Establishment of a WHO Reference Reagent for anti-Mullerian hormone.

BACKGROUND: There is a need for a reference material to support the development and ensure the quality of immunoassays for human AMH. A batch of ampoules, coded 16/190, containing lyophilised recombinant AMH was evaluated in a WHO Collaborative Study. The aims of the study were to determine the AMH content in terms of the calibration of each immunoassay method, to predict long-term stability and to assess the suitability of the preparation to calibrate AMH immunoassays. METHODS: Study participants were asked to report the AMH content of specific dilutions of coded ampoules of 16/190 and a comparator preparation containing approximately half the AMH content. In each assay, participants also reported the AMH content of 22 patient samples to assess commutability. A robust all-laboratory geometric mean of the content estimates was determined using the laboratory geometric mean estimates. Commutability was assessed using a difference in bias approach. Stability was predicted by the measurement of thermally accelerated degradation samples. RESULTS: Seven laboratories performed twenty-one immunoassay method-platform combinations, sixteen of which provided data which met the validity criteria, giving a consensus geometric mean estimate of AMH content of 511 ng/ampoule (95% CI, 426-612, n = 16, GCV 42%) and a robust geometric mean of 489 ng/ampoule. By contrast, the GCV% for the all-laboratory geometric mean of the relative content estimates for the comparator sample to 16/190 was 12%. Commutability was assessed using 20 of the 22 representative patient samples. Of the valid assays, 16/190 was within the limits of acceptable commutability for 6 methods, partially commutable for a further 3 methods and non-commutable when measured by 7 methods. The preparation was predicted to be highly stable when stored at - 20 °C. CONCLUSION: The majority of methods met the validity criteria. Content estimates showed a high between-method variability, yet assays exhibited a similar proportionality of response as demonstrated using the comparator sample. 16/190 was commutable in some but not all methods. On the basis of these results, it was agreed by the WHO Expert Committee on Biological Standardization to establish 16/190 as a WHO Reference Reagent for AMH with a content defined by consensus immunoassay of 489 ng/ampoule.

Vacuum-Induced Surface Freezing for the Freeze-Drying of the Human Growth Hormone: How Does Nucleation Control Affect Protein Stability?

In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum-induced surface freezing technique has been compared to conventional freezing, both with and without an annealing step. Size exclusion chromatography and cell-based potency assays have been used to characterize the formation of soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that controlled nucleation has a positive effect on both cycle performance and product homogeneity because of the formation of bigger ice crystals, and characterized by a narrower dimensional distribution. From the point of view of hGH stability, we observed that vacuum-induced surface freezing is not detrimental to the biological activity of the protein, or aggregate formation. In addition, the effect of 2 different formulations, including trehalose or cellobiose, on protein preservation was also considered for this study.

The first World Health Organization International Standard for in vitro biological activity of darbepoetin.

The expiry of patents protecting the manufacture and sale of therapeutic darbepoetin products is expected to lead to the emergence of biosimilar products. In response to this, the first World Health Organization (WHO) International Standard (IS) for darbepoetin has been developed. A lyophilized preparation of darbepoetin, coded 17/204, was evaluated in an international collaborative study, the results of which suggest that the candidate preparation is suitable to serve as an IS. This material defines the International Unit (IU) of in vitro biological activity of darbepoetin and should be used to calibrate of in vitro potency assays of darbepoetin preparations. It is envisaged that widespread use of the IS will promote the consistency and harmonization of darbepoetin in vitro bioassay measurements in laboratories worldwide. Each ampoule contains 100,000 IU of darbepoetin activity. The IU is not intended to revise product labelling or dosing requirements, decisions regarding which lie solely with the regulatory authority. Additionally, the IS is not intended to define the specific activity of darbepoetin, as this may differ between products in the future. Finally, the IS is not intended to serve any regulatory role in defining biosimilarity (i.e. as a reference medicinal product).

Continued provision of WHO International Standards for total and free PSA: Content and commutability of replacement preparations.

OBJECTIVES: Replacements are required for the WHO International Standards (IS) for free PSA, coded 96/668 and total PSA (90:10), coded 96/670, which were established in 1999 to support efforts to harmonise PSA assays and address non-equimolarity. An important consideration is that the introduction of the replacements should have minimal impact on PSA measurements. DESIGN AND METHODS: We report the development of a replacement strategy, informed by field assessment of preparations through an external quality assessment scheme and the subsequent evaluation of the candidate ISs in worldwide collaborative studies. RESULTS: By immunoassay, data from participants confirmed the value assigned to the current standards. Robust geometric mean estimates of the free PSA content of the candidate replacement for 96/668 coded 17/102 was 0.533 µg/ampoule (n = 21). The ratio of the content estimates of 17/102:96/668 was 0.516 (GCV 12.5%, n = 21). Robust geometric mean estimates of the total PSA content of the candidate replacement for 96/670, coded 17/100, was 0.505 µg/ampoule (n = 22). The ratio of the content estimates of 17/100:96/670 was 0.490 (GCV 5.3%, n = 22). Through concomitant measurement of a panel of 15 representative patient samples, the candidate ISs were shown to exhibit commutability with patient samples that was comparable with that of the current ISs. CONCLUSION: On the basis of these results, the preparations coded 17/102 and 17/100 were established by the WHO Expert Committee on Biological Standardization as the 2nd ISs for free and total PSA (PSA-ACT+free PSA) respectively, with assigned contents of 0.53 µg/ampoule and 0.50 µg/ampoule.

HIV infection after prenatal screening: an open window leading to perinatal infection.

Despite a dramatic decrease in vertical transmission of HIV in the developed world, maternal HIV infection acquired after negative prenatal screening still leaves a window of vulnerability. Through quality assurance programs in two Canadian Provinces, five cases where perinatal HIV transmission occurred despite negative prenatal screening were identified between 2005 and 2015. Maternal risk factors such as intravenous drug use, high-risk sexual behavior, hepatitis C virus co-infection, and belonging to high prevalence minority groups were common. Two mothers had their negative HIV test performed in the first trimester and three mothers had negative testing in the third trimester. All babies were clinically healthy at delivery with a normal weight. Three babies were tested following subsequent identification of maternal HIV infection and two babies presented with opportunistic infections leading to their diagnoses. The characteristics of these cases suggest that to achieve complete elimination of vertical HIV transmission, selective and innovative clinical management of mothers at high risk for HIV may be required.

Towards international standardization of immunoassays for Müllerian inhibiting substance/anti-Müllerian hormone.

RESEARCH QUESTION: Is formulated and lyophilized, recombinant human Müllerian inhibiting substance, also known as anti-Müllerian hormone (AMH), suitable for the preparation of a WHO international standard to calibrate AMH immunoassays? DESIGN: The AMH content of a trial preparation, coded SS-581, was determined by five laboratories using seven immunoassay methods. Participants were requested to report the content of the preparation in terms of their method calibrators through the measurement of a minimum of five concentrations in the linear part of the dose-response curve. Participants were also asked to measure, concomitantly, a panel of six serum samples containing AMH at concentrations of 0.1-13.0 ng/ml. RESULTS: Across all assays, including two automated assays in development, the geometric mean content was 361.76 ng/ampoule with a geometric coefficient of variation (GCV%) of 39.95%. When measured by immunoassays that were commercially available at the time of the study, the mean content was 423.08 ng/ampoule, with a GCV% of 26.67%. The inter-method geometric means of five serum samples with an AMH concentration >0.3 ng/ml and measured concomitantly with dilutions of SS-581 varied with a range of GCV% of 14.90-22.35%, which may reflect the use of serum sample value transfer to calibrate current immunoassays, some of which use non-human AMH calibrators. The AMH in trial preparation SS-581 was shown to be biologically active in the Müllerian duct regression assay. CONCLUSIONS: A reference material prepared using human recombinant AMH is a promising candidate for the preparation of an international standard for AMH for immunoassays calibrated to recombinant human AMH.

Quantification of Müllerian Inhibiting Substance/Anti-Müllerian Hormone polypeptide by isotope dilution mass spectrometry.

Measurement of serum concentrations of Müllerian inhibiting substance (MIS), also known as anti-Müllerian Hormone (AMH) by immunoassay is gaining clinical acceptance and widespread use for the diagnosis of ovarian conditions and for prediction of the response to ovarian stimulation protocols as part of assisted reproductive therapies. Provision of an International Standard to harmonize immunoassay methods is required. It is desirable for the content of a future International Standard to be assigned in mass units for consistency with the units reported by current methods. Isotope dilution mass spectrometry (IDMS), a physicochemical method with traceability to the SI (Système International d'Unités) unit of mass, is a candidate approach to provide orthogonal data to support this mass assignment. Here, we report on the development of an IDMS method for quantitation of AMH using three peptides from different regions of the AMH monomer as surrogates for the measurement of AMH. We show the sensitivity and linearity of the standard peptides and demonstrate the reproducibility and consistency of the measurement amongst the three peptides for determining the AMH content in buffered preparations and in trial preparations of recombinant AMH, lyophilised in the presence of an excess of bovine casein.

Preparation, calibration and evaluation of the First International Standard for human C-peptide.

BACKGROUND: Measurement of C-peptide by immunoassay contributes to the diagnosis of a number of disorders related to ß cell function. Stocks of the current international reference reagent (IRR) for C-peptide, used to calibrate these immunoassays, are exhausted, and this report summarises the international study to establish a replacement World Health Organization (WHO) international standard (IS) to maintain the availability of a globally available reference material and support efforts to standardise C-peptide assays. METHODS: The study was conducted in three phases; phase I involved the assignment of a value to a primary calibrant in mass units by amino acid analysis and phase II applied this value to the calibration of a candidate standard, 13/146, by reversed phase high-performance liquid chromatography (RP-HPLC) assay. In phase III, the candidate standard was compared to the first IRR by current immunoassays to assess its suitability to serve as an IS. RESULTS: Calibration of the candidate standard by RP-HPLC gave a final estimated content of 8.64 µg/ampoule with expanded uncertainty of 8.21-9.07 µg/ampoule (95% confidence; k=2.45). The candidate standard also appears sufficiently stable to serve as an IS, based on HPLC analysis of accelerated thermal degradation samples of 13/146, and was also shown to have appropriate immunological activity. A difference in bias approach was used to assess the commutability of 13/146 with human serum and urine samples. With the exception of two laboratories, the candidate standard demonstrated commutability with respect to the serum and urine samples included in this study. CONCLUSIONS: The candidate standard, 13/146, is suitable to serve as the First International Standard for human C-peptide, and it has been formally adopted by the Expert Committee on Biological Standardisation of the WHO.

Applications of cell-based bioassays measuring the induced expression of endogenous genes.

Cell-based bioassays are used to determine the biological activity of complex biotherapeutic products, to assign potency and to assure the quality and consistency of the manufacturing process. Clinically, these assays are used to assess bioactivity in patient samples, particularly for the detection of antidrug neutralizing antibodies. Owing to their versatility, cellular assays that measure endogenous gene expression by quantitative reverse transcription PCR offer a rapid and automatable alternative to assays measuring functional, late-stage responses. Notably, detection of immediate early gene expression represents a direct response of the cell to receptor ligation by the biotherapeutic. We review current developments in the use of this approach and demonstrate its application to the detection of receptor-binding autoantibodies using, as a case study, the detection of autoantibodies to the thyroid-stimulating hormone receptor.

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.

Detection of neutralizing antibodies to erythropoietin by inhibition of rHuEPO-stimulated EGR1 gene expression in the UT-7/EPO cell line.

Recombinant erythropoietin (rHuEPO) is used extensively to treat anaemia associated with chronic kidney disease. However, the development of neutralizing antibodies (NAbs) to rHuEPO can result in the development of antibody-mediated pure red cell aplasia (PRCA). The detection of NAb in patient sera by in vitro bioassay relies on the inhibition of a cellular response to rHuEPO. Current bioassays for rHuEPO measure proliferation in responsive cell lines such as the erythroleukaemic cell lines, UT-7 and UT-7/EPO, the latter sensitized to EPO. Using these cell lines, we show the dose-responsive induction of both PIM1 and EGR1 gene expression in UT-7 cells and of EGR1 in UT-7/EPO cells. The expression of EGR1 in UT-7/EPO cells in response to rHuEPO was comparable to the proliferative response measured by (3)H-thymidine incorporation and could be inhibited by serum from a patient with NAb-mediated PRCA in a dilution-dependent manner. Bioassays based on the induction of endogenous gene expression are comparable to current bioassays but are considerably quicker given that incubation time is decreased from 2-3 days to 50 min. Measurement of EGR1 gene expression in response to rHuEPO in UT-7/EPO cells offers a rapid, non-radioactive and automatable alternative to current assays for the detection of rHuEPO NAbs.