An ECVAM pre-validation study of physicochemical methods for predicting the biological potency of recombinant follicle stimulating hormone.

A pre-validation study was carried out, by six laboratories from six countries, of two physicochemical methods for predicting the in vivo biological potency of recombinant follicle stimulating hormone (follitropin beta), based on quantitative measures of isoform distribution by isoelectric focusing (IEF) and by capillary zone electrophoresis. Each of these methods was used to estimate the predicted bioactivities of four preparations of follitropin beta differing widely in their isoform compositions and specific bioactivities. The results of this study indicate that these methods, and particularly IEF, are transferable between laboratories, and produce results which are sufficiently accurate, precise, and reproducible, for them to be used for predicting the bioactivity of follitropin beta, especially if used with a standard preparation. The performance of these two methods for predicting the bioactivity of other types of follicle stimulating hormone, such as follitropin alfa, would need to be assessed separately, and might involve quantitatively different relationships between the responses measured in the physicochemical method and the bioactivities of preparations estimated by bioassay.

The fourth International Standard for Human Urinary FSH and LH: specificities of LH seminal vesicle weight gain assays in the collaborative study differ between laboratories.

The fourth International Standard for Human Urinary FSH and LH (IS; in ampoules coded 98/704) was compared with the third International Standard for Urinary FSH and LH (IS 71/264) by 10 laboratories in nine countries, using FSH and LH in vivo bioassays. Estimates of the FSH content of the IS by augmented ovarian weight gain assays were homogeneous within each laboratory and over all laboratories. The combined weighted geometric mean estimate of FSH content of the IS (with 95% fiducial limits) in terms of IS 71/264 was 71.9 (69.0-74.9) IU/ampoule. Although estimates by seminal vesicle weight gain (SVW) assays of the relative LH activities of the IS and IS 71/264 were homogeneous within laboratories, estimates were heterogeneous between laboratories. This indicated differences between the spectrum of LH isoforms in the IS and IS 71/264, which were obtained from different manufacturers, and differences between the specificities of SVW assays performed in different laboratories. The differences between the specificities of SVW assays appeared to be related to interactions among mean laboratory seminal vesicle weights, age and genetic strain of rat. The finding of inter-laboratory differences in the specificities of SVW assays is of some significance, as this assay method has been generally adopted by Pharmacopoeias for the control of the LH content of therapeutic products. The combined unweighted geometric mean estimate of LH content of the IS (with 95% fiducial limits) in terms of IS 71/264 by SVW and ovarian ascorbate depletion assays was 70.2 (61.7-80.0) IU/ampoule. Estimates of the FSH and LH content of ampoules of the IS kept at increased temperatures suggested that the IS would be adequately stable under normal storage conditions. On the basis of these results, the World Health Organization Expert Committee on Biological Standardization established the preparation in ampoules coded 98/704 as the fourth International Standard for Human Urinary FSH and LH, and assigned to the contents of each ampoule an activity of 72 International Units of urinary FSH and an activity of 70 International Units of urinary LH.

Epoetin alfa and beta differ in their erythropoietin isoform compositions and biological properties.

Epoetin alfa and beta are the two forms of recombinant DNA-derived erythropoietin (rEPO), both synthesized in Chinese hamster ovary cells, which are used for the treatment of erythropoietin (EPO)-responsive anaemias. Several batches of each of these rEPOs were compared for differences in their EPO isoform compositions by isoelectric focusing (IEF) and in a range of lectin-binding assays, and for differences in their EPO activities by in-vivo and in-vitro mouse bioassays and by immunoassay. Epoetin beta was found to differ from epoetin alfa in containing: (a) a greater proportion of more basic isoforms, (b) a greater proportion of EPO binding to Erythrina cristagalli agglutinin (which binds N-glycans with nonsialylated outer Gal beta1-4GlcNAc moieties), and (c) isoforms with higher in-vivo:in-vitro bioactivity ratios. Epoetin beta also contained slightly more than epoetin alfa of EPO binding to Lycopersicon esculentum agglutinin (which binds N-glycans containing repeating Gal beta1-4GlcNAc sequences), to the leucoagglutinin of Phaseolus vulgaris (which binds tetraantennary and 2,6-branched triantennary N-glycans) and to Agaricus bisporus agglutinin (which binds Gal beta1-3GalNAc containing O-glycans). No differences were found between the two rEPOs in their binding to a further five lectins. The differences between the isoform composition of epoetin alfa and beta, and the smaller inter-batch differences appear to be due to differences in glycosylation. The higher murine in-vivo:in-vitro bioactivity ratio of epoetin beta compared to epoetin alfa could not be explained in terms of differences in their degrees of sialylation, but was consistent with differences in their pharmacokinetics and pharmacodynamics observed in human subjects. There have been no reports that epoetin alfa differs from epoetin beta in its clinical efficacy, but the differences between epoetin alfa and beta in some analytical systems suggest that there might be a need for separate international standards for these two types of rEPO.

Lectin-binding assays for the isoforms of human erythropoietin: comparison of urinary and four recombinant erythropoietins.

Assays have been developed for the isoforms of erythropoietin (EPO) based on their binding to eight different lectins. These assays were used to compare the isoform compositions of two preparations of human urinary EPO (uEPO) and four preparations of recombinant DNA-derived human EPO (rEPO), which had been shown to differ in their biological and immunological properties and in their isoform composition as judged by isoelectric focusing and electrophoresis. Agarose-bound Ricinus communis agglutinin I (RCA), Erythrina cristagalli agglutinin (ECA), Maackia amurensis leukoagglutinin (MAL), Sambucus nigra agglutinin (SNA), Lycopersicon esculentum agglutinin (LEA), concanavalin A (Con A), Phaseolus vulgaris agglutinin-L4 (L-PHA) and Agaricus bisporus agglutinin (ABA) were used to bind EPO isoforms possessing: N-glycans containing non-sialylated outer Gal beta 1-4GlcNAc (RCA and ECA), NeuAc alpha 2-3Gal beta 1-4GlcNAc (MAL), NeuAc alpha 2-6Gal (SNA), or repeating Gal beta 1-4GlcNAc sequences (LEA); biantennary N-glycans (Con A); tetraantennary and 2,6-branched triantennary N-glycans (L-PHA); and O-glycans containing NeuAc alpha 2-6GalNAc (SNA) and Gal beta 1-3GalNAc (ABA). Free EPO was measured by mouse spleen cell bioassay or immunoassay. Estimates from most lectin-binding assays were reproducible between assays and batches of lectin-agarose, although batches of MAL- and ABA-agarose, and to a lesser extent LEA-agarose, differed in their EPO-binding. Lectin-binding assays showed differences between the isoform compositions of all EPOs, including the two Chinese hamster ovary cell-derived rEPOs, with RCA- and ECA-binding assays being the most discriminating. Lectin-binding estimates provided evidence that uEPO differs from these rEPOs in its lower content of isoforms with biantennary N-glycans and higher content of those with multiantennary N-glycans, and in its lower content of isoforms with N-glycans possessing repeating Gal beta 1-4GlcNAc sequences and of those with O-glycans containing Gal beta 1-3GalNAc. Lectin-binding estimates also indicated that, contrary to some reports, uEPO possesses Gal beta 1-3GalNAc-containing O-glycans but not NeuAc alpha 2-6GalNAc-containing O-glycans or NeuAc alpha 2-6Gal-containing N-glycans. Most groups of lectin-bound EPO isoforms did not differ in their relative bioactivities and immunoreactivities. However, estimates for ABA-bound EPO isoforms suggested that O-glycans might influence the bioactivity of EPO differently to its immunoreactivity. Furthermore, the bioactivities of some ECA-bound EPO isoforms were higher, and those of some of the MAL-bound EPO isoforms lower, than their immunoreactivities, consistent with the reported enhancement of EPO in vitro bioactivity by desialylation.

Application of IUPAC-IFCC recommendations on quantities and units to WHO biological reference materials for diagnostic use. International Union of Pure and Applied Chemistry (IUPAC) and International Federation of Clinical Chemistry (IFCC).

This document deals with the nature of WHO biological reference materials, their development for the control of therapeutic substances and recommendations to improve their application in diagnosis. The nature of international units specified by WHO biological reference materials is contrasted with that of SI units, and the method for assigning values in international units to such reference materials is described. The document recommends the use of SI units (mole) with existing and proposed WHO biological reference materials whenever the elementary entity of the stated component can be recognized. It also recommends that the description of quantities having no recognized kind-of-quantity with a definable dimension should be clearly distinguished by the term "arbitrary" and include a reference to the procedure and calibrator used. The WHO is urged to involve appropriate non-governmental organizations in advising on the need for, and the suitability of international reference materials.

Application of IUPAC-IFCC recommendations on quantities and units to WHO biological reference materials for diagnostic use: recommendations 1994.

This document deals with the nature of WHO biological reference materials, their development for the control of the therapeutic substances, and recommendations to improve their application in diagnosis. The nature of international units specified by WHO biological reference materials is contrasted with that of SI units, and the method for assigning values in international units to such reference materials is described. The document recommends the use of SI units (mole) with existing and proposed WHO biological reference materials whenever the elementary entity of the stated component can be recognized. It also recommends that the description of quantities having no recognized kind-of-quantity with a definable dimension should be clearly distinguished by the term "arbitrary" and include a reference to the procedure and calibrator used. The WHO is urged to involve appropriate nongovernmental organizations in advising on the need for, and the suitability of, international reference materials.

The Second International Standard for Human Pituitary LH: its collaborative study by bioassays and immunoassays.

The second International Standard for Human Pituitary LH (in ampoules coded 80/552; 2nd IS) and LH 81/535 (prepared in the same way as the 2nd IS from the same LH preparation) were compared with the International Reference Preparation of Human Pituitary LH for Immunoassay (IRP 68/40) by 19 laboratories in 11 countries, using in-vivo and in-vitro bioassays, a receptor assay and immunoassays. Geometric mean estimates of the LH content of the 2nd IS (with 95% fiducial limits) in terms of IRP 68/40 were: 34.6 (29.1-41.0) IU/ampoule by in-vivo bioassays; 35.8 (27.0-47.4) IU/ampoule by in-vitro bioassays; 58.6 IU/ampoule by one receptor assay; and 36.8 (35.5-38.1) IU/ampoule by immunoassays. The close agreement between the relative activities of the 2nd IS and IRP 68/40 in the wide range of assay systems studied appears to reflect the fact that both standards contain highly purified LH with similar isoform compositions as judged by isoelectric focusing. Estimates of the LH content of LH 81/535 in terms of IRP 68/40 and in terms of the 2nd IS tended to be lower than those for the 2nd IS across all methods, but the differences were not statistically significant. The 2nd IS was found to be as suitable as IRP 68/40 as a standard for the in-vitro bioassay and immunoassay of LH in the two serum samples studied. However, the mean estimates of serum LH in terms of either of these standards were more than 150% larger by in-vitro bioassays than by immunoassays and more than 50% larger by one-site than by two-site immunoassays.(ABSTRACT TRUNCATED AT 250 WORDS)

Assaying glycoprotein hormones--the influence of glycosylation on immunoreactivity.

The epitopes of the human glycoprotein hormones (follicle-stimulating hormone [hFSH], luteinizing hormone [hLH], chorionic gonadotrophin [hCG], thyroid-stimulating hormone [hTSH] and erythropoietin [hEPO]) appear to consist only of peptide components. Their interactions with antibodies, however, are influenced by their bulky and often highly charged carbohydrate moieties. Thus, isoforms of these hormones (the majority of which are glycoforms) differ in their specific immunoreactivities as well as in their specific in vivo and in vitro bioactivities. This can create difficulties for the standardization of immunoassays as the isoform composition of a hormone depends both on its source and method of isolation.

The International Standard for Recombinant DNA-derived Erythropoietin: collaborative study of four recombinant DNA-derived erythropoietins and two highly purified human urinary erythropoietins.

The International Standard (IS) for Recombinant DNA-Derived (rDNA) Erythropoietin (EPO) (in ampoules coded 87/684) and three other rDNA EPO preparations in ampoules coded 87/690, 87/696 and 88/574 respectively, were compared with two preparations of highly purified human urinary (HU) EPO and the 2nd International Reference Preparation of Human Urinary Erythropoietin for Bioassay (2nd IRP) by 26 laboratories in 11 countries using a wide range of in-vivo and in-vitro bioassays and immunoassays. These EPO preparations were also compared by electrophoresis and isoelectric focusing. Estimates of EPO content in terms of the 2nd IRP by all in-vivo bioassay methods gave combined unweighted geometric means (with 95% fiducial limits) of: 86 (75-99) IU/ampoule for the IS, 81 (70-94) IU/ampoule for 87/690, 58 (48-71) IU/ampoule for 87/696 and 120 (100-143) IU/ampoule for 88/574. Mean estimates of EPO content in terms of the 2nd IRP by in-vitro bioassays (except receptor assays) were larger than, and those by immunoassays were similar to, the mean estimates by in-vivo bioassays. The use of purified rDNA or HU EPO as standards in place of the 2nd IRP reduced the inter-laboratory variability of estimates of purified EPO preparations by in-vivo and in-vitro bioassays and by immunoassays, and reduced the variability of overall mean estimates for each of these preparations between the three types of method. The inter-laboratory variability of immunoassay estimates of human serum EPO was similar whether the 2nd IRP or one of the purified EPOs was used as standard. Significant differences in in-vivo and in-vitro biological, immunological and physicochemical properties were found between these four rDNA EPO preparations and between them and the HU EPO in the two purified preparations and in the 2nd IRP. There were also differences between the immunoreactivities of the two serum EPO samples included in the study, and between them and the immunoreactivities of the purified EPOs. The differences between rDNA EPOs appeared to be related to differences between the cells used for their biosynthesis, but may also be the result of differences in purification methods and of inter-batch variations. Significant differences in assay specificity were observed within each of the three general types of method. The specificity of the in-vivo bioassays was influenced by the route of hormone administration. The specificities of the mouse spleen cell in-vitro bioassays differed from that of the mouse spleen receptor-binding assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparisons of human, rat and mouse erythropoietins by isoelectric focusing: differences between serum and urinary erythropoietins.

Isoelectric focusing (IEF) in the pH range 2.5-5.0 has been used to compare the immunoreactive (ir) erythropoietin (Epo) in paired samples of serum and urine from three patients, two with idiopathic aplastic anaemia and one with paroxysmal nocturnal haemoglobinuria and also from three anaemic rats. Serum samples only were also examined from two further patients with aplastic anaemia and from three mice, made anaemic (like the rats) by irradiation and phenylhydrazine treatment. Most of the ir-Epo recovered after IEF was found in the pH range 2.5-3.9. For the sera, the proportion of more acidic ir-Epo with pI less than 3.0 recovered after IEF increased from human to rat to mouse. Human sera contained a greater proportion of ir-Epo with pI greater than 3.4 than rat or mouse sera. For the urines, the distribution of ir-Epo by IEF was similar between human and rat. For both species, the proportion of ir-Epo with pI less than 3.0 recovered after IEF was greater in urine than in the paired serum samples. The Second International Reference Preparation of Human Urinary Epo differed from the Epo in unextracted human urine in that there was a lower proportion of ir-Epo with pI less than 3.0. The differences observed between serum and urinary Epo are of particular interest because only the urinary form of native human Epo has ever been purified, and because this was used to compare native with rDNA-derived Epo.

The International Standard for Pituitary FSH: collaborative study of the Standard and of four other purified human FSH preparations of differing molecular composition by bioassays, receptor assays and different immunoassay systems.

The International Standard for Pituitary FSH (IS; in ampoules coded 83/575) was assayed in terms of the Second International Reference Preparation of Human Pituitary FSH and LH for Bioassay (IRP 78/549) by 27 laboratories in 13 countries using bioassays, receptor assays and immunoassays. Estimates of the FSH content of the IS by in-vivo bioassay were homogeneous both within and between laboratories and gave a combined geometric mean (with 95% fiducial limits) of 79.9 (74.6-85.4) i.u./ampoule. Estimates by different in-vitro bioassays and receptor assays were also homogeneous between assays and laboratories, and gave a combined geometric mean (with 95% fiducial limits) of 31.2 (28.8-33.9) i.u./ampoule. However, estimates by the 19 different immunoassay systems were heterogeneous and varied between 5 and 31 i.u./ampoule. The material in ampoules coded 83/575 was established by the World Health Organization as the International Standard for Pituitary FSH. It was assigned a unitage of 80 i.u./ampoule on the basis of its calibration by in-vivo bioassay, because this assay best identifies and defines the hormone. However, the introduction of the new IS will necessitate the recalibration of immunoassay kits. FSH 84/530, prepared in the same way as the IS from the same FSH preparation, did not differ significantly from the IS in any of the assay systems studied and appeared to be equally suitable as a standard. Four highly purified preparations of human FSH (FSH A-D), differing in their isoform compositions and in their in-vivo: in-vitro bioactivity ratios, were also studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma clearance in the rat of the LH bioactivity of two human LH standards of differing molecular composition.

The LH biological potency of the International Reference Preparation (IRP) of Human Pituitary LH for Immunoassay (IRP 68/40) relative to that of the 2nd IRP of Human Pituitary FSH and LH for Bioassay (IRP 78/549) is markedly greater when estimated by in-vitro interstitial cell testosterone production (TICT) bioassay than by in-vivo bioassay, and by the 4-h ovarian ascorbate depletion (OAAD) assay than by the 4-day seminal vesicle weight gain assay. Other preparations of human LH which, like IRP 68/40, were highly purified, showed a similar spectrum of bioactivity in these assay systems and also contained a higher proportion of more basic LH isoforms than are found in crude pituitary extracts such as IRP 78/549. In an attempt to explain these differences, a comparison was made of the plasma survival in rats of the LH bioactivity (by TICT assay) of these two preparations. Contrary to expectation, their relative plasma clearance rates over a 4-h period did not account for their differing bioactivities. The plasma half-life of the LH bioactivity (with 95% confidence limits) was estimated to be 42.4 (35.3-49.5) min for IRP 68/40 and 41.3 (31.5-51.0) min for IRP 78/549. Furthermore the time-course of action in vivo of IRP 78/549 did not appear to be more prolonged than that of IRP 68/40. Thus their plasma testosterone responses during the course of these 4-h plasma clearance studies were similar, and estimates of the LH potency of IRP 68/40 relative to that of IRP 78/549 were no greater by 2-h than by 4-h OAAD assay.(ABSTRACT TRUNCATED AT 250 WORDS)

A radioassay for nonoxidized methionine in peptides. A method for identifying (after isoelectric focusing) and for estimating biologically active forms of corticotropin and other hormones.

A radioassay for nonoxidized methionine in peptides is described; it has advantages over other methods currently used because of its simplicity, sensitivity, accuracy, and applicability to individual peptide components in mixtures and to many samples at a time. Methionyl residues were S-carboxymethylated with iodo[2-14C]acetic acid; iodo[2-3H]acetic acid did not provide a stable radioactive tracer. The labeled peptide was isolated by carboxymethylcellulose chromatography or by isoelectric focusing (IEF) or electrophoresis in polyacrylamide gel, and its radioactivity measured. The assay was applied to corticotropins, alpha-melanotropin, bombesin, glucagon, substance P, parathormone, and calcitonin. Twenty-four to thirty samples were conveniently analyzed at a time with a lower detection limit of less than 1 nmol of methionine per sample. The accuracy of the assay, assessed also by reverse-phase high-performance liquid chromatography, is a consequence of its precision, the specificity of the reaction with iodoacetic acid, and the use of an appropriate standard of the peptide being assayed. Methionine was identified, and could be estimated, in individual peptide components of a mixture by using IEF to separate simultaneously the labeled peptide from iodo[2-14C]acetic acid and from other peptide and protein components. This was facilitated by a convenient method for detecting and quantifying these peptides after IEF. The assay is particularly useful for several peptide hormones whose biological activity depends on their sole methionine residue being in a nonoxidized state. It can be used for monitoring their isolation or synthesis and their stability during processing and storage, as well as for evaluating differences in biological potency between preparations and analogues.

The International Reference Preparation of Tetracosactide for Bioassay: characterization and estimation of its (1-24)corticotrophin-tetracosapeptide content by physicochemical and biological methods.

The preparation and nature of the International Reference Preparation of Tetracosactide for Bioassay (IRP; in ampoules coded 80/590) are described. The IRP was studied by six laboratories in five countries using in-vivo and in-vitro bioassays and various physicochemical methods. The bulk (1-24)corticotrophin-tetracosapeptide (batch 000179) from which the IRP was prepared contained 10.4% (w/w) acetic acid and 8.3% (w/w) water; its (1-24)corticotrophin-tetracosapeptide content was estimated to be 71.7% (w/w) by amino acid analysis, 74.2% (w/w) by high performance liquid chromatography (HPLC) and 77.5% (w/w) by spectrophotometry. (1-24)Corticotrophin-tetracosapeptide accounted for more than 90% (w/w) of the total peptide in the IRP as judged by HPLC, thin-layer chromatography, carboxymethyl-cellulose chromatography, isoelectric focusing (IEF) and electrophoresis. The homogeneity of the peptide in the IRP was similar by all methods to that in batch 000179 from which it was prepared. The (1-24)corticotrophin-tetracosapeptide content of the IRP (with 95% confidence limits), in terms of batch 000179, was found to be 491 micrograms/ampoule by HPLC and spectrophotometry, 473 (433-513) micrograms/ampoule by IEF and 505 (473-539) micrograms/ampoule by the in-vitro rat adrenocortical cell assay. A comparison in the same bioassay system of the IRP with a laboratory house standard of (1-24)corticotrophin-tetracosapeptide, which originated from a different manufacturer, gave similar results. Accelerated thermal degradation studies of the IRP by adrenocortical cell assay, HPLC and IEF suggested that more than 99.9% of its original content of (1-24)corticotrophin-tetracosapeptide would remain after 10 years under normal storage conditions of -20 degrees C in the dark. Bioassay estimates of samples of the IRP which had undergone significant degradation were higher than estimates by HPLC, indicating that molecular species other than (1-24)corticotrophin-tetracosapeptide contributed to their corticotrophic activity. The corticotrophic activity of the IRP was demonstrated by cytochemical bioassay and by in-vivo bioassays as well as by the adrenocortical cell assay. After consideration of these data, the Expert Committee on Biological Standardization of the World Health Organization established the ampoule d preparation, coded 80/590, as the International Reference Preparation of Tetracosactide for Bioassay and assigned to it a potency of 490 i.u./ampoule; thus the i.u. is represented by 1 microgram (1-24)corticotrophin-tetracosapeptide.

The International Reference Preparation of Gonadorelin for Bioassay: a comparison with different preparations of synthetic luteinizing hormone releasing hormone using physicochemical methods of analysis, different bioassays and immunoassay.

The preparation and nature of the International Reference Preparation of Gonadorelin for Bioassay (IRP; coded 77/596) are described. The IRP was studied by four laboratories in four countries and compared, using physicochemical methods of analysis, various bioassay procedures and immunoassay, with preparations of synthetic luteinizing hormone releasing hormone (LH-RH) produced by different manufacturers. Analyses by thin-layer chromatography and by reverse-phase high-performance liquid chromatography (HPLC) indicated some heterogeneity of the peptide present in most of these preparations of synthetic LH-RH, including that of the IRP; the latter preparation appeared to be 88.3% (w/w) pure, judged by HPLC. The data from the collaborative study suggested that each ampoule of the IRP contains approximately 31 nmol LH-RH. The IRP appeared to be suitable to serve as an international reference preparation for bioassay since its behaviour was similar in different bioassays, so far as this could be examined, to that of the other preparations of LH-RH with which it was compared. Furthermore, the biological activities of different preparations of LH-RH, assessed in terms of the IRP, appeared to correlate with their degrees of purify assessed by physicochemical methods, suggesting that the peptides other than LH-RH present in the IRP did not contribute significantly to the biological activity of the preparation in these assay procedures. The limited data available suggested that the IRP might also be suitable as a reference preparation for immunoassay. The ampouled preparation, coded 77/596, was therefore established by the World Health Organization as the International Reference Preparation of Gonadorelin for Bioassay and assigned a unitage of 31 i.u./ampoule on the basis that the i.u. is represented by 1 nmole of LH-RH.