A radioreceptor assay for pharmaceutical preparations of insulin

A radioreceptor assay has been developed that is suitable for the measurement of the potency of crystalline insulin and pharmaceutical insulin formulations. It utilizes the well characterized and widely available IM-9 human lymphocyte cell line as the source of receptor. Bovine, porcine and human crystalline and formulated insulins have been assayed against the 4th International and European Standards for Insulin and the potencies compared with those obtained by the mouse blood glucose method. Results with bovine insulin were in full correspondence with the in vivo results. Porcine and human insulins were 15-20% more potent by the radioreceptor assay than by the in vivo method when the mixed bovine and porcine insulin 4th International and European Standards were used, but were equivalent when compared with like materials. Average 95% confidence limits for formulated insulins in two assays were +/- 6% of the mean. The coefficient of variation on repeated assay of the same sample was 3.8%. The three dose parallel line radioreceptor assay with appropriate species species standards is a candidate biological test capable of international adoption as an alternative to in vivo animal testing of insulin.     

WHO international reference reagents for bovine and porcine proinsulins

Candidate preparations for International Reference Reagents (IRR) for immunoassays of bovine and porcine proinsulin were evaluated in an international collaborative study. With the authorization of the Expert Committee on Biological Standardization of WHO, the following preparations were established as IRRs: bovine proinsulin (code 84/514, defined ampoule content 25 micrograms) and porcine proinsulin (84/528, 20 micrograms). The content of ampoules of these materials is defined in terms of mass rather than international units of activity, therefore they are IRR rather than International Standards. Both preparations are intended as primary reference reagents for the calibration of immunoassays.     

Collaborative study for the calibration of a replacement International Standard for Tetanus Toxoid Adsorbed

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for Tetanus Toxoid Adsorbed. Two candidate preparations were included in the study, one of which was established as the 4th IS for Tetanus Toxoid Adsorbed at the WHO Expert Committee on Biological Standardization meeting in October 2010. This preparation was found to have a unitage of 490 IU/ampoule, based on calibration in guinea pig challenge assays. Results from mouse challenge assays suggest that the relative performance of two candidate preparations may differ significantly between guinea pigs and mice. The authors note that the number of laboratories that performed guinea pig challenge assays, which are used to calibrate and assign IU, is much lower than in previous collaborative studies and this may have implications for calibration of replacement standards in the future. The issue of assigning separate units to the IS for guinea pig and mouse assays is discussed. The study also assessed performance of the replacement standard in serological assays which are used as alternative procedures to challenge assays for tetanus potency testing. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays.     

Collaborative study for the calibration of a replacement international standard for diphtheria toxoid adsorbed

We present the results of a collaborative study for the characterization of a preparation of diphtheria toxoid adsorbed, and its calibration in terms of the 3rd International Standard (IS) for Diphtheria Toxoid Adsorbed. Calibration was performed using established World Health Organization (WHO) and European Pharmacopoeia (Ph. Eur.) protection models. Two candidate toxoid preparations were included in the study, one of which was adopted as a replacement Ph. Eur. Biological Reference Preparation (BRP, batch 4) in February 2009. The second candidate preparation was found to have a unitage of 213 IU/ampoule based on the calibration by in vivo bioassay in 19 laboratories in 16 countries, and was established as the 4th IS for Diphtheria Toxoid Adsorbed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2009.The study also assessed performance of the replacement standard in mouse and guinea pig serological assays which are used as alternative procedures for diphtheria potency testing. Participants tested both candidate preparations and potency was expressed in relative terms only. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays and that the Vero cell assay may be suitable for calibration of future replacement standards.     

Evaluation of Fourth International Standard for Whole Cell Pertussis Vaccine

Whole cell pertussis vaccine is still widely used in many countries. An International Standard is needed for its potency control. The Third International Standard for Pertussis Vaccine was prepared about 40 years ago and its replacement was recommended by the Expert Committee for Biological Standardisation (ECBS) of the WHO. Material in ampoules coded 94/532 was prepared as a candidate replacement and has been evaluated in international collaborative studies which consisted of two parts. The first part, to assess the suitability of the candidate standard by comparing it with the Second International Standard for Pertussis Vaccine (IS2) involved 14 laboratories in 11 countries. The second part to compare the candidate standard with the Third International Standard for Pertussis Vaccine (IS3) involved 16 laboratories in 14 countries. Since 1995 various other studies have included the international standards and the results of these are also considered in assessing likely continuity of the IU for potency of whole cell pertussis vaccine. The preparation in ampoules coded 94/532 was adopted by the WHO ECBS in October 2006 as the 4th International Standard for whole cell pertussis vaccine and assigned an activity of 40 IU per ampoule on the basis of the studies reported here.     

Correlation between HbA1c, purified insulins, diabetic control, and insulin antibodies in diabetic children

Insulin antibodies were determined in sera from 38 children diagnosed as having juvenile diabetes for a duration of 0.7-15.2 years (median = 4.9 years). 8 children were treated with purified porcine insulins from the beginning of their disease, 16 children with bovine insulin NPH alone, and 14 children with non-purified, of whom 9 were later transferred to purified insulins. Serum insulin antibodies were measured by non-specific and specific methods using beef (B) and pork (P) antigens as described by Welborne and Sebriakova, respectively. 12/38 children had insulin binding levels similar to those of normal children, irrespective of the type of insulin used. The concentration of antibodies using radiolabelled B or P insulins as antigens were strongly correlated, by both the non-specific (p less than 0.01) and the specific (p less than 0.01) methods. Children with better score for diabetic control had significantly lower levels of insulin antibodies against B (p less than 0.05) and P (p less than 0.05) than those with poor diabetic control. There was also a significant positive correlation between mean HbA1c concentration and both B and P mean insulin antibody concentration (p less than 0.01). Finally, patients treated with purified porcine insulin had significantly lower levels of antibodies than patients with non-purified bovine insulin (p less than 0.05).     

Autoantibodies against human insulin.

Sera from 680 non-diabetic subjects with suspected autoimmune disease were screened for 13 different antibodies. Of the 582 sera found to contain these antibodies, nine bound insulin in an IgG specific enzyme linked immunosorbent assay (micro ELISA). Four of the sera bound human, porcine, and bovine insulins and five bound exclusively human insulin. "Cold" human, porcine, and bovine insulins each displaced, in a dose dependent manner, the four sera which bound all three insulins, but only human insulin displaced the remaining five, porcine and bovine insulins having little or no effect in concentrations up to 1000 U/1. These observations point to the existence of autoantibodies specifically against human insulin in some subjects with established autoimmunity.     

Transfer from porcine insulin to human insulin in insulin-dependent diabetes mellitus: effects on insulin binding to IgG and glycemic control

Thirty type I diabetic patients who were treated for at least 2 years with a combination of regular and lente monocomponent porcine insulins were allocated in a double-blind study to either continued porcine insulin treatment or a transfer to the corresponding semi-synthetic human insulins. Insulin binding to IgG measured by an immunoelectrophoretic method, was followed at 3-month intervals for 1 year, and did not change after the transfer. The glycemic control, as assessed by hemoglobin A1 levels, tended to deteriorate in the human insulin group during the first 3 months of the trial and then return to the baseline level. It is concluded that a transfer from highly purified porcine insulin to human insulin apparently does not change the insulin binding to IgG in already sensitized patients.     

Third International Standard for pertussis vaccine: international confirmation study of activity of British Standard for pertussis vaccine, coded 66/303.

The first British Standard (BS) (Code 66/303) for pertussis whole cell vaccine was prepared from the same suspension of Bordetella pertussis as the now exhausted Second International Standard for Pertussis Vaccine (2ndIS). The BS and the 2ndIS were compared and calibrated in a previous international study. This report describes a small international study, which included the BS, the 1stIS and the 2ndIS so that the present relationship of these preparations to one another could be assessed. The results of this study show that the relationship of the BS to the 1stIS is consistent with its established unitage of 46 IU per ampoule. The results further show that the potency of the BS is broadly consistent with that of the 2ndIS and that the BS has not lost activity relative to the 2ndIS (from which it was previously found to be indistinguishable). Based on its original calibration and supported by the results of this present study, the BS has been established as the Third International Standard for Pertussis Vaccine, with an assigned unitage of 46 IU per ampoule.     

An international collaborative study on the First International Standard of bovine luteinizing hormone for immunoassay

An ampouled freeze-dried preparation of bovine pituitary luteinizing hormone (bLH), coded EHC-bLH-1, has been evaluated in an international collaborative study and shown to be suitable and sufficiently stable to serve as a standard for bLH. Eight laboratories provided immunoassay data, one laboratory provided receptor assay data, and bioassay data were obtained from 4 laboratories. The geometric mean potency estimate obtained by immunoassays, expressed as milliunits of the USDA bLH-B-5 preparation per ampoule, was 25.6, which is consistent with the result obtained by in-vivo bioassays. The geometric mean estimate obtained by receptor assays or by in-vitro bioassays was lower, i.e. 13.2 milliunits per ampoule. The reason for this discrepancy is currently under investigation. With the authorization of the Expert Committee on Biological Standardization of the World Health Organization this preparation was established in 1985 as the International Standard for Luteinizing Hormone, Bovine, for Immunoassay with a unitage of 25 mi.u. per ampoule.     

Rabies vaccine standardization: International collaborative study for the characterization of the fifth international standard for rabies vaccine

A collaborative study was carried out to establish a replacement for the International Standard for Rabies Vaccine, the stocks of which are exhausted. Three rabies vaccines for human use derived from different rabies virus strains and prepared on different cell culture substrates were compared with the International Standard for Rabies Vaccine using in vivo and in vitro assay methods in a collaborative study involving 14 participants. The proposed fifth International Standard (PISRAV) which was derived from the same virus strain as the present international standard preparation, the Pitman Moore (PM) strain, was found to be approximately twice as potent relative to the International Standard in immunogenicity assays as in antigenicity assays. On the other hand another vaccine, derived from the LEP strain, was considerably more potent in antigenicity assays than in immunogenicity assays. The glycoprotein of the proposed replacement standard measured in antigenicity assays appeared to be stable at +37 degrees C for 245 days, whereas the immunogenicity of the proposed replacement vaccine was sensitive to this heat treatment and the vaccine lost 66% of its immunogenic potency. The results of this study indicate that the NIH protection test should continue to form the primary basis for potency assay of rabies vaccine as glycoprotein content does not appear to correlate with immunogenic potency for different types of vaccine. The vaccine coded PISRAV has been established as the fifth International Standard for Rabies Vaccine and a potency of 16 International Units of Rabies Vaccine (based on the immunogenicity assays) assigned to the contents of each ampoule. Each ampoule has also been assigned a unitage of 10 IU of PM Rabies Virus Glycoprotein and 135 IU of PM Rabies Virus Ribonucleoprotein.     

International collaborative study by in vitro bioassays of the first international standard for porcine inhibin.

A lyophilized preparation of inhibin from porcine ovarian follicular fluid, ampoule code 86/690, was made internationally available as a research standard for in vitro bioassays in 1987. A study involving ten participants in eight countries assessed the stability and suitability of this research standard to serve as an international standard. Each of the participants used in vitro assays, the majority of which depended upon the inhibition of release of follicle-stimulating hormone from dispersed rat anterior pituitary cells. The research standard 86/690 was compared with coded ampoules of 86/690 stored under conditions of accelerated thermal degradation and with inhibins from different species. Intra- and interlaboratory variation for estimates of potency of a coded duplicate ampoule of the research standard provided the basis for comparisons of non-identical inhibins, but the fourfold variability of potency estimates for identical ampoules was such that no conclusions about the differences seen for non-identical inhibins could be made. Predictions of stability from consensus estimates of potency of ampoules that have undergone accelerated thermal degradation indicated that the research standard had satisfactory stability. On the basis of this study, the research standard 86/690 was deemed sufficiently stable and suitable to serve as a standard for in vitro bioassays and was established by the World Health Organization Expert Committee on Biological Standardization as the First International Standard for Porcine Inhibin. The possible presence, in biological extracts (standard or sample), of other bioactive proteins, such as activin and follistatin, complicates the quantitative interpretation of bioassay data. A standard of highly purified human inhibin is now required as a standard for immunoassays used for clinical research purposes; sufficient quantities of recombinant human inhibins have recently been donated for ampouling and evaluation by bio- and immunoassay in the subsequent phase of the standardization of inhibins.     

The Second International Standard for somatropin (recombinant DNA-derived human growth hormone): preparation and calibration in an international collaborative study.

A preparation of somatropin (recombinant DNA-derived human growth hormone) was prepared as lyophilised ampoules according to WHO procedures for international biological standards. The candidate preparation (98/574) was evaluated in an international collaborative study (16 laboratories, nine countries), with the following aims: (i) to determine the suitability of the preparation to serve as the International Standard for somatropin by studying its performance in the current range of physico-chemical and biological assay methods employed for somatropin; (ii) to assign a content in terms of the existing (first) International Standard for somatropin, using the currently recognised assay procedure (Size Exclusion High Performance Liquid Chromatography, SE HPLC); (iii) to confirm the specific biological activity of the candidate preparation; (iv) to confirm the stability of the candidate preparation. On the basis of the collaborative study WHO agreed that: the preparation in ampoules coded 98/574 is suitable to serve as the next WHO International Standard for somatropin; the preparation in ampoules coded 98/574 should be established as the second International Standard for somatropin, with a defined ampoule content of 1.95 mg total somatropin plus somatropin-related proteins per ampoule; the specific activity of the preparation should be defined as 3.0 IU/mg somatropin.     

Very pure porcine insulin in clinical practice.

In a one-year follow-up study the insulin dose in diabetic patients using very pure porcine insulin was compared with that in patients using conventional preparations. The dose of insulin used to obtain diabetic control was reduced by 7% in 108 patients treated solely with very pure porcine insulin from the start of insulin treatment when compared with 108 matched patients who had received conventional insulins. In 117 patients whose treatment had been changed from conventional bovine or bovine-porcine insulin to very pure porcine insulin the dose was reduced by 9%. A further 511 patients receiving conventional insulins were examined for local cutaneous or subcutaneous abnormalities at insulin injection sites. Lipoatrophy was found in 49 of these patients (10%), but not in patients using very pure porcine insulin. The results confirm that very pure porcine insulin reduces the insulin dose needed to maintain diabetic control and may resolve or prevent local reactions such as lipoatrophy. Long-term advantages in reduced antigenicity to insulin and contaminating peptides remain to be established.     

The International Standard for sisomicin

An International Standard for Sisomicin has been established on the basis of a collaborative assay. There were seven participating laboratories in seven countries. The activity of the contents of each ampoule of the International Standard of Sisomicin is defined as 35,200 International Units of Sisomicin.     

Standardization of therapeutic,urinary gonadotrophins: An update on the use and availability of International Standards for Menotrophin

The potencies of therapeutic preparations of gonadotrophins of human, urinary origin, which comprise a heterogenous mix of isoforms with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) bioactivities, are standardized by WHO International Standards (IS). We report here, the evaluation, through an international collaborative study, of a candidate preparation, coded 10/286, to replace the 4th IS, 98/704, for human, urinary FSH and LH (Menotrophin) which has been used for many years for the potency assignment of therapeutic preparations using bioassays. The mean FSH and LH bioactivities of 10/286, determined by in vivo bioassays in terms of 98/704, were 183 IU per ampoule (95% confidence limits 165–202) and 177 IU per ampoule (95% confidence limits 159–197), respectively.     

The International Standard for Netilmicin

An International Standard for Netilmicin has been established on the basis of a collaborative assay. There were five participating laboratories in five countries. The activity of the contents of each ampoule of the International Standard for Netilmicin is defined as 4810 IU of netilmicin.     

Evaluation of two human plasma pools as candidate international standard preparations for syphilitic antibodies

A collaborative study was designed to asses two freeze-dried human plasma preparations containing anti-Treponema pallidum antibodies, 05/132 and 05/122, for their suitability as international reference reagents for syphilis serology. Both preparations are intended as replacements of the first international standard (IS) for syphilitic serum antibodies (HS). Samples were tested by eight laboratories using the T. pallidum passive particle agglutination assay (TPPA), the venereal disease research laboratory test (VDRL) and the rapid plasma reagin test (RPR). In addition a range of immunoassays was also used. The outcome of the collaborative study revealed that candidate standard 05/132 contains T. pallidum-specific IgG and IgM and is reactive in VDRL or RPR, and that 05/122 contains T. pallidum-specific IgG but is not reactive in either the VDRL or RPR test. Both 05/132 and 05/122 are reactive in the TPPA. On the basis of these results the Expert Committee on Biological Standardization of the World Health Organization designated 05/132 as the 1st IS for human syphilitic plasma IgG and IgM with a unitage of 3 IU per ampoule relative to HS and 05/122 as the 1st IS for human syphilitic plasma IgG with a unitage of 300 mIU per ampoule relative to 05/132.     

Human insulin: study of safety and efficacy in man.

The safety and efficacy of a new highly purified neutral soluble human insulin produced by conversion of porcine insulin was compared with a highly purified neutral soluble porcine insulin in six normal men. The insulins were administered by subcutaneous injection at a dose of 0.075 U/kg body weight. Somatostatin was infused during the experiment to suppress endogenous insulin secretin. No difference was found in the plasma glucose, insulin, or metabolite responses. Thus the potency, onset, and duration of effect were identical with the two insulins. No short-term side effects to either insulin were observed. Highly purified, semi-synthetic human insulin offers a safe and effective means to explore the possible advantages of homologous human insulin in the management of diabetes mellitus.     

A review of WHO International Standards for botulinum antitoxins.

Clostridium botulinum produces the most potent known toxins, with seven distinct serotypes currently defined (A-G). These toxins can cause a life threatening systemic toxicity whether through natural causes such as food poisoning, infant botulism, wound botulism, or through use as bio-terror agents (e.g. inhalational botulism). It was realised early on that standard reference botulinum antitoxins were required to reduce the variation between assays and ensure a consistent potency of therapeutic antitoxins and vaccines, and to define the serotype. This led to the International Unit being defined by the World Health Organisation (WHO) in the 1960s with the establishment of the first International Standards (IS) for serotypes A-F. Since then botulinum antitoxin ISs have been used world wide as the 'yard stick' to measure the neutralising potency of antitoxins. These primary WHO ISs are used to calibrate in house working reagents that are more extensively utilised. A definition of the International Unit for serotype G antitoxin has yet to be defined or accepted by the WHO and urgently needs addressing. However, before September 11th 2001 there was very little interest in botulinum antitoxin IS and as a result stocks of most of the original preparations are now completely exhausted or depleted and replacements long overdue. We have reviewed the extensive history and availability of the primary WHO ISs and interim materials. All type A and B antitoxin materials were recently assayed and their relative activities confirmed against the original IS preparations. The recent increase in demand for these materials has further exacerbated the shortage. We describe here the production and characterization of stable freeze dried potential candidate replacements along with a new prospective first IS for type G antitoxin. Available toxin A reference preparations are also briefly reviewed.