who/bs/2012.2196 english only expert committee on

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

[email protected] or [email protected]

Note:

This document has been prepared for the purpose of inviting comments and suggestions on the

proposals contained therein, which will then be considered by the Expert Committee on

Biological Standardization (ECBS). Comments MUST be received by 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: [email protected]

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WHO/BS/2012.2196

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

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

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

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

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


per the US Pharmacopeia (6)



3


per the British Pharmacopoeia (7)


the British Pharmacopoeia (7)

4


per the European Pharmacopoeia (8)



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


7




the British Pharmacopoeia (7)

8




British Pharmacopoeia (7)

9 In vivo FSH bioassay Augmentation of ovary weight in immature female rats as


10





11





WHO/BS/2012.2196

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

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

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

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

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

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 7 estimates were calculated using log10 (organ weight) response, as no data for body weights

was available.

WHO/BS/2012.2196

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

N

um

be

r o

f A

ssa

ys

0

2

4

6

8

10


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


log transformation.

WHO/BS/2012.2196

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


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




was available.

WHO/BS/2012.2196

N

um

be

r o

f A

ssa

ys

0

2

4

6

8

10


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




was available.

WHO/BS/2012.2196

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


log transformation.

WHO/BS/2012.2196

Figure 4: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using

log10(organ weight) as assay response


log transformation.

Nu

mb

er

of A

ssa

ys

0

2

4

6

8

10


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

Appendix 1: Individual Assay Results

Table A1.1: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704


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

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




available.

WHO/BS/2012.2196

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


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

* = Non-Linearity of at least one sample with 0.01 < p < 0.05






available.

WHO/BS/2012.2196

Table A1.4: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704


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



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

Table A1.5: Fitted slopes for FSH, calculated using log10(organ weight) as assay response


(-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


NL = Non-Linearity of Sample with p < 0.01


transformation.

WHO/BS/2012.2196

Table A1.6: Fitted slopes relative to IS 98/704 for FSH, calculated using


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





transformation.

WHO/BS/2012.2196

Table A1.7: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704


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




† = Non-Parallelism with 0.01 < p < 0.05




available.

WHO/BS/2012.2196

Table A1.8: Fitted slopes for LH, calculated using log10(organ weight/body weight) as

assay response


(-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






available.

WHO/BS/2012.2196

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


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






available.

WHO/BS/2012.2196

Table A1.10: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704


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




† = Non-Parallelism with 0.01 < p < 0.05


transformation.

WHO/BS/2012.2196

Table A1.11: Fitted slopes for LH, calculated using log10(organ weight) as assay response


(-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




transformation.

WHO/BS/2012.2196

Table A1.12: Fitted slopes relative to IS 98/704 for LH, calculated using


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




transformation.

WHO/BS/2012.2196

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

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

For further information, please contact:

Dr. Jackie Ferguson,

Senior Scientist, Endocrinology,

Biotherapeutics,

NIBSC

Tel: +44 (0) 1707 641135

Email: [email protected]

Or

Dr. Chris Burns

Principal Scientist, Endocrinology,

Biotherapeutics,

NIBSC

Tel: +44 (0) 1707 641247

Email: [email protected]



WHO/BS/2012.2196

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

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

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:

[email protected]

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

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

[email protected]

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

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

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