Tracing recombinant bovine somatotropin ab(use) through transcriptomics: the potential of bovine somatic cells in a multi-dose longitudinal study.

In the European Union, the use of recombinant bovine somatotropin (rbST) in dairy cattle is forbidden. Monitoring rbST (ab)use by its direct detection in animal matrices still remains a challenging task. New monitoring methods based on indirect detection of the substance are necessary. A new transcriptomic system based on the use of high-throughput real-time PCR in combination with somatic cells was developed to control rbST administration in dairy animals. A total of nine cows, separated into control and rbST-treated groups, were included in the study. A subcutaneous injection containing 500 mg of rbST was administered to the treated group every 14 days, up to a total of 12 doses. Milk somatic cells (MSCs) were sampled from each animal at different time points throughout 8 months of study. It was possible to obtain the transcriptomic profile of 18 genes in MSCs of rbST-treated and control groups, and using univariate and multivariate statistical analysis control and treated animals were discriminated. The transcription of CCND1, IGF-1R, TNF and IL-1ß genes resulted strongly influenced by rbST treatment. The combination of MSCs, transcriptomic tools and statistical analysis has allowed the selection of four genes as potential biomarkers that could be used in a transcriptomic panel for monitoring rbST administration in cows.

[What doctors need to know about biosimilar medicinal products?]. / Sto bi lijecnici trebali znati o bioloski slicnim lijekovima?

Biological drug is a drug containing one or more active substances produced or secreted from a biological source. Some of them may be previously present in the human body, and examples include proteins such as insulin, growth hormone or erythropoetin. Biosimilar drug is a medical product that is a copy of the original approved drug whose patent has expired. Strict rules apply to similar biological medicines: 1) it is unable to support extrapolation of data on safety and efficacy between individual indications, except in the case of appropriate, science-based evidence; 2) biosimilar drugs must meet the requirements associated with testing the immunogenicity and safety monitoring afterthe introduction of the drug in clinical practice, including the risk management program; 3) each biosimilar drug has to be labeled under its own name in order to allow clear traceability of all medications; and 4) the principle of automatic substitution cannot apply to biosimilar drugs because they are not interchangeable.

International collaborative study for the calibration of the Ph. Eur. somatropin chemical reference substance batches 3 and 4 (BSP108).

An international collaborative study was carried out for the establishment of replacement batches for the European Pharmacopoeia (Ph. Eur.) Somatropin Chemical Reference Substance (CRS) batch 2. The study was organised within the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the European Commission. Seventeen laboratories from Europe, North America, South America and Australia took part in the collaborative study. The study aimed at calibrating the somatropin content of 2 candidate preparations and demonstrating their suitability to serve as a reference substance in the tests for identification, for related proteins, for dimers and related substances of higher molecular mass (HMM), for charged variants distribution and for the assay of somatropin, as prescribed by the current Ph. Eur. monographs 0950 Somatropin bulk solution, 0951 Somatropin and 0952 Somatropin for injection. Based on the results summarised herein the Ph. Eur. Commission adopted in January 2012 candidate preparation b (cCRS-b, Sample D) as somatropin CRS batch 3 with an assigned content of 3.86 mg of somatropin monomer per vial, and candidate preparation a (cCRS-a, Sample C) as somatropin CRS batch 4 with an assigned content of 2.59 mg of somatropin monomer per vial.

Consensus statement on the standardization and evaluation of growth hormone and insulin-like growth factor assays.

Growth hormone (GH) and insulin-like growth factor I (IGF-I) measurements are widely used in the diagnosis of disorders of GH secretion, evaluation of children with short stature from multiple causes, management of disorders that lead to nutritional insufficiency or catabolism, and monitoring both GH and IGF-I replacement therapy. Therefore, there is an ongoing need for accurate and precise measurements of these 2 peptide hormones. Representatives of the Growth Hormone Research Society, the IGF Society, and the IFCC convened an international workshop to review assay standardization, requirements for improving assay comparability, variables that affect assay interpretation, technical factors affecting assay performance, assay validation criteria, and the development and use of normative data. Special attention was given to preanalytical conditions, the use of international commutable reference standards, antibody specificity, matrix requirements, QC analysis, and interference by binding proteins. Recommendations for each of these variables were made for measurements of each peptide. Additionally, specific criteria for IGF-I were recommended for age ranges of normative data, consideration of Tanner staging, and consideration of the effect of body mass index. The consensus statement concludes that major improvements are necessary in the areas of assay performance and comparability. This group recommends that a commutable standard for each assay be implemented for worldwide use and that its recommendations be applied to accomplish the task of providing reliable and clinically useful results.

Similar biological medicinal products containing recombinant human growth hormone: European regulation.

The concept of similar biological medicinal products ('biosimilar' medicinal products) allows pharmaceutical companies to develop products based on an abridged dossier once the marketing protection of the 'reference' biological medicinal product has expired. A biosimilar medicinal product can be granted a marketing authorization provided that its similarity to a reference product is established in terms of quality, safety and efficacy (step-wise comparability exercise). A decision to launch a biosimilar medicinal product on the market is taken if it has a similar efficacy and comparable or better (less) immunogenicity than the chosen reference biological medicinal product. However, this decision is based on limited data and the comparability program may detect substantial differences in immunogenicity profiles but is likely incapable of detecting rare events. This is why clinical experience, through clinical trials and extensive pharmacovigilance programs, remains the most reliable way to assess the immunogenicity and tolerance profile of recombinant therapeutic proteins. Substitution of one biological medicinal product by a biosimilar medicinal product is not currently recommended before long-term clinical efficacy and safety have been acquired in all relevant populations. Here we review recent regulatory guidelines provided by EMEA and comment on the marketing authorizations and risk management plans of two recently approved biosimilar somatropins.

International harmonization issues.

With the globalization of trade in many product sectors occurring at a rapid pace, interest in international harmonization issues relating to veterinary products continues to grow. Because of the potential impact on trade, there has been a focus for several years on harmonization of maximum residue limits (MRLs) or tolerances on a global basis. There has also been interest, especially on the part of pharmaceutical companies, to facilitate the approval of a veterinary product in multiple countries. This article discusses two major international initiatives that have been formed to address these harmonization areas of concern for veterinary products.

Immune complex transfer two-site chemiluminescent immunoassay for serum growth hormone in alevin chum salmon.

An immune complex transfer two-site chemiluminescent immunoassay (CLIA) for salmon growth hormone (GH) was developed to measure serum GH in alevin chum salmon (Oncorhynchus keta) using a chemiluminescent acridinium ester as a label. The immune complex transfer method dramatically reduced non-specifically bound of acridinium ester labelled antibody without a decrease in the specific binding. Consequently, we could detect lower levels of GH than achieved previously in a two-site CLIA for salmon GH. The detection limit of the assay was 7.8 fg/ml and the standard curve was linear up to 250 fg/ml. Coefficients of variation were 2.2-7.7% within-assay and 5.3-91% between-assay. We have developed a highly sensitive and reproducible GH method and applied it to measurement of GH in alevin chum salmon.

Novel assays based on human growth hormone receptor as alternatives to the rat weight gain bioassay for recombinant human growth hormone.

Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as 'receptor' in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyzed the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the Kd (approximately 0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC-P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16-20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of < or = 5% vs. an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4-16%. Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitro alternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.

WHO activities towards the three Rs in the development and control of biological products.

WHO supports the concept of replacement, reduction and refinement of the use of in vivo methods for biologicals production and control, and regularly conducts reviews of its recommended procedures to allow reduction in the use of animals. The coordination of collaborative studies, publication of standardized methods, and holding of workshops on the use of these methods contributes to their use. The neurovirulence test for oral poliovaccine is probably the single most visible animal test for which alternative methods are sought. Collaborative studies on alternative methods for screening products are currently being sponsored by WHO. The use of Vero cells rather than primary monkey kidney cells for poliovaccine production can avoid the use of many monkeys. Cell banks of Vero and HEp-2 cells have been developed by WHO, tested for virus sensitivity and freedom from adventitious agents, and are available for vaccine production and control, replacing primary animal cells. For the future, final product testing will increasingly be directed towards establishment of consistency of production rather than potency. By supporting the validation and use of this approach, WHO can effectively influence more rational animal use in biological production and control.

Review of Turkish patients with growth hormone insensitivity (Laron type).

Clinical spectrum and endocrine details of thirteen Turkish children (age 0.3-14.2 years; eight females and five males; ten prepubertal, three pubertal) with growth hormone insensitivity are presented. All patients display phenotypical features of severe growth hormone deficiency. The diagnosis based on height standard deviation score (SDS), basal growth hormone (GH), basal insulin-like growth factor I (IGF-I, IGF-I response in an IGF generation test and growth hormone binding protein (GHBP) measurements. The median height SDS was -7.4 (range -3.2 to -10), weight for height index was 100 (range 81-152) and bone age/height age ratio was 2 (range 1.6-3.3). Endocrine investigations showed a median basal GH concentration of 61.4 mU/l (range 23.5-120 mU/l). Basal IGF-I level was below 10 ng/ml in all patients except one. None of the patients showed a significant IGF-I response to injections of GH (0.1 U/kg body weight for 4 days). The median IGFBP-3 level was 0.23 mg/l (range 0.1-0.56 mg/l). The GHBP level was undetectable in all of 10 patients. The high number of patients in our center may be due to the high rate of consanguinity among the Turkish population and the referral facility of our center in the area. These patients may benefit from the new therapy with recombinant human IGF-I.

The use of recombinant gilthead sea bream (Sparus aurata) growth hormone for radioiodination and standard preparation in radioimmunoassay.

A gilthead sea bream growth hormone (sbGH) obtained by cloning and expression of sbGH cDNA was used to develop a sensitive and specific radioimmunoassay (RIA). Iodination of recombinant sbGH (rsbGH) was performed by the classical Chloramine-T method. Specific antiserum, raised in rabbits, was added in a final dilution of 1/36,000. The minimum detectable dose was 30 pg, and the midrange of the assay (ED50) was 275 pg. Intra- and inter-assay coefficients of variation (CV) were 3.3 and 5.8% at ED50 levels. Human GH (hGH), ovine GH (oGH), carp gonadotropin (cGtH), chinook salmon gonadotropin (sGtH), ovine prolactin (oPRL) and recombinant tilapia prolactin (rtiPRL) did not show cross-reactivity. Serial dilutions of chinook salmon GH (sGH) and recombinant rainbow trout GH (rtGH) showed a low but significant cross-reactivity. A good parallelism between rsbGH standard and serial dilutions of native sbGH, plasma and pituitary extracts was observed. In addition, when plasma and pituitary samples were analyzed for GH quantification, non-significant differences were observed within this and previous RIA for native sbGH. Therefore, it appears conclusive that our rsbGH can be used successfully as a standard and radioiodinated hormone in GH assays for gilthead sea bream, which is extensively cultured in the Mediterranean area.

Comparative effectiveness of somatotropin and anabolic steroids in feedlot steers.

Crossbred steers (n = 252, BW = 379 +/- 28 kg) were allotted to 42 pens in a 2 x 3 factorial arrangement of treatments: control or steroid implant (STR; estradiol benzoate+progesterone [three lighter blocks reimplanted on d 84] and trenbolone acetate [reimplanted on d 63]), and either 0, 80, or 160 mg/wk of recombinant bovine somatotropin (bST). Steers were adapted to the finishing diet (12% roughage equivalent, 13% CP) before the start of the experiment and fed for 84 or 119 d. Blood samples were taken on d 0, 14, 28, 56, and 84 for plasma urea N (PUN), serum somatotropin (ST), plasma insulin-like growth factor I (IGF-I), and plasma amino acid assay. Few interactions were noted (P > .1). Gain was increased by both treatments: 1.30 vs 1.66 kg/d for control vs. STR (P < .001) and 1.44, 1.49, and 1.51 kg/d (linear, P = .07) for 0, 80, and 160 mg of bST/wk, respectively. Gain efficiency was also improved: 169 vs 205 g/kg (P < .001) and 177, 189, and 195 g/kg (linear, P < .001), respectively. Average PUN was decreased (P < .001) 29% by STR and decreased 17 and 29% by 80 and 160 mg of bST/wk, respectively (linear, P < .001). Somatotropin decreased mean serum ST compared with controls; STR increased ST 36% compared with controls. Average plasma IGF-I was increased (P < .001) 12% by STR and 13 and 19% (linear, P < .001) by 80 and 160 mg of bST/wk, respectively. Both STR and bST influenced (P < .05) plasma amino acid profiles. Indicators of carcass fatness were decreased linearly (P < .05) by bST; STR implant tended to decrease carcass fatness and increase longissimus muscle area, which was related to carcass weight. The anabolic effects of STR and bST were found to be additive and possibly independent in feedlot steers.

The role of bioassays in the assessment of recombinant proteins.

Although the need is diminishing, biological assays still have an important place in the characterization and quality control of therapeutic peptides prepared by recombinant DNA technology. This role needs to be assessed on a case-by-case, product-by-product basis. It includes as a minimum the need to establish during product development the range and quantitative nature of its biological activities, particularly those relevant to its intended clinical use and potential side effects. For those products whose quality and consistency can be established by alternative approaches, then a biological assay may no longer be necessary on a batch basis. For others, bioassays will be required until adequate and appropriate alternatives can be shown, through international collaborative studies, to be sufficient to assure the product's efficacy and safety in clinical use. So-called "bio-identity" tests are inappropriate and should not be used.

Human growth hormone: the dilemma of expanded use in children.

In the specialized area of pediatric endocrinology, the use of human growth hormone (hGH) both for children who have a growth hormone abnormality and for the treatment of non-hGH-deficient children who are short is a current clinical reality that raises important ethical questions. Generally speaking, the use of hGH for those children who are clearly lacking it is an efficacious intervention based upon established clinical criteria. The use of hGH for children who are short, but have no growth hormone abnormality is ethically and clinically more controversial. The moral conundrum of how to gain knowledge about new medical treatments that may be beneficial to children while, at the same time, ethically enrolling them in clinical trials with placebo arms in order to gain such knowledge will continue to be a contentious issue in the conduct of research and in the delivery of health care. In addition, there are questions about what ought to be studied and whether a physical characteristic such as short stature ought to be viewed as a circumstance of less than optimal health.

Transmissible spongiform encephalopathies and the safety of naturally-derived biologicals.

Late in 1986, a new neurodegenerative disease, referred to as bovine spongiform encephalopathy (BSE), was recognized in domestic cattle in southern England. Since then, tens of thousands of cases have been confirmed throughout the U.K. and the disease has also appeared sporadically outside of the British Isles. BSE belongs to a group of rare, progressive and fatal disorders of the central nervous system of animals and man caused by anomalous infectious agents whose properties are not yet full understood. Consequently, fears were raised about the possibility that the disease might constitute a possible health risk for humans. Minimization of potential contamination has thus become an issue of growing interest. This short review summarizes the main aspects of the problem together with the procedures developed to sterilize preparation of biologicals even from sources containing potentially high levels of contamination.

Analysis of therapeutic growth hormone preparations: report of an interlaboratory collaborative study on growth hormone assay methodologies.

Recombinant DNA-derived human growth hormone (somatotropin) is widely used to treat growth hormone-deficient children. The potency of this product is determined by in-vivo bioassay in hypophysectomized rats, which is imprecise, costly and invasive, and there have been suggestions that it could safely be replaced with in-vitro or physico-chemical alternatives. In this report we present the results of a collaborative study designed to test this proposal. Somatotropin was modified by mild or severe proteolysis, mild or severe oxidation or treatment at high pH, and compared in a multi-centre collaborative study with unmodified somatotropin or with dimerized somatotropin. Participating laboratories included manufacturers and national control laboratories, and pharmacopoeial bioassays were compared with in-house in-vitro and physico-chemical bioassays. Although performing adequately with untreated somatotropin, for degraded samples the in-vivo bioassays were relatively unresponsive to changes in the growth hormone molecule. In contrast, the physico-chemical assays, in particular the reverse-phase HPLC, performed with a high degree of selectivity. We conclude that in the case of somatotropin, the in-vivo bioassay can be removed from the routine product specification with an acceptable degree of security. This however does not obviate the requirement rigorously to demonstrate biological activity in-vivo during product development, nor may the conclusions of this study be applied to other therapeutic recombinant proteins without similar collaborative investigations.