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EXPERT CCM-1I'ITEE ON BIOUx;TCAL STANIAZI.RDIZATION Geneva, 12 - 18 Nove~r 1985

F.NGL 1 :~H ONLY

PARATHYROID HOIM:JNE, EUIINE, FOR BIOASSAY Draft Repxt

WHO International Laboratory for Biological Standards

J.M. Zanelli, Division of Hormones and R.E. Gaines~Das, Section of Statistics National Instit'..lte for Biological Standards and Control

London M'13 6 RB, UK

The first International Reference Preparation of Parathyroid Hormone, Bovine, for Bio- assay (IRP of bPTH in ampoules coded 67/342) was ampouled in 1967 and initially had the status of 'Research Standard A'; it was established as the IRP in 1974 (26th ECBS Report, 1975). It was then already apparent that the ampouled preparation also contained substances which interfered with certain in vivo and in vitro bioassays, and the establishment of the

IRP was therefore qualified bya staterrentthat it had been found unsuitable for use in certain assay systems (WHO/BS/74.1078). At that time, it was recommended that a suitable house standard, consisting of highly purified bovine parathyroid hormone, should be used for such assay systems until the IRP was replaced by a standard of rrore highly purified hormone.

Because bPTH was very scarce and costly, such replacement could not be foreseen for several years.

The need for replacement of the IRP, and the difficulties in obtaining suitable material were reviewed in 1981 (WHO/BS/81.1325). In 1983 it was reported (WHO/BS/83.1405) that two candidate preparations (fran two research sources) had been prepared and tested. ^As both of them appeared to be satisfactory, they had been pooled and ampouled. As recommended

(WHO/BS/82.1360) two batches of ampoules have been prepared, 250 ampoules (coded 82/515) each containing approximately 120~1g of the extract and 2500 of ampoules (coded 82/632), each containing approximately lO~g extract.

The international collaborative study involving ll laboratories in 4 countries, including four manufacturers, has now been carried out, the results have bee~ analysed and are summarized in the following pages. This report is to be circulated to the participants.

Preparation of bovine parathyroid horrrone (bPTH) for the proposed replacement IRP

Approximately 25mg purified bPTH was made available through the good offices of Dr Henry Keutmann, Massachusetts General Hospital, Boston, USA. The material was part of a batch extracted in 1980 from pooled fresh frozen bovine parathyroid glands (purchased from a source

in Toronto, Canada), and purified (estimated to be 95% pure) by gel and ion-exchange

chromatographic procedures. The identity and purity of the extract at successive stages of extraction and purification were monitored by an in vitro bioassay (rat renal membrane adenylate cyclase) and in vivo bioassay (chick hypercalcaemia), amino acid Compo6ition analysis and end-terminal analysis (Keutmann et al, 1974). The second donation consisted of 95mg of purified bPTH extracted and purified by Dr J.S. Woodhead, University Hospital, Cardiff, Wales and Dr J .M. Zanelli and Dr N. Kent at NIBSC. The material was part of a batch, extracted in 1982 from fresh frozen bovine parathyroid glands (also obtained from Toronto, Canada). Identity and purity at successive stages of extraction and purification were rronitored by similar bioassays and by high performance liquid chromatography HPU:::

rrethods ( Zanelli et al, 1981). The final product purified by chromatographic procedures was also assessed by amino-acid composition and end-terminal analysis (kindly carried out by Dr Keutmann) and was estimated to be approximately 90% pure.

The issue of this document does not constitute formal publication. It should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone are responsible for v1ews expressed in s1gned articles.

Ce document ne const1tue pas une publication.

II ne dolt faire l'objet d'aucun compte rendu ou resume n1 d'aucune CitatiOn sans l'autorisation de I'Organisat1on monoiale de Ia Sante Les opmions expr1mees dans les art1cles s1gnes n'engagent que leurs auteurs.

WHO/BS/85.1490 page 2

Both batches ~re retested by HPLC and polyacrylamide gel electrophoresis at NIBSC. The batches appeared to be similar and had the characteristics expected of bPl'H extracted and purified by the conventional methods used (Aurbach, 1959). Both contained trace axoounts of the major non-honoonal peptide contaminant <a-chain of bovine haemoglobin> and internediate OKidation products (~OKimately 10-15% of total peptide). The smaller batch assayed at 2040 IU/Jtg of powder (95% confidence limits 1499-2779 IU) after gel chranatography but before final ion exchange chranatography~ the purified larger batch bioassayed at 3243 IU/ng of protein measured by optical density at 276rm, <con£. lim. 2507-4196) when both were included in the same in vivo chick hypercalcaemia bioassays carried out at NIBSC.

In October 1982, the batches of bPl'H were dissolved in O.OlM acetic acid to give a con- centration of lOng/ml. The solutions were pooled, passed through a Millipore membrane filter

< 0. 45lJIIl pore size> and diluted in a pre-£ il tered solution of 0.1% mannitol in 0. OlM acetic acid. In view of the limited cuoount of honrone available, and as previously recaroended

(WfD/BS/82.1360), 250 anp:>ules <code nunbered 82/515) ~e filled by hand with l.Oml. of the solution to give an estimated 300 IU per anpoule, ~oximately 120llg of peptide. This was expected to provide sufficient bPl'H/arlp)ule for the most widely used in vivo (chick hyper- calcaemia> bioassay. The residual solution was then diluted with a solution of mannitol to give a final concentration of 1% mannitol in O.OlM acetic acid~ 2500 anp:>ules <code n\Dber 82/632> were filled with 0.:2ml of solution to give an estimated 25 IU/anpoule, approximately lOllg, assuming that loss of up to 25% could occur because of the extra manipulations and lower concentration of peptide in solution without protein carrier. This was expected to provide sufficient bPl'H/anp:>Ule for the most widely used in vitro <rat renal membrane adenylate cyclase) bioassay. The choice of diluent and bulking agent for 82/515 and 82/632 was the same as that used for the widely used, stable research standard, anpoule code 72/286, details of which are given below. The mean ~ight of solution in each of 50 ~ighed anpoules was 0. 20385g (variance 0. 64%, range 0. 2024-0. 2050g) • Both batches were tested for leaks and

then stored in the dark at -20°C.

All other details of aRiX)uling are as recarmended for preparing international biological standards (Annex 4, 29th EX:BS Report, 1978).

Preparations included in the collaborative stugy

The first IRP for bPTH, ampoule code 67/342, which consists of partly purified bPTH, is known to given ananalous results in the in vivo and in vitro assay systems which were devel- oped during 1972-1974 (\<iiO/BS/74.1078). Although various in vivo and in vitro bioassay systans were used for the current international collaborative study, none of the expert laboratories currently carries out assays with intact or thyroidectomized dogs or parathy- roidectanized rats, as were used in the collaborative study to calibrate the first IRP in 1967-1972. These assay systems are costly, require a high degree of skilled expertise and, being relatively insensitive, are extravagant with Pl'H. OVer the last 15 years they have been replaced by alternative bioassay methods which are less costly, si.npler and more practical, precise and sensitive, but for which the first IRP is not a suitable standard.

For these reasons, as recarmended in WHO/BS/74.1078, a research standard for purified bPl'H has been provided for the newer bioassay systems pending the availability of sufficient purified bPTH for a replacement IRP.

In 1972 a preparation of more highly purified bPTH was ampouled for administration to man for the EllStNOrth Howard diagnostic provocation test. This preparation was distributed

into ampoules with mannitol, freeze-dried and sealed in ampoules by fusion of the glass with the equi{Xllent, precision and freeze-drying conditions as used for preparing biolOgical standards (Annex 4, 29th ECBS Report 1978). It was assayed against 67/342 by i.v. and s.c.

bioassays in chicks and in the Munson rat assay. The estimated potency was 520 units/- ampoule, ( 95% conf. lim. 460-580 > • The preparation (coded 72/286) was found to be sui table for use in those bioassay systems for which the IRP was unsatisfactory. It has since been used widely and extensively as an unofficial international standard with the assigned unitage of 520 IU/arrp for calibration of fiouse standards. The material has apparently remained stable, as judged by HPLC analysis and biological activity in several different bioassay systems in expert laboratories around the world since 1972. Supplies of this batch of ampoules are now virtually finished, the final stock was retained for this collaborative

WHO/BS/85.149(]

page 3 study. i'\ccelerated therma1 degradation bioassay studies carried out after 6 years 1n 1978 gave a predicted rate of Loss of potency of 0.198% per year at -20°C, and 1.4% per year at +4°C.

This research standard, ampoule code 72/286, was therefore included as the standard of bovine P'I'H l-84 for the collo.borative study.

Other preparations intended to be used as research standards and included in the study were synthesized l-34 fragments of human and bovine PTH. The hl-34 PTH (ampoule code 82/508) was donated by Revlon He__alth Care (Tuckahoe, N~ York, USi'\l and the bl-34 PTH (ampoule ccrle 82/512) by Beckman (Palo i'\lto, California, USi'\). Both preparations were of commercial grade,

~re 85-90% pure by amino acid analysis, and were heterogeneous on HPLC. 1\mpouling was carried out as recanrrended for biological standards (i'\nnex 4, 29th ECBS Rep::>rt, 1978).

There has been no previous research or house standard for international use for bl-34 PTH and manufactured batches for clinical use are tested for bioactivity relative to inter- nationally available research standards for the intact hormone extracted from bovine

parathyroid glands, bPTH.

A house standard for the human l-34 PTH fragment was set up jointly in 1975 by Armour Pharmaceutical Company (Kankakee, Illinois, USA) and the Medical Research Council, Division of Biological Standards (Mill Hill, London, UK) to calibrate manufactured batches of hl-34 PTH for a long term multicentre clinical trial. This standard (ampoule code 75/596), was ampouled under the conditions used for preparing international biological standards and compared with the bPTH research standard, 72/286, in the chick hypercalcaemia bioasay. The potency was 520 units/ampoule, (95% conf. lim. 430-630), and 75/595 was assigned a u.nitage of 500 U/ampoule. This peptide appears to be stable and significant loss of biological p::>tency in ampoules stored for up to l year at +40° and +20°C was not detected. Subsequent degrad- ation studies have predicted a loss of potency of approximately 0.4% per year at -20°C.

i'\ summary of the estimates of ampoule contents is given in Table 1. Prior to the collaborative study, the ampouled preparations 82/515, 82/632, 82/508 and 82/512 were tested by in vivo bioassay at NIBSC or by Laboratory 3 (see Table 1). Comparison of bPTH bulk and ampouled products 82/515 and 82/632 by HPLC showed no loss of peptide and slight differences in the peptide profile also found in other ampouled preparations, including the research standard 72/286. These differences may be due to peptide deamidation which occurs during ampouling <Zanelli et al, 1981). The extensive international use and excellent stability of the research standard 72/286 over the last 13 years indicates that these minor impurities are unlikely to cause problems in bioassays. For this reason, amp::>uling conditions for the prep- arations 82/515 and 82/632 were based on those used for 72/286. No changes in HPLC profiles for the bulk and amp::>uled h and b l-34 PTH peptides were detected.

Design of the collaborative study

The participants in the study are listed in alphabetical order in Table 2. The order of listing does not correspond to the code numbers assigned to the laboratory. Where a laoora- tory has contributed data from more than one bioassay system, an additional letter is used to indicate the different systems, eg. 7A, 7B and 7C indicate the systems used by laboratory 7.

Part1c1pants ~re asked to assay the preparations with which they were issued in their routine assay procedure, to include each preparation at three or more doses to test for assumptions of linearity and parallelism, and to use at least 5 animals (for in vivo bioassay) or 3-4 replicates (for in vitro assays) for each dose.

As the number of preparations that could be included in a single assay differed with the bioassay system used by a participant, the priorities for design of assay and type of prepar- ations ~re agreed with each participant. For this reason not all preparations have been compared in all bioassay systems. i'\ summary of the different bioassay systems is given in Table 3.

Statistical analysis

For each assay log dose-response lines ~re examined. In some assays transformation of

WHO/BS/85.1490 page 4

responses gave inproved linearity <legit transfonnation for laboratories 4,5,6,8 and 9, log transformation for laboratory 7A, no transfonnation in the remaining laboratories). Log chse-(transfo~) response lines were examined for linearity and their slopes carpared within assays. In a few instances (sare assays fran laboratories 5,6 and 9) carparisons were limited to the rrore linear central part of the response range so that carparisons were for doses of the different preparations which gave carparable responses. Log potency estimates were cc:aputed as the displacement of parallel log oose-response lines and for each estimate a weight based oo the residual error within assays was calculated (except for laboratories 5 and 9 where weights were based on variation between replicate potency estimates for the same preparation). Potency estimates were examined for heterogeneity using a chi-squared test.

Hanogeneous estimates were canbined as weighted gearetric rreans and heterogeneous estimates were canbined as unweighted gearetric rreans except as otherwise noted.

RESULTS

Ipg dose-response lines: Slopes of log oose-(transformed) response lines are given in Table 4. Where carparison within assays was possible, slopes of log dose-response lines for the bovine Pl'H 1-84 preparations (82/632, 82/515, 72/286 and 67/342) were similar except in lab- oratory 6 where slopes of lines for 72/286 were consistently steeper than those for 82/632.

Slopes of log cbse response lines for the human Pl'H 1-34 preparations ( 75/596 and 82/508) were also similar to one another. There were too few carparisons between the

different types of Pl'H preparation for any general conclusion to be drawn. In sare, but not all, assay systEIIIS there was evidence of non-parallelism of log cbse-response lines for the different preparations (Table 3) •

Potency estimates: the estimates of potency of the preparations examined in this study in terms of the research standard 72/286 are given in Table 5.

The laboratory gearetric rreans for aJrtX>ule content of the proposed standard for bovine PTH 82/515, in terms of research standard 72/286, in in vivo bioassays in laboratories 7A, 7C, and in in vitro bioassays in laboratories 5 and 9 are shown in Table 6. Although limited in nmtber the results appear consistent, are statistically hanogeneous and give a canbined nea.n potency estimate of 292 international units per arcpoule, 92% confidence limits 252-337 IU. This value is in close agreement with the predicted value of 300 IU and the assumption that anpoules contained approximately 120J.Jg of the pooled bulk preparation of 2500 IU/ng.

The laboratory nea.n estimates for anp:>ule content of the proposed standard for bovine PTH 82/632, in terms of research standard 72/286 in variouS in vitro bioassay systems

(laboratories 5,6,8,9,10,12) and in one in vivo bioassay <laboratory 7C) are rore variable, as shown in Table 6. Holo1lever, with the exceptions of laboratories 6,10 and 12, the remainder of the results fran in vitro systems and the in vivo bioassay can be canbined to give a nea.n estimate of 38. 9 IU/aJ'Ii)Oule, 95% con£ idence limits 35. 6-42.6 IU. This value is larger than the predicted value of 25 IU which had allowed for loss of approximately 25% during anpouling of bPTH without protein carrier; the estimates on the finished product indicate that such losses did not cxx:ur and the revised estimate of anpoule content is approximately 15.6lJg of the bulk preparation.

The potency estimates of the aJrtX>Uled synthetic human ( 82/508) and bovine ( 82/512) 1-34 PTH relative to the research standard of intact bovine PTH ( 72/286) were generally consistent but not statistically haoogeneous in the chick hypercalcaemia assay <laboratories 3 and 7C>, by an in vivo xoouse renal cyclic AMP assay <laboratory 7A) and by in vitro renal adenylate cyclase systems using renal nenlxanes fran rat (laboratories 4, > 00g <laboratory 6 and 8 > and man (laboratory 9) (See table 5). In marked contrast, tw::> out of three bioassay systems, one in vivo and one in vitro system (laboratories 7B and 10 respectively> which use a response in bone tissue, showed that both the bovine and human 1-34 Pl'H fragments gave potency estimates in terms of the bPTH research standard 72/286 that were 4-10 times higher than those obtained in the other in vivo and in vitro bioassay systems (Table 5). As noted, the slopes of the log chse-response lines were markedly non-parallel in laboratories 7B and 10. These results are in agreement with observationS reported by Martin et al < 1981 > and Zanelli et al < 1985

>.

The third "bone" assay system, using cloned rat osteogenic sarcana cells <laboratory 5) gave results in agreement with the other in vivo and in vitro bioassay system;. emitting results fran laboratories 7B and 10, the unweighted gearetric nea.n potency estimate for 82/508 (hl-34

WHO/BS/85. 1490 pag0 5

Pill) relative to bPI'H <72/286) was 293 units/ampoule, (95% con£. lim. 250-345). Assswning an ampoule content of 34 pg, the results were in close agreement with the original estimate of potency of bulk material of 10,000 'units'/mg peptide in the chick hypercalcaemia bioassay).

Similarly, omitting results from laboratories 7B and 10, the unweighted geometric mean

potency estimate for 82/512 (bl-34 PTHl relative to bPTH (72/286) was 503 units/ampoule, (95%

conf. lim. 415-608). Assuming an ampoule content of 60 )Jg peptide, the results are in good agreement with the original estimate of potency of approximately 8500 'units'/mg peptide (as obtained in the chick hypercalcaemia bioassay).

The potency estimates for the preparation of hl-34 PTH, 82/508, relative to the original hoQse standard of hl-34 PTH, 75/596, carried out in both in vivo and in vitro bioassay

systems were homogeneous giving a weighted geometric mean potency estimate of 355 units/- ampoule, ( 95% limits 325-388), in agreement with the predicted value (see Table 7). It is also reassuring to note that the estimate relative to bPTH 72/286, which was originally used to assign a potency to the house standard 75/596 in 1975, was not markedly different at 293 units, (95% limits 250-345).

Comparisons of ampoule 82/512 <approximately 60 wg bovine l-34 PTH) with ampoule 82/508 (approximately 34 !Jg ht.nnan l-34 PI'H), are given in Table 9. There are consistent differences in the ratio of the potency estimates - laboratories 1,3 and 7C (chick hypercalcaemia bio- assays) gave higher values for hl-34 relative to bl-34, whereas in contrast, laboratory 4, (in vitro rat renal membrane adenylate cyclase bioassay), gave a higher esteimate for the bovine than for the ht.nnan l-34 peptide. These results confirm published observations which relate to the presence of serine (human) or alanine (bovine) at position l of the PTH rrolecule (Rosenblatt, 1981).

Stability of the proposed standards

Ampoules of bPTH 82/632 which had been stored for 38 months at 45°C or 20°C were com- pared with ampoules of 82/632 stored continuously at -20°C in two in vivo bioassays (system Al in laboratory 7. Using the results from these assays and assuming dependence of the rate of degradation on temperature to be described by the Arrhenius equation, the predicted loss of activity for ampoules stored at -20°C was found to be 0.06% per year. Two assays were also carried out comparing ampoules of 82/515 stored at 37°C for 38 months with ampoules of 82/515 stored continuously at -20°C. Results for this sample were consistent with degrad- ation rates predicted for 82/632. These results are also consistent with degradation rates predicted for the research standard 72/286 as given earlier in this report.

Ampoules of hl-34 PTH 82/508 which had been stored for 38 months at 45°C or 20°C were compared with ampoules of 82/508 stored continuously at -20°C in two bioassays (system C) in laboratory 7. Estimates of relative potency for these did not differ significantly from one another or from l. and no rate of degradation could be predicted. Similar results were obtained when ampoules of bl-34 PTH 82/512 stored for 35 months at 45°C or 20°C were compared with ampoules of 82/512 stored continuously at -20°C.

DISCUSSION

Both of the preparations of bovine PTH (coded 82/515 and 82/632) included in the study, compared with the research standard of bovine PTH (72/286) in a variety of in vivo and in vitro bioassay systems, have given relative potency estimates which are in good agreement with the values predicted from evaluation of the bulk materials.

The preparation of human l-34 PTH fragment (coded 82/508) compared with the previous house standard for this peptide (75/596) and with the research standard of bovine PTH (72/286) gave reasonably consistent results in the bioassay systems based on hypercalcaemia in chicks and activation of renal adenylate cyclase or cAMP production in vivo and in vitro.

There was marked discrimination between the l-34 fragments (human and bovine) and the l-84 intact bPTH rrolecule in the two bone tissue bioassay systems, showing that bone tissue in these systems responded to the whole peptide quite differently from its response to the amino terminal fragmenLs.

The estimates for the bovine l-34 PTH <82/512) in terms of the human l-34 PTH 75/596,

WHO/BS/85. 1490 page 6

<Nere rrore variable than those for the hwmn 1-34 PI'H ( 82/5 08), showing that sare bioassay systems discriminated between the bovine and human l-34 PrH fragments.

Proposed standard and assignment of potency

It is proposed that the material in ampoules coded 82/632 is suitable to be established as the International Standard for parathyroid hormone, bovine. It is proposed that a unitage of 39 International Units be assigned to each ampoule of the preparation 82/632 bovine PTH for in vitro bioassay. It is further proposed that a unitage of 290 International Units be assigned to each ampoule of the preparation 82/515, bovine PTH for in vivo bioassay in order to maintain continuity of the International Unit as measured in current in vivo bioassay systems.

1-34 PTH fragments

It is recognized that synthetic preparations of hwmn and bovine 1-34 fragments have been available fran several manufacturers for several years, but that in the absence of an international standard for either peptide, the potency of manufactured batches has been expressed in "International Units" relative to internationally available standards of intact bovine PTH 1-84. Continued use of such inappropriate "units" will cause great confusion. It is recarrrended that each peptide should be assayed against its own standard. As synthetic peptides which are essentially rronocanponent are now becaning available, it is reccmrended that the ampouled preparations of hl-34 (82/508) and of bl-34 PTH (82/512) should be made available as calibrated research standards for international distribution, pending the availability of rrore highly purified materials as candidate international standards. (Since carrying out this study, a sufficient arrount of highly purified human 1-34 PI'H has been offered for this purpose).

Acknowledgenents

Grateful acknowledgements are due to Dr H. Keutmann (MassachQsetts General Hospital, Boston, USA) and Dr J.S. Wcxxihead (University Hospital, Cardiff, UK) for donating the two batches of bovine parathyroid horrrone; Ms Jane Malone (Beckman Bioproducts, California, USA) for donating the synthetic bl-34 PTH fragment~ Dr C. Bassinger (Revlon Health Care, Tuckahoe, NY, USA) for donating the synthetic hl-34 PTH fragment~ and to all the participants of the collatxxativ•:: study.

References

Aurbach GD ( 1959). Isolation of parathyroid honnone after extraction with phenol. ,J Biol Chern 234:3170-81.

Keutmann HT (1974). The cnemistry of parathyro1d honnone. Clin Endocrinol Metab 3:173-197.

Martin KJ, Bellorin-Font E, Freitag J, Rosenblatt M & Slatopolsky E tl98l). The arterio- venoQs difference for immunoreactive parathyroid hormone and the production of adenosine 3'5' rronophosphate by isolated perfused bone: studies with analogues of parathyroid hormone.

Endocrinology 109:956-959.

Rosenblatt M (1981). Parathyroid horrrone: chemistry and structure-activity relations. In:

Pathobiology Annual Vol II (Ed. HL loachim) Raven Fress:New York, pp53-85.

Zanelli ,JM, O'Hare MJ, Nice EC & Corran PH <l98ll. Purification and assay of bovine para- thyroid honnone by rev;~rsed phase high performance Liquid chraratography. J Chranatog 223:59-67.

Table 1 Materials for the study ule

82/632

(in vitro bioassay) 82/515

(in vivo bioassay) 82/512 82/508 72/286

Research Standard 67/342 IRPbPTH for Bioassay

75/596 MRC Clinical Reference Preparation

Assurred ule content Awrox 1 rurole ( l0Jlg l bPTH, with 2mg mannitol.

Awrox 12 ruroles ( l20)1g l bPTH, with 2mg mannitol.

Approx 15 ruroles ( 6011g l bl-34 PTH fragment, 2mg mannitol.

Approx 8.5 ruroles (34wgl hl-34 PTH fragment, 2mg mannitol.

Awrox 200!Jg ( 20 ruroles l bPTH, 2.5mg mannitol.

Awrox 0.6mg TCA bPTH powder, 5mg lactose. NB: This material is known to give anomalous results in in vivo and in vitro systems in current use Awrox l0011g ( 25 ruroles l hl-34 PTH fragment, 5mg mannitol.

Biol

Expected 25 IU Expected 300 IU

WHO/BS/85. 1490 page 7

Expected* 300 "units"

Expected 300 "units"

Assigned 520 IU Defined 200 IU

Assigned 500 "units"

*Expected bioactivity may only be relevant to the in vivo chick hypercalcaemia (intravenous injection), bioassay; all samples were bioassayed relative to 72/286 bPTH or 75/596 hl-34 fragment in this bioassay system prior to issue in the collaborative study.

Table 2 Participants in the collaborative study

Dr J P Aldred, Arrrour Pharmaceutical Canpany, Kankakee, Illinois 60901, USA.

Dr B Auf'mkolk, Abteilung Klinische Endokrinologie, Medizinische Hochschule, D-3000 Hannover 61, West Germany.

Dr C G Dacke, Physiology Department, University of Aberdeen, Marischal College, Aberdeen AB9 lAS, Scotland.

Dr J Dietrich, Revlon Health Care Group, Tuckahoe, New York 10707, USA.

Dr J Elting & Dr A Lindall, ImmunoNuclear Corporation, Stillwater, Minnesota 55082, USA (Dr Elting is now at Molecular Diagnostics Inc, West Haven, Connecticut 06516, USA.

Dr H T Keutmann, Endocrine Unit, Massachusetts General Hospital, Boston, Mass. 02114, USA.

Dr N Loveridge, Division of Cellular Biology, Mathilda & Terence Kennedy Institute for Rheumatology, London W6 7[J(J, UK.

Dr R Nissenson, Department of Medicine, Veterans Administration Hospital, San Francisco, California 94121, USA.

Dr L G Raisz, Endocrinology & Metabolism, University of Connecticut Health Centre, School of Medicine, Farmington, Connecticut 06032, USA.

Dr H. Yamauchi, Product Planning Department, Toyo-Jozo Company, Minato-ku, Tokyo 108, Japan.

Dr J M Zanelli & Miss E Lane, National Institute for Biological Standards & Control, Hampstead, London NW3 6RB, UK.

\-'lHO/BS/85.1490 page 8

Table 3 Bioassay systems used

r

Type of bioassay In vivo

Chick hypercalcaemia, subcutaneous dose administration Chick hypercalcaemia, intravenous dose administration Japanese Quail hypercalcaemia iv dose administration M::>use renal cAMP

~use bone (calvaria) cAMP In vitro

Rat bone resorption

Rat osteogenic sarccma cloned cell line Rat renal membrane adenylate cyclase Canine renal membrane adenylate cyclase Hunan renal nanbrane adenylate cyclase Guinea-pig renal glucose-6-phosphate

dehydrogenase cytochemical bioassay

Reference

a a b c c

d e f g,h

j k,l

Laboratory

l 7C, 11

3 7A 78

10 5 4 6, 8

9 12

L ________________________________________

References

a Parsons, Reit & Robinson. Endocrinology 92:454, 1973 (modified) b Dacke & Kenny. Endocrinology 92:463, 1973 (modified)

c Zanelli, Lane, Kimura & Sakakibara. Endocrinology, in press.

d Raisz & Niemann. Endocrinology 85:446, 1969 (modified)

e Lindall, Elting, Ells & Roos. J Clin Endo Metab 57:1007, 1983 f Marcus & Aurbach. Endocrinology 85:801, 1969 (modified)

g Galt~, Callahan, Tregear & Potts. Endocrinology 103:1352, 1978

h Nissenson, Abbott, Teitelbaum, Clark & Arnaud. J Clin Endo Metab 52:840, 1981 j Niepel, Radeke, Atkinson, Jueppner & Hesch. J lmmunoasay 4:21, 1983

k Chambers, Dunham, Zanelli, Parsons, Bitensky & Chayen. Clin Endocrino1 9:375, 1978 1 Golt~n, Henderson & Loveridge. J Clin Invest 65:1309, 1980

WHO/BS/85.1490 Table 4. Slopes of log dose-( transfonred l response lines page 9

Laboratory Bovine PI'H l-84 Bovine PI'H l-34 Hwnan PI'H 1-34

Assay 72/286 82/632 82/515 67/342 82/512 75/596 82/508

l 1 3.60 3. 38 3.05

2 2.95 3.27 3.08

3 1.33 2. 98 2.91

4 2.87 4. 79 3.40

3 l 0.60 0.92 1.10

2

o.

3 0. 73 0.80 0. 72

4 A 0.99 0.86

B 0.86 0.97 0. 84

l 0.88 0.66 0. 77

2 0. 84 ^l. 20 1.11

5 1 -1.64 ( -1.75 -1.31 -1.91 -1.90 -1.52 ( -1.76

( -1.70 (-1. 51

6 1 1.64 1.17 1.29 1.37

2 2.13 l. 27 1.39 l. 28

3 2.41 1.45 1.36 1.26

7A 1 0.50 0.23

2 0.42 0.41 0.43

3 0.57 0. 29 0.34

4 0.55 0.52 0.60

5 0.47 0.49 0.51

6 0.47 0.40 0.46 0.56

7 0. 51 0.45 0.56 0.57

7B l 30 80 147

2 24 61 73

7C 1 0.68 0.42 0.62

2 0.31 0.24 0.48

3 0.59 0.75

4 0.52 0.57

o.

5 0.52 0.40 0.63

6 0.63 0.84 0. 74 0.86

7 0.70 0.37 0.11 0.34 0.22

8 0.61 0.55

8 l 1.20 1.21 1.12 1.08

2 l. 23 l. 22 ^l. 34 1.05

9 1 0.86 0.51 0. 72 0. 77 0.79 0.74 0.74

2 0.98 l. 08 0.88 (0.93 1.21 0.88 l. 06

( 0.99

10 1 14.2 20.4 11.6 14.1

2 37.6 44.2

3 39.4 69.3

11 l 6.48* 2.89

2 2.49* 2. 76

3 4.07* 3.89

12 l 11.8 12.0

2 15.0 13.1

3 21.1 19.9

* In house standard calibrated in terms of 75/596

WHO/BS/85.1490 page 10

Table 5 Potency of ampouled materials as units of 72/286 per ampoule

Laboratory Assay 83/632 82/515 67/342 75/596 82/508 82/512

3 ^l S73 503

2 310 254

3 311

225

4 B 224 154

5 63.5 227 228 531 360 938

28 469

6 l 68.0 223 592

2 79.7 285 821

3 97.7 188 630

7A l 29.4

2 171 680

3 281 461

4 35.1 339

5 33.6 279

6 46.5 361 663

7 44.0 258 620

78 l 797** 1110**

2 1730** 1900**

7C 2 363 534

3 395

4 427 387

5 415 277

6 337 612 377

7 522 468 198 322

8 30.1

8 ^l 43.3 235 561

2 39.2 159 457

9 ^l 26 .l 289 78.4 247 374 672

2 38.2 203 66.9 358 383 624

84.7

10 l 81.5 6090* 7640*

2397

12 ll. 7

2 15.8

3 15.4

* D1fferent response range from 82/286

** calibration l::Bsed on non-parallel log dose-response lines

WHO/BS/85.1490

page 11 Table 6 Laboratory geometric mean estimates for ampoule content of 82/515 and 82/632 expressed in units of 72/286 per ampoule.

Laboratory 82/515

Lab. mean No. assays Weight

5 277

6

7A 298

7C 364

8

9 240

10 12

Canbined 292 95% Confidence 252-337 Interval

* Onitted fran canbined rrean

l 100

2 539

2 l34

2 190

7 963

82/632

Lab. mean No. assays Weight

42.2 1 100

80.8* 3 760

38.4 5 998

30.1 1 40

40.4 2 1450

31.9 2 190

81.5* l 40

14. 7* 3 495

38.9 11 2780

35.6-42.6

Table 7 Laboratory geometric rreans for ampoule content of 82/508 as units of 75/596 (taken to be 500 units per ampoule).

Labor a ton: Lab. mean No. assays Weight

l 334 4 860

4 343 l 500

5 387 l 100

7C 313 3 169

9 630 2 190

l l 344* 3 780

Canbined 355 14 2600

95% Limits 325-388

* Units of laboratory standard calibrated in tenns of 75/596

::;

of arrpouh:s of 82/Sfl<i --: ^c.:;,

Table 8 Potency of arrpoules 82/515 and Table 9 Potency estinates ^--~ _to

82/508 expressed as units of 75/596 (taken to expressed as equivalent arrpou1es of 82/512

-·

contain 500 units per aJllX)ule). ^co· _lJl

Laboratory Assay

Laboratory Assay 82/508 82/512 _1.0 ^~

(h1..1Il'ai1) (bovine) 1 1 1.680 ⁰

2 3.140

1 1 280 167 3 1.440

2 384 122 4 2.390

3 342 237

4 336 141 3 1 1.140

2 l.220

4 ^B 343 3 1.380

5 l (339 883 4 1 0.124

( 441 2 0.152

7C 1 414

s

6 309

7 212 344 6 1 0. 377

2 0.348

9 1 756 1360 3 0.299

2 535 873

7A 2 0.250

l l 1 314* 3 0.609

2 322* 6 0.545

3 380* 7 0.416

• Units labor a lory standard calibrated 78 1 0.286

in tenrt, of 75/)96 2 0.4lL

7C 2 0.681

4 1.100

5 1.495

7 0.617

8 1 0.420

2 0.349

9 1 0.556

2 0.613

10 1 0.797