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SummaryAn international collaborative study involving seven laboratories was undertaken to assess the suitability of a freeze- dried preparation of human plasmin to replace the current International Reference Preparation (IRP) for plasmin. Chromogenic and fibrinolytic assays were used by all participating laboratories to assess the potencies of the Proposed International Reference Preparation (PIRP) and two other freeze-dried plasmins, one of human and one of porcine originThe data suggest that the PIRP is a more suitable standard for plasmin than the IRP in that the former binds to fibrin whereas only 50% of the latter binds. The PIRP compared well to other plasmin preparations and the potency assays were independent of the assay procedure and substrate used. Degradation studies indicated that the PIRP was far more stable than the glycerol solution of the IRP, surviving for 12 months at 37° C with no significant loss in either amidolytic or fibrinolytic activity. The International Committee for Thrombosis and Haemostasis (Bergamo, 1982) has recommended the use of this material as a standard and it has been established by the Expert Committee on Biological Standardization of the World Health Organization as the second International Reference Preparation for Plasmin with a defined potency of 10 International Units of Plasmin per ampoule.

The stability of the (W.H.O.) 4th International Standard for Insulin, has been assessed by accelerated thermal degradation studies. This is a crystalline preparation of insulin, freed from proteolytic enzymes, sealed in ampoules containing air and with a moisture content of 5--6%. Of the original biological activity 95.8 (92.8--98.9;P = 0.95)% was retained after storage for 12 years in the dark at 20 degrees C and 65.7 (63.4--68.1;P = 0.95)% after 14 years at 37 degrees C. Degradation rate constants were calculated from these data for the Standard at 20 degrees C and 37 degrees C, assuming first order kinetics. The degradation constant at 37 degrees C did not differ significantly from those found in earlier degradation studies at 37 degrees C over shorter periods, thereby supporting the assumption that the degradation of crystalline insulin, at least at 37 degrees C, is a first order reaction. Extrapolation of these data suggest that the Standard stored at -20 degrees C for 20 years would have retained at least 99.93% (P = 0.95) of its original activity and so for practical purposes can be considered to be stable.

An international collaborative study involving seven laboratories was undertaken to assess the suitability of a freeze-dried preparation of human plasmin to replace the current International Reference Preparation (IRP) for plasmin. Chromogenic and fibrinolytic assays were used by all participating laboratories to assess the potencies of the Proposed International Reference Preparation (PIRP) and two other freeze-dried plasmins, one of human and one of porcine origin. The data suggest that the PIRP is a more suitable standard for plasmin than the IRP in that the former binds to fibrin whereas only 50% of the latter binds. The PIRP compared well to other plasmin preparations and the potency assays were independent of the assay procedure and substrate used. Degradation studies indicated that the PIRP was far more stable than the glycerol solution of the IRP, surviving for 12 months at 37 degrees C with no significant loss in either amidolytic or fibrinolytic activity. The International Committee for Thrombosis and Haemostasis (Bergamo, 1982) has recommended the use of this material as a standard and it has been established by the Expert Committee on Biological Standardization of the World Health Organization as the second International Reference Preparation for Plasmin with a defined potency of 10 International Units of Plasmin per ampoule.

The second International Reference Preparation of Procaine Benzylpenicillin in Oil with Aluminium Monostearate was established in replacement of the first international reference preparation. The blood-level duration test (WHO Requirements for Biological Substances, No. 9), used to evaluate batches of manufactured material in relation to this reference preparation, was revised to relate to the new reference preparation. Thus a batch now passes the test if the lower confidence limit of the ratio is greater than 0.45, instead of 0.50—the value that related to the first international reference preparation. This change will ensure that material passing the test in the future will be of the same quality as in the past.

A collaborative study to determine the suitability of establishing an Author's Preparation of dextran sulfate as an international standard has been carried out. It was concluded from the results that the existing Author's Preparation should not be used for this purpose. The factors governing this decision were (a) that only a small amount of the preparation is now available, and (b) that the amount of material in the ampoules is not as accurately known as is desirable for an international standard owing to the degree of hydration of the original material.The International Unit of Dextran Sulfate has been defined as the activity contained in 0.04 mg of the Author's Preparation.

In 1952, the Department of Biological Standards, National Institute for Medical Research, London, was authorized by the WHO Expert Committee on Biological Standardization to proceed with the establishment of an International Standard for Bacitracin. Thirty grams of bacitracin were obtained and it was decided to compare the proposed International Standard with the existing Working Standard for Bacitracin of the United States Food and Drug Administration.Six laboratories in five countries participated in the collaborative assay. A total of 52 assays was carried out, but the overall weighted mean potency was calculated from only the 48 biological assays, the 4 assays by the Hiscox method being excluded. The biological assays were done by the plate, the cylinder, or the disc method, using Streptococcus pyogenes, Staphylococcus aureus, Corynebacterium xerosis, or Micrococcus flavus.After analysing the results by standard statistical methods, the authors established the batch of bacitracin examined as the International Standard. The Standard contains 55 International Units of activity per mg, and the International Unit has therefore been defined as the activity contained in 0.0182 mg of the International Standard for Bacitracin.

In 1950, the Department of Biological Standards, National Institute for Medical Research, London, was authorized by the WHO Expert Committee on Biological Standardization to proceed with the establishment of an International Standard for Aureomycin. A 100-g batch of aureomycin was obtained and was compared with the Standard Preparation of Aureomycin of the United States Food and Drug Administration (FDA) in a collaborative assay in which six laboratories in five countries participated.In all, 30 assays were carried out; 26 of these were done by biological methods, using Sarcina lutea, Bacillus pumilus, Staphylococcus aureus, or Bacillus cereus, and the remaining four by physicochemical methods. The results were subjected to standard methods of analysis, and the overall weighted mean potency (calculated from the biological assays only) was 1.0139, with limits of error of 99.5% to 100.5%.Since the International Standard is 1.39% more potent than the FDA Standard Preparation, it is probable that the latter contains a small amount of inert material; it is also possible that the International Standard itself is not 100% pure. For most practical purposes, however, both preparations may be regarded as substantially pure, and it is considered that to alter the present practice of quoting aureomycin dosage in metric units of weight would be inadvisable. Nevertheless, since the International Standard may not be a pure substance, a unit notation-for use where required in bioassays-is desirable, and the International Unit of Aureomycin has therefore been defined as the activity contained in one microgram of the International Standard.

In 1953, the Department of Biological Standards, National Institute for Medical Research, London, was authorized by the WHO Expert Committee on Biological Standardization to proceed with the establishment of an International Standard for Thyrotrophin. A number of laboratories were requested to assist in the collaborative study of the proposed new standard, and those which agreed were sent a short note (annexed to the paper) explaining the basis of the study. In this paper, the authors describe the assay methods used by ten laboratories and discuss the results obtained.

In 1950, the Department of Biological Standards, National Institute for Medical Research, London, was authorized by the WHO Expert Committee on Biological Standardization to proceed with the establishment of an International Standard for Dihydrostreptomycin. A 50-g batch of dihydrostreptomycin was obtained and was compared with the Standard Preparation of Dihydrostreptomycin of the Food and Drug Administration (FDA), United States Department of Health, Education and Welfare, in a collaborative assay in which seven laboratories in five countries participated.In all, 76 assays were carried out; 62 of these were done by biological methods, using Staphylococcus aureus, Bacillus subtilis, or Klebsiella pneumoniae, and the remaining 14 by physicochemical methods. The results were subjected to standard methods of analysis, and the overall weighted mean potency (calculated from the results of biological assays only) was 0.958, with limits of error of 99.6% to 100.4%.Since the FDA Standard Preparation is considered to be substantially pure dihydrostreptomycin sulfate, the content of dihydrostreptomycin base (after allowing for known moisture content) is assumed to be 79.2%. The potency of the International Standard has been established as 760 International Units per mg, so that, on the foregoing assumption, one International Unit may be regarded as equivalent to the activity of one microgram of dihydrostreptomycin base.

The authors describe the steps taken to establish the Second International Standard for Corticotrophin and discuss the results of the collaborative assay in which it was compared with the First International Standard. Sixty-one assays of the blended material from two batches of crude corticotrophin prepared by the Astwood procedure, but not subjected to oxycellulose purification, were carried out by 12 laboratories in 5 countries. Almost exclusively, the assay methods used were the thymus involution method and the subcutaneous and intravenous Sayers methods. It was estimated from the statistical analysis of the results that the new standard is 1.14 times as potent as the existing standard, and the International Unit has therefore been re-defined as 0.88 mg of the new standard.

The International Reference Preparation for Vitamin B(12) was prepared in tablet form from a mixture of anhydrous cyanocobalamin and sodium chloride. In an international collaborative study, in which eleven laboratories participated, this preparation was examined qualitatively and quantitatively by spectrophotometric and microbiological methods. It was concluded that the International Reference Preparation is substantially pure and has a potency of 9.3 mug of cyanocobalamin per 20 mg of tablet.

In October 1955, stocks of the Second International Standard for Posterior Pituitary were running low and the Department of Biological Standards of the National Institute for Medical Research, London, was asked to proceed with the arrangements for an international collaborative assay of material for the Third Standard. A single 142-g batch of posterior-pituitary-lobe powder was obtained and distributed in ampoules, in approximately 30-mg quantities. Samples were sent to 19 laboratories in 10 countries. In all, 185 assays were carried out, 122 for oxytocic activity, 53 for vasopressor activity and 10 for antidiuretic activity.On the basis of the results, which were analysed statistically at the National Institute for Medical Research, it was agreed that the potency of the Third Standard (re-named International Standard for Oxytocic, Vasopressor and Antidiuretic Substances in 1956, in view of the recent synthesis of oxytocin and vasopressin) should be expressed as 2.0 International Units per milligram. The International Unit therefore remains unchanged as 0.5 mg of the dry powder.

The study of procaine benzylpenicillin in oil with aluminium monostearate (PAM) described in this paper was undertaken to devise laboratory assay methods, based on animal tests, to evaluate those properties of PAM responsible for the persistence of penicillin in the circulation, as a preferable substitute for the existing test in man. Comparative studies of a number of batches of PAM in man proved, with statistical certainty, that the duration of the blood level of penicillin which they produced varied from batch to batch. The chemical and physical properties of the batches gave inadequate characterization and were of no value in assessing material for clinical use. A blood level duration test in rabbits was developed which reflected the blood level duration properties shown in man. The test was incorporated in the Requirements for Procaine Benzylpenicillin in Oil with Aluminium Monostearate that were set up by the WHO Expert Committee on Biological Standardization to ensure the suitability of batches for clinical use. An International Reference Preparation was necessary and was established. Batches of PAM in use in the field by WHO in anti-yaws campaigns were examined by the test described and found to be satisfactory.

A collaborative study of the second international Reference Preparation of Erythropoietin, Human, Urinary, for Bioassay was carried out in 10 laboratories. Combined potency estimates obtained by comparison with the International Reference Preparation, indicate that ampoules of the second Preparation contain 10.0 IU (weighted mean potency) or, taking the unweighted mean potency, 9.8 IU, with fiducial limits (P=0.95) of 8.4-11.5 IU. The second Preparation could be used as a standard in estimating the potency of a preparation of sheep plasma erythropoietin (68/307) although, as with the International Reference Preparation, there was a tendency for the sheep plasma preparation to produce log-dose-log-response regression lines that were steeper than those produced by the second Preparation.In accelerated degradation studies of the second Preparation stored as the dry product in ampoules for up to 1 year, there was no consistent trend to indicate instability of the preparation.Following its establishment in 1971, the Second International Reference Preparation was allocated a potency of 10 IU/ampoule.

As supplies of the International Standard for Chlortetracycline were practically exhausted, it was replaced. The potency of the second international standard was defined on the basis of an international collaborative assay comprising 157 individual assays performed in 9 laboratories in 6 countries. The mean potencies obtained in the participating laboratories, although they varied by only 7%, were heterogeneous. However, the mean potency for all the laboratories combined did not differ significantly from that of the first international standard. The International Unit for chlortetracycline was therefore defined as the activity contained in 0.001 mg of the second international standard, corresponding to a potency of 1 000 IU/mg.

A batch of highly purified phenoxymethylpenicillin has been examined by eight laboratories in seven different countries, and has been assayed against the phenoxymethylpenicillin standard of the Food and Drug Administration of the US Department of Health, Education, and Welfare. The material examined has been established as the International Standard for Phenoxymethylpenicillin, and the International Unit of Phenoxymethylpenicillin is defined as the activity contained in 0.000590 mg of the International Standard.