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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
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WHO/BS/2012.2196
Page 2
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 Follicle-
stimulating 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 cost-
effective 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:
WHO/BS/2012.2196
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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.,
WHO/BS/2012.2196
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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
Urinary FSH and urinary LH
preparation
Ampoule unitage and nominal
content
Not coded 4th
International Standard (98/704) 70 IU FSH per ampoule and 72 IU
LH per ampoule
D Candidate 5
th International
Standard (10/286) stored at -20°C
Nominally 58µg (assumed to be
approximately 200 IU FSH per
ampoule and 200 IU LH per
ampoule)
A, F and B
Accelerated thermal degradation
(ATD) samples of 10/286 stored at
+20°C, +37°C and +45°C for
6 months
Content assumed identical to
10/286 stored at -20ºC
WHO/BS/2012.2196
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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.
WHO/BS/2012.2196
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Table 3: Assay methods used
Lab.
No.
Assay type Comments
1
In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the US Pharmacopeia (6)
In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
the US Pharmacopeia (6)
2
In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the US Pharmacopeia (6)
In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
the US Pharmacopeia (6)
3
In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the British Pharmacopoeia (7)
In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
the British Pharmacopoeia (7)
4
In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the European Pharmacopoeia (8)
In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
the US Pharmacopeia (6)
5 In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
the Pharmacopoeia of the People’s Republic of China (9)
6 In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the European Pharmacopoeia (8)
7
In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the European Pharmacopoeia (8)
In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
the British Pharmacopoeia (7)
8
In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the British Pharmacopoeia (7)
In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
British Pharmacopoeia (7)
9 In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the European Pharmacopoeia (8)
10
In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the British Pharmacopoeia (7)
In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
British Pharmacopoeia (7)
11
In vivo FSH bioassay Augmentation of ovary weight in immature female rats as
per the British Pharmacopoeia (7)
In vivo LH bioassay Seminal vesicle weight gain in immature male rats as per
British Pharmacopoeia (7)
WHO/BS/2012.2196
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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.
WHO/BS/2012.2196
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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
WHO/BS/2012.2196
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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. WHO. Expert Committee on Biological Standardization. World Health Organ Tech Rep
Ser. 2002;910:25.
2. Fertility: Assessment and Treatment for People with Fertility Problems. London UK:
National Collaborating Centre for Women's and Children's Health; 2004.
3. WHO. Expert Committee on Biological Standardization. World Health Organ Tech Rep
Ser. 1990;800:181-214.
4. Steelman SL, Pohley FM. Assay of the follicle stimulating hormone based on the
augmentation with human chorionic gonadotropin. Endocrinology. 1953;53:604-16.
5. Hell V, R. Matthijsen R, Overbeek G. Effects of human menopausal gonadotrophin
preparations in different bioassay methods. Acta Endocrinol (Copenh). 1964;47:409-18.
6. United States Pharmacopeia. Rockville, Maryland, USA: United States Pharmacopeial
Convention Inc.; 2011.
7. British Pharmacopeia. London: The Stationary Office; 2011.
8. European Pharmacopoeia. Strasbourg: Council of Europe; 2011.
9. Pharmacopoeia of the People’s Republic of China. China: Pharmacopoeia Commission of
the Ministry of Health of the People’s Republic of China; 2010
10. Finney DJ. Statistical Method in Biological Assay 3rd Edition. London: Charles Griffin;
1978.
11. 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.
WHO/BS/2012.2196
Page 10
12. Statistical analysis of results of biological assays and tests, general chapter 5.3. European
Pharmacopoeia. Strasbourg, France: Council of Europe; 2008.
13. International Reference Preparation for human menopausal gonadotrophin. Bull World
Health Organ. 1960;22:563-4.
14. 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.
WHO/BS/2012.2196
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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 D
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 178 161
2 155 148
3 240
4 195 155 96
6 165 162
7 198 207 198
8 150 176 112
9 191
10 210 187 141
11 183 97
GM
185 168 178 110
95% C.I. 168 – 206 133 – 211 151 – 210 83 – 146
GCV 15% 16% 11% 20%
n 10 4 4 4
Excluding Laboratory 7
GM
183 157 172 110
95% C.I. 164 – 206 138 – 178 136 – 217 83 – 146
GCV 16% 5% 10% 20%
n 9 3 3 4
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.
WHO/BS/2012.2196
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N
um
be
r o
f A
ssa
ys
0
2
4
6
8
10
Potency (IU per ampoule)
90 180 360
11 8
11
2
3
6
8
2 4 1
1
9
1
1
4
4
6
7
7
9
7
7
10
4
10
11
11 3 3
Figure 1: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704
using log10(organ weight/body weight) as assay response
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.
WHO/BS/2012.2196
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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 D
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 181 164
2 156 147
3 232
4 186 164 127
6 159 155
7 198 207 198
8 146 177 116
9 188
10 209 170 135
11 190 99
GM
183 167 177 118
95% C.I. 165 – 202 131 – 211 155 – 201 96 – 146
GCV 15% 16% 9% 14%
n 10 4 4 4
Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using
log transformation.
WHO/BS/2012.2196
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N
um
be
r o
f A
ssa
ys
0
2
4
6
8
10
Potency (IU per ampoule)
90 180 360
11 8 6 2
3
8
2
11
4 1
4
4
6
9
1
1
1
7
7
9
4
7
7
10
10 11 11 3 3
Figure 2: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704
using log10(organ weight) as assay response
Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using
log transformation.
WHO/BS/2012.2196
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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 D
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 192
2 124 159 113
3 199
4 180 196 137
5 180 177
7 195 201 203
8 139 178 102
10 196 189 150
11 178 93
GM
174 179 188 117
95% C.I. 153 – 198 42 – 771 175 – 203 92 – 150
GCV 18% n/a 6% 22%
N 9 2 5 5
Excluding Laboratory 7
GM
171 159 185 117
95% C.I. 148 – 198 n/a 171 – 200 92 – 150
GCV 19% n/a 5% 22%
N 8 1 4 5
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.
WHO/BS/2012.2196
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N
um
be
r o
f A
ssa
ys
0
2
4
6
8
10
Potency (IU per ampoule)
90 180 360
2 8
11
8 3
5
4
4
4
11
5
7
11
1
1
10
3
4
7
7
7
3 11
Figure 3: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using
log10 (organ weight/body weight) as assay response
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.
WHO/BS/2012.2196
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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 D
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 191
2 139 158 118
3 207
4 189 169 139
5 162 185
7 195 201 203
8 145 172 104
10 195 180 152
11 181 93
GM
177 178 181 119
95% C.I. 159 – 197 n/a 166 – 199 93 – 153
GCV 15% n/a 8% 22%
N 9 2 5 5
Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using
log transformation.
WHO/BS/2012.2196
Page 18
Figure 4: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using
log10(organ weight) as assay response
Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using
log transformation.
Nu
mb
er
of A
ssa
ys
0
2
4
6
8
10
Potency (IU per ampoule)
90 180 360
11 2
8
8 3
5
11
4 4
7
1
10
10
11
3
7
7
7
3
4
11
WHO/BS/2012.2196
Page 19
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 Assay D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 182 164
1 2 174 158
1 3 181 158
1 4 177 159
2 1 150 151 NL(B)
2 2 162 146 NP
3 1 156 NDR
3 2 284 294
4 1 181 215 163 NP
4 2 169 183* 133 96
6 1 149 170
6 2 186 156
7 1 201 194 212 209
7 2 202 194 201 189
8 1 155 182 116
8 2 143 171 103
9 1 199
9 2 175*
9 3 0.8321
9 4 0.7101
10 1 205 187 135
10 2 212 186 144
11 1 211* 227
11 2 139 104
11 3 143 86
NL(X) = Non-Linearity of Sample X with p < 0.01
NP = Non-Parallelism with p < 0.01
NDR = 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 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.
WHO/BS/2012.2196
Page 20
Table A1.2: Fitted slopes for FSH, calculated using log10(organ weight/body weight) as
assay response
Lab Assay IS 98/704 D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 0.538 0.557 0.511
1 2 0.388 0.447 0.459
1 3 0.494 0.495 0.530
1 4 0.541 0.517 0.528
2 1 0.004 0.004 0.004 NL
2 2 0.004 0.003 0.004 0.002
3 1 0.220 0.348 0.173
3 2 0.319 0.495 0.531
4 1 0.801 0.815 0.797 0.646 0.510
4 2 0.690 0.684 0.645* 0.545 0.408
6 1 0.390 0.440 0.408
6 2 0.368 0.362 0.472
7 1 0.403 0.367 0.385 0.308 0.324
7 2 0.333 0.336 0.358 0.359 0.361
8 1 0.496 0.485 0.434 0.419
8 2 0.658 0.534 0.583 0.454
9 1 0.634 0.639
9 2 0.637 0.555*
9 3 0.249
9 4 0.197
10 1 0.375 0.402 0.464 0.367
10 2 0.380 0.339 0.441 0.376
11 1 0.570 0.550* 0.545
11 2 0.422 0.389 0.535
11 3 0.502 0.417 0.377
* = 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 log10(organ weight) response, as no data for body weights was
available.
WHO/BS/2012.2196
Page 21
Table A1.3: Fitted slopes relative to IS 98/704 for FSH, calculated using
log10 (organ weight/body weight) as assay response
Lab Assay D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 1.035 0.950
1 2 1.154 1.185
1 3 1.002 1.073
1 4 0.956 0.977
2 1 0.975 1.075 NL(B)
2 2 0.773 0.886 0.523
3 1 1.582 0.786
3 2 1.553 1.665
4 1 1.017 0.994 0.806 0.636
4 2 0.991 0.935* 0.790 0.592
6 1 1.128 1.044
6 2 0.984 1.283
7 1 0.912 0.957 0.764 0.805
7 2 1.011 1.076 1.081 1.085
8 1 0.976 0.875 0.844
8 2 0.811 0.886 0.689
9 1 1.008
9 2 0.871*
10 1 1.072 1.238 0.979
10 2 0.892 1.161 0.989
11 1 0.965* 0.957
11 2 0.922 1.266
11 3 0.831 0.750
GM 1.003 1.026 1.022 0.942 0.779
95% C.I. 0.971 – 1.035 0.932 – 1.120 0.976 – 1.068 0.876 – 1.007 0.684 – 0.873
GCV 19% 26% 16% 20% 33%
n 23 7 10 8 9
Excluding Laboratory 7
GM
1.007 1.031 1.052 0.944 0.779
95% C.I. 0.972 – 1.042 0.878 – 1.120 1.009 – 1.096 0.857 – 1.032 0.684 – 0.873
GCV 19% 33% 13% 21% 33%
n 21 5 8 6 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 log10(organ weight) response, as no data for body weights was
available.
WHO/BS/2012.2196
Page 23
Table A1.4: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704
using log10(organ weight) as assay response
Lab Assay D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 183 167
1 2 175 158
1 3 183 161
1 4 184 165
2 1 152* 152 NL(B)
2 2 160 145 NP
3 1 154
3 2 259 291
4 1 176 204 169 133
4 2 172 175 146 99
6 1 147 158
6 2 177 152†
7 1 201 194 212 209
7 2 202 194 201 189
8 1 153 193 122
8 2 135 168 103
9 1 194
9 2 177
9 3 0.8281
9 4 0.7121
10 1 211 169 141
10 2 207 171 124
11 1 220* 232
11 2 158 107
11 3 134 84
NL(X) = Non-Linearity of Sample X with p < 0.01
NP = Non-Parallelism with p < 0.01
NDR = 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.
WHO/BS/2012.2196
Page 24
Table A1.5: Fitted slopes for FSH, calculated using log10(organ weight) as assay response
Lab Assay IS 98/704 D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 0.544 0.555 0.527
1 2 0.382 0.447 0.450
1 3 0.493 0.507 0.495
1 4 0.536 0.533 0.534
2 1 0.233 0.233 0.251 NL
2 2 0.262 0.199
0.226 0.134
3 1 0.259 0.349 0.125
3 2 0.361 0.513 0.409
4 1 0.794 0.875 0.723 0.655 0.586
4 2 0.675 0.694 0.663 0.577 0.437
6 1 0.394 0.453 0.427
6 2 0.364 0.394 0.480
7 1 0.403 0.367 0.385 0.308 0.324
7 2 0.333 0.336 0.358 0.359 0.361
8 1 0.473 0.473 0.445 0.446
8 2 0.678 0.536 0.624 0.442
9 1 0.617 0.637
9 2 0.633 0.528
9 3 0.242
9 4 0.196
10 1 0.401 0.401 0.443 0.391
10 2 0.377 0.328 0.433 0.330
11 1 0.435 0.412* 0.557
11 2 0.446 0.398 0.518
11 3 0.590 0.535 0.365
* = 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.
WHO/BS/2012.2196
Page 25
Table A1.6: Fitted slopes relative to IS 98/704 for FSH, calculated using
log10(organ weight) as assay response
Lab Assay D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 1.021 0.970
1 2 1.170 1.179
1 3 1.027 1.003
1 4 0.993 0.995
2 1 0.999* 1.078 NL(B)
2 2 0.760 0.863 0.512
3 1 1.347 0.480
3 2 1.420 1.133
4 1 1.103 0.911 0.825 0.738
4 2 1.028 0.982 0.854 0.648
6 1 1.149 1.084
6 2 1.083 1.319
7 1 0.912 0.957 0.764 0.805
7 2 1.011 1.076 1.081 1.085
8 1 1.000 0.941 0.944
8 2 0.790 0.920 0.652
9 1 1.032
9 2 0.835
10 1 1.000 1.106 0.976
10 2 0.872 1.149 0.876
11 1 0.946* 0.944
11 2 0.893 1.190
11 3 0.906 0.819
GM 1.002 0.899 1.023 0.952 0.794
95% C.I. 0.974 – 1.030 0.783 – 1.014 0.975 – 1.071 0.902 – 1.003 0.709 – 0.880
GCV 16% 33% 17% 15% 29%
n 23 7 10 8 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.
WHO/BS/2012.2196
Page 26
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 Assay D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 192*
1 2 191*
2 1 124 146 115
2 2 NP 167 111
3 1 217 NP
3 2 149 205†
4 1 174 178 NL(F) 137
4 2 173 200 196 NL(B)
5 1 189 177†
5 2 150 177
7 1 204 199 215 205
7 2 181 200 184 200
8 1 145 186 102
8 2 133 167 103
10 1 196* 189
* 154
10 2 NL(IS,D1) NL(IS) 146*
11 1 246 181
11 2 130 93
11 3 174 NP
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 log10(organ weight) response, as no data for body weights was
available.
WHO/BS/2012.2196
Page 27
Table A1.8: Fitted slopes for LH, calculated using log10(organ weight/body weight) as
assay response
Lab Assay IS 98/704 D1
(-20° C)
D2
(-20° C)
A
(+20° C)
F
(+37° C)
B
(+45° C)
1 1 0.740* 0.666
1 2 0.750* 0.756
*
2 1 2.8 × 10-4
3.3 × 10-4
3.2 × 10-4
3.5 × 10-4
2 2 5.0 × 10-4
2.0 × 10-4
4.1 × 10-4
3.8 × 10-4
3 1 0.509 0.349 0.163
3 2 0.324 0.304 0.537
4 1 0.480 0.388 0.557 NL 0.417
4 2 0.478 0.500 0.386 0.598 NL
5 1 0.657 0.691 0.396
5 2 0.406 0.368 0.206
7 1 0.391 0.362 0.425 0.372 0.347
7 2 0.340 0.419 0.437 0.413 0.329
8 1 0.489 0.470 0.374 0.492
8 2 0.357 0.389 0.392 0.313
10 1 0.478* 0.447 0.443 0.552
10 2 NL NL 0.466
11 1 0.365 0.264 0.473
11 2 0.399 0.220 0.257
11 3 0.632 0.401 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 log10(organ weight) response, as no data for body weights was
available.
WHO/BS/2012.2196
Page 28
Table A1.9: Fitted slopes relative to IS 98/704 for LH, calculated using
log10(organ weight/body weight) as assay response
Lab Assay D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 0.900*
1 2 1.008*
1 3
1 4
2 1 1.179 1.143 1.250
2 2 0.400 0.820 0.760
3 1 0.687 0.321
3 2 0.940 1.658
4 1 0.809 1.161 NL(F) 0.868
4 2 1.046 0.807 1.251 NL(B)
5 1 1.052 0.602
5 2 0.906 0.508
7 1 0.926 1.086 0.952 0.887
7 2 1.233 1.285 1.214 0.967
8 1 0.961 0.765 1.006
8 2 1.091 1.100 0.877
10 1 0.934* 0.926
* 1.154
10 2 NL(IS,D1) NL(IS) 1.083*
11 1 0.723 1.296
11 2 0.552 0.644
11 3 0.634 0.285
GM 0.858 0.985 1.020 0.843 0.820
95% C.I. 0.796 – 0.919 0.768 – 1.202 0.897 – 1.144 0.734 – 0.953 0.670 – 0.969
GCV 33% 72% 20% 35% 56%
n 18 7 4 8 9
Excluding Laboratory 7
GM 0.834 0.916 0.968 0.817 0.820
95% C.I. 0.768 – 0.901 0.571 – 1.262 n/a 0.658 – 0.976 0.670 – 0.969
GCV 33% 90% n/a 42% 56%
n 16 5 2 6 9
WHO/BS/2012.2196
Page 29
* = 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.
WHO/BS/2012.2196
Page 30
Table A1.10: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704
using log10(organ weight) as assay response
Lab Assay D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 191*
1 2 NL(IS)
2 1 139 142 120
2 2 NP 168 117
3 1 228* NP
3 2 164 207
4 1 NL(D1) 178* 135 135
4 2 185 226 207 141
5 1 NL(IS) 187*†
5 2 162 178
7 1 204 199 215 205
7 2 181 200 184 199
8 1 150 193 104
8 2 141 154 103
10 1 198 191 159
10 2 194* 173
* 146
*
11 1 267 193
11 2 126 93
11 3 162 NP
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.
WHO/BS/2012.2196
Page 31
Table A1.11: Fitted slopes for LH, calculated using log10(organ weight) as assay response
Lab Assay IS 98/704 D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 0.736 0.665
1 2 NL(IS) 0.759*
2 1 0.018 0.024 0.020 0.023
2 2 0.031 0.014 0.029 0.025
3 1 0.503* 0.346 0.196
3 2 0.351 0.294 0.530
4 1 0.484 0.449 0.537* 0.391 0.457
4 2 0.511 0.511 0.379 0.584 0.527
5 1 0.658 0.689 0.394
5 2 0.396 0.389 0.229
7 1 0.391 0.362 0.425 0.372 0.347
7 2 0.340 0.419 0.437 0.413 0.329
8 1 0.499 0.501 0.395 0.486
8 2 0.343 0.461 0.354 0.311
10 1 0.498 0.445 0.433 0.574
10 2 0.450* 0.462 0.474 0.508
11 1 0.344 0.241 0.509
11 2 0.626 0.410 0.299
11 3 0.364 0.247 0.185
* = 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.
WHO/BS/2012.2196
Page 32
Table A1.12: Fitted slopes relative to IS 98/704 for LH, calculated using
log10(organ weight) as assay response
Lab Assay D1
(-20°C)
D2
(-20°C)
A
(+20°C)
F
(+37°C)
B
(+45°C)
1 1 0.902*
1 2 NL(IS)
2 1 1.333 1.117 1.283
2 2 0.450 0.914 0.783
3 1 0.688* 0.390
*
3 2 0.837 1.509
4 1 NL(D1) 1.110* 0.808 0.945
4 2 0.999 0.742 1.142 1.031
5 1 NL(IS) 0.598*
5 2 0.981 0.577
7 1 0.926 1.086 0.952 0.887
7 2 1.233 1.285 1.214 0.967
8 1 1.004 0.791 0.973
8 2 1.341 1.029 0.905
10 1 0.893 0.869 1.153
10 2 1.026* 1.054 1.129
11 1 0.701 1.398
11 2 0.655 0.869
11 3 0.678 0.295
GM 0.882 0.992 1.042 0.853 0.883
95% C.I. 0.815 – 0.949 0.802 – 1.182 0.950 – 1.135 0.782 – 0.924 0.755 – 1.011
GCV 34% 60% 14% 26% 51%
n 16 7 4 10 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.
WHO/BS/2012.2196
Page 33
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).
WHO/BS/2012.2196
Page 34
Table 1.
Human, urinary FSH/LH Preparation Ampoule content
Human Urinary FSH/LH 4th
International
Standard (98/704)
72 IU per ampoule of urinary FSH
70 IU per ampoule of urinary LH
Candidate 5th
International Standard
(10/286) stored at -20ºC
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
WHO/BS/2012.2196
Page 35
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
WHO/BS/2012.2196
Page 36
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 UNITS of FSH and 177
INTERNATIONAL UNITS of LH
WHO/BS/2012.2196
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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 approximately 58µg
Human serum albumin 0.2 % (w/v)
Lactose 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.
WHO/BS/2012.2196
Page 38
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. REFERENCES
10. 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:
WHO/BS/2012.2196
Page 39
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.
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