ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 15 to 19 October 2012
WHO International Collaborative Study of the proposed 3
rdInternational Standard for Erythropoietin, recombinant, for bioassay.
Chris. Burns*, Richard. Tiplady and Jason. Hockley National Institute for Biological Standards and Control,
Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK
*Corresponding Author Phone: +44 (0)1707 641247 Email: Chris.Burns@nibsc.hpa.org.uk
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 01 October 2012 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:
Quality Safety and Standards (QSS). Comments may also be submitted electronically to the Responsible Officer: Dr Jongwon Kim at email: kimjon@who.int
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Summary
The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) has recognized (2010) the need for a replacement International Standard for Erythropoietin (EPO) for the assignment of potency to therapeutic preparations of recombinant human EPO used in the treatment of anaemia.
We report here the characterization of a candidate standard for EPO in an International
Collaborative Study carried out by fifteen laboratories in seven countries, and a comparison by bioassay with the existing International Standard coded 88/574.
The mean estimate of the EPO content of the candidate International Standard, coded 11/170, is 1648 IU per ampoule (95% confidence limits 1562 – 1738) and it is proposed that it be
established as the Third International Standard for Erythropoietin, recombinant, for bioassay, with an assigned content of 1650 IU per ampoule.
Introduction
Erythropoietin (EPO) is a glycoprotein hormone produced in the kidneys which plays a major role in the regulation of red blood cell production. Recombinant preparations of EPO are widely used as therapeutic products in the treatment of anaemia. The second International Standard (IS) for EPO, recombinant, for bioassay, in ampoules coded 88/574, was established in 2003 and has been widely used for the calibration of therapeutic preparations of EPO by in vivo bioassay.
Stocks of this standard are now exhausted and since the IS defines the International Unit (IU) for EPO activity and is essential for the correct potency labelling of therapeutic EPO products, there is a requirement for a replacement standard.
With this in mind, a new preparation of EPO was filled into ampoules (NIBSC code 11/170), following procedures recommended by WHO (1) and an international collaborative study was set up with expert laboratories to aid in the value assignment of the proposed IS.
The pressing requirement for a replacement IS meant that there was insufficient time, prior to the start of the collaborative study, to complete a programme of accelerated degradation on the candidate standard to determine its long term stability. As a result, the study was conducted in two phases with the following three primary aims:
Phase 1 – all participants
1) To calibrate the candidate standard (11/170) relative to the 2nd IS for EPO, recombinant, for bioassay (88/574) by in vivo bioassays.
2) To assess the suitability of the candidate preparation 11/170 to serve as the 3rd International Standard for the calibration of therapeutic preparations of recombinant EPO by in vivo bioassay.
Phase 2 – selected participants
3) To determine the stability of the preparation 11/170 by comparison with ampoules stored at elevated temperatures as part of an accelerated degradation stability study.
In addition to these primary aims, the study had the secondary aim of calibrating (in terms of the IS) a national EPO standard on behalf of NIFDC. Participants were therefore also invited to include this preparation, coded 11/172, in the assays they contributed to the collaborative study, where resources permitted. Ensuring the correct calibration of national “secondary” reference materials is critical in ensuring the quality of therapeutic preparations of EPO. The in vivo nature of these calibration exercises means that every effort should be made to reduce the number of assays performed to a minimum, hence the request for inclusion of this standard in the study.
Participants
Fifteen laboratories in seven countries took part in the study and are listed alphabetically, by country, in Table 1. Throughout the study each participating laboratory is referred to by a code number. These code numbers were randomly assigned and do not reflect the order of listing.
Table 1: List of participants.
Dr. Kevin Grant, Department of Health and Ageing, Therapeutic Goods Administration, P.O.
Box 100 Woden, ACT 2606, AUSTRALIA.
Dr. Martin Schiestl and Dr. Jana Hribar, Sandoz Biopharmaceuticals, Biochemiestrasse 10, A- 6250 Kundl / Tirol, AUSTRIA
Dr. Sergio Luiz Dalmora, Department of Industrial Pharmacy, Federal University of Santa Maria, 97.105-900.Santa Maria, RS, BRAZIL.
Dr. A.Winkler, LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Redderweg 8, 21147 Hamburg, GERMANY.
Dr. Sven Michael Cords, Bioassay GmbH, Im Neuenheimer Feld 515, D-69120, Heidelberg, GERMANY.
Dr. Gerd Zimmerman, Roche Diagnostics GmbH, Sandhofer Strasse 116, 68305 Mannheim GERMANY.
Dr. Yingguo Long, Beijing Four Rings Bio-Pharmaceutical Co., Ltd. No. 5 Jian’an Street BDA Beijing P.R. CHINA.
Dr. Zhou Yong, National Institutes for Food and Drug Control, 2#Tiantan Xili, Beijing, P.R.CHINA.
Dr. Ding Mansheng, Shanghai Chemo Wanbang Biopharma Co., Ltd. No.1289 Yishan Road, Shanghai, P.R. CHINA.
Dr. Jiaoe Zhang, Shenyang Sunshine Pharmaceutical Co., Ltd. 3A1, Road 10, Shenyang Economy ＆ Technology Development Zone, Shenyang, P.R. CHINA.
Dr. Zhang Xiangrong, Shenzhen Sciprogen Bio-Pharmaceutical Co., Ltd. YaYuan Rd, Bantian Petrochemical Industry Zone, Longgang District, ShenZhen, P.R. CHINA.
Dr. Yang Meihua, Xiamen Amoytop Biotech Co., Ltd., No.330 Wengjiao Road, Xinyang Industry Zone, Haicang, Xiamen, Fujian, P.R. CHINA.
Mr Richard Tiplady, NIBSC, Biotherapeutics, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, UK.
Dr Graham Molineux, AMGEN Inc, Mailstop 15-2-A, One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
Dr. Joe Albanese and Dr. Harry Walker, Johnson & Johnson, 930W Rt 202 South Raritan, New Jersey, 08869, USA.
Materials
Bulk materials and preparation of ampoules of EPO.
For the candidate International Standard, a bulk preparation of highly purified, recombinant human EPO alpha was generously donated to the WHO by a manufacturer of therapeutic EPO.
The bulk preparation was provided as a solution and was combined and formulated with human serum albumin (0.2% w/v; tested and found to be negative for HBsAg, anti-HIV, HCV NAT), trehalose (1% w/v) and NaCl (0.12% w/v), dispensed in 0.5 ml aliquots into glass ampoules on the 18th August 2011, lyophilised and sealed on the 22nd August 2011. The candidate National Standard was prepared in an identical manner, following a donation of EPO from NIFDC.
Ampoules containing EPO were lyophilised and sealed under nitrogen according to procedures described by WHO for International Biological Standards (1) and stored at -20C in the dark at NIBSC. For the candidate International Standard, a final total of 6,228 ampoules, each coded 11/170, were obtained, with a mean fill weight of 0.507g (CV 0.15%; n=692), a mean dry weight of 0.06g (CV 1.89%), a residual moisture content (Karl Fischer titration) of 0.99% (CV 52.38%) and a mean oxygen of 0.35% (CV 41.13%), determined using a non-destructive, Lighthouse laser
headspace analyser,
The materials for this study, which were identified only by code letter in the case of the
degradation samples, are listed in Table 2. Where appropriate, each participant was allocated a set of core preparations and a further selection of samples based on assay capacity and sample availability (some thermally accelerated degradation samples were only available in limited numbers and will be sent out to selected participants as part of phase 2 – the stability study, described above).
Table 2. Ampouled materials provided
Recombinant EPO Preparation Ampoule content Core samples
2nd International Standard for EPO,
recombinant, for bioassay (88/574) 120 IU per ampoule Candidate 3rd International Standard
(11/170) stored at -20ºC Assumed to be approximately 1500 IU per ampoule Other samples
Accelerated thermal degradation samples of 11/170 stored at +4°C, +20°C, +37°C and +45°C
Content assumed identical to 11/170 stored at -20ºC
Candidate NIFDC National Standard
(11/172) Assumed to be approximately 600IU per ampoule
Design of the study and assay methods contributed Bioassay of the candidate standards 11/170 and 11/172.
Participants were requested to carry out the in vivo bioassay method(s) for EPO normally in use in their laboratory, and where possible, to perform at least two independent assays, using fresh ampoules (not a stored aliquot) for each. It was also requested that each assay should include all of the preparations allocated, at preferably no less than three dose levels in the linear part of the dose-response curve in order to provide information on parallelism. In instances where there was not a fresh ampoule for subsequent assays, it was suggested that fresh dilutions were made from frozen stock solutions, and where this was the case, participants were requested to provide details of freeze-thaw steps.
The ampoule contents of the test preparations are listed in Table 2. On receipt, ampoules were to be stored at -20°C until use. It was recommended that the contents of each ampoule were
reconstituted in appropriate assay diluent (eg. phosphate-albumin buffered saline, pH 7.2) according to the protocol normally used. Participants were requested to make every effort to ensure that all of the ampoule contents were removed. It was recommended that appropriate dilutions should be made from this stock using assay diluent according to the assay protocol used.
Participants were asked to provide details of the assay methods used, including detail of the dilution steps made and all raw assay data in electronic excel spreadsheet format if possible for central computation at NIBSC. Participants’ own estimates of activity as calculated by the method normally used in their laboratory were also requested.
Assay methods contributed
Summaries of the methods used are given in Table 3. In the fifteen laboratories contributing data to the study, thirteen contributed normocythemic (NM) in vivo assays and two contributed
polycythemic (P) in vivo assays.
Table 3. Assay methods used.
Lab No. Assay type Comments
1 NM
European Pharmacopoeial method
2 P
European Pharmacopoeial method
3 NM
European Pharmacopoeial method
4 NM
European Pharmacopoeial method
5 NM
European Pharmacopoeial method
6 NM
European Pharmacopoeial method
7 P
European Pharmacopoeial method
8 NM Chinese Pharmacopoeial method with the exception that blood was sampled 96 hours post injection instead of 72 hours 9 NM Chinese Pharmacopoeial method with the exception that blood
was sampled 96 hours post injection instead of 72 hours
10 NM
European Pharmacopoeial method
11 NM
Chinese Pharmacopoeial method
12 NM
Chinese Pharmacopoeial method
13 NM
Chinese Pharmacopoeial method
14 NM
European Pharmacopoeial method
15 NM European Pharmacopoeial method with the exception that Acridine Orange was used instead of Thiazol Orange
Statistical analysis
An independent statistical analysis of all bioassay data was performed at NIBSC. Potency estimates for 11/170 and 11/172 were calculated relative to IS 88/574 by fitting a parallel-line model comparing assay response to log concentration (2). Assay validity was assessed by
analysis of variance with non-linearity and non-parallelism considered significant at the 1% level (p<0.01). Analysis has been performed using log10 of the assay response (reticulocyte count or reticulocyte percentage) in all laboratories except for laboratories 2 and 7, which performed polycythemic assays and recorded gamma counts in cpm/ml. An in-house program (3) was used to determine any outlier responses. Any outliers were omitted from the calculation of relative potency.
Laboratory means were calculated as weighted geometric means except in cases where the individual assay estimates were found to be heterogeneous (p<0.1 in χ2 test for homogeneity) and a semi-weighted geometric mean was calculated (4). 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).
Results
Data returned for analysis
Data were contributed by 15 laboratories. All laboratories except for laboratory 2 and laboratory 7 performed normocythemic assays. Laboratories 2 and 7 performed polycythemic assays. A total of 42 assays provided data for 11/170 and 39 assays provided data for 11/172. Mean potency estimates are summarised in Table 4, and Figures 1 and 2. Results from individual assays for both are given in Appendix 1 in Tables A1 and A3.
Assay validity
The majority of assays allowed statistically valid estimates of relative potency to be calculated, although one assay from laboratory 7 was excluded from further analysis due to significant non- linearity. This is indicated in the table of results. Tables showing the ratio of the slopes of the assayed test samples to the IS 88/574 are included in Appendix 1 in Tables A2 and A4 for information.
Potencies of 11/170 and 11/172 calculated relative to IS 88/574
Analysis gave geometric mean potency estimates of 1648 IU per ampoule (n=15; 95%
confidence limits 1562 - 1738; GCV 10.1%) for sample 11/170 and 628 IU per ampoule (n=15;
95% confidence limits 590 - 668; GCV 11.9%) for sample 11/172. An unpaired 2-tailed t-test of the laboratory means did not detect any significant difference between the potencies calculated using the normocythemic and the polycythemic assay methods.
Stability of 11/170
The pressing requirement for a replacement standard has meant that there has been insufficient time to complete an accelerated degradation study prior to submission of this report to ECBS.
Estimates of the potency of ampoules stored at elevated temperatures, for a period of 225 days, will be provided as an addendum to this report prior to its consideration for establishment at the ECBS meeting.
Conclusions and recommendations
Recombinant preparations of EPO are widely used as therapeutic products in the treatment of anaemia. International Standards for EPO have been widely used for the calibration of
therapeutic preparations of EPO by in vivo bioassay. Stocks of the current IS (2nd) are now exhausted and since the IS defines the International Unit (IU) for EPO activity and is essential for the correct potency labelling of therapeutic EPO products, there is a requirement for a replacement standard. This report describes a collaborative study to establish a replacement IS.
The 2nd IS consisted of a donation of recombinant human EPO from a manufacturer and this approach has been repeated for the proposed 3rd IS.
As expected for similar materials, analysis of the fitted slopes for the dose-response of the candidate standard 11/170 and the 2nd IS 88/574 allowed statistically valid estimates of relative potency to be calculated from all laboratories, demonstrating the requirement of a replacement international standard in terms of parallelism of assay response with the existing IS. The
geometric mean potency for the candidate standard was 1648 IU per ampoule (n=15; 95%
confidence limits 1562 - 1738; GCV 10.1%)
Analysis of the fitted slopes for the dose-response of the candidate NIFDC National Standard also allowed statistically valid estimates of relative potency to be calculated from all
laboratories. The geometric mean potency for the candidate NIFDC National Standard was 628 IU per ampoule (n=15; 95% confidence limits 590 - 668; GCV 11.9%).
Proposal
It is recommended that the preparation in ampoules coded 11/170 be established as the Third International Standard for Erythropoeitin, recombinant, for bioassay, with an assigned content of 1650 IU per ampoule.
Acknowledgements
We gratefully acknowledge the important contributions of all the participants and the Centre for Biological Reference Materials, NIBSC for the preparation of the ampouled materials.
References
1. WHO Technical Report Series No.800, (1990). 181-214.
2. Finney DJ. Statistical Method in Biological Assay. 3rd Edition. London: Charles Griffin 1978.
3. Gaines Das RE, Rice LR. (1985) SCAN, an exploratory program for preliminary analysis of bioassay and immunoassay data. Comput. Methods Programs Biomed. 21(1): 25-33.
4. Statistical analysis of results of biological assays and tests, general chapter 5.3. Ph. Eur. 7th edition. Strasbourg, France: Council of Europe; 2008.
Table 4: Laboratory mean potency estimates (in IU/ampoule), calculated relative to 88/574
Lab 11/170 11/172
1 1754 615
2 1691 574
3 1550 511
4 1760 639
5 1780 711
6 1553 531
7 1443 640
8 1964 708
9 1459 579
10 1589 739
11 1910 754
12 1550 614
13 1470 618
14 1602 612
15 1751 629
GM 1648 628
95% C.I. 1562 – 1738 590 – 668
GCV 10.1% 11.9%
n 15 15
Excluding Polycythemic assays (Laboratories 2 and 7) Laboratories
2 and 7
GM 1661 631
95% C.I. 1568 – 1761 588 – 678
GCV 10.1% 12.6%
n 13 13
Figure 1: Potency estimates for 11/170 (in IU/ampoule), calculated relative to 88/574
Number of Assays
0 2 4 6 8 10
Potency (IU/ml)
800 1600 3200
2 7 7 13
7 9 9 13
3 5 6 10 12 12 13 14
3 6 7 10 12 13
4 14 14 15
1 1 2 7 11
4 4 8 10 15
5 8 11
2 11
Figure 2: Potency estimates for 11/172 (in IU/ampoule), calculated relative to 88/574
Number of Assays
0 2 4 6 8 10
Potency (IU/ml)
300 600 1200
2 3
6 3 4
15 9 13 14
1 2 2 6 7 9 12 13 14
4 7 8 12 12 13
1 10 13
5 7 11
14 15
4 5 10
8 11 11
10
Appendix 1: Individual assay results
Table A1: Potency estimates of 11/170 (in IU/ampoule), calculated relative to IS (88/574)
Lab Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6 GCV
1 1744 1759* n/a
2 1322* 1757 2170 28.3%
3 1589 1515 n/a
4 1801 1855† 1682 5.2%
5 1514 2048 n/a
6 1607 1503 n/a
7 1369 1777† 1469* 1358 1581 NL(IS) 11.9%
8 1870† 2064 n/a
9 1468 1456 n/a
10 1869 1617 1482* 12.4%
11 2383 2056 1768 16.1%
12 1502 1537 1609 3.6%
13 1360 1509* 1409 1615 7.9%
14 1672 1529 1654 5.0%
15 1701 1799 n/a
NL(X) = Non-Linearity of Sample X with p < 0.01
* = Non-Linearity of at least one sample with 0.01 < p < 0.05
† = Non-Parallelism with 0.01 < p < 0.05
Table A2: Fitted slopes for 11/170 relative to IS (88/574)
Lab Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6
1 0.739 0.969*
2 0.983* 1.095 0.994
3 0.854 1.135
4 1.089 0.679† 0.904
5 1.214 1.212
6 1.186 1.507
7 1.113 0.624† 1.113* 0.998 1.267 NL(IS)
8 0.709† 0.954
9 1.182 1.332
10 0.880 1.901 1.061*
11 0.900 3.244 0.726
12 1.083 0.954 0.991
13 0.995 0.833* 0.720 0.860
14 1.197 0.846 1.111
15 0.909 0.822
* = Non-Linearity of Sample with 0.01 < p < 0.05
† = Non-Parallelism with 0.01 < p < 0.05 NL(X) = Non-Linearity of Sample X with p < 0.01
Table A3: Potency estimates of 11/172 (in IU/ampoule), calculated relative to IS (88/574)
Lab Assay 1 Assay 2 Assay 3 Assay 4 GCV
1 646 584 n/a
2 424 597 595 21.6%
3 499 527 n/a
4 761 635 530 19.8%
5 676 768 n/a
6 497† 604 n/a
7 601 627 680* 6.5%
8 793 633* n/a
9 611 562 n/a
10 824 662 775* 11.9%
11 697 782 795 7.4%
12 611 620 612 0.8%
13 559 597 671 622* 7.9%
14 590 575 736 14.5%
15 549 713 n/a
* = Non-Linearity of at least one sample with 0.01 < p < 0.05
† = Non-Parallelism with 0.01 < p < 0.05
Table A4: Fitted slopes for 11/172 relative to 88/574
Lab Assay 1 Assay 2 Assay 3 Assay 4
1 0.892 1.044
2 0.890 0.982 1.028
3 1.082 1.045
4 0.989 0.781 0.789
5 1.062 0.873
6 1.400† 1.204
7 0.951 1.022 1.036*
8 0.931 1.049*
9 1.187 1.300
10 1.120 1.881 0.806*
11 1.400 3.129 0.952
12 1.049 0.902 1.015
13 1.015 0.757 0.860 0.993*
14 0.902 0.869 1.007
15 0.838 0.902
* = Non-Linearity of at least one sample with 0.01 < p < 0.05
† = Non-Parallelism with 0.01 < p < 0.05
Appendix 2: Study protocol
WHO International Collaborative Study of the proposed 3
rdInternational Standard for Erythropoietin, recombinant, for bioassay.
Introduction
Erythropoietin (EPO) is a glycoprotein hormone produced in the kidneys which plays a major role in the regulation of red blood cell production. Recombinant preparations of EPO are widely used as therapeutic products in the treatment of anaemia.
The second International Standard (IS) for EPO, recombinant, for bioassay, in ampoules coded 88/574, was established in 2003 and has been widely used for the calibration of therapeutic preparations of EPO by in vivo bioassay. Stocks of this standard are now exhausted and since the IS defines the International Unit (IU) for EPO activity and is essential for the correct potency labelling of therapeutic EPO products, there is a requirement for a replacement standard.
With this in mind, a new preparation of EPO has been filled into ampoules (NIBSC code 11/170), following procedures recommended by WHO. It is now intended to set up an international
collaborative study with expert laboratories to aid in the value assignment of the proposed IS.
The pressing requirement for a replacement IS means that there will not be sufficient time, prior to the start of the collaborative study, to complete a programme of accelerated degradation on the candidate standard to determine its long term stability. As a result, the study will be conducted in two phases with the following three primary aims:
Phase 1 – all participants
1) To calibrate the candidate standard (11/170) relative to the 2nd IS for EPO, recombinant, for bioassay (88/574) by in vivo bioassays.
2) To assess the suitability of the candidate preparation 11/170 to serve as the 3rd International Standard for the calibration of therapeutic preparations of recombinant EPO by in vivo bioassay.
Phase 2 – selected participants
3) To determine the stability of the preparation 11/170 by comparison with ampoules stored at elevated temperatures as part of an accelerated degradation stability study.
In addition to these primary aims, the study has the secondary aim of calibrating (in terms of the IS) a national EPO standard on behalf of NIFDC. Participants will therefore also be invited to include this preparation in the assays they contribute to the collaborative study, where resources permit. Ensuring the correct calibration of national “secondary” reference materials is critical in ensuring the quality of therapeutic preparations of EPO. The in vivo nature of these calibration exercises means that every effort should be made to reduce the number of assays performed to a minimum, hence the request for inclusion of this standard in the study.
Materials
Preparations supplied to participants in collaborative study.
A bulk preparation of highly purified, recombinant human EPO alpha was generously donated to the WHO by a manufacturer of therapeutic EPO. The bulk preparation was provided as a
solution and was combined and formulated with human serum albumin (0.2% w/v; tested and found to be negative for HBsAg, anti-HIV, HCV NAT), trehalose (1% w/v) and NaCl (0.12%
w/v), dispensed in 0.5 ml aliquots into glass ampoules, lyophilised and sealed. The candidate National Standard was prepared in an identical manner, following a donation of EPO from NIFDC.
The materials for this study, which may be identified only by code letter in the case of the degradation samples, are listed in Table 1. Where appropriate, each participant will be allocated a set of core preparations and a further selection of samples based on assay capacity and sample availability (some thermally accelerated degradation samples are only available in limited numbers and will be sent out to selected participants as part of phase 2 – the stability study, described above).
Tests requested
Participants are requested to carry out the in vivo bioassay method(s) for EPO normally in use in their laboratory, and where possible, to perform at least two independent assays, using fresh ampoules (not a stored aliquot) for each. Each assay should include all of the preparations allocated, at preferably no less than three dose levels in the linear part of the dose-response curve in order to provide information on parallelism. In instances where there is not a fresh ampoule for subsequent assays, it is suggested that fresh dilutions are made from frozen stock solutions, and where this is the case, participants are requested to provide details of freeze-thaw steps.
The ampoule contents of the test preparations are listed in Table 1. On receipt, ampoules should be stored at -20°C until use. It is recommended that the contents of each ampoule are
reconstituted in appropriate assay diluent (eg. phosphate-albumin buffered saline, pH 7.2) according to the protocol normally used. Please make every effort to ensure that all of the
ampoule contents are removed. Appropriate dilutions should be made from this stock using assay diluent according to the assay protocol used.
Participants are asked to provide details of the assay methods used, including detail of the dilution steps made and all raw assay data in electronic excel spreadsheet format if possible for central computation at NIBSC. Participants’ own estimates of activity as calculated by the method normally used in their laboratory are also requested.
Table 1.
Recombinant EPO Preparation Ampoule content Core samples
2nd International Standard for EPO,
recombinant, for bioassay (88/574) 120 IU per ampoule Candidate 3rd International Standard
(11/170) stored at -20ºC Assumed to be approximately 1500 IU per ampoule
Other samples
Accelerated thermal degradation samples of 11/170 stored at +4°C, +20°C, +37°C and +45°C
Content assumed identical to 11/170 stored at -20ºC
Candidate NIFDC National Standard Assumed to be approximately 600IU per ampoule
Report
A preliminary report will be prepared and circulated to all participants for comment before 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 requests to treat information in confidence will be respected. For further information, please contact:
Dr. Chris Burns
Principal Scientist, Endocrinology, Biotherapeutics, NIBSC Tel: 01707 641247
Email: Chris.Burns@nibsc.hpa.org.uk
Appendix 3: Draft Instructions for use
3
rdWHO International Standard for Erythropoietin, recombinant, for bioassay.
NIBSC Code 11/170.
Instructions for use (November 2012, first version)
This material is not for in vitro diagnostic use
1. INTENDED USE
The second International Standard (IS) for Erythropoietin (EPO) in ampoules coded 88/574 has been widely used for the calibration of preparations of recombinant DNA-derived EPO by bioassay. Stocks of the 2nd IS are exhausted and the World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) has recognized (2010) the need for a replacement International Standard for EPO for the assignment of potency to therapeutic preparations of recombinant human EPO used in the treatment of anaemia.
A new preparation of recombinant EPO has been filled into ampoules (NIBSC Code 11/170) and has been characterized by in vivo bioassay in an international collaborative study with expert laboratories and was established as the 3rd International Standard at the 63rd meeting of the ECBS. This material replaces the 2nd IS.
2. CAUTION
This preparation is not for administration to humans.
The preparation contains material of human origin and either the final product, or the source materials from which it is derived, have been tested and found negative for HBsAg, anti-HIV and HCV RNA. As with all materials of biological origin, this preparation should be regarded as potentially hazardous to health. It should be used and discarded according to your own laboratory's safety procedures. Such safety procedures should include the wearing of protective gloves and avoiding the generation of aerosols. Care should be exercised in opening ampoules to avoid cuts.
3. UNITAGE
Each ampoule contains 1650 IU of EPO
4. CONTENTS
Country of origin of biological material: USA
Each ampoule contains the residue after freeze-drying of 0.5 ml of a solution that contained:
Recombinant human EPO approximately 11µg
Human serum albumin 0.2 % (w/v)
Trehalose 1.0 % (w/v)
NaCl 0.12 % (w/v)
5. STORAGE
Unopened ampoules should be stored at -20°C.
Please note: because of the inherent stability of lyophilized material, NIBSC may ship these materials at room temperature.
6. DIRECTIONS FOR OPENING
DIN ampoules have an ‘easy-open’ coloured stress point, where the narrow ampoule stem joins the wider ampoule body.
Tap the ampoule gently to collect the material at the bottom (labeled) end. Ensure that the disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the disposable ampoule breaker covering the ampoule stem between the thumb and first finger of the other hand. Apply a bending force to open the ampoule at the coloured stress point, primarily using the hand holding the plastic collar.
Care should be taken to avoid cuts and projectile glass fragments that might enter the eyes, for example, by the use of suitable gloves and an eye shield. Take care that no material is lost from the ampoule and no glass falls into the ampoule. Within the ampoule is dry nitrogen gas at slightly less than atmospheric pressure. A new disposable ampoule breaker is provided with each DIN ampoule.
7. USE OF THE MATERIAL
No attempt should be made to weigh out any portion of the freeze-dried material prior to reconstitution.
For practical purposes, each ampoule contains the same quantity of recombinant human EPO. The entire content of each ampoule should be completely dissolved in an accurately measured amount of buffer solution. The use of water to reconstitute ampoule contents is not recommended. The material has not been sterilized and the ampoules contain no bacteriostat.
COLLABORATIVE STUDY
The preparation was evaluated in a collaborative study in which fifteen laboratories in seven countries took part, organized with the following aims:
1) To calibrate the candidate preparation, 11/170 relative to the 2nd IS (88/574) for EPO by in vivo bioassays.
2) To determine the stability of the candidate preparation 11/170 by comparison with ampoules stored at elevated temperatures as part of an accelerated degradation stability study.
The geometric mean potency for the candidate standard was 1648 IU per ampoule (n=15;
95% confidence limits 1562 - 1738; GCV 10.1%)
The candidate preparation 11/170 is sufficiently stable to serve as an International Standard. Analysis of the thermally accelerated degradation samples in this study gave a predicted xx (dependent on the results of phase 2) loss of potency per year for EPO when stored at -20°C. This suggests that 11/170 is likely to be highly stable under long term storage at -20°C.
8. STABILITY
It is the policy of WHO not to assign an expiry date to their international reference materials. They remain valid with the assigned potency and status until withdrawn or amended.
Reference materials are held at NIBSC within assured, temperature-controlled storage facilities. Reference materials should be stored on receipt as indicated on the label. For information specific to a particular biological standard, contact the Technical Information Officer or, where known, the appropriate NIBSC scientist.
In addition, once reconstituted, diluted or aliquoted, users should determine the stability of the material according to their own method of preparation, storage and use. Users who have data supporting any deterioration in the characteristics of any reference preparation are encouraged to contact NIBSC.
9. REFERENCES
10. ACKNOWLEDGEMENTS
We gratefully acknowledge the important contributions of all the participants and the manufacturer of the therapeutic EPO for the kind donation of material.
11. FURTHER INFORMATION
Further information can be obtained as follows:
This material:
enquiries@nibsc.hpa.org.uk WHO Biological Standards:
http://www.who.int/biologicals/en/
JCTLM Higher order reference materials:
http://www.bipm.org/en/committees/jc/jctlm/
Derivation of International Units:
http://www.nibsc.ac.uk/products/biological_reference_materials/frequently_asked_questio ns/how_are_international_units.aspx
Ordering standards from NIBSC:
http://www.nibsc.ac.uk/products/ordering_information/frequently_asked_questions.aspx NIBSC Terms and Conditions:
http://www.nibsc.ac.uk/terms_and _conditions.aspx
12. CUSTOMER FEEDBACK
Customers are encouraged to provide feedback on the suitability or use of the material provided or other aspects of our service. Please send any comments to enquiries@nibsc.hpa.org.uk
13. CITATION
In all publications, including data sheets, in which this material is referenced, it is important that the preparation’s title, its status, the NIBSC code number and the name and address of NIBSC are cited and cited correctly.
14. MATERIAL SAFETY SHEET
Physical properties (at room temperature) Physical appearance Freeze dried powder
Fire hazard None
Chemical properties
Stable: Yes Corrosive: No
Hygroscopic: Yes Oxidising: No
Flammable: No Irritant: No
Other (specify) Contains material of human origin Handling: See caution, section 2
Toxicological properties
Effects of inhalation: Not established, avoid inhalation Effects of ingestion: Not established, avoid ingestion
Effects of skin absorption: Not established, avoid contact with skin Suggested First Aid
Inhalation Seek medical advice Ingestion Seek medical advice
Contact with eyes Wash with copious amounts of water. Seek medical advice.
Contact with skin Wash thoroughly with water.
Action on Spillage and Method of Disposal
Spillage of ampoule contents should be taken up with absorbent material wetted with an appropriate disinfectant. Rinse area with an appropriate disinfectant followed by water.
Absorbent materials used to treat spillage should be treated as biologically hazardous waste.
15. LIABILITY AND LOSS
Information provided by the Institute is given after the exercise of all reasonable care and skill in its compilation, preparation and issue, but it is provided without liability to the Recipient in its application and use.
It is the responsibility of the Recipient to determine the appropriateness of the standards or reference materials supplied by the Institute to the Recipient (“the Goods”) for the proposed application and ensure that it has the necessary technical skills to determine that
they are appropriate. Results obtained from the Goods are likely to be dependant on conditions of use by the Recipient and the variability of materials beyond the control of the Institute.
All warranties are excluded to the fullest extent permitted by law, including without limitation that the Goods are free from infectious agents or that the supply of Goods will not infringe any rights of any third party.
The Institute shall not be liable to the Recipient for any economic loss whether direct or indirect, which arise in connection with this agreement.
The total liability of the Institute in connection with this agreement, whether for negligence or breach of contract or otherwise, shall in no event exceed 120% of any price paid or payable by the Recipient for the supply of the Goods.
If any of the Goods supplied by the Institute should not prove to meet their specification when stored and used correctly (and provided that the Recipient has returned the Goods to the Institute together with written notification of the alleged defect within seven days of the time when the Recipient discovers or ought to have discovered the defect), the Institute shall either replace the Goods or, at its sole option, refund the handling charge provided that the performance of either one of the above options shall constitute an entire discharge of the Institute’s liability under this Condition.
16. INFORMATION FOR CUSTOMS USE ONLY
Country of origin for customs purposes*: United Kingdom
*Defined as the country where the goods have been produced and/or sufficiently processed to be classed as originating from the country of supply, for example a change of state such as freeze drying.
Net weight: 6 mg
Toxicity statement: Non-toxic
Veterinary certificate or other statement if applicable.
Attached: No
