WHO/BS/2012.2196 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 15 to 19 October 2012 WHO International Collaborative Study of the proposed 5th International Standard for human, urinary Follicle-Stimulating Hormone and human, urinary Luteinizing Hormone, for bioassay. Jackie Ferguson*, Jason Hockley, Richard Tiplady and Chris Burns National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts, EN6 3QG, UK *Corresponding author: Jackie Ferguson +44 (0) 1707 641000 Jackie.Ferguson@nibsc.hpa.org.uk or 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 © World Health Organization 2012 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site (http://www.who.int/about/licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.
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Summary The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) has recognized (2011) the need for a replacement International Standard for human urinary Folliclestimulating hormone (FSH) and urinary Luteinizing hormone (LH) for the assignment of potency to therapeutic preparations of human urinary FSH and urinary LH (menotrophin, human menopausal gonadotrophins) used in the treatment of infertility. We report here the characterization of a candidate standard for urinary FSH and urinary LH in an International Collaborative Study carried out by eleven laboratories in ten countries, and a comparison by bioassay with the existing International Standard coded 98/704. The mean estimate of the FSH bioactivity of the candidate standard, coded 10/286, is 183 IU per ampoule (95% confidence limits 165 - 202). The mean estimate of the LH bioactivity of the candidate standard, coded 10/286, is 177 IU per ampoule (95% confidence limits 159 - 197). It is proposed that it is established as the fifth International Standard for human urinary FSH and urinary LH with an assigned bioactivity of 183 IU FSH and 177 IU LH 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.
Introduction Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) are glycoprotein hormones, produced in the anterior pituitary gland, which play a major role in the regulation of reproductive processes and pubertal maturation. Human urinary FSH and urinary LH, known as menotrophin, is widely used as a therapeutic product to stimulate ovulation in women and to achieve controlled ovarian hyperstimulation as part of assisted reproductive technologies. It is also used to treat male infertility caused by hypogonadotropic hypogonadism. The fourth International Standard (IS) for human urinary FSH and urinary LH, in ampoules coded 98/704, was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2002 (1) and has been widely used for the calibration of preparations of human urinary FSH and urinary LH by bioassay. Menotrophin continues to be considered a costeffective alternative to recombinant products in women undergoing assisted reproductive technologies (2). The global requirement for such a standard is evidenced by the continued demand for the current standard and the continued manufacture of urinary FSH and urinary LH products. Stocks of the current IS, 98/704, are exhausted and there is an urgent requirement to replace the standard. A new preparation of human urinary FSH and urinary LH has been filled into ampoules (NIBSC Code 10/286), following procedures recommended by WHO (3) and an international collaborative study has been organized with expert laboratories to aid in the value assignment of the proposed 5th International Standard. The aims of this study were therefore:
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1) To calibrate the candidate standard,10/286, relative to the 4th IS, 98/704, for urinary FSH bioactivity and urinary LH bioactivity by in vivo bioassays 2) To assess the suitability of the candidate standard,10/286, to serve as the 5th IS for the calibration of therapeutic human urinary FSH and urinary LH products by bioassay. 3) To determine the stability of the candidate standard, 10/286, by comparison with ampoules stored at elevated temperatures as part of an accelerated degradation stability study.
Participants Eleven laboratories in ten 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. The code numbers were randomly assigned and do not reflect the order of listing.
Table 1: List of participants Dr Claudio Wolfenson, Instituto Massone, Arias 4431, 1430 Buenos Aires, ARGENTINA. Dr Kevin Grant and Dr Tursun Kerim, Therapeutic Goods Administration, P.O. Box 100 Woden, ACT 2606, AUSTRALIA. Dr Sergio Luiz Dalmora, Department of Industrial Pharmacy, Federal University of Santa Maria, 97.105-900.Santa Maria, RS, BRAZIL. Dr Jan Rohde, Minapharm Pharmaceuticals, El-Bardissi Street, 2T Takseem Assmaa Fahmy Street, Heliopolis, Cairo, EGYPT and Dr Sven-Michael Cords, Bioassay - Labor für biologische Analytik GmbH, Im Neuenheimer Feld 515, 69120 Heidelberg, GERMANY. Dr Gundel Hager and Marta Leis, Aurigon Life Science GmbH, Bahnhofstraße 9-15, D-82327, Tutzing, GERMANY. Dr A. Winkler, LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Redderweg 8, 21147 Hamburg, GERMANY. Dr Cinzia Ciampolillo, Merck Serono Ivrea – RBM S.p.A, Via Ribes 1, 10010 Colleretto Giacosa (TO), ITALY. Ms Yuan Zhang, National Institutes for Food and Drug Control, Pharmacology Division, Tiantan Xi Li 2#, Dongcheng District, Beijing, 100050, P.R. CHINA. Dr Tiziano Fossati, IBSA Institut Biochimique SA, Via Al Ponte 13, 6900 Massagno, SWITZERLAND. Mr Richard Tiplady, NIBSC, Biotherapeutics Department, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, UK. Dr Elizabeth Raike, Qualtech Laboratories, 104 Green Grove Road, Ocean, NJ 07712, USA.
Materials Bulk materials and preparation of ampoules of human urinary FSH and urinary LH. Bulk preparations of highly purified, human urinary FSH and urinary LH were generously donated to the WHO by Instituto Massone S.A., Argentina and IBSA Institut Biochimique S.A.,
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Switzerland. The bulk preparation received from Instituto Massone S.A., Argentina (Batch No. 3626365910) comprised of 0.133 g of purified FSH and LH that had been precipitated with alcohol and dried under vacuum, with reported bioactivities of 3018 IU/mg FSH and 3057 IU/mg LH. The preparation had been tested by the manufacturer and found negative for HBsAg, anti-HIV and HCV NAT.
The bulk preparation of purified FSH and LH received from IBSA Institut Biochimique S.A., Switzerland (Batch No. WHO 01/2010) comprised a frozen solution of 88.4 ml of 1.43 mg/ml protein with reported bioactivities of 3875 IU/ml FSH and 4587 IU/ml LH. The preparation had been tested by the manufacturer and found negative for HBsAg, anti-HIV and HCV NAT. Combined, 259.41 mg of the material was formulated with 0.2% (w/v) human plasma albumin (also tested and found to be negative for HBsAg, anti-HIV, anti-HCV and HCV NAT) and 0.5% (w/v) lactose and 1.0 ml was dispensed into glass ampoules (nominally, 58µg protein) on the 8th April 2011, lyophilised and sealed on the 12th April 2011. Ampoules containing human urinary FSH and urinary LH were lyophilized and sealed under nitrogen according to procedures described by WHO for International Biological Standards (3) and stored at -20°C in the dark at NIBSC. Ampoules were checked visually for ampoule integrity. A final total of 4247 ampoules of human urinary FSH and urinary LH, each coded 10/286, were obtained, with a mean fill weight of 1.0081 g (n = 166; CV 0.18%), a mean dry weight of 0.0082 g (CV 3.77%), a residual moisture content (Karl Fischer titration) of 2.84% (CV 22.12%) and a mean oxygen content of 0.21% (CV 53.96%), determined using a non-destructive, Lighthouse laser headspace analyser, The materials for this study, which may be identified only by code letter, 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).
Table 2: Preparations supplied to participants in collaborative study Ampoule Code Not coded Urinary FSH and urinary LH preparation 4th International Standard (98/704) Ampoule unitage and nominal content 70 IU FSH per ampoule and 72 IU LH per ampoule Nominally 58µg (assumed to be approximately 200 IU FSH per ampoule and 200 IU LH per ampoule) Content assumed identical to 10/286 stored at -20ºC
D
Candidate 5th International Standard (10/286) stored at -20°C
Accelerated thermal degradation (ATD) samples of 10/286 stored at A, F and B +20°C, +37°C and +45°C for 6 months
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Design of the study and assay methods contributed Bioassay of the candidate standard 10/286 Participants were requested to carry out the assay(s) normally in use in their laboratory and, where possible, to perform at least two independent assays, using fresh ampoules, each assay to include all of the preparations allocated. Handling instructions for the materials were included in the study protocol. In instances where there was not a fresh ampoule for subsequent assays, it was suggested that fresh dilutions be made from frozen stock solutions. Where dilutions of a stored stock solution were used, participants were asked to provide details of its storage and identification of the initial preparation. Participants were asked to provide details of the assay method used, including dilution steps, together with all raw assay data in the form of clearly annotated organ and body weights 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 Participants were requested to perform in vivo bioassays of FSH based on the bioassay described by Steelman and Pohley (1953) of augmentation of ovary weight in immature female rats (4) and/or in vivo bioassays of LH based on the bioassay described by Hell et al., (1964) of seminal vesicle weight gain in immature male rats (5). Participants were asked to follow protocols normally used in their laboratory. The assays contributed by each laboratory are listed in Table 3.
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Table 3: Assay methods used Lab. No. Assay type Comments
In vivo FSH bioassay 1 In vivo LH bioassay In vivo FSH bioassay 2 In vivo LH bioassay In vivo FSH bioassay 3 In vivo LH bioassay In vivo FSH bioassay 4 In vivo LH bioassay 5 6 In vivo LH bioassay In vivo FSH bioassay In vivo FSH bioassay 7 In vivo LH bioassay In vivo FSH bioassay 8 In vivo LH bioassay 9 In vivo FSH bioassay In vivo FSH bioassay 10 In vivo LH bioassay In vivo FSH bioassay 11 In vivo LH bioassay
Augmentation of ovary weight in immature female rats as per the US Pharmacopeia (6) Seminal vesicle weight gain in immature male rats as per the US Pharmacopeia (6) Augmentation of ovary weight in immature female rats as per the US Pharmacopeia (6) Seminal vesicle weight gain in immature male rats as per the US Pharmacopeia (6) Augmentation of ovary weight in immature female rats as per the British Pharmacopoeia (7) Seminal vesicle weight gain in immature male rats as per the British Pharmacopoeia (7) Augmentation of ovary weight in immature female rats as per the European Pharmacopoeia (8) Seminal vesicle weight gain in immature male rats as per the US Pharmacopeia (6) Seminal vesicle weight gain in immature male rats as per the Pharmacopoeia of the People’s Republic of China (9) Augmentation of ovary weight in immature female rats as per the European Pharmacopoeia (8) Augmentation of ovary weight in immature female rats as per the European Pharmacopoeia (8) Seminal vesicle weight gain in immature male rats as per the British Pharmacopoeia (7) Augmentation of ovary weight in immature female rats as per the British Pharmacopoeia (7) Seminal vesicle weight gain in immature male rats as per British Pharmacopoeia (7) Augmentation of ovary weight in immature female rats as per the European Pharmacopoeia (8) Augmentation of ovary weight in immature female rats as per the British Pharmacopoeia (7) Seminal vesicle weight gain in immature male rats as per British Pharmacopoeia (7) Augmentation of ovary weight in immature female rats as per the British Pharmacopoeia (7) Seminal vesicle weight gain in immature male rats as per British Pharmacopoeia (7)
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Statistical analysis An independent statistical analysis of all bioassay data was performed at NIBSC. Potency estimates for the candidate standard, 10/286, and the accelerated thermal degradation samples were calculated relative to IS 98/704 by fitting a parallel-line model comparing assay response to log concentration (10). 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(organ weight/body weight) as assay response in all laboratories except for laboratory 7, where no data on animal body weights was available, and using log10(organ weight) as assay response in all laboratories. An in-house program (11) was used to determine any outlier responses and assess homogeneity of variance across treatment groups. Any outliers were omitted from 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 (12). Overall means were calculated as the unweighted geometric mean of laboratory means. 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 potency estimates).
Results Data returned for analysis Data were contributed by 11 laboratories. Laboratory 7 provided data for organ weights only. All other laboratories provided data for both organ weights and body weights. A total of 23 assays were performed for FSH, giving 30 sets of results for sample D. A total of 19 assays were performed for LH, giving 26 sets of results for sample D. Mean potency estimates for 10/286 are summarised in Tables 4 and 5 and Figures 1 and 2 for FSH, and Tables 6 and 7 and Figures 3 and 4 for LH. Results from individual assays for both are given in Appendix Tables A1.1, A1.4, A1.7 and A1.10.
Assay validity Excluding Laboratory 7, significant heterogeneity of variance (p < 0.05 in Bartlett’s test) was found in 25/92 assays (27.2%) when using organ weight as assay response, 13/92 assays (14.1%) when using log10(organ weight) as assay response, in 31/92 assays (33.7%) when using (organ weight/body weight) as assay response and 11/92 assays (12.0%) when using log10(organ weight/body weight) as assay response, suggesting that log10(organ weight/body weight) and log10(organ weight) provide better agreement with the variance homogeneity required for parallel-line analysis. Further analysis was performed using these assay responses only. The majority of assays allowed statistically valid estimates of relative potency to be calculated, although some samples were excluded from further analysis due to significant non-linearity or non-parallelism, or a lack of significant dose-response. These are indicated in the tables of results. Tables showing the slopes of the assayed samples and the ratio of the slopes of the test samples to IS 98/704 are included in Appendix Tables A1.2, A1.3, A1.5, A1.6, A1.8, A1.9, A1.11 and A1.12 for information.
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Potency of 10/286 calculated relative to IS 98/704 Analysis incorporating the animal body weight data (excluding laboratory 7) gave geometric mean potency estimates for sample D of 183 IU per ampoule (n = 9; 95% confidence limits 164 206; GCV 16%) for FSH and 171 IU per ampoule (n = 8; 95% confidence limits 148 - 198; GCV 19%) for LH. Using organ weight data only, the geometric mean potency estimate for sample D was calculated to be 183 IU per ampoule (n = 10; 95% confidence limits 165 - 202; GCV 15%) for FSH and 177 IU per ampoule (n = 9; 95% confidence limits 159 - 197; GCV 15%) for LH.
Stability of 10/286 Estimates of the potency of ampoules stored at elevated temperatures for a period of 6 months are summarized in Tables 4 - 7 and Appendix Tables A1.4, A1.7, A1.9, A1.10. Analysis of the thermally accelerated degradation samples in this study gave a predicted 0.001% loss of potency per year for FSH when stored at -20°C, but no consistent loss of activity was detected for the samples stored at +20°C or +37°C for LH.
Conclusions and recommendations Therapeutic human urinary FSH and LH continues to be marketed by both innovator and biosimilar manufacturers and is considered a cost-effective option for the treatment of infertility and as part of assisted reproductive technologies. International Reference Preparations (13) and International Standards (14) for urinary derived human menopausal gonadotrophins have been available since the 1960s and are widely used for the determination of FSH and LH potency of therapeutic preparations of human urinary FSH and LH. As a result, stocks of the current, 4th IS, (NIBSC 98/704) are exhausted. This report describes a collaborative study to establish a replacement IS for human urinary FSH and LH. In order to prepare a sufficiently large stock of the replacement IS, two manufacturers generously agreed to donate bulk preparations of human urinary FSH and LH which were pooled. Both manufacturers provided highly purified urinary FSH and LH, thereby allowing the candidate standard to be filled at a higher potency than previous standards which will decrease storage and despatch costs and reduce the number of ampoules required per assay. The bulk material was formulated with human serum albumin and lactose to promote long term stability of the ampouled material. Quality analysis of the candidate ampoules confirmed that mean fill weight, mean dry mass and mean oxygen head space were within the expected values. The mean residual moisture content of the candidate standard, 10/286, was higher than expected (2.83% (CV 22.12%)). This has been observed previously with glycoprotein hormone preparations and although stability was not affected, further bioassays of the candidate standard, 10/286 and the accelerated thermal degradation samples are recommended to ascertain stability. The collaborative study participants comprised eleven laboratories who provided 30 sets of results for the determination of FSH bioactivity and 26 sets of results for the determination of LH bioactivity. Analysis of the fitted slopes for the dose-response of the candidate standard 10/286 (samples coded D) and the 4th IS 98/704 allowed statistically valid estimates of relative potency to be calculated from all laboratories, fulfilling the requirement of a replacement international standard in terms of parallelism of assay response with the existing IS. Analysis of assay responses of log10(organ weight/body weight) and log10(organ weight) provided geometric
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mean potency determinations that were in agreement. However, in order to use data from all laboratories, log10(organ weight) was selected as the assay response for assignment of potency. Using log10(organ weight) the geometric mean potency for the candidate standard was 183 IU per ampoule (n = 10; 95% confidence limits 165 - 202; GCV 15%) for FSH bioactivity and 177 IU per ampoule (n = 9; 95% confidence limits 159 - 197; GCV 15%) for LH bioactivity. The candidate preparation, 10/286, appears to be sufficiently stable to serve as an international standard. Although these results suggest that 10/286 is likely to be highly stable under long terms storage conditions at -20°C, it is noted that because of the short duration of this study and the lack of detectable degradation for LH, it is impossible to predict the degradation rate of the proposed standard. As a result, it will be a future requirement to complete the assessment of FSH and LH stability in the residual ampoules that have remained stored at elevated temperatures.
Proposal It is recommended that the preparation in ampoules coded 10/286 be established as the fifth International Standard for human urinary FSH and urinary LH for bioassay, with an assigned potency of 183 IU FSH per ampoule and 177 IU LH per ampoule.
Acknowledgements We gratefully acknowledge the important contributions of all the participants, Instituto Massone S.A and IBSA Institut Biochimique S.A. who kindly donated the urinary FSH and urinary LH and the Centre for Biological Reference Materials, NIBSC for the preparation of the ampouled materials.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. WHO. Expert Committee on Biological Standardization. World Health Organ Tech Rep Ser. 2002;910:25. Fertility: Assessment and Treatment for People with Fertility Problems. London UK: National Collaborating Centre for Women's and Children's Health; 2004. WHO. Expert Committee on Biological Standardization. World Health Organ Tech Rep Ser. 1990;800:181-214. Steelman SL, Pohley FM. Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology. 1953;53:604-16. Hell V, R. Matthijsen R, Overbeek G. Effects of human menopausal gonadotrophin preparations in different bioassay methods. Acta Endocrinol (Copenh). 1964;47:409-18. United States Pharmacopeia. Rockville, Maryland, USA: United States Pharmacopeial Convention Inc.; 2011. British Pharmacopeia. London: The Stationary Office; 2011. European Pharmacopoeia. Strasbourg: Council of Europe; 2011. Pharmacopoeia of the People’s Republic of China. China: Pharmacopoeia Commission of the Ministry of Health of the People’s Republic of China; 2010 Finney DJ. Statistical Method in Biological Assay 3rd Edition. London: Charles Griffin; 1978. Gaines Das RE, Rice LR. SCAN, an exploratory program for preliminary analysis of bioassay and immunoassay data. Comput Methods Programs Biomed. 1985;21:25-33.
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12. 13. 14.
Statistical analysis of results of biological assays and tests, general chapter 5.3. European Pharmacopoeia. Strasbourg, France: Council of Europe; 2008. International Reference Preparation for human menopausal gonadotrophin. Bull World Health Organ. 1960;22:563-4. Storring PL, Dixon H, Bangham DR. The first international standard for human urinary FSH and for human urinary LH (ICSH), for bioassay. Acta Endocrinol (Copenh). 1976;83:700-10.
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Table 4: Laboratory mean potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704 using log10 (organ weight/body weight) as assay response Lab 1 2 3 4 6 7 8 9 10 11 GM 95% C.I. GCV n GM 95% C.I. GCV n D (-20°C) 178 155 240 195 165 198 150 191 210 183 185 168 – 206 15% 10 183 164 – 206 16% 9 168 133 – 211 16% 4 Excluding Laboratory 7 157 138 – 178 5% 3 172 136 – 217 10% 3 110 83 – 146 20% 4 178 151 – 210 11% 4 187 141 97 110 83 – 146 20% 4 162 207 198 176 112 155 96 A (+20°C) 161 148 F (+37°C) B (+45°C)
Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log10(organ weight) response, as no data for body weights was available.
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Figure 1: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight/body weight) as assay response
10
8
Number of Assays
6 6
4
8 6
4 9 4 9 10 11 1 1 7 7 10 3 3
2
11 3
11 8 2 2 4 1 1 7 7 4 11
0 90 180 Potency (IU per ampoule) 360
Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log10 (organ weight) response, as no data for body weights was available.
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Table 5: Laboratory mean potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704, using log10(organ weight) as assay response Lab 1 2 3 4 6 7 8 9 10 11 GM 95% C.I. GCV n D (-20°C) 181 156 232 186 159 198 146 188 209 190 183 165 – 202 15% 10 167 131 – 211 16% 4 177 155 – 201 9% 4 170 135 99 118 96 – 146 14% 4 155 207 198 177 116 164 127 A (+20°C) 164 147 F (+37°C) B (+45°C)
Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Figure 2: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight) as assay response
10
8
Number of Assays
6 9
4 8
6
10
4 1 9 7 4 1 7 7 3 3
2
3 11
11 8 6 2 2 4 1 1 7 4 10 11 11
0 90 180 Potency (IU per ampoule) 360
Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Table 6: Laboratory mean potency estimates for LH (IU per ampoule), calculated relative to IS 98/704, using log10(organ weight/body weight) as assay response Lab 1 2 3 4 5 7 8 10 11 GM 95% C.I. GCV N GM 95% C.I. GCV N D (-20°C) 192 124 199 180 180 195 139 196 178 174 153 – 198 18% 9 171 148 – 198 19% 8 179 42 – 771 n/a 2 Excluding Laboratory 7 159 n/a n/a 1 185 171 – 200 5% 4 117 92 – 150 22% 5 188 175 – 203 6% 5 201 196 177 203 178 189 102 150 93 117 92 – 150 22% 5 137 A (+20°C) 159 F (+37°C) B (+45°C) 113
Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log10(organ weight) response, as no data for body weights was available.
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Figure 3: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using log10 (organ weight/body weight) as assay response
10
8
Number of Assays
6 7
4
11
7
4 11 10 7
2
11 2 8
5 8 3
4 7 1 4 4 5 1 3 3 11
0 90 180 Potency (IU per ampoule) 360
Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log10(organ weight) response, as no data for body weights was available.
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Table 7: Laboratory mean potency estimates for LH (IU per ampoule), calculated relative to IS 98/704, using log10 (organ weight) as assay response Lab 1 2 3 4 5 7 8 10 11 GM 95% C.I. GCV N D (-20°C) 191 139 207 189 162 195 145 195 181 177 159 – 197 15% 9 178 n/a n/a 2 181 166 – 199 8% 5 201 169 185 203 172 180 104 152 93 119 93 – 153 22% 5 139 A (+20°C) 158 F (+37°C) B (+45°C) 118
Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Figure 4: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight) as assay response
10
8
Number of Assays
6
4 11
11 7 10 7 7 10 7 4 4 1 3 4 3 11
2 11
8 2
5 8 3
0 90 180 Potency (IU per ampoule) 360
Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Appendix 1: Individual Assay Results Table A1.1: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight/body weight) as assay response Lab 1 1 1 1 2 2 3 3 4 4 6 6 7 7 8 8 9 9 9 9 10 10 11 11 11 NL(X) NP NDR * 1
Assay 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 4 1 2 1 2 3
D1 (-20°C) 182 174 181 177 150 162 156 284 181 169 149 186 201 202 155 143 199 175*
D2 (-20°C)
A (+20°C) 164 158 158 159 151 146
F (+37°C)
B (+45°C)
NL(B) NP
NDR 294 215 183 *
163 133 170 156
NP 96
194 194
212 201
209 189 182 171 116 103
0.8321 0.7101 205 212 211 *
187 186 227
135 144 104 86
139 143
= Non-Linearity of Sample X with p < 0.01 = Non-Parallelism with p < 0.01 = No dose-response for one or more of the samples = Non-Linearity of at least one sample with 0.01 < p < 0.05 = Potency relative to 10/286 noted, and excluded from overall calculations, as no IS included in this
assay Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log10(organ weight) response, as no data for body weights was available.
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Table A1.2: Fitted slopes for FSH, calculated using log10(organ weight/body weight) as assay response Lab 1 1 1 1 2 2 3 3 4 4 6 6 7 7 8 8 9 9 9 9 10 10 11 11 11 Assay 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 4 1 2 1 2 3 0.375 0.380 0.570 0.422 0.502 0.402 0.339 0.550* 0.389 0.417 0.545 0.535 0.377 IS 98/704 0.538 0.388 0.494 0.541 0.004 0.004 0.220 0.319 0.801 0.690 0.390 0.368 0.403 0.333 0.496 0.658 0.634 0.637 D1 (-20°C) 0.557 0.447 0.495 0.517 0.004 0.003 0.348 0.495 0.815 0.684 0.440 0.362 0.367 0.336 0.485 0.534 0.639 0.555* 0.249 0.197 0.464 0.441 0.367 0.376 0.385 0.358 0.173 0.531 0.797 0.645* 0.408 0.472 0.308 0.359 0.324 0.361 0.434 0.583 0.419 0.454 0.646 0.545 0.510 0.408 D2 (-20°C) A (+20°C) 0.511 0.459 0.530 0.528 0.004 0.004 NL 0.002 F (+37°C) B (+45°C)
* = Non-Linearity of Sample with 0.01 < p < 0.05 NL = Non-Linearity of Sample with p < 0.01 Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log 10(organ weight) response, as no data for body weights was available.
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Table A1.3: Fitted slopes relative to IS 98/704 for FSH, calculated using log10 (organ weight/body weight) as assay response Lab 1 1 1 1 2 2 3 3 4 4 6 6 7 7 8 8 9 9 10 10 11 11 11 GM 95% C.I. GCV n GM 95% C.I. GCV n Assay 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 D1 (-20°C) 1.035 1.154 1.002 0.956 0.975 0.773 1.582 1.553 1.017 0.991 1.128 0.984 0.912 1.011 0.976 0.811 1.008 0.871* 1.072 0.892 0.965 *
D2 (-20°C)
A (+20°C) 0.950 1.185 1.073 0.977 1.075 0.886
F (+37°C)
B (+45°C)
NL(B) 0.523
0.786 1.665 0.994 0.935* 1.044 1.283 0.957 1.076 0.764 1.081 0.805 1.085 0.875 0.886 0.844 0.689 0.806 0.790 0.636 0.592
1.238 1.161 0.957
0.979 0.989 1.266 0.750
0.922 0.831 1.003 0.971 – 1.035
1.026 0.932 – 1.120
1.022 0.976 – 1.068
0.942 0.876 – 1.007
0.779 0.684 – 0.873
19% 23 1.007 0.972 – 1.042
26% 7 1.031 0.878 – 1.120
16% 10 1.052 1.009 – 1.096
20% 8 0.944 0.857 – 1.032
33% 9 0.779 0.684 – 0.873
Excluding Laboratory 7
19% 21
33% 5
13% 8
21% 6
33% 9
WHO/BS/2012.2196 Page 22
* = Non-Linearity of at least one sample with 0.01 < p < 0.05 NL(X) = Non-Linearity of Sample X with p < 0.01 NP = Non-Parallelism with p < 0.01 Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log 10(organ weight) response, as no data for body weights was available.
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Table A1.4: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight) as assay response Lab 1 1 1 1 2 2 3 3 4 4 6 6 7 7 8 8 9 9 9 9 10 10 11 11 11 NL(X) NP NDR *
Assay 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 4 1 2 1 2 3
D1 (-20°C) 183 175 183 184 152 *
D2 (-20°C)
A (+20°C) 167 158 161 165 152 145
F (+37°C)
B (+45°C)
NL(B) NP
160 154 259 176 172 147 177 201 202 153 135 194 177 194 194 291 204 175
169 146 158 152† 212 201 209 189 193 168
133 99
122 103
0.8281 0.7121 211 207 220 *
169 171 232
141 124 107 84
158 134
= Non-Linearity of Sample X with p < 0.01 = Non-Parallelism with p < 0.01 = No dose-response for one or more of the samples = Non-Linearity of at least one sample with 0.01 < p < 0.05 1 = Potency relative to sample D noted, and excluded from overall calculations, as no IS included in this assay Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Table A1.5: Fitted slopes for FSH, calculated using log10(organ weight) as assay response Lab 1 1 1 1 2 2 3 3 4 4 6 6 7 7 8 8 9 9 9 9 10 10 11 11 11 Assay 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 4 1 2 1 2 3 0.401 0.377 0.435 0.446 0.590 0.401 0.328 0.412 *
IS 98/704 0.544 0.382 0.493 0.536 0.233 0.262 0.259 0.361 0.794 0.675 0.394 0.364 0.403 0.333 0.473 0.678 0.617 0.633
D1 (-20°C) 0.555 0.447 0.507 0.533 0.233 0.199 0.349 0.513 0.875 0.694 0.453 0.394 0.367 0.336 0.473 0.536 0.637 0.528
D2 (-20°C)
A (+20°C) 0.527 0.450 0.495 0.534 0.251 0.226
F (+37°C)
B (+45°C)
NL 0.134
0.125 0.409 0.723 0.663 0.427 0.480 0.385 0.358 0.308 0.359 0.324 0.361 0.445 0.624 0.446 0.442 0.655 0.577 0.586 0.437
0.242 0.196 0.443 0.433 0.557 0.518 0.365 0.391 0.330
0.398 0.535
* = Non-Linearity of Sample with 0.01 < p < 0.05 NL = Non-Linearity of Sample with p < 0.01 Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Table A1.6: Fitted slopes relative to IS 98/704 for FSH, calculated using log10(organ weight) as assay response Lab 1 1 1 1 2 2 3 3 4 4 6 6 7 7 8 8 9 9 10 10 11 11 11 GM 95% C.I. GCV n Assay 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 D1 (-20°C) 1.021 1.170 1.027 0.993 0.999 *
D2 (-20°C)
A (+20°C) 0.970 1.179 1.003 0.995 1.078 0.863
F (+37°C)
B (+45°C)
NL(B) 0.512
0.760 1.347 1.420 1.103 1.028 1.149 1.083 0.912 1.011 1.000 0.790 1.032 0.835 1.000 0.872 0.946 *
0.480 1.133 0.911 0.982 1.084 1.319 0.957 1.076 0.764 1.081 0.805 1.085 0.941 0.920 0.944 0.652 0.825 0.854 0.738 0.648
1.106 1.149 0.944
0.976 0.876 1.190 0.819
0.893 0.906 1.002 0.974 – 1.030
0.899 0.783 – 1.014
1.023 0.975 – 1.071
0.952 0.902 – 1.003
0.794 0.709 – 0.880
16% 23
33% 7
17% 10
15% 8
29% 9
* = Non-Linearity of at least one sample with 0.01 < p < 0.05 NL(X) = Non-Linearity of Sample X with p < 0.01 NP = Non-Parallelism with p < 0.01 Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Table A1.7: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight/body weight) as assay response Lab 1 1 2 2 3 3 4 4 5 5 7 7 8 8 10 10 11 11 11 Assay 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 D1 (-20°C) 192* 191* 124 NP 217 149 174 173 189 150 204 181 145 133 196 *
D2 (-20°C)
A (+20°C)
F (+37°C)
B (+45°C)
146 167 NP 205† 178 200 NL(F) 196 177 199 200 215 184 †
115 111
137 NL(B)
177 205 200 186 167 189 181 93 NP *
102 103 154 146*
NL(IS,D1) 246 130 174
NL(IS)
NL(X) = Non-Linearity of Sample X with p < 0.01 NP = Non-Parallelism 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 Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log 10(organ weight) response, as no data for body weights was available.
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Table A1.8: Fitted slopes for LH, calculated using log10(organ weight/body weight) as assay response Lab 1 1 2 2 3 3 4 4 5 5 7 7 8 8 10 10 11 11 11 Assay 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 IS 98/704 0.740* 0.750* 2.8 × 10-4 5.0 × 10-4 0.509 0.324 0.480 0.478 0.657 0.406 0.391 0.340 0.489 0.357 0.478 NL 0.365 0.399 0.632 *
D1 (-20° C) 0.666 0.756* 3.3 × 10-4 2.0 × 10-4 0.349 0.304 0.388 0.500 0.691 0.368 0.362 0.419 0.470 0.389 0.447 NL 0.264 0.220 0.401
D2 (-20° C)
A (+20° C)
F (+37° C)
B (+45° C)
3.2 × 10-4 4.1 × 10-4 0.163 0.537 0.557 0.386 NL 0.598 0.396 0.206 0.425 0.437 0.372 0.413 0.347 0.329 0.374 0.392 0.443 0.466 0.473
3.5 × 10-4 3.8 × 10-4
0.417 NL
0.492 0.313 0.552
0.257 0.180
* = Non-Linearity of Sample with 0.01 < p < 0.05 NL(X) = Non-Linearity of Sample X with p < 0.01 Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log 10(organ weight) response, as no data for body weights was available.
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Table A1.9: Fitted slopes relative to IS 98/704 for LH, calculated using log10(organ weight/body weight) as assay response Lab 1 1 1 1 2 2 3 3 4 4 5 5 7 7 8 8 10 10 11 11 11 GM 95% C.I. GCV n GM 95% C.I. GCV n Assay 1 2 3 4 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 1.179 0.400 0.687 0.940 0.809 1.046 1.052 0.906 0.926 1.233 0.961 1.091 0.934 *
D1 (-20°C) 0.900* 1.008*
D2 (-20°C)
A (+20°C)
F (+37°C)
B (+45°C)
1.143 0.820 0.321 1.658 1.161 0.807 NL(F) 1.251 0.602 0.508 1.086 1.285 0.952 1.214 0.887 0.967 0.765 1.100 0.926 1.296 *
1.250 0.760
0.868 NL(B)
1.006 0.877 1.154 1.083* 0.644 0.285
NL(IS,D1) 0.723 0.552 0.634 0.858 0.796 – 0.919
NL(IS)
0.985 0.768 – 1.202
1.020 0.897 – 1.144
0.843 0.734 – 0.953
0.820 0.670 – 0.969
33% 18 0.834 0.768 – 0.901
72% 7 0.916 0.571 – 1.262
20% 4 0.968 n/a
35% 8 0.817 0.658 – 0.976
56% 9 0.820 0.670 – 0.969
Excluding Laboratory 7
33% 16
90% 5
n/a 2
42% 6
56% 9
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* = Non-Linearity of at least one sample with 0.01 < p < 0.05 NL(X) = Non-Linearity of Sample X with p < 0.01 Laboratory 2 estimates were calculated using (organ weight/body weight) response due to assay invalidity when using log transformation. Laboratory 7 estimates were calculated using log10(organ weight) response, as no data for body weights was available.
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Table A1.10: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight) as assay response Lab 1 1 2 2 3 3 4 4 5 5 7 7 8 8 10 10 11 11 11 Assay 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 D1 (-20°C) 191* NL(IS) 139 NP 228 *
D2 (-20°C)
A (+20°C)
F (+37°C)
B (+45°C)
142 168 NP 207 178* 226 135 207 187 199 200 215 184 *†
120 117
164 NL(D1) 185 NL(IS) 162 204 181 150 141 198 194 *
135 141
178 205 199 193 154 191 173 193 93 NP *
104 103 159 146*
267 126 162
NL(X) = Non-Linearity of Sample X with p < 0.01 NP = Non-Parallelism 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 Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Table A1.11: Fitted slopes for LH, calculated using log10(organ weight) as assay response Lab 1 1 2 2 3 3 4 4 5 5 7 7 8 8 10 10 11 11 11 Assay 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 IS 98/704 0.736 NL(IS) 0.018 0.031 0.503 *
D1 (-20°C) 0.665 0.759* 0.024 0.014 0.346 0.294 0.449 0.511 0.689 0.389 0.362 0.419 0.501 0.461 0.445 0.462 0.241 0.410 0.247
D2 (-20°C)
A (+20°C)
F (+37°C)
B (+45°C)
0.020 0.029 0.196 0.530 0.537* 0.379 0.391 0.584 0.394 0.229 0.425 0.437 0.372 0.413 0.347 0.329 0.395 0.354 0.433 0.474 0.509
0.023 0.025
0.351 0.484 0.511 0.658 0.396 0.391 0.340 0.499 0.343 0.498 0.450 *
0.457 0.527
0.486 0.311 0.574 0.508 0.299 0.185
0.344 0.626 0.364
* = Non-Linearity of Sample with 0.01 < p < 0.05 NL(X) = Non-Linearity of Sample X with p < 0.01 Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Table A1.12: Fitted slopes relative to IS 98/704 for LH, calculated using log10(organ weight) as assay response Lab 1 1 2 2 3 3 4 4 5 5 7 7 8 8 10 10 11 11 11 GM 95% C.I. GCV n Assay 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 D1 (-20°C) 0.902* NL(IS) 1.333 0.450 0.688 *
D2 (-20°C)
A (+20°C)
F (+37°C)
B (+45°C)
1.117 0.914 0.390 *
1.283 0.783
0.837 NL(D1) 0.999 NL(IS) 0.981 0.926 1.233 1.004 1.341 0.893 1.026 *
1.509 1.110* 0.742 0.808 1.142 0.598 1.086 1.285 0.952 1.214 *
0.945 1.031
0.577 0.887 0.967 0.791 1.029 0.869 1.054 1.398 0.869 0.295 0.992 0.802 – 1.182
0.973 0.905 1.153 1.129
0.701 0.655 0.678 0.882 0.815 – 0.949
1.042 0.950 – 1.135
0.853 0.782 – 0.924
0.883 0.755 – 1.011
34% 16
60% 7
14% 4
26% 10
51% 10
* = Non-Linearity of at least one sample with 0.01 < p < 0.05 NL(X) = Non-Linearity of Sample X with p < 0.01 Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.
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Appendix 2: Study Protocol Collaborative study to establish the 5th WHO International Standard for human urinary Follicle-stimulating hormone and urinary Luteinizing hormone for bioassay Introduction Follicle-stimulating hormone (FSH) and Luteinizing Hormone (LH) are glycoprotein hormones produced in the anterior pituitary gland which play a major role in the regulation of reproductive processes and pubertal maturation. Human urinary FSH and LH, also known as Menotrophin, is widely used as a therapeutic product in the treatment of fertility disorders. The fourth International Standard (IS) for human, urinary FSH/LH (NIBSC Code 98/704) was established in 2000 and has been widely used for the calibration of therapeutic preparations of human, urinary FSH/LH by bioassay. Stocks of the 4th IS are almost exhausted and there is an urgent requirement to replace the standard. A new preparation of human urinary FSH/LH has been filled into ampoules (NIBSC Code 10/286), following procedures recommended by WHO (1). It is intended that this international collaborative study with expert laboratories will aid in the value assignment of the proposed 5th International Standard. The aims of this study are therefore: 1. To calibrate the candidate standard 10/286 relative to the 4th IS for human urinary FSH/LH (98/704) by in vivo bioassays for FSH and LH. 2. To assess the suitability of the candidate preparation 10/286 to serve as the 5th International Standard for the calibration of therapeutic preparations of human urinary FSH/LH by bioassay. 3. To determine the stability of the preparation 10/286 by comparison with ampoules stored at elevated temperatures as part of an accelerated degradation stability study.
Materials Preparations supplied to participants in collaborative study. A bulk preparation of highly purified, human urinary FSH/LH was generously donated to the WHO by IBSA Institut Biochimique S.A., Lugano, Switzerland and Instituto Massone S.A., Buenos Aires, Argentina. The bulk preparations were provided as a frozen solution (IBSA S.A.) and a lyophilized powder (Instituto Massone S.A.) and were tested and found to be negative for HBsAg, anti-HIV and HCV NAT. The material was combined and formulated with human plasma albumin (0.2% w/v) and lactose (0.5% w/v), dispensed in 1 ml aliquots into glass ampoules, lyophilized and sealed. The materials for this study, which may be identified only by code letter, 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).
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Table 1. Human, urinary FSH/LH Preparation Human Urinary FSH/LH 4th International Standard (98/704) Candidate 5th International Standard (10/286) stored at -20ºC Ampoule content 72 IU per ampoule of urinary FSH 70 IU per ampoule of urinary LH
Nominally 58 µg FSH/LH (assumed to be approx 200 IU per ampoule of urinary FSH and 200 IU per ampoule of urinary LH)
Accelerated thermal degradation (ATD) samples of 10/286 stored at +4°C, +20°C, +37°C and +45°C
Content assumed identical to 10/286 stored at -20ºC
Tests Requested Participants are requested to carry out the bioassay method(s) 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. The candidate standard 10/286 and its degradation samples will be coded, in random order by letter, in the final protocol. 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. PBS or saline, preferably with 0.05 – 0.1% added protein to reduce adsorption) according to the protocol used. 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.
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.
References 1. WHO Tech Rep Ser No 800, 1990 181-214
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For further information, please contact: Dr. Jackie Ferguson, Senior Scientist, Endocrinology, Biotherapeutics, NIBSC Tel: +44 (0) 1707 641135 Email: Jackie.Ferguson@nibsc.hpa.org.uk Or Dr. Chris Burns Principal Scientist, Endocrinology, Biotherapeutics, NIBSC Tel: +44 (0) 1707 641247 Email: Chris.Burns@nibsc.hpa.org.uk
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Appendix 3: Draft Instructions for use WHO International Standard 5th International Standard for Urinary Follicle Stimulating Hormone and Urinary Luteinizing Hormone for Bioassay NIBSC Code: 10/286 Instructions for use (November 2012, first version) This material is not for in vitro diagnostic use.
1.
INTENDED USE The fourth International Standard (IS) for human urinary Follicle Stimulating hormone (FSH) and urinary Luteinizing Hormone (LH) in ampoules coded 98/704 was established in 2002 and has been widely used for the calibration of preparations of human urinary FSH and urinary LH by bioassay. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) has recognized (2011) the need for a replacement International Standard for human urinary FSH and urinary LH for the assignment of potency to therapeutic preparations of human urinary FSH and urinary LH (menotrophin, human menopausal gonadotrophins) used in the treatment of infertility. The 5th IS, coded 10/286, was established at the 63rd Meeting of the ECBS. This material replaces the 4th IS which is discontinued.
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 183 INTERNATIONAL INTERNATIONAL UNITS of LH UNITS of FSH and 177
WHO/BS/2012.2196 Page 37
4.
CONTENTS Country of origin of biological material: Argentina and Switzerland Each ampoule contains the residue after freeze-drying of 1 ml of a solution that contained: Purified proteins from human menopausal urine Human serum albumin Lactose approximately 58µg 0.2 % (w/v) 0.5 % (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 ambient 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 human urinary FSH and urinary LH. 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 eleven laboratories in ten countries took part, organized with the following aims: 1) To calibrate the candidate preparation,10/286 relative to the 4th IS (98/704) for urinary FSH and urinary LH by in vivo bioassays 2) To assess the suitability of the candidate preparation 10/286 to serve as the 5th IS for the calibration of therapeutic human urinary FSH and urinary LH products by bioassay.
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3) To determine the stability of the candidate preparation 10/286 by comparison with ampoules stored at elevated temperatures as part of an accelerated degradation stability study. The geometric mean potency calculated from all laboratories using log10(organ weight) as assay response was 183 IU FSH per ampoule (n=10; 95% confidence limits 165 - 202; GCV 15%) and 177 IU LH per ampoule (n = 9; 95% confidence limits 159 - 197; GCV 15%). The candidate preparation 10/286 is sufficiently stable to serve as an International Standard. Analysis of the thermally accelerated degradation samples in this study gave a predicted 0.001% loss of potency per year for FSH when stored at -20°C, but no consistent loss of activity was detected for the samples stored at +20°C or +37°C for LH. This suggests that 10/286 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. 10.
REFERENCES ACKNOWLEDGEMENTS We gratefully acknowledge the important contributions of all the participants and Instituto Massone S.A. and IBSA Institut Biochimique S.A. for the kind donation of urinary FSH and urinary LH.
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:
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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 Fire hazard Stable: Yes Hygroscopic: Yes Flammable: No Other (specify) Handling: Freeze dried powder None Chemical properties Corrosive: Oxidising: Irritant: Contains material of human origin See caution, section 2 No No No
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.
WHO/BS/2012.2196 Page 40
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: 7 mg Toxicity statement: Non-toxic Veterinary certificate or other statement if applicable. Attached: No