigshafen, Germany

WHO/BS/2013.2219

Page 28

Qian Weizhu, National Engineering Research Center of Antibody Medicine and National

Key Laboratory of Antibody Medicine, Libing Rd. 301#, ZhangJiang Hitech Park,

Pudong, Shanghai 201210, P.R. China

Michael Li, Shanghai CP Guojian pharmaceutical Co. Ltd, No 399 Libing Road

ZhangJiang High-tech Park, Shanghai 201210, P.R. China

Yanjun Liu and Fang Wu, Genetic Engineering Department, Shanghai FuDan-

ZhangJiang Biopharmaceutical Co., Ltd, 308 Cailun Road, ZhangJiang High-Tech Park,

Shanghai 201210, P.R .China

Brian Hassett, Pfizer, Grange Castle Development Facility, Clondalkin, Dublin 22,

Ireland.

Murali Pasumarthy, Amgen, ARI Building 7 Room 1320, 40 Technology Way, West

Greenwich RI 02817, USA

WHO/BS/2013.2219

Page 29

Appendix 2

COLLABORATIVE STUDY FOR 3rd

International Standard (IS) for HUMAN TNF-alpha

(TNF-

Study Protocol for TNF-

1. AIMS OF THE STUDY

i. To assess the suitability of ampouled preparations of human TNF-rd

IS for the

bioassay of human TNF- by assaying their biological activity in a range of routine,

'in-house' bioassays.

ii. To assess the activity of the ampouled preparations in different assays (e.g., bioassays,

immunoassays etc) in current use for these materials and to calibrate the candidate IS

against the 2nd IS (88/786).

iii. To compare the ampouled preparations with characterised 'in-house' laboratory

standards where these are available.

2. MATERIALS INCLUDED IN THE STUDY

Participants will be sent

A set of samples coded by letter A to C (5 ampoules for each preparation) for testing in

TNF-bioassays. Each sample contains approximately 1 g of TNF-.

5 ampoules of the current IS (88/786). The current IS contains approximately

1 g of TNF-.

3. RECONSTITUTION AND STORAGE OF PREPARATIONS

Prior to initiating the study, please read the Instructions for Use provided with the

collaborative study. Please note the statements regarding safety and that these

preparations are not for human use.

Lyophilized preparations provided should be stored at -20oC or below until used.

All preparations, A to C should be reconstituted with 1ml of sterile distilled

water and used immediately.

The IS coded 88/786 should also be reconstituted with 1ml of sterile distilled

water and used immediately. This solution will contain TNF- at a

concentration of 46,500 International Units/ml. Use carrier protein where

extensive dilution is required. Use immediately after reconstitution.

4. ASSAY STRUCTURE

1. Participants are asked to include all samples A to C and the current IS (88/786) in each

TNF- assay. In addition, we request that participants include their own in house standard

in each assay, where available.

WHO/BS/2013.2219

Page 30

2. For this study, please use a freshly prepared ampoule of each preparation, A to C and of

the current IS (88/786) in each of the assays. An assay is considered independent if the

assay is carried out on different days/occasions.

3. For each assay method used, participants are asked to perform an assay initially (a pilot assay)

to ensure that all preparations (A to C, 88/786 and in-house standard) are diluted such that

the concentration range falls within the working range of the assay. Please include dilution

series of all preparations (A to C, 88/786 and if available an in-house standard) in the

assay.

4. Following the pilot assay (as in step 2 above), perform at least 3 independent assays for

each of the preparations (A to C, 88/786 and in-house standard) using the most

appropriate dilutions (those giving responses in the linear portion of the dose response

curve) derived from the pilot assay for the different preparations tested.

5. Irrespective of the assay to be performed, participants are requested to include dilution

series for each preparation in duplicate in each assay. Each plate must include 88/786

and samples A-C. Therefore, samples A-C will be split across plates as shown in the

example layout, and replicated (at least three times if performing a bioassay). Include

blank control wells in the assay. Please try to ensure that assays are not susceptible to

edge or positional effects.

For bioassay, follow the suggested bioassay layout provided if possible. Include blank

control wells (cells with culture medium but no TNF-) as indicated. The layout can be

amended if performing an immunoassay. However, it is important to ensure that each

plate includes a dose response curve of 88/786 and samples A-C.

5. INFORMATION TO BE SUPPLIED AND PRESENTATION OF RESULTS

1. We have provided an Excel template (separate excel file) for returning the data obtained

from 3 bioassays for the samples tested. For immunoassays, this will need to be

amended as per the layout of the assay.

2. Please let us know, as clearly as possible, how the assay was carried out, especially how the

stock solutions were diluted and what dilutions were entered into the assay (and at what

positions, if microtitre plates were used). We have provided an example for a microtitre

plate format data sheet at the end of this protocol for diagrammatically illustrating the

assay format, dilutions and results.

IT IS VITAL TO INDICATE THE PREDILUTIONS (starting dilutions) OF THE

ORIGINAL PREPARATION IN EACH ASSAY, along with the working dilutions on

the plate.

Please PROVIDE ALL RAW DATA (microtitre plate readout CPM/OD, Response Units

etc) as direct analysis of the raw data provided by the assays permits data from all

participants to be handled, as far as possible by uniform procedures .

We request participants to follow the example provided and enter data as indicated in

the Excel template (that has been provided separately). Please return all data relating

to the 3 assays electronically in the same format as the Excel template provided.

WHO/BS/2013.2219

Page 31

3. Please provide information regarding your local in-house standard on the sheet provided.

4. Please provide information regarding your assay on the sheet provided.

PLEASE PROVIDE ALL INFORMATION REQUESTED AS THIS IS NEEDED FOR

COMPILATION OF THE STUDY REPORT AND SEND TO:

Meenu.Wadhwa@nibsc.hpa.org.uk

6. CALCULATION OF RESULTS BY PARTICIPATING LABORATORY

Although NIBSC will calculate relative potencies from the raw data provided by the

participants, participants are requested (if possible) to calculate the contents of each

preparation using their own in-house methods relative to the IS (88/786) and their in-house

standard.

PLEASE PROVIDE INFORMATION OF ALL METHODS USED TO CALCULATE

RESULTS

7. REPORTING OF RESULTS

A draft report of the results will be sent to participants so that they will have an opportunity

to comment on it. Participants in the collaborative study are asked to note that they do so with

the understanding that they agree not to publish or circulate information concerning the

materials sent to them without the prior consent of the organisers.

WHO/BS/2013.2219

Page 32

COLLABORATIVE STUDY FOR HUMAN TNF-ALPHA

Laboratory identification……

Local standard information

1. What is the nature of your local standard?

Please state expression system ___________

2. How did you obtain the standard?

Bought ____ Source _____________

Made in-house ____ (please give reference if available)

3. What units do you use with the standard?

Mass ________

Units _________

International Units _________

4. If units or international units, please provide information on how it was derived

__________________________________________________________________

______________________________________________________

WHO/BS/2013.2219

Page 33

COLLABORATIVE STUDY FOR HUMAN TNF-ALPHA

Laboratory identification……

Assay information

Outline the assay methods used (provide full protocol on separate sheets if available):

WHO/BS/2013.2219

Page 34

COLLABORATIVE STUDY FOR HUMAN TNF-ALPHA

Example Layout Plate 1. Sample Layout:

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

A blank CS* CS IH* IH A A B B C C blank

B blank CS CS IH IH A A B B C C blank

C blank CS CS IH IH A A B B C C blank

D blank CS CS IH IH A A B B C C blank

E blank CS CS IH IH A A B B C C blank

F blank CS CS IH IH A A B B C C blank

G blank CS CS IH IH A A B B C C blank

H blank CS CS IH IH A A B B C C blank

Optional Sample Pre-dilution: reciprocal e.g. 10 for 1/10 or 100 for 1/100.

CS: IH:

A:

B:

C:

Sample On-plate Dilutions (reciprocal e.g. 2 for 1 /2, 10 for 1/10 etc).

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

A blank 10 10 10 10 10 10 10 10 10 10 blank

B blank 20 20 20 20 20 20 20 20 20 20 blank

C blank 40 40 40 40 40 40 40 40 40 40 blank

D blank 80 80 80 80 80 80 80 80 80 80 blank

E blank 160 160 160 160 160 160 160 160 160 160 blank

F blank 320 320 320 320 320 320 320 320 320 320 blank

G blank 640 640 640 640 640 640 640 640 640 640 blank

H blank 1280 1280 1280 1280 1280 1280 1280 1280 1280 1280 blank

Response e.g. OD / cpm (with duplicates listed vertically)

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

A

B

C

D

E

F

G

H

*CS=Current Standard *IH=In-house Standard *Blank=Blank Control Wells

WHO/BS/2013.2219

Page 35

Plate 2. Sample Layout:

Sample Pre-dilution: reciprocal e.g. 10 for 1/10, 100 for 1/100 etc.

CS: IH:

A:

B:

C:

Sample On plate Dilutions (reciprocal e.g. 2 for 1 /2, 10 for 1/10 etc).

Response e.g. OD / cpm

*CS=Current International Standard *IH=In-house Standard Blank=Blank Control Wells

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

A blank B B C C CS* CS IH* IH A A blank

B blank B B C C CS CS IH IH A A blank

C blank B B C C CS CS IH IH A A blank

D blank B B C C CS CS IH IH A A blank

E blank B B C C CS CS IH IH A A blank

F blank B B C C CS CS IH IH A A blank

G blank B B C C CS CS IH IH A A blank

H blank B B C C CS CS IH IH A A blank

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

A

blan

k 10 10 10 10 10 10 10 10 10 10

blan

k

B

blan

k 20 20 20 20 20 20 20 20 20 20

blan

k

C

blan

k 40 40 40 40 40 40 40 40 40 40

blan

k

D

blan

k 80 80 80 80 80 80 80 80 80 80

blan

k

E

blan

k 160 160 160 160 160 160 160 160 160 160

blan

k

F

blan

k 320 320 320 320 320 320 320 320 320 320

blan

k

G

blan

k 640 640 640 640 640 640 640 640 640 640

blan

k

H

blan

k

128

0

128

0

128

0

128

0

128

0

128

0

128

0

128

0

128

0 1280

blan

k

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

A

B

C

D

E

F

G

H

WHO/BS/2013.2219

Page 36

Plate 3. Sample Layout:

Sample Pre-dilution: reciprocal e.g. 10 for 1/10, 100 for 1/100 etc.

CS: IH:

A:

B:

C:

Sample On plate Dilutions (reciprocal e.g. 2 for 1 /2, 10 for 1/10 etc).

Response e.g. OD / cpm

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

A blank IH* IH A A C C CS* CS B B blank

B blank IH IH A A C C CS CS B B blank

C blank IH IH A A C C CS CS B B blank

D blank IH IH A A C C CS CS B B blank

E blank IH IH A A C C CS CS B B blank

F blank IH IH A A C C CS CS B B blank

G blank IH IH A A C C CS CS B B blank

H blank IH IH A A C C CS CS B B blank

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

A

blan

k 10 10 10 10 10 10 10 10 10 10

blan

k

B

blan

k 20 20 20 20 20 20 20 20 20 20

blan

k

C

blan

k 40 40 40 40 40 40 40 40 40 40

blan

k

D

blan

k 80 80 80 80 80 80 80 80 80 80

blan

k

E

blan

k 160 160 160 160 160 160 160 160 160 160

blan

k

F

blan

k 320 320 320 320 320 320 320 320 320 320

blan

k

G

blan

k 640 640 640 640 640 640 640 640 640 640

blan

k

H

blan

k

128

0

128

0

128

0

128

0

128

0

128

0

128

0

128

0

128

0 1280

blan

k

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

A

B

C

D

E

F

G

H

WHO/BS/2013.2219

Page 37

Appendix 3: Instructions for Use

(Please see following page)

WHO/BS/2013.2219

Page 38

WHO/BS/2013.2219

Page 39