WHO International Collaborative Study of the proposed 5

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EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 15 to 19 October 2012

WHO International Collaborative Study of the proposed 5

^th

International Standard for human, urinary Follicle-Stimulating Hormone and human,

urinary Luteinizing Hormone, for bioassay.

Jackie Ferguson*, Jason Hockley, Richard Tiplady and Chris Burns National Institute for Biological Standards and Control,

Blanche Lane, South Mimms, Potters Bar, Herts, EN6 3QG, UK

*Corresponding author: Jackie Ferguson +44 (0) 1707 641000

Jackie.Ferguson@nibsc.hpa.org.uk or Chris.Burns@nibsc.hpa.org.uk

Note:

This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Comments MUST be received by 01 October 2012 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:

Quality Safety and Standards (QSS). Comments may also be submitted electronically to the Responsible Officer: Dr Jongwon Kim at email: kimjon@who.int

© World Health Organization 2012

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Summary

The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) has recognized (2011) the need for a replacement International Standard for human urinary 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 5^th International Standard.

The aims of this study were therefore:

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1) To calibrate the candidate standard,10/286, relative to the 4^th 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 5^th IS for the calibration of therapeutic human urinary FSH and urinary LH products by bioassay.

3) To determine the stability of the candidate standard, 10/286, by comparison with ampoules stored at elevated temperatures as part of an accelerated degradation stability study.

Participants

Eleven laboratories in ten countries took part in the study and are listed alphabetically, by country, in Table 1. Throughout the study, each participating laboratory is referred to by a code number. The code numbers were randomly assigned and do not reflect the order of listing.

Table 1: List of participants

Dr Claudio Wolfenson, Instituto Massone, Arias 4431, 1430 Buenos Aires, ARGENTINA.

Dr Kevin Grant and Dr Tursun Kerim, Therapeutic Goods Administration, P.O. Box 100 Woden, ACT 2606, AUSTRALIA.

Dr Sergio Luiz Dalmora, Department of Industrial Pharmacy, Federal University of Santa Maria, 97.105-900.Santa Maria, RS, BRAZIL.

Dr Jan Rohde, Minapharm Pharmaceuticals, El-Bardissi Street, 2T Takseem Assmaa Fahmy Street, Heliopolis, Cairo, EGYPT and Dr Sven-Michael Cords, Bioassay - Labor für biologische Analytik GmbH, Im Neuenheimer Feld 515, 69120 Heidelberg, GERMANY.

Dr Gundel Hager and Marta Leis, Aurigon Life Science GmbH, Bahnhofstraße 9-15, D-82327, Tutzing, GERMANY.

Dr A. Winkler, LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, Redderweg 8, 21147 Hamburg, GERMANY.

Dr Cinzia Ciampolillo, Merck Serono Ivrea – RBM S.p.A, Via Ribes 1, 10010 Colleretto Giacosa (TO), ITALY.

Ms Yuan Zhang, National Institutes for Food and Drug Control, Pharmacology Division, Tiantan Xi Li 2#, Dongcheng District, Beijing, 100050, P.R. CHINA.

Dr Tiziano Fossati, IBSA Institut Biochimique SA, Via Al Ponte 13, 6900 Massagno, SWITZERLAND.

Mr Richard Tiplady, NIBSC, Biotherapeutics Department, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, UK.

Dr Elizabeth Raike, Qualtech Laboratories, 104 Green Grove Road, Ocean, NJ 07712, USA.

Materials

Bulk materials and preparation of ampoules of human urinary FSH and urinary LH.

Bulk preparations of highly purified, human urinary FSH and urinary LH were generously donated to the WHO by Instituto Massone S.A., Argentina and IBSA Institut Biochimique S.A.,

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Switzerland. The bulk preparation received from Instituto Massone S.A., Argentina (Batch No.

3626365910) comprised of 0.133 g of purified FSH and LH that had been precipitated with alcohol and dried under vacuum, with reported bioactivities of 3018 IU/mg FSH and 3057 IU/mg LH. The preparation had been tested by the manufacturer and found negative for HBsAg, anti-HIV and HCV NAT.

The bulk preparation of purified FSH and LH received from IBSA Institut Biochimique S.A., Switzerland (Batch No. WHO 01/2010) comprised a frozen solution of 88.4 ml of 1.43 mg/ml protein with reported bioactivities of 3875 IU/ml FSH and 4587 IU/ml LH. The preparation had been tested by the manufacturer and found negative for HBsAg, anti-HIV and HCV NAT.

Combined, 259.41 mg of the material was formulated with 0.2% (w/v) human plasma albumin (also tested and found to be negative for HBsAg, anti-HIV, anti-HCV and HCV NAT) and 0.5% (w/v) lactose and 1.0 ml was dispensed into glass ampoules (nominally, 58µg protein) on the 8^th April 2011, lyophilised and sealed on the 12^th 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 4^th 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

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Design of the study and assay methods contributed Bioassay of the candidate standard 10/286

Participants were requested to carry out the assay(s) normally in use in their laboratory and, where possible, to perform at least two independent assays, using fresh ampoules, each assay to include all of the preparations allocated. Handling instructions for the materials were included in the study protocol. In instances where there was not a fresh ampoule for subsequent assays, it was suggested that fresh dilutions be made from frozen stock solutions. Where dilutions of a stored stock solution were used, participants were asked to provide details of its storage and identification of the initial preparation. Participants were asked to provide details of the assay method used, including dilution steps, together with all raw assay data in the form of clearly annotated organ and body weights for central computation at NIBSC. Participants’ own estimates of activity as calculated by the method normally used in their laboratory were also requested.

Assay methods contributed

Participants were requested to perform in vivo bioassays of FSH based on the bioassay described by Steelman and Pohley (1953) of augmentation of ovary weight in immature female rats (4) and/or in vivo bioassays of LH based on the bioassay described by Hell et al., (1964) of seminal vesicle weight gain in immature male rats (5). Participants were asked to follow protocols normally used in their laboratory. The assays contributed by each laboratory are listed in Table 3.

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Table 3: Assay methods used

Lab.

No.

Assay type Comments

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)

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)

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 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

11

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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 = {10^s-1} × 100% where s is the standard deviation of the log₁₀-transformed potency estimates).

Results

Data returned for analysis

Data were contributed by 11 laboratories. Laboratory 7 provided data for organ weights only. All other laboratories provided data for both organ weights and body weights. A total of 23 assays were performed for FSH, giving 30 sets of results for sample D. A total of 19 assays were performed for LH, giving 26 sets of results for sample D. Mean potency estimates for 10/286 are summarised in Tables 4 and 5 and Figures 1 and 2 for FSH, and Tables 6 and 7 and Figures 3 and 4 for LH. Results from individual assays for both are given in Appendix Tables A1.1, A1.4, A1.7 and A1.10.

Assay validity

Excluding Laboratory 7, significant heterogeneity of variance (p < 0.05 in Bartlett’s test) was found in 25/92 assays (27.2%) when using organ weight as assay response, 13/92 assays (14.1%) when using log₁₀(organ weight) as assay response, in 31/92 assays (33.7%) when using (organ weight/body weight) as assay response and 11/92 assays (12.0%) when using log10(organ weight/body weight) as assay response, suggesting that log10(organ weight/body weight) and log₁₀(organ weight) provide better agreement with the variance homogeneity required for parallel-line analysis. Further analysis was performed using these assay responses only.

The majority of assays allowed statistically valid estimates of relative potency to be calculated, although some samples were excluded from further analysis due to significant non-linearity or non-parallelism, or a lack of significant dose-response. These are indicated in the tables of results. Tables showing the slopes of the assayed samples and the ratio of the slopes of the test samples to IS 98/704 are included in Appendix Tables A1.2, A1.3, A1.5, A1.6, A1.8, A1.9, A1.11 and A1.12 for information.

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Potency of 10/286 calculated relative to IS 98/704

Analysis incorporating the animal body weight data (excluding laboratory 7) gave geometric mean potency estimates for sample D of 183 IU per ampoule (n = 9; 95% confidence limits 164 - 206; GCV 16%) for FSH and 171 IU per ampoule (n = 8; 95% confidence limits 148 - 198; GCV 19%) for LH. Using organ weight data only, the geometric mean potency estimate for sample D was calculated to be 183 IU per ampoule (n = 10; 95% confidence limits 165 - 202; GCV 15%) for FSH and 177 IU per ampoule (n = 9; 95% confidence limits 159 - 197; GCV 15%) for LH.

Stability of 10/286

Estimates of the potency of ampoules stored at elevated temperatures for a period of 6 months are summarized in Tables 4 - 7 and Appendix Tables A1.4, A1.7, A1.9, A1.10. Analysis of the thermally accelerated degradation samples in this study gave a predicted 0.001% loss of potency per year for FSH when stored at -20°C, but no consistent loss of activity was detected for the samples stored at +20°C or +37°C for LH.

Conclusions and recommendations

Therapeutic human urinary FSH and LH continues to be marketed by both innovator and

biosimilar manufacturers and is considered a cost-effective option for the treatment of infertility and as part of assisted reproductive technologies. International Reference Preparations (13) and International Standards (14) for urinary derived human menopausal gonadotrophins have been available since the 1960s and are widely used for the determination of FSH and LH potency of therapeutic preparations of human urinary FSH and LH. As a result, stocks of the current, 4^th 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 4^th IS 98/704 allowed statistically valid estimates of relative potency to be calculated from all laboratories, fulfilling the requirement of a replacement international standard in terms of parallelism of assay response with the existing IS. Analysis of assay responses of log10(organ weight/body weight) and log10(organ weight) provided geometric

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mean potency determinations that were in agreement. However, in order to use data from all laboratories, log₁₀(organ weight) was selected as the assay response for assignment of potency.

Using log10(organ weight) the geometric mean potency for the candidate standard was 183 IU per ampoule (n = 10; 95% confidence limits 165 - 202; GCV 15%) for FSH bioactivity and 177 IU per ampoule (n = 9; 95% confidence limits 159 - 197; GCV 15%) for LH bioactivity.

The candidate preparation, 10/286, appears to be sufficiently stable to serve as an international standard. Although these results suggest that 10/286 is likely to be highly stable under long terms storage conditions at -20°C, it is noted that because of the short duration of this study and the lack of detectable degradation for LH, it is impossible to predict the degradation rate of the proposed standard. As a result, it will be a future requirement to complete the assessment of FSH and LH stability in the residual ampoules that have remained stored at elevated temperatures.

Proposal

It is recommended that the preparation in ampoules coded 10/286 be established as the fifth International Standard for human urinary FSH and urinary LH for bioassay, with an assigned potency of 183 IU FSH per ampoule and 177 IU LH per ampoule.

Acknowledgements

We gratefully acknowledge the important contributions of all the participants, Instituto Massone S.A and IBSA Institut Biochimique S.A. who kindly donated the urinary FSH and urinary LH and the Centre for Biological Reference Materials, NIBSC for the preparation of the ampouled materials.

References

1. 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.

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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.

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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.

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Number of Assays

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.

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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.

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Number of Assays

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

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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

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

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Number of Assays

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

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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

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Figure 4: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using log₁₀(organ weight) as assay response

Number of Assays

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

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Appendix 1: Individual Assay Results

Table A1.1: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight/body weight) as assay response

Lab 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.

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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

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Table A1.3: Fitted slopes relative to IS 98/704 for FSH, calculated using log10 (organ weight/body weight) as assay response

(-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

(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

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Table A1.4: Potency estimates for FSH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight) as assay response

(-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

1 = Potency relative to sample D noted, and excluded from overall calculations, as no IS included in this assay

Laboratory 2 estimates were calculated using (organ weight) response due to assay invalidity when using log transformation.

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

Lab 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

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Table A1.6: Fitted slopes relative to IS 98/704 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 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

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Table A1.7: Potency estimates for LH (IU per ampoule), calculated relative to IS 98/704 using log10(organ weight/body weight) as assay response

(-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

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

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

Lab 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