WHO/BS/2015.2257

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 12 to 16 October 2015

Report on a Collaborative Study for Proposed 1st International

Standard for TNF receptor II Fc fusion protein (Etanercept)

Meenu Wadhwa1, Chris Bird, Paula Dilger and Peter Rigsby

National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters

Bar, HERTS EN6 3QG, UK 1Email 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 14 September 2015 and

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

Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to

the Responsible Officer: Dr Kai Gao at email:gaok@who.int

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WHO/BS/2015.2257

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Summary

Three candidate preparations of human sequence recombinant TNF receptor II Fc fusion protein

(etanercept) were formulated and lyophilized at NIBSC prior to evaluation in a collaborative

study for their suitability to serve as an international standard for the potency of TNF receptor II

Fc fusion proteins (etanercept). The preparations were tested in twenty eight laboratories using

different in vitro cell-based bioassays.

The results of this study indicated that the candidate preparation, coded 13/204 was suitable to

serve as an international standard for etanercept based on the data obtained for biological

activity. Since etanercept inhibits the biological activity of TNF-, this study also provides an

indication of the inhibitory activity of etanercept in terms of the biological activity of TNF-

based on ED50 data derived from a limited number of laboratories using a cytotoxicity assay.

Therefore, it is proposed that the candidate standard, coded 13/204 is established as the first

International Standard for TNF receptor II Fc fusion protein (etanercept) with an assigned in

vitro bioactivity of 10,000 IU per ampoule.

Responses from study participants

Responses were obtained from twenty-seven of the twenty-eight participants of the study. Minor

comments were received relating to typographical errors or corrections in the names of

participants and address details. A minor correction relating to the amount of TNF- used in the

assay was reported by a participant. All of these minor amendments have been corrected in the

report. The proposed IS 13/204 was arbitrarily assigned a value of 50, 000 IU per ampoule

initially but to ensure harmonization with the currently marketed product, this value was changed

to 10, 000 IU. All responses received were in agreement with the proposal regarding suitability

of 13/204 as the WHO 1st IS for TNF receptor II Fc fusion protein (etanercept) and with an

assigned in vitro bioactivity of 10,000 IU per ampoule.

Introduction

The recombinant human tumor necrosis factor (TNF) receptor Fc fusion protein, etanercept is a

dimer engineered by fusing the extracellular ligand binding domain of human tumor necrosis

factor receptor-2 (TNFR2/p75) to the Fc domain of human IgG1 which contains the hinge, CH2

and CH3 regions, but not the CH1 region of IgG1. It is a large glycoprotein with a molecular

weight of approximately 150 kilodaltons containing 934 amino acids and several N-linked and

O-linked glycosylation sites (1,2).

Etanercept acts as a competitive inhibitor of TNF and prevents it from binding to its cell surface

receptors, thereby reducing the biological activity of TNF. As a result, it has potential in

treatment of various autoimmune diseases or disorders associated with increased TNF and excess

inflammation. Current therapeutic indications include rheumatoid arthritis, juvenile idiopathic

arthritis, psoriatic arthritis, ankylosing spondylitis and plaque psoriasis (2-4).

WHO/BS/2015.2257

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Currently, there is only one approved etanercept product (Enbrel, Amgen/Pfizer) in USA and

Europe. Ranked in the top ten biological products in US, etanercept (Enbrel) achieved sales of ~

9 billion US dollars in 2013. With recent patent expiration in Europe (August 2015) although not

in US until 2028 (November), it is a lucrative target for manufacturers worldwide. It is not

unexpected, therefore, that several ‘intended copy’ versions of etanercept are already approved in

poorly regulated countries while biosimilar medicines are under regulatory evaluation or in

clinical trials in various countries (5-6).

Etanercept product is dosed in mass units and the label does not provide any information relating

to its biological activity (i.e., international unit or specific activity of protein). Determination of

the bioactivity in vitro is routinely performed for lot release and stability assessment against

proprietary reference material by the licence holders. Availability of an international reference

standard would facilitate determination of biological activity of intended copies or potential

biosimilars and enable availability of products with similar biological activities thus ensuring

patient access to products which are consistent in quality and effectiveness.

We have therefore evaluated in an international collaborative study, three candidate TNFR II-Fc

fusion protein (etanercept) preparations with the aim of selecting a suitable standard for

bioactivity of these products.

Based on its categorization as a TNF antagonist, this etanercept project was endorsed by the

WHO Expert Committee on Biological Standardisation in October 2012.

Aims of the Study The purpose of the study was to characterize a candidate WHO 1

st IS for the bioassay of human

etanercept and assign a unitage for in vitro biological activity. To achieve this, the study sought

To assess the suitability of ampouled preparations of human etanercept to serve as the 1st

International Standard (IS) for the bioassay of human etanercept by assaying their biological

activity in a range of routine, 'in-house' bioassays.

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

immunoassays etc) in current use for these materials and to determine, if possible,

concentrations of etanercept required to neutralise specific amounts of TNF-α IS.

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

these are available.

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Materials and Methods

Two preparations of human sequence recombinant etanercept, both pure and expressed in CHO

cells were kindly donated to WHO (see Acknowledgement). Trial fills were conducted and the

biological activity of the lyophilized preparations compared with the bulk material in two different

bioassays; 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- (7-8) 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 (9).

Based on bioassay data with trial lyophilizations of etanercept, a formulation was selected and final

lyophilizations of different etanercept preparations carried out at NIBSC as per the procedures used

for International Biological Standards (ECBS guidelines - WHO Technical Report Series 932,

2006).

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 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 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 both preparations, a solution at a concentration predicted as 5g/ml etanercept was

distributed in 1.0ml aliquots, giving the theoretical protein content per ampoule 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 coulometric Karl-Fischer method (Mitsubishi CA100), 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.

WHO/BS/2015.2257

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Participants

Samples were dispatched in November 2014 to 29 laboratories in 15 different countries. The

participants included 1 academic, 8 control, 2 pharmacopoeial, 15 manufacturers’ and 2 contract

research organisation laboratories. 28 participants submitted data and are listed in Appendix 1.

Assay Methods and Study Design

A summary of the assay methods used in the study is given in Table 3. A majority of participants

used procedures for assessing the inhibitory effect of etanercept based on cell-lines that are

commonly used for TNF- bioassays (10). Some of these were used previously in development

of the 3rd

IS for TNF- (11).

Bioassays which measure the cytotoxic effect of TNF- in the murine fibroblast cell-line L929

were used by numerous laboratories although murine or human fibrosarcoma or

rhabdomyosarcoma cell-lines were also used (10) as shown in Table 3A. The readouts for

assessing the cytotoxic effect varied between laboratories. In some laboratories, bioassays based

on the apoptotic effect of TNF- were used, however all laboratories performing this assay used

the human histiocytic lymphoma cell-line, U937 in the apoptosis assays and employed the

caspase 3/7 reagent for apoptosis evaluation. In rare instances, reporter gene assays using the

K562 cells (9) or the HEK-293 over transfected with the TNF- responsive NFκB regulated

Firefly luciferase (FL) reporter-gene construct were used. In addition to bioassays, two

laboratories also performed binding assays (Table 3B).

Participating laboratories were sent five sets of six study samples coded A-D along with the 3rd

TNF- IS (12/154) as detailed in Table 1. Samples A, D (code, 13/192) and C (code, 13/260)

are preparations lyophilized on different occasions from the same batch of the active substance.

Samples A and D are coded duplicates of the same candidate standard (code, 13/192). Sample B

(code, 13/204) is a lyophilized preparation of the protein provided by a different manufacturer.

Since etanercept acts by inhibiting the biological activity of TNF- α, study participants were

requested to use a fixed amount of the 3rd

TNF-α IS (coded 12/554; supplied along with the

ampoules of etanercept) instead of the in routine use in their bioassays and advised that the final

dose should give a biological response similar to the dose of the TNF-α reagent used routinely in

assays in-house. All participants were advised to perform a pilot assay for selection of a suitable

dose of TNF-α and for evaluating the dose response curve of etanercept.

Participants were asked to assay all samples 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 3).

It was requested that participants perform at least 8 dilutions of each preparation using freshly

reconstituted ampoules for each assay and include their own in-house standard where available

on each plate.

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

and also their own calculations of potency of the study samples relative to preparation A or their

own in-house standard.

Statistical Analysis

Raw data from all participants were analysed with a four-parameter logistic model (sigmoid

curves) using EDQM CombiStats Software Version 5.0 (12). The fitted models were used to

calculate estimates of ED50 (dilution of ampoule reconstituted in 1 ml required to achieve 50%

response) and potency of samples coded B, C and D relative to sample coded A.

All mean results shown in this report are unweighted geometric means (GM). Variability

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

1}×100% where s is the standard deviation of the log10 transformed laboratory GM estimates).

Individual assay estimates of ED50 or relative potency were log transformed and a mixed effects

model was fitted using statistical software R (version 3.1.3) with the R package ‘lme4’ (version

1.1-7) in order to determine between-assay and between-plate (within-assay) variance

components (by restricted maximum likelihood) which were also expressed as %GCV.

The relative contents of the accelerated thermal degradation samples were used to fit an

Arrhenius equation relating degradation rate to absolute temperature assuming first-order decay

and hence predict the degradation rates when stored at -20°C (13).

Results

Data returned for analysis

Results were received from 28 laboratories. Participating laboratories have been assigned code

numbers allocated at random, and not necessarily representing the order of listing in Appendix 1

to retain confidentiality in the report.

The majority of participants tested all samples in 3 assays with 3 plates per assay. Exceptions

were lab 1 (U937: 2 plates per assay), lab 3 (U937: 1 plate per assay), lab 10 (U937: 2 plates per

assay), lab 14 (L929: 1 plate per assay) and lab 27 (Reporter Gene: 1 plate per assay).

Assay validity and model fit

The goodness of fit for the sigmoid curves model varied between different laboratories and assay

types. The majority of L929 plates (97%) gave R2 values ≥0.97 with only laboratory 13 giving

R2<0.97 for 3 plates. U937 assays showed a lower proportion (61%) of plates with R

2≥0.97 and

only laboratories 1, 2, 9 and 10 demonstrated this on all plates. Almost all other cytotoxicity

plates (96%) gave R2≥0.97 and this was also the case in all reporter gene and binding assays.

WHO/BS/2015.2257

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Parallelism as measured by the ratio of fitted slopes for samples B, C and D relative to the slope

for sample A also varied between different laboratories and assay types. For U937 plates, slope

ratios outside the range 0.67 to 1.50 were noted in 8%, 10% and 8% of plates for samples B, C

and D respectively with slope ratios outside the range 0.80 to 1.25 in 21%, 30% and 29% of

plates for samples B, C and D. Better parallelism was observed in L929 assays where only 11%,

9% and 10% of plates gave slope ratios outside 0.80 to 1.25 for samples B, C and D. These

figures were similar for other cytotoxicity assays (11%, 7% and 7%) and there were no apparent

parallelism issues (using the 0.80 to 1.25 criteria) in the reporter gene and binding assays.

Following visual assessment of assay data, a small number of plates were excluded from further

analysis. These were from lab 6 (all U937 plates on day 2) and lab 8 (L929 plate on day 2 and

two L929 plates on day 3). Results from all other assays have been included in this report.

Estimates of ED50

Geometric mean estimates of ED50 for each laboratory are shown in Appendix 2, Tables 1- 4.

As expected, ED50 values showed an inverse relationship with the dose of TNF-α used by

participants (Figures 1-4). Variance components (expressed as %GCV) for between-assay and

between-plate (within-assay) variability were considerably different between different

laboratories and assay methods, ranging up to 19.5% in U937 assays and were generally higher

in L929 assays, ranging up to 37.1%. A summary of ED50 estimates for laboratories performing

U937 assays or L929 assays with a fixed TNF-α concentration is given in Table 4A.

Estimates of relative potency

Geometric mean estimates of potency for samples B, C and D relative to sample A are shown in

Appendix 2 Tables 1-5 and Figure 5. A summary of relative potency estimates is given in Table

5.

Overall estimates showed good agreement between assay methods with mean potencies of 0.93 –

0.95 being obtained for samples B and C relative to sample A in U937, L929, other cytotoxicity

and reporter gene assays. Between-laboratory GCV values were lower for U937 assays (5-7%)

than for L929 assays (8-10%). Variance components (expressed as %GCV) for between-assay

and between-plate (within-assay) variability were mostly lower than those observed for ED50

estimates, ranging up to 19.2% in U937 assays and up to 33.7% in L929 assays. A summary of

relative potency estimates for laboratories performing U937 assays or L929 assays with a fixed

TNF-α concentration is given in Table 4B.

Geometric mean relative potency results for sample D were 1.00, 1.01 and 0.99 in U937, L929

and other cytotoxicity assays respectively, showing good agreement with the expected value of

1.00 as sample D is a coded duplicate of sample A. Individual plate results for the potency of D

relative to sample A were within the range 0.80 to 1.25 in 92% of U937 assays, 91% of L929

assays and 91% of other cytotoxicity assays.

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Stability studies

Accelerated Degradation Studies

Samples of the candidate standards 13/192 and 13/204 were stored at elevated temperatures

(4°C, 20°C, 37°C and 45°C) for a period of 14-19 months and assayed at NIBSC using the KLJ

reporter gene and KD4 cytotoxicity assays. Samples were tested concurrently with those stored

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

potencies of all samples were expressed relative to the appropriate -70°C baseline samples and

the results are summarised in Table 6. No loss in activity was detected at any of the elevated

temperatures for both 13/192 and 13/204 and therefore no predicted loss in activity can be

calculated.

Stability after reconstitution and on freeze-thaw

Samples of the candidate standards 13/192 and 13/204 were reconstituted and left at 4°C or 20°C

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

concurrently against a freshly reconstituted ampoule. The potencies of all samples were

expressed relative to the freshly reconstituted samples and the results are summarised in Table 7.

There is no evidence that the potency of either standard is diminished after a week of storage at

either at 4°C or 20°C.

Samples of the candidate standards 13/192 and 13/204 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. The potencies of all samples were expressed relative to the freshly

reconstituted samples and the results are summarised in Table 8. From the results it is concluded

that the potency of these preparations does not decrease with repeated freeze-thaw cycles (up to

4).

Discussion

In this study, we focused on the development of an international standard for the in vitro

biological activity of etanercept following a demand from manufacturers for availability of this

standard. Currently, there is only one approved etanercept product in Europe and USA but with

patent expiry and intense development worldwide, it is likely that multiple products will become

available worldwide. Although etanercept is dosed in mass units and the label does not provide

any information relating to its biological activity (i.e., international unit or specific activity of

protein), it is a regulatory requirement to determine the bioactivity in vitro for lot release and

stability assessment using an appropriate reference standard.

Etanercept is a dimeric fusion protein comprising the extracellular ligand-binding domains of

human TNFR2 and the Fc portion of a human IgG1 antibody. Like naturally occurring soluble

TNF receptors, etanercept is highly specific for TNF and lymphotoxin-However, compared

WHO/BS/2015.2257

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with the monomeric soluble TNF receptors, it has a higher affinity for TNF (1) and the Fc

portion extends the half-life of the protein by approximately five to eight-fold in vivo.

The fusion protein just like the approved monoclonal antibodies e.g., infliximab, adalimumab

targets the availability of TNF, however unlike the monoclonal antibodies which also have

significant Fc effector functions, the mechanism of action of etanercept appears to be

predominantly via neutralization of TNF activity (4;14)

Since etanercept functions as a TNF-antagonist by competitively inhibiting TNF from binding

to its cell surface receptors, bioassays which quantify the blocking of TNF-to specific TNF cell

surface receptors are used to assess the functional activity of etanercept. Such bioassays include,

for example, blocking of TNF-inducedproliferation (orcytotoxicity), blocking of TNF-

mediated apoptosis or blocking of TNF-induced NF-κB (nuclear factor kappa-light-chain-

enhancer of activated B cells) activation. All participating laboratories adopted one or more of

these cell-based assays to assess the biological activity of the different etanercept preparations. A

majority of laboratories measured inhibition of TNF mediated cytotoxicity using either murine

fibroblast (L929) or murine (WEHI 164/13 VAR) or human rhabdomyosarcoma (KD4Cl21) cell-

lines (Meager and Gaines Das, 1994). Twelve laboratories performed assays based on the ability

of etanercept to inhibit TNF- induced apoptosis by measuring the activities of Caspase 3 and 7

(members of the cysteine aspartic acid-specific protease - caspase family) which play key

effector roles in cell apoptosis (15) in a human histiocytic lymphoma cell-line, U937 which

exhibits various properties typical of macrophages (16). In rare instances, reporter gene assays

that measure luciferase activity on activation of NF-κB transcription factor have been used (9).

Results from this study showed that the goodness of fit for the sigmoid curves model and

parallelism (as measured by the ratio of fitted slopes for samples B, C and D relative to the slope

for sample A) varied between different laboratories and assay types. A majority of cytotoxicity

assays (L929 - 97% of the plates; other assays - 96%) gave R2 values ≥0.97. This was also the

case in reporter gene and binding assays. For U937 assays, however, R2≥0.97 was evident in a

lower proportion (61% of plates) of assays. Better parallelism was observed for samples B, C

and D in cytotoxicity assays (L929-11%, 9% and 10% of plates gave slope ratios outside 0.80 to

1.25; other assays - 11%, 7% and 7%) as opposed to U937 assays (21%, 30% and 29%). No

parallelism issues (using the 0.80 to 1.25 criteria) were apparent in the reporter gene and binding

assays.

For assessing the inhibitory activity of etanercept, geometric mean estimates of ED50 were

derived for each laboratory. As expected, an inverse relationship was seen between the ED50

values and the amount of TNF- used in the assay. So in assays using high amounts of TNF, the

inhibition as reflected by the ED50 values was low. While the variability differed between assays

and laboratories, the cytotoxicity assays in general showed more variability compared with

apoptosis assays. However, for ED50 estimates, many laboratories (60%) achieved a GCV of

less than 20% (derived from between plate and between assay variability) in L929 based

cytotoxicity assays as opposed to all laboratories (100%) performing apoptosis assays. Of the

five laboratories performing cytotoxicity assays using WEHI or KD4 cell-lines, large variability

was seen with data from two laboratories showing GCV less than 19%. Only two laboratories

performed the reporter gene assays - the GCV was low, less than 10%.

WHO/BS/2015.2257

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Despite differences in the principles of the bioassay methods used for assessing the activity of

etanercept, similar results for potencies were obtained for samples, B and C relative to A. So,

geometric mean potency for sample B was 0.93 and between 0.93 – 0.95 for sample C regardless

of the bioassay used. The inter-laboratory variability was lower for apoptosis assays (5-7%)

relative to L929 based assays (8-10%) or other cytotoxicity (7-15%) assays. The assay variance

(both between plate and between assay) was lower than that seen for ED50 estimates and was

about 19% in apoptosis and up to 34% or 29% in L929 based or other cytotoxicity assays. For

reporter gene assays, the variance was lower at about 15%.

Data for coded duplicates, A and D were also highly consistent. The mean potency estimates for

D derived from different assays ranged between 0.99-1.03 with a mean value of 1.00.

Slightly lower potencies were seen for samples B (0.91), C (0.89) and D (0.96) relative to A in

the immunoassays compared with bioassays (B – 0.93, C – 0.94, D - 1.00) although this data was

contributed by only two labs.

Stability studies over 14 – 19 months indicated that the candidate preparations (coded 13/204 and

13/192) are stable for long term storage at -20˚C and the potencies are not diminished after 1

week of storage at either 4˚C or 20˚C following reconstitution or after repeated freeze-thaw

cycles. As no loss in activity was detected at any of the elevated temperatures for these

preparations no predicted loss in activity can be calculated.

Although both candidate preparations (coded 13/204 and 13/192) are stable and suitable for use

in TNF-neutralization assaysas reference standards, the preparation coded 13/204 is

selectedas the 1st International Standard (IS) for etanercept for in vitro bioactivity determination

of etanercept products. It is proposed that the preparation (code 13/204) be established as the

WHO 1st International Standard for etanercept with an assigned value for biological activity of

10,000 IU/ampoule.

Since the other candidate preparation (13/192) behaved similarly in the bioassays, 13/192 would

serve as a suitable replacement standard when stock of the proposed IS, coded 13/204 is

exhausted on the provision that the preparation is sufficiently stable. Taking the potency of

13/204 to be 10,000 IU/ampoule gives an estimated potency for 13/192 of 10,753 IU/ampoule

based on relative potency of 0.93 obtained in all bioassays for 13/204.

Since etanercept inhibits activity of TNF-the inhibitory activity should be expressed in terms

of TNF-. Therefore, an indication of inhibitory activity has been determined for the proposed

IS based on the ED50 data derived for L929 cytotoxicity assays (for the fixed amount of TNF-

used) from laboratories using these assays by the following equation;

Amount of etanercept (IU) inhibiting X amount of TNF- (IU) = potency of preparation

ED50 value

WHO/BS/2015.2257

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Therefore, based on ED50 responses from data of nine laboratories (using a fixed concentration

of TNF-IS, 2.4 IU of etanercept (sample B, code 13/204) inhibits the cytotoxic effect of 10-20

IU of TNF-IS (code 12/154) in L929 cytotoxicity assays.

Conclusions and Proposal

Based on the results of this study, it is clear that the etanercept preparation (code 13/204) is

suitable to serve as the WHO 1st IS for in vitro bioactivity measurement of etanercept products. It

is proposed that the candidate preparation 13/204 be accepted as the WHO 1st IS for etanercept

with an assigned value for biological activity of 10,000 IU/ampoule.

Acknowledgements

We are very grateful to the manufacturers (Sandoz, Austria, Pfizer, Ireland) for the supply of

etanercept preparations for use as candidate materials and to the participating laboratories for

performing the laboratory tests. Thanks are also extended to some of the laboratories involved in

medicines control, particularly those in Europe for facilitating in the optimization of the study

design. We are grateful to Paul Matejtschuk and Kiran Malik for assistance with pilot fills of

etanercept preparations, staff of SPD for lyophilizing and dispatching the candidate materials of

the study and to Adrian Bristow and Robin Thorpe for their continuous support and helpful

discussions.

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standards for human tumour necrosis factors alpha (c) and beta (hTNF-beta) and for

murine tumour necrosis factor alpha (mTNF-alpha). J Immunol Methods.170(1):1-13

12. CombiStats v5.0, EDQM – Council of Europe, www.combistats.eu.

13. Kirkwood TBL (1977) Predicting the stability of biological standards and products.

Biometrics, 33: 736-742.

14. Arora T, Padaki R, Liu L, Hamburger AE, Ellison AR, Stevens SR, Louie JS, Kohno T

(2009) Differences in binding and effector functions between classes of TNF antagonists.

Cytokine. 45(2):124-31

15. Thornberry NA and Lazebnik Y (1998) Caspases: enemies within. Science. 281 (5381):

1312-6.

16. Minafra L, Di Cara G, Albanese NN, Cancemi P (2011) Proteomic differentiation pattern

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

WHO/BS/2015.2257

Page 13

Table 1: Materials used in study

Ampoule

code

Fill

date

Study

code

No Of

Ampoules

in Stock

Protein

(Predicted

Mass - g)

Protein

Expression

System

Excipients

13/192

10/10/13

A, D

4,500

5

TNFR II-Fc

CHO

1% Sucrose

4% Mannitol

10mM Tris

HCL

0.2% Human

Serum

Albumin

13/204

24/10/13

B

4,700

5

13/260

20/03/14

C

6,601

5

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

Ampoule

Code

Mean

Fill weight g

(n)

Coefficient of

Variation

Fill weight %

Mean Residual

Moisture%(n)

Coefficient

of Variation

Residual

Moisture %

Mean

Headspace

Oxygen %

(n)

Coefficient of

Variation

Headspace

Oxygen %

13/192

1.0084 (333)

0.140

0.945 (12)

31.55

0.24 (12)

46.9

13/204

1.0082 (330)

0.192

0.659 (12)

18.05

0.20 (12)

38.98

13/260

1.0068 (232)

0.249

0.550 (12)

18.5

0.36 (12)

33.07

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/2015.2257

Page 14

Table 3A: Brief details of bioassays contributed to the study

Lab

Code

Bioassay

Cell Line

Assay

Type

TNF-

(IU/ml)+

Assay

Period

(hrs)

Assay Readout Readout Reagent

1 U937 Apoptosis 107.5 2 - 2.5 Luminescence Caspase Glo 3/7

2 U937 Apoptosis 40.87 2.25 Luminescence Caspase Glo 3/7

3 U937 Apoptosis 20.0 2.5 Luminescence Caspase Glo 3/7

4 U937 Apoptosis 151.5 2.5 Luminescence Caspase Glo 3/7

5 U937 Apoptosis ** 2.5 Luminescence Caspase Glo 3/7 £

6 U937 Apoptosis ** 2.5 Luminescence Caspase Glo 3/7 $

7 U937 Apoptosis 64.5 2.5 Luminescence Caspase Glo 3/7

8 U937 Apoptosis ** 2.5 Luminescence Caspase Glo 3/7£

9 U937 Apoptosis 37.5 2 Luminescence Caspase Glo 3/7

10 U937 Apoptosis 53.8 2 - 2.5 Luminescence Caspase Glo 3/7

11 U937 Apoptosis ** 2.5 Luminescence Caspase Glo 3/7

12 U937 Apoptosis ** 2.5 Luminescence Caspase Glo 3/7

13 L929 Cytotoxicity 20 18 - 24 Absorbance MTS

14 L929 Cytotoxicity 10 20 - 24 Luminescence ATPLite Luciferase

15 L929 Cytotoxicity 10 18.5 – 19.5 Absorbance MTS

16 L929 Cytotoxicity 86 18 - 22 Fluorescence Alamar Blue

17 L929 Cytotoxicity 25 19 - 21 Fluorescence Alamar blue

8 L929 Cytotoxicity 20 24 Absorbance Alamar Blue

9 L929 Cytotoxicity 10 18 Absorbance CCK-8

18 L929 Cytotoxicity 20 22 Fluorescence Resazurin

19 L929 Cytotoxicity 20 18 Absorbance CCK-8

20 L929 Cytotoxicity

10

14 - 18 Absorbance

Cell Titer 96 AQueous

One (MTS)

21 L929 Cytotoxicity 20 18 - 22 Fluorescence Resazurin

22 L929 Cytotoxicity 15 14 - 16 Absorbance CCK-8

23

WEHI-13

VAR Cytotoxicity

5

18 - 20 Absorbance MTS

24

WEHI-13

VAR Cytotoxicity

2.5

16 - 26 Absorbance

Cell Titer 96 AQueous

One (MTS)

25

WEHI-13

VAR Cytotoxicity

1.505

24 Absorbance CCK-8

26 WEHI-164 Cytotoxicity 100 24 Absorbance MTS

8 KD4 Cl21 Cytotoxicity

4.3

24 Absorbance

Cell Titer 96 AQueous

One (MTS)

8 KLJ Reporter Gene 40 4 Luminescence Steadylite plus

27 HEK 293 Reporter Gene 172 16 Luminescence Steady Glo

28* Kphi 13.2 Reporter Gene 4 Luminescence Dual Glo

$ - For test 3 dil 1:3; £ - 1: 4 dilution; + - the IU refers to the final amounts of the 3rd

IS for TNF- used in assay, * - received late and not included in analysis;

** - information not disclosed

WHO/BS/2015.2257

Page 15

Table 3B: Brief details of binding assays contributed to the study

Lab

Code TNF-

amount

Source of

TNF- Detection reagent

Readout

17 250ng/ml R&D Systems Protein A-HRP Absorbance

21 150ng/ml R&D Systems Anti-Hu IgG Fc-HRP Absorbance

WHO/BS/2015.2257

Page 16

Table 4A. Summary of ED50 estimates for selected assays using same fixed amounts of

TNF-α

Sample Assay TNF-α

(IU/ml) GM

95% Confidence

Limits Between-lab GCV (%) n

A U937 ** 3903 3226 4722 16.6 5*

L929 20 4514 3244 6282 30.5 5

L929 10 4644 4125 5228 7.7 4

B U937 ** 3657 2938 4550 19.3 5*

L929 20 4145 3213 5348 22.8 5

L929 10 4247 3584 5032 11.3 4

C U937 ** 3706 3040 4518 17.3 5*

L929 20 4196 3138 5612 26.4 5

L929 10 4346 3866 4884 7.6 4

D U937 ** 3984 3247 4889 17.9 5*

L929 20 4444 3276 6029 27.8 5

L929 10 4608 4013 5290 9.1 4

*excludes lab 1; ** - information not disclosed

Table 4B. Summary of relative potency estimates for selected assays using same fixed

amounts of TNF-α

Sample Assay TNF-α

(IU/ml) GM

95% Confidence

Limits Between-lab GCV (%) n

B U937 ** 0.94 0.89 0.98 3.9 5*

L929 20 0.92 0.84 1.00 7.5 5

L929 10 0.92 0.86 0.98 4.1 4

C U937 ** 0.95 0.93 0.97 1.9 5*

L929 20 0.93 0.86 1.01 6.9 5

L929 10 0.94 0.84 1.04 7.1 4

D U937 ** 1.02 0.93 1.12 7.8 5*

L929 20 0.98 0.89 1.09 8.5 5

L929 10 0.99 0.93 1.06 4.3 4

*excludes lab 11, ** - information not disclosed

WHO/BS/2015.2257

Page 17

Table 5. Summary of potency estimates relative to sample A

Assay Sample GM 95% Confidence

Limits

Between-lab GCV

(%) n

U937 B 0.93 0.90 0.97 5.5 12

C 0.93 0.91 0.96 4.7 12

D 1.00 0.96 1.04 6.8 12

L929 B 0.93 0.88 0.97 8.0 12

C 0.95 0.89 1.01 10.4 12

D 1.01 0.95 1.08 10.4 12

Other cytotoxicity B 0.93 0.78 1.10 14.5 5

C 0.95 0.87 1.03 7.4 5

D 0.99 0.89 1.09 8.7 5

Reporter Gene B 0.93 . . . 2

C 0.94 . . . 2

D 1.03 . . . 2

Binding B 0.91 . . . 2

C 0.89 . . . 2

D 0.96 . . . 2

Potency (all assays)

B

C

D

0.93

0.94

1.00

0.90

0.92

0.97

0.95

0.96

1.03

7.8

7.6

8.3

33

33

33

Potency (excluding

binding assays)

B

C

D

0.93

0.94

1.00

0.90

0.92

0.98

0.96

0.97

1.03

7.9

7.4

8.2

31

31

31

WHO/BS/2015.2257

Page 18

Table 6. Summary of results from assays of accelerated degradation samples

Assay Sample Storage

Temperature oC

Potency relative to -70 o

C

Estimate 95% Confidence Limits n

KLJ

13/1921

-20 1.05 1.02 1.08 4

4

1.02 0.95 1.10 4

20 1.05 0.97 1.14 4

37

1.02 1.00 1.05 4

45

0.99 0.94 1.04 4

13/2042

-20 1.03 0.98 1.07 4

4

0.99 0.86 1.13 4

20 1.03 0.89 1.19 4

37

0.99 0.87 1.13 4

45

1.00 0.92 1.09 4

KD4

13/1923

-20 0.95 0.85 1.07 9

4

1.03 0.86 1.24 6

20 1.00 0.89 1.12 6

37

1.00 0.90 1.11 9

45

0.93 0.82 1.05 4

13/2041

-20 1.02 0.90 1.16 8

4

1.00 0.91 1.09 7

20 1.08 1.01 1.15 7

37

1.03 0.93 1.15 7

45

0.94 0.82 1.09 5

1,2,3 indicate storage for 14, 16 and 19 months respectively

Table 7. Summary of results from reconstitution stability studies using KLJ reporter gene

assay

Sample Storage

Temperature oC

Potency relative to fresh

Period (day) Estimate 95% Confidence Limits n

13/192 1 +4 1.03 0.98 1.08 6

7 +4 0.99 0.94 1.04 6

1 +20 1.04 0.99 1.10 6

7 +20 1.01 0.99 1.03 6

13/204 1 +4 0.95 0.91 1.00 6

7 +4 0.99 0.95 1.02 4

1 +20 0.97 0.93 1.02 6

7 +20 0.96 0.92 1.00 6

WHO/BS/2015.2257

Page 19

Table 8. Summary of results from freeze-thaw studies using KLJ reporter gene assay

Sample Storage Potency relative to fresh

Estimate 95% Confidence Limits n

13/192 1X 0.92 0.98 1.05 4

2X 0.91 0.97 1.03 4

3X 0.88 0.97 1.06 4

4X 1.01

2

13/204 1X 0.91 0.98 1.05 5

2X 0.89 0.94 1.00 5

3X 0.87 0.93 0.99 5

4X 0.95

2

WHO/BS/2015.2257

Page 20

Figure 1. Laboratory geometric mean ED50 estimates for sample A

Figure 2. Laboratory geometric mean ED50 estimates for sample B

256128643216842

16000

8000

4000

2000

1000

500

TNF-α (IU/ml)

Sam

ple

A E

D5

0

U937

L929

Other Cytotoxity

Reporter Gene

256128643216842

16000

8000

4000

2000

1000

500

TNF-α (IU/ml)

Sam

ple

B E

D5

0

U937

L929

Other Cytotoxity

Reporter Gene

WHO/BS/2015.2257

Page 21

Figure 3. Laboratory geometric mean ED50 estimates for sample C

Figure 4. Laboratory geometric mean ED50 estimates for sample D

256128643216842

16000

8000

4000

2000

1000

500

TNF-α (IU/ml)

Sam

ple

C E

D5

0

U937

L929

Other Cytotoxity

Reporter Gene

256128643216842

16000

8000

4000

2000

1000

500

TNF-α (IU/ml)

Sam

ple

D E

D5

0

U937

L929

Other Cytotoxity

Reporter Gene

WHO/BS/2015.2257

Page 22

Figure 5. Box-plot summary of individual assay potency estimates relative to sample A

WHO/BS/2015.2257

Page 23

Appendix 1 - List of Participants

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

a number that is not related to this order of listing.

Cecilia Medrano, Eduardo Cioppi, Gema Biotech S.A., Fray Justo Sarmiento 2350 edificio 2B 5

piso B1636AXK, Olivos, Buenos Aires, Argentina

Anne-Marie Wilkes and Keith Mortimer, Biochemistry Section, Office of Laboratory &

Scientific Services, Therapeutic Goods Administration,136 Narrabundah Lane, Symonston,

Canberra ACT 2602, Australia.

Haibin Wang, Lei Li and Yong Yang, Zhejiang Hisun Pharmaceutical Co Ltd, 46 Waisha Rd. Jiaojiang, Taizhou, Zhejiang, P.R. China

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

Haicang, Xiamen, Fujian, P.R.China

Gao Kai and Yu Chuanfei, Division of Monoclonal Antibodies, NIFDC, No2.Tiantan Xili,

Beijing, 100050, P.R.China

Qian Weizhu, Sheng Hou, Shanghai Zhangjiang Biotechnology Co., Ltd, State Key laboratory of

Antibody Medicine and Targeted therapy, 99 Libing Rd. Pudong, Shanghai 201203, P.R. China

Zhihui Zhai, Mei Guo and Song Zhao, Shanghai CP Guojian Pharmaceutical Co. Ltd, No 399

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

Luochun Wang and Fang Wu, Genetic Engineering Department, Shanghai FuDan-ZhangJiang

Biopharmaceutical Co., Ltd, 308 Cailun Road, ZhangJiang High-Tech Park, Shanghai 201210,

P.R .China

Erik Ostergaard, Danish Health and Medicines Authority, Laboratory and Inspection, Axel

Heides Gade 1, 2300 Copenhagen S, Denmark

Jaana Vesterinen, Finnish Medicines Agency, Mannerheimintie 166, P.O.Box 55,00300 Helsinki,

Finland

Sylvie Jorajuria, Biological Section Laboratory Department, European Directorate for the Quality of Medicines and HealthCare (EDQM) Council of Europe, 7 allée Kastner CS 30026, F-67081 Strasbourg –France

Jean-Claude Ourlin, ANSM, 635 Rue de la Garenne,CS 60007, 34740 Vendargues Cedex,

France

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

WHO/BS/2015.2257

Page 24

Christine Hölbling, Karin Schmidt, Boehringer Ingelheim Pharma GmbH & Co. KG,

Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany

Ulrike Herbrand, Simone Scotti, Charles River Biopharmaceutical Services GmbH, Max-Planck-

Str. 15A, 40699 Erkrath, Germany

Priya Darshini P, Shubrata Khedkar, Prabhavathy Munagala, Disha Dadke and Ranjan

Chakrabarti, Biologics & Biotechnology Division, United States Pharmacopeia-India (P) Ltd,

Plot No. D6 & D8, IKP Knowledge Park, Genome Valley, Shameerpet, Hyderabad – 500078,

R.R. District, Telangana, India

Shubhangi Argade and Gargi Seth, Intas Pharmaceuticals Ltd., Plot No. 423/P/A, Sarkhej-Bavla

Highway, Village-Moraiya, Taluka –Sanand, Ahmedabad, Pin-382213, Gujarat, India

Veena Raiker and Lalita Bisht, BioAnalytical team, Lupin Ltd, Biotechnology Division, 1st Floor,

G-O Square Mall, Survey No 249+250, Wakad, Pune - 411057, Maharashtra, India

Sridevi Khambhampaty, Manish Kumar, Kamala Bhavaraju, Rakesh Kumar, Biologics

Development Centre, Dr Reddy’s Laboratories, Survey No: 47, Bachupally, Qutubullapur, R R

Dist 500090, Andhra Pradesh, India

Victoria Hayes, Brian Hassett, QC immunology and MSAT, Pfizer, Grange Castle, Clondalkin,

Dublin 22, Ireland.

Ana Urmal, Infarmed IP, Parque de Saude Avenida do Brasil 53, 1749 004 Lisboa

Portugal

Ezra Mulugeta, Medical Products Agency, P.O. Box 26,SE-751 03 Uppsala, Sweden

Cornelius Fritsch and Sandrine Linder, Biologics Process R&D, Novartis Pharma AG, CH-4052

Basel, Switzerland

Chris Bird, Paula Dilger and Haiyan Jia, Cytokines and Growth Factors Section, Biotherapeutics

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

Stuart Dunn, Covance Laboratories Ltd, BioCMC, Otley Road, Harrogate HG3 1PY, UK.

Andrea López Barragán, Alvaro Alberti, Laboratorio control biológico, Laboratorios Clausen

S.A., Bv. Artigas 3896, Montevideo CP 11700, Uruguay

Guoping Wu, Bioassay, R&D Systems, Inc.614 McKinley Place NE, Minneapolis, MN 55413,USA

Danielle Paul, Murali Pasumarthy, Amgen Rhode Island, Building 7 Room 1320, 40 Technology

Way, West Greenwich RI 02817, USA

WHO/BS/2015.2257

Page 25

Appendix 2 - Table 1 - Summary of results from U937 assays

Sample Lab TNF-α

(IU/ml)

ED50 Potency relative to sample A

GM

Variance

Components

(as %GCV) GM

Variance

Components

(as %GCV)

Assay Plate Assay Plate

A 1 107.5 1213 9.1 2.1

A 2 40.87 2876 16.6 6.6

A 3 20.0 8693 4.7

A 4 151.5 930 0.0 5.6

A 5 ** 3540 11.3 7.9

A 6 ** 4853 0.0 17.1

A 7 64.5 2201 3.6 6.7

A 8 ** 3805 15.1 5.0

A 9 37.5 3279 0.0 13.5

A 10 53.8 2246 9.7 6.1

A 11 ** 1075 8.6 5.9

A 12 ** 4224 12.5 18.3

B 1 107.5 1143 6.2 1.7 0.94 2.5 2.2

B 2 40.87 2669 9.2 2.6 0.93 6.3 7.2

B 3 20.0 7224 12.2

0.83 9.0

B 4 151.5 843 0.0 5.2 0.91 0.0 8.7

B 5 ** 3367 0.0 8.4 0.95 7.9 7.1

B 6 ** 4590 6.4 6.3 0.95 0.0 14.0

B 7 64.5 2121 5.1 6.2 0.96 0.0 8.3

B 8 ** 3685 13.6 8.2 0.97 3.5 9.1

B 9 37.5 2875 9.4 12.4 0.88 0.0 13.8

B 10 53.8 2113 14.6 1.6 0.94 2.5 4.1

B 11 ** 1113 1.9 5.9 1.04 3.9 10.1

B 12 ** 3992 13.8 17.0 0.95 10.4 12.3

C 1 107.5 1144 2.9 0.8 0.94 5.7 2.7

C 2 40.87 2660 10.3 5.1 0.93 2.8 8.8

C 3 20.0 7107 10.1

0.82 7.4

C 4 151.5 848 1.3 3.9 0.91 0.0 7.9

C 5 ** 3325 3.9 6.7 0.94 4.4 8.3

C 6 ** 4688 0.0 11.4 0.97 0.0 10.3

C 7 64.5 2131 2.0 4.4 0.97 4.2 7.1

C 8 ** 3687 7.5 7.4 0.97 4.7 7.1

C 9 37.5 3103 11.7 13.0 0.95 0.0 17.7

C 10 53.8 2115 17.4 4.6 0.94 6.0 2.5

C 11 ** 1037 2.3 5.3 0.96 3.9 11.5

C 12 ** 3920 1.4 8.8 0.93 9.0 19.2

WHO/BS/2015.2257

Page 26

Appendix 2 - Table 1 - Summary of results from U937 assays

Sample Lab TNF-α

(IU/ml)

ED50 Potency relative to sample A

GM

Variance

Components

(as %GCV) GM

Variance

Components

(as %GCV)

Assay Plate Assay Plate

D 1 107.5 1228 1.7 2.1 1.01 10.4 2.8

D 2 40.87 2797 6.6 3.5 0.97 8.1 8.7

D 3 20.0 8113 3.9

0.93 0.8

D 4 151.5 922 0.0 8.7 0.99 0.0 11.1

D 5 ** 3990 18.6 15.5 1.13 19.1 15.8

D 6 ** 5037 0.0 19.5 1.04 0.0 5.4

D 7 64.5 2400 7.5 5.1 1.09 3.9 9.1

D 8 ** 4012 12.2 9.9 1.05 0.0 13.1

D 9 37.5 3161 17.0 12.3 0.97 6.8 17.7

D 10 53.8 2030 16.8 5.5 0.91 6.4 2.5

D 11 ** 1066 0.0 3.7 0.99 9.0 5.4

D 12 ** 3939 5.8 11.9 0.93 17.3 8.9

** information not disclosed

WHO/BS/2015.2257

Page 27

Appendix 2 - Table 2 - Summary of results from L929 assays

Sample Lab TNF-α

(IU/ml)

ED50 Potency relative to sample A

GM

Variance

Components

(as %GCV) GM

Variance

Components

(as %GCV)

Assay Plate Assay Plate

A 8 20 4667 17.7 2.5

A 9 10 4320 13.2 6.2

A 13 20 5534 16.8 11.5

A 14 10 5023 16.4

A 15 10 4398 7.5 11.4

A 16 86 1026 9.8 6.6

A 17 25 2438 19.0 9.1

A 18 20 6126 0.0 31.9

A 19 20 3492 16.7 6.5

A 20 10 4873 7.7 8.4

A 21 20 3392 9.4 9.2

A 22 15 4651 10.1 3.5

B 8 20 4458 25.3 8.6 0.95 6.0 8.9

B 9 10 3904 17.0 8.7 0.90 2.4 6.7

B 13 20 4693 0.0 24.4 0.85 7.0 23.3

B 14 10 4848 11.9

0.97 4.3

B 15 10 3886 0.0 11.1 0.88 6.3 3.9

B 16 86 1141 2.4 9.2 1.11 12.6 6.1

B 17 25 2102 28.2 9.1 0.86 4.6 11.8

B 18 20 5236 0.0 37.1 0.85 8.9 23.8

B 19 20 3294 8.7 11.1 0.94 12.4 7.8

B 20 10 4422 8.0 4.0 0.91 7.2 5.3

B 21 20 3392 0.0 10.1 1.00 0.0 11.3

B 22 15 4185 12.4 4.4 0.90 1.8 1.9

C 8 20 4285 26.6 1.9 0.92 6.9 4.0

C 9 10 4299 15.6 12.1 1.00 0.0 13.7

C 13 20 5399 26.0 14.1 0.98 16.3 9.7

C 14 10 4836 18.7

0.96 2.1

C 15 10 4153 0.0 8.2 0.94 5.8 6.0

C 16 86 1264 12.9 4.4 1.23 23.8 5.2

C 17 25 2225 25.0 11.2 0.91 0.0 14.4

C 18 20 5142 0.0 33.0 0.84 4.4 20.7

C 19 20 3242 15.6 5.3 0.93 7.3 5.1

C 20 10 4131 0.0 16.9 0.85 0.0 15.5

C 21 20 3374 3.9 13.4 0.99 0.0 10.6

C 22 15 4205 20.9 7.4 0.90 10.9 5.7

WHO/BS/2015.2257

Page 28

Appendix 2

Table 2 - Summary of results from L929 assays

Sample Lab TNF-α

(IU/ml)

ED50 Potency relative to sample A

GM

Variance

Components

(as %GCV) GM

Variance

Components

(as %GCV)

Assay Plate Assay Plate

D 8 20 4793 24.9 5.7 1.03 4.7 5.2

D 9 10 4196 17.5 4.0 0.97 5.3 7.6

D 13 20 5829 26.8 16.8 1.05 13.0 20.6

D 14 10 5174 15.9

1.03 2.6

D 15 10 4514 2.9 8.8 1.03 4.0 4.2

D 16 86 1322 18.5 11.7 1.32 33.7 7.0

D 17 25 2431 23.4 16.8 1.00 0.0 14.7

D 18 20 5253 12.7 21.7 0.86 11.5 14.1

D 19 20 3539 17.6 7.3 1.01 7.1 3.4

D 20 10 4599 11.8 7.3 0.94 9.2 5.1

D 21 20 3336 7.8 7.1 0.98 0.0 10.3

D 22 15 4674 16.8 5.4 1.00 10.5 3.8

WHO/BS/2015.2257

Page 29

Appendix 2 - Table 3 - Summary of results from other cytotoxicity assays

Sample Lab TNF-α

(IU/ml)

ED50 Potency relative to sample A

GM

Variance

Components

(as %GCV) GM

Variance

Components

(as %GCV)

Assay Plate Assay Plate

A 8 4.3 3025 154.5 13.2

A 23 5.0 10483 34.0 40.8

A 24 2.5 13560 9.6 8.9

A 25 1.505 5920 0.0 88.4

A 26 100 1166 0.5 3.6

B 8 4.3 3110 144.7 12.2 1.03 0.0 11.0

B 23 5.0 7715 43.1 47.4 0.74 0.0 22.4

B 24 2.5 12785 8.4 16.2 0.94 0.0 10.5

B 25 1.505 6011 0.0 96.7 1.02 6.0 6.4

B 26 100 1107 2.5 2.2 0.95 0.0 4.3

C 8 4.3 2958 153.0 9.3 0.98 0.0 6.6

C 23 5.0 8980 40.7 31.2 0.86 0.0 29.2

C 24 2.5 12482 18.6 6.3 0.92 5.6 10.1

C 25 1.505 6149 0.0 87.6 1.04 1.5 7.5

C 26 100 1101 3.4 2.5 0.94 2.4 4.5

D 8 4.3 3062 157.5 9.8 1.01 6.2 8.5

D 23 5.0 8984 46.4 32.9 0.86 0.0 23.0

D 24 2.5 13500 12.3 6.9 1.00 3.9 5.9

D 25 1.505 6339 0.0 90.0 1.07 4.5 7.2

D 26 100 1174 1.9 1.4 1.01 0.0 3.0

WHO/BS/2015.2257

Page 30

Appendix 2 - Table 4 - Summary of results from reporter gene assays

Sample Lab TNF-α

ED50 Potency relative to sample A

GM

Variance

Components

(as %GCV) GM

Variance

Components

(as %GCV)

Assay Plate Assay Plate

A 8 40 IU/ml 4213 4.2 0.7

A 27 172 IU/ml 846 2.6

B 8 40 IU/ml 3979 8.1 3.4 0.94 4.2 3.4

B 27 172 IU/ml 767 3.8

0.91 6.0

C 8 40 IU/ml 4026 2.7 3.2 0.96 0.0 3.0

C 27 172 IU/ml 775 4.1

0.92 4.8

D 8 40 IU/ml 4409 4.8 3.9 1.05 0.0 14.9

D 27 172 IU/ml 856 1.6

1.01 3.7

Appendix 2 - Table 5 - Summary of results from binding assays

Sample Lab TNF-α

ED50 Potency relative to sample A

GM

Variance

Components

(as %GCV) GM

Variance

Components

(as %GCV)

Assay Plate Assay Plate

A 17 25 ng/well 287 0.0 9.3

A 21 15 ng/well 1287 29.2 4.5

B 17 25 ng/well 247 0.0 11.9 0.86 0.0 10.9

B 21 15 ng/well 1243 29.1 5.9 0.97 0.0 7.3

C 17 25 ng/well 237 6.7 6.7 0.82 0.9 7.6

C 21 15 ng/well 1233 27.6 5.9 0.96 3.9 5.4

D 17 25 ng/well 254 2.8 5.8 0.88 0.0 14.9

D 21 15 ng/well 1339 23.2 7.4 1.04 0.0 9.8

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Appendix 3

WHO COLLABORATIVE STUDY FOR 1ST International Standard (IS) for Human TNF Receptor type II Fusion protein (TNFR II-Fc, Etanercept)

Study Protocol – 14 Nov’14

Project leader : Meenu Wadhwa

1. AIMS OF THE STUDY

To assess the suitability of ampouled preparations of human TNF Receptor type II

Fusion protein (TNFR II-Fc, Etanercept) to serve as 1st WHO IS for the bioassay of

human TNFR II-Fc by assaying their biological activity in a range of bioassays.

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

bioassays, immunoassays etc) in current use for these materials, and to determine, if

possible, concentrations of TNFR II-Fc required to neutralise specific amounts of

TNF- α IS.

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, B, C, D (5 ampoules for each preparation)

for testing in TNFR II-Fc bioassays. Each sample contains approximately 5 µg

of TNFR II-Fc.

4 ampoules of the current IS for TNF-alpha (12/154), containing 43,000 IU of

TNF-α.

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 D should be reconstituted with 1ml of sterile distilled

water. Allow contents to dissolve prior to use.

Reconstitute the IS for TNF-α coded 12/154 with 1ml of sterile distilled water.

Allow the contents to dissolve prior to use. This solution contains TNF-α at a

concentration of 43,000 International Units/ml. Use carrier protein (e.g. assay

medium containing foetal bovine serum) where extensive dilution is required.

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4. ASSAY STRUCTURE

Cell based assays: These are based on the inhibitory action of TNFR II-Fc on the

biological activity of TNF- α. To achieve the aims of the study participants are requested to

use a fixed dose of the TNF- α IS. The concentration used should provide a biological

response similar to the dose of TNF- α currently used for in house cell-based assays for

Etanercept.

For L929 assays – we suggest use of a final dose of 10 or 20 IU/ml of TNF- α

IS (please contact us for further guidance if you feel neither of the doses would

provide suitable data)

For U937 assays –we suggest using a suitable working dilution of TNF-α IS

which will provide a final dose corresponding to the dose of the TNF- α

reagent used routinely in your in-house assay

a) Use a freshly reconstituted ampoule of each preparation, A to D in each of the

assays. An assay is considered independent if the assay is carried out on different days/occasions.

b) Participants are asked to perform a pilot assay for each assay method used, to ensure that all preparations (A to D) are diluted such that the concentration range falls within the working range of the assay.

c) If the pilot assay (step b above) provides suitable dose response curves and an

adequate signal above background ratio, perform at least 3 independent assays for each of the preparations A to D (and in-house standard where available) using the most appropriate dilutions derived from the pilot assay for the different preparations tested.

d) Participants are requested to include dilution series for each preparation in duplicate

in each assay. A suggested assay layout using 3 plates is attached. It is important to ensure that each plate includes a dose response curve of the samples A-D.

e) Include appropriate control wells in the assays. For bioassays, these are blank

control wells (cells with culture medium but no TNF-α) and also wells containing cells with TNF-α only, at the fixed concentration used for the assay.

Binding assays: For performing these, follow each of the steps a-e as above. However, the plate layout can be amended if needed. Please note that the IS for TNF-α 12/154, contains 0.6% Human serum albumin as an excipient and therefore is unsuitable for use in binding assays; please use TNF-α from a commercial supplier.

5. INFORMATION TO BE SUPPLIED AND PRESENTATION OF RESULTS We have provided an Excel template (separate excel file) for returning bioassay data for

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the samples tested. For binding assays, this will need to be amended as per plate layout used for the assay Please use assay information sheets to give brief information regarding your

1) Assay, and how it was performed

2) In-house TNFR II-Fc standard

3) TNF-α preparation

4) Dilutions used – stock solutions and final dilutions in assay

We have included example layouts (A & B) for microtitre plate format data sheets at the end of this protocol for illustrating assay format, dilutions and results.

Please PROVIDE ALL RAW DATA (microtitre plate readouts e.g. O.D., Luminescence Units etc) as direct analysis of the raw data provided by the assays permits data from all participants to be handled consistently, as far as possible.

We request participants to follow the examples 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.

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 potencies of each preparation using their own in-house methods relative to their in-house standard. Please provide information of all methods used for calculating results.

7. DATA SUBMISSION Please provide all information requested as this is needed for compilation of the study report following instructions given in Sections 5 and 6 by using the provided data reporting sheets and Excel file Deadline for data submission – 16 January ’15 at the latest.

8. REPORTING OF RESULTS A draft report will be prepared and circulated to all participants for comment prior to submission to the Expert Committee on Biological Standardization of WHO. In the report, participating laboratories will be identified by a laboratory number only and any request to treat information in confidence will be respected. For submission of study results and for any further information please email: Dr Meenu Wadhwa Tel: +44 (0)1707 641472 Email: Meenu.Wadhwa@nibsc.org

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NOTE: Participants in the collaborative study are asked to note that

1. they participate in the study 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.

2. their results may be shared anonymously with non-for-profit public health bodies in the interests of global harmonisation.

WHO Collaborative Study for Human TNF Receptor type II Fusion protein (TNFR II-Fc, Etanercept)

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 _________

5. If units, please provide information on how they were derived

___________________________________________________________

___________________________________________________________

__

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WHO Collaborative Study for Human TNF Receptor type II Fusion protein (TNFR II-Fc, Etanercept)

Assay methodology information Briefly outline the assay methods used (provide full protocol on separate sheets if available): Cell-based assay Please report details of the cell line, source, seeding density and passage number of cells used in each assay, if applicable: Please report details of any pretreatment of the cells before the assay (eg. removal of growth factors): Details of the ampoule reconstitution, dilution steps and dilution buffer used:

TNF IS, A, B, C, D, In-house standard Please also provide details of any blanks if applicable: Dose range of Etanercept used in the assay and the plate layout (separately). Concentration of TNFα used in the assay: Indicate the concentration of the initial working solution of TNF-α, the concentration used in neutralization of etanercept samples, and also the final concentration in the assay after addition of the cells as appropriate for the assay. For example in some assays - TNF- α working solution = X IU/ml; diluted 1:1 with etanercept samples = X/2 IU/ml; further diluted 1:1 with cells = X/4 IU/ml. Length of incubation of the cells with Etanercept +TNFα: Report details of the readout of the assay (e.g Absorbance, Luminescence) and the equipment used to obtain this readout:

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Additional comments: Binding assay Details of the ampoule reconstitution, dilution steps and dilution buffer used:

TNF IS, A, B, C, D, In-house standard Please also provide details of any blanks if applicable: Dose range of Etanercept used in the assay and the plate layout (separately). Concentration and Source/Supplier of TNFα used in the assay Please report details of the readout of the assay (e.g Absorbance, Luminescence) and the equipment used to obtain this readout: Additional comments

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Example Layout A

WHO Collaborative Study for Human TNF Receptor type II Fusion protein (TNFR II-Fc, Etanercept)

Plate 1. Sample Layout:

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

A blank IH IH A A B B C C D D TNFα

B blank IH IH A A B B C C D D TNFα

C blank IH IH A A B B C C D D TNFα

D blank IH IH A A B B C C D D TNFα

E blank IH IH A A B B C C D D TNFα

F blank IH IH A A B B C C D D TNFα

G blank IH IH A A B B C C D D TNFα

H blank IH IH A A B B C C D D TNFα

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

IH A

B

C D

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 TNFα

B blank TNFα

C blank TNFα

D blank TNFα

E blank TNFα

F blank TNFα

G blank TNFα

H blank TNFα

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

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

A

B

C

D

E

F

G

H

IH=In-house Standard; Blank=Cells only Control Wells; TNFα= cells+TNFα Control Wells

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Example Layout A

Plate 2. Sample Layout:

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

B C

D

IH

A

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

Response e.g. OD / LUMINESCENCE

IH=In-house Standard; Blank=Cells only Control Wells; TNFα= cells+TNFα Control Wells

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

A blank B B C C D D IH IH A A TNFα

B blank B B C C D D IH IH A A TNFα

C blank B B C C D D IH IH A A TNFα

D blank B B C C D D IH IH A A TNFα

E blank B B C C D D IH IH A A TNFα

F blank B B C C D D IH IH A A TNFα

G blank B B C C D D IH IH A A TNFα

H blank B B C C D D IH IH A A TNFα

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

A blank TNFα

B blank TNFα

C blank TNFα

D blank TNFα

E blank TNFα

F blank TNFα

G blank TNFα

H blank TNFα

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

A B C D E F G H

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Example Layout A

Plate 3. Sample Layout:

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

C D

IH

A

B

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

Response e.g. OD / LUMINESCENCE

IH=In-house Standard; Blank=Cells only Control Wells; TNFα= cells+TNFα Control Wells

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

A blank C C D D IH IH A A B B TNFα

B blank C C D D IH IH A A B B TNFα

C blank C C D D IH IH A A B B TNFα

D blank C C D D IH IH A A B B TNFα

E blank C C D D IH IH A A B B TNFα

F blank C C D D IH IH A A B B TNFα

G blank C C D D IH IH A A B B TNFα

H blank C C D D IH IH A A B B TNFα

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

A blank TNFα

B blank TNFα

C blank TNFα

D blank TNFα

E blank TNFα

F blank TNFα

G blank TNFα

H blank TNFα

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

A B C D E F G H

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Example Layout B

WHO Collaborative Study for Human TNF Receptor type II Fusion protein (TNFR II-Fc, Etanercept)

Plate 1. Sample Layout:

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

A blank A A A A A A A A A A A

B blank A A A A A A A A A A A

C blank B B B B B B B B B B B

D blank B B B B B B B B B B B

E TNFα C C C C C C C C C C C

F TNFα C C C C C C C C C C C

G TNFα D D D D D D D D D D D

H TNFα D D D D D D D D D D D

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

A B

C

D

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

B blank

C blank

D blank

E TNFα

F TNFα

G TNFα

H TNFα

Response e.g. OD / LUMINESCENCE

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

A

B

C

D

E

F

G

H

Blank=Cells only Control Wells; TNFα= cells+TNFα Control Wells

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Example Layout B

Plate 2. Sample Layout:

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

B C

D

A

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

Response e.g. OD / LUMINESCENCE

Blank=Cells only Control Wells; TNFα= cells+TNFα Control Wells

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

A blank B B B B B B B B B B B

B blank B B B B B B B B B B B

C blank C C C C C C C C C C C

D blank C C C C C C C C C C C

E TNFα D D D D D D D D D D D

F TNFα D D D D D D D D D D D

G TNFα A A A A A A A A A A A

H TNFα A A A A A A A A A A A

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

A blank

B blank

C blank

D blank

E TNFα

F TNFα

G TNFα

H TNFα

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

A B C D E F G H

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Example Layout B Plate 3. Sample Layout:

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

C D

A

B

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

Response e.g. OD / LUMINESCENCE

Blank=Cells only Control Wells; TNFα= cells+TNFα Control Wells

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

A blank C C C C C C C C C C C

B blank C C C C C C C C C C C

C blank D D D D D D D D D D D

D blank D D D D D D D D D D D

E TNFα A A A A A A A A A A A

F TNFα A A A A A A A A A A A

G TNFα B B B B B B B B B B B

H TNFα B B B B B B B B B B B

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

A blank

B blank

C blank

D blank

E TNFα

F TNFα

G TNFα

H TNFα

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

A B C D E F G H

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Example Layout B

Plate 4. Sample Layout:

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

IH A

B

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

Response e.g. OD / LUMINESCENCE

*IH=In-house Standard; Blank=Cells only Control Wells; TNFα= cells+TNFα Control Wells

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

A blank IH IH IH IH IH IH IH IH IH IH IH

B blank IH IH IH IH IH IH IH IH IH IH IH

C blank A A A A A A A A A A A

D blank A A A A A A A A A A A

E TNFα IH IH IH IH IH IH IH IH IH IH IH

F TNFα IH IH IH IH IH IH IH IH IH IH IH

G TNFα B B B B B B B B B B B

H TNFα B B B B B B B B B B B

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

A blank

B blank

C blank

D blank

E TNFα

F TNFα

G TNFα

H TNFα

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

A B C D E F G H

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Appendix 4 – Instructions for Use

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