WHO/BS/2013.2219 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 21 to 25 October 2013
Report on a Collaborative study for proposed 3
rdInternational standard for Tumor Necrosis Factor -alpha (TNF-)
Meenu Wadhwa1, 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: [email protected]
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: [email protected].
© World Health Organization 2013
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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.
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
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.
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
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%).
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.
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).
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
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.
References
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The 2nd International Standard for human tumour necrosis factor alpha WHO/BS/03.1981 WHO Technical Report Series 927, 2005, pg 24
Finney DJ (1978) Statistical methods in biological assay. 3rd edition Charles Griffin. London.
Duncan DB (1975) T-tests and intervals for comparisons suggested by the data. Biometrics 31, 339-359
Kirkwood TBL & Tydeman MS (1984) Design and analysis of accelerated degradation tests for the stability of biological standards II. A flexible computer program for data analysis. J Biol Standardisation 12; 207-14.
CombiStats v5.0, EDQM – Council of Europe, www.combistats.eu.
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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)
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
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
Table 4: Potencies of sample A relative to IS 88/786 (in IU/ml)
Laboratory Laboratory GM1 n1 GCV1 Laboratory GM2 n2 GCV2
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
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
Table 6: Potencies of sample C relative to IS 88/786 (in IU/ml)
Laboratory Laboratory GM1 n1 GCV1 Laboratory GM2 n2 GCV2
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
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
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
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
Table 9C: Potencies of sample C relative to IS 88/786 (in IU/ml)
Laboratory Laboratory GM1 n1 GCV1
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
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%
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
18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3b 3a 2 1b 1a 2.0
1.50 1.25
1.0
0.80 0.67
0.50
Laboratory
Ratio of Slopes
18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3b 3a 2 1b 1a 2.0
1.50 1.25
1.0
0.80 0.67
0.50
Laboratory
Ratio of Slopes
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
18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3b 3a 2 1b 1a 2.0
1.50 1.25
1.0
0.80 0.67
0.50
Laboratory
Ratio of Slopes
18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3b 3a 2 1b 1a 2.0
1.50 1.25
1.0
0.80 0.67
0.50
Laboratory
Ratio of Slopes
Figure 5: Laboratory mean potencies of samples A and C calculated relative to IS 88/786 (IU/ml)
Number 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 18 A
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
Figure 6: Laboratory mean potencies of sample B calculated relative to IS 88/786 (IU/ml)
Number 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
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
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
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.
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.
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:
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.
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 __________________________________________________________________
______________________________________________________
COLLABORATIVE STUDY FOR HUMAN TNF-ALPHA
Laboratory identification……
Assay information
Outline the assay methods used (provide full protocol on separate sheets if available):
