An overview of the strengths and limitations of biological assays in quality control

Biological assays have been an integral part of the regulation of biological products in the United States since licensing of the first product in 1902. Bioassays have been used along with other methodologies in the assessment of identity, purity, safety, potency and stability. The technology of production, i.e. normal or recombinant, has not altered the use or value of these assays. Although many scientists and regulators would prefer to replace all in vivo bioassays with other assays, there is a reluctance to agree that this will be possible, at least in the near future. Scientific efforts are continuing to be focussed on the development of more precise, reliable and less cumbersome assay methods. This paper presents an overview of the strengths and limitations of bioassays as used in quality control, followed by a discussion of specific biological products as models for these principles.     

Structure-function studies on human growth hormone

The relationship between the conformation of human pituitary growth hormone (hGH), biological activity, and ligand binding activity was studied by comparing conformational details previously published on in vivo and in vitro studies of identical samples of hGH and its known derivatives. In vivo assays included the rat tibia test for somatotropic activity and the pigeon crop-sac assay for lactogenic hormone activity. Relative binding affinities were compared in radioimmuno-assays using ¹²⁵I-hGH as tracer with 1) anti-human chorionic somatomammotropin (hCS) serum (low discriminatory hybrid assay), 2) anti-hGH sera (in conventional assays), 3) monospecific anti-hGH serum (absence of cross-reaction with hCS) and 4) human anti-hGH sera obtained from GH-deficient patients on replacement therapy. In addition, binding affinities were examined in two receptor-binding assays, one specific for somatotropic activity (rabbit liver membranes, ¹²⁵I-hGH), and the other, for lactogenic hormones (rabbit mammary membranes, ¹²⁵I-oPRL). The conformational properties of native hGH and various chemically and enzymatically modified derivatives of the hormone were evaluated primarily from circular dichroism spectra, while conformational stabilities were estimated from the relative rates of tryptic digestion. Unfragmented, but chemically modified derivatives, exhibited good parallelism between retention or loss of native conformation and the in vivo potencies and in vitro binding affinities. None of the fragments of hGH showed activity in any of the radioreceptor assays or radioimmunoassays. Two derivatives of hGH, which contain gaps of 6 or 12 residues in the polypeptide backbone produced by partial enzymatic digestion, had full or increased in vivo potencies, full activities in the radioimmunoassays, and were the most active derivatives in both radio-receptor assays. One of these, missing the hexapeptide corresponding to residues 135–140, was also found to retain nearly all the conformational properties of native hGH. These studies proved further evidence that 1) retention by modified forms of hGH of a high degree of in vivo biological potency or in vitro binding affinity is causally related to the retention of most of the conformation and conformational stability of the molecule, and 2) the biologically acitve, receptor-binding and immunoreactive sites on the hGH molecule are 3-dimensional in nature.     

Assays for human growth hormones

Human growth hormone is, in effect, defined by its activity in an in vivo bioassay and the standard used with it, growth being measured as the increase in body weight in hypophysectomised immature rats. The assay reflects the hormone's survival and metabolism in vivo, its cell-cell interactions, the activation and effects of its secondary hormones, such as GF1 and GF2, and various feedback mechanisms. Although it is insensitive, imprecise, easily influenced by contaminants TSH and vasopressin, it is the only practical assay that reflects all the in vivo properties of "hGH". The in vivo tibial epiphysis bioassay is more sensitive and precise, but the response reflects only the elongation of bone. Both these bioassays are well established. By contrast, in vitro receptor assays do not reflect in vivo properties; there may be different natural forms of receptor molecules, they may be altered during their extraction, and the measured response (like those of immunoassays) is not relevant to the biological action of the hormone. The validity of a bioassay depends on the use of a suitable standard. The collaborative study of the International Standard for human growth hormone (in 1984) revealed marked disparities between results with different assay methods. When a growth hormone protein (such as somatotropin, 191 amino acids) is produced in quantity, reproducibly, and with physicochemical properties consistently related to in vivo bioassay results, it may then be reasonable to use physico-chemical tests for control purposes. Many such tests require international reference materials for comparison purposes.     

Pharmacokinetics of human growth hormone administered subcutaneously with two different injection systems

The bioavailability of recombinant human growth hormone (somatropin, CAS 12629-01-5) was compared between a transcutaneous jet injection device and subcutaneous cannula injection. Thirteen healthy male subjects received 8.64 IU somatropin once with jet and once with cannula injection in a randomized cross-over study. Baseline-corrected somatropin serum concentrations were evaluated with non-compartmental and compartmental methods. The 90% confidence intervals with two one-sided t-tests around the ratios of injection devices were 91-120% for maximum concentration, 94-110% for area-under-curve until 14 h, and 92-103% for area-under-curve to infinity. Somatropin has a known metabolic half-life of ca. 20-30 min while the observed terminal half-lives were 2-4 h. Absorption and elimination rate constants were similar. Times of maximum concentrations, terminal half-lives and lag times to start of absorption appeared to be shorter and the absorption rate constant appeared to be larger for jet than for cannula injection. In conclusion, the kinetics of somatropin from subcutaneous tissue had a "flip-flop" characteristic. Bioavailability of somatropin after jet injection was equivalent to cannula injection.     

Microbial toxicity assays used for water quality evaluation in South Africa

A number of microbial assays have been developed for the detection of toxic chemicals in water. These include bacterial and protozoan oxygen uptake assays, a bacterial growth test, a colony formation test, and a microassay. The bioassays are rapid and relatively simple, cost effective and sensitive, and have been applied to the toxicity evaluation of drinking water and wastewaters. These tests have been used as a valuable part of a battery of bioassays that also include other, more time-consuming biological tests.     

News in brief …

The availability of somatropin [recombinant human growth hormone] has afforded the opportunity for achievement of normal physical stature to thousands of growth-deficient children. However, a major downside of the therapy has been the need for daily injections of the hormone. An advance in drug delivery may greatly improve the acceptability and compliance with somatropin regimens. A microencapsulated formulation of somatropin has been developed to reduce the need for injections from daily to once or twice a month. Results of a phase III multicentre clinical trial showed that the microencapsulated formulation achieved growth improvement comparable to that of conventional daily injections, according to a report at the 81st Annual Meeting of the Endocrine Society [ San Diego, US; June 1999 ].     

Growth hormone antibodies formation in patients treated with recombinant human growth hormone

Human growth hormone (hGH) is normally produced by acidophilic cells of the anterior lobe of the pituitary gland. Recombinant DNA technology has made it possible to produce rhGH. There have been reports of immunological reactions in patients treated with rhGH. For this reason, it is necessary to check sera of patients for presence of antibody against rhGH. Forty-seven children were treated for up to 6 months with recombinant human growth hormone (rhGH-Novo), 0.1 IU/Kg body weight, subcutaneously, three times weekly. The magnitude of growth response was similar to those expected from clinical experience with pituitary growth hormone. We examined sera for specific antibodies against rhGH by ELISA methods. Four patients developed serum antibodies against growth hormone. The analysis of these four sera by Dot blotting method also showed presence of antibodies against rhGH. In the sera of treated patients, pre-incubated with different concentration of rhGH, specific antibodies were detected by neutralizing assay. This finding was confirmed by ELISA technique. In conclusion, the main concern with anti-GH antibodies could be their ability to neutralize circulating growth hormone and inhibition its growth promoting effect.     

Activity-based reporter assays for the screening of abused substances in biological matrices

Abstract

The (ab)use of designer drugs and steroid hormones has gained popularity due to the lower chance of getting caught, as routine drug or doping tests may miss these (novel) compounds. Current analytical approaches mostly make use of targeted, structure-based techniques, such as immunoassays or mass spectrometry (MS)-based methods. However, these approaches have limitations, including a lack of cross-reactivity and the need for prior knowledge of molecular identity. This has initiated considerable interest in the so-called “untargeted” screening strategies to detect these compounds. The use of “untargeted” MS-based screening methods (e.g. gas chromatography MS and especially high-resolution MS) has gained considerable interest to detect and identify novel compounds. However, due to their expensive and time-consuming character, very sophisticated analytical methods are not ideal as a first-line screening method and are not routinely implemented in most laboratories. Given the above, it is clear that there lies potential in novel “untargeted” screening approaches, which are less expensive, more high-throughput-amenable and more routinely applicable. Activity-based assays, capable of monitoring the biological activity of an abused substance in a biological matrix, have been proposed as an alternative. These biological assays do not require knowledge about a compound’s structure and could be used as a first-line screening tool to identify potentially positive samples. In this review, we focus on activity-based reporter bioassays for the detection of steroids and drugs of abuse in biological matrices. As for drugs of abuse, only bioassays for detecting cannabinoid or opioid activity in biological matrices are available, only (synthetic) cannabinoid receptor agonists and opioids are discussed.     

PEGylation of somatropin (recombinant human growth hormone): impact on its clearance in humans

1. PHA-794428 is a PEGylated version of somatropin (human growth hormone). The pharmacokinetics of PHA-794428 have been studied in humans following single subcutaneous administration (dose range 10-500 microg kg(-1)). In the same study the pharmacokinetics of somatropin were also determined following a 3.6 mg (51 microg kg(-1)) subcutaneous dose. Comparison of the pharmacokinetics of both molecules indicates that PEGylation of somatropin with a 40 kD PEG results in a ten- to 20-fold increase in area under the curve and a similar increase in half-life when compared with somatropin in human (at equivalent subcutaneous doses). 2. Literature data indicate that somotropin is cleared by two mechanisms. The first processes is clearance by glomerular filtration. This is a passive, non-capacity-limited process. A second, capacity-limited, process is mediated by interaction with growth hormone receptors present in a number of tissues including the liver. It is hypothesized that PHA-794428 shares the same clearance mechanisms. However, the addition of the PEG moiety has modulated the clearance by both of these processes. Pharmacokinetic modelling of human serum concentration data obtained for these molecules strongly supports this hypothesis. The renal clearance is reduced due to the increased size of the molecule (Cl/F reduced from 9.6 to 0.1 l h(-1) for somatropin and PHA-794428, respectively). In addition, the reduction in growth hormone receptor affinity has reduced the clearance mediated by interaction with this receptor (somatropin Km = 3.6 microg l(-1) and Vmax = 104 microg h(-1)/PHA-794428 Km = 53 microg l(-1) and Vmax = 84 microg h(-1)).     

Bioassays in whole animals

The basis for whole animal bioassays as practised today was laid down by Paul Ehrlich in 1894. He introduced the concepts of a stable standard preparation and of the unit of activity as the activity of a defined mass of that standard preparation in the assay performed. Such assays have often provided a way of quantifying newly discovered active principles of biological origin, so that they could be applied in clinical medicine. Whole animal bioassays can be applied not only for the quantitative analysis of a biological product (analytical assays), but also for the comparison of different products intended for the same clinical indication (comparative or research assays). As such they have been the model for controlled clinical trial. For some products many different types of bioassay have been developed. They may produce heterogeneous results when more than one active principle is involved, and these are present in standard preparation and in the unknown preparation in different relative concentrations. In addition the precision of different assay methods for the same substance may vary markedly. An important source of variation in whole animal bioassays is the influence of some environmental factors on the individual animals during the assay. Thus the number of rats per cage markedly influences the variation of the response of rats to serum gonadotrophin. Careful studies are required to detect the discriminatory environmental factors in a particular whole animal bioassay. Keeping these discriminatory factors constant at their optimal level may increase the precision of the assay markedly and thus reduce the number of animals required to attain the precision specified, e.g. in pharmacopoeial tests.     

Progress in the Use of Biological Assays During the Development of Biotechnology Products

The complexity of the structure and function of many biotechnology derived products necessitates a wide range of analytical procedures to adequately characterize the product. In-depth characterization is required for the assessment of several criteria vital to the success of product development such as consistency, purity, stability, and potency. More recently, the concern over the immunogenicity of biologics has increased the need to develop assays to detect neutralizing anti-product antibodies. Although many physicochemical tests are available to characterize the structure of a protein and detect the presence of contaminants, they provide little, if any, information regarding biological potency or the neutralizing capacity of antibody responses in immunogenicity studies. There is a continual need to refine biological assays to increase their accuracy and reproducibility, in particular to replace in vivo bioassays with appropriate in vitro assays. There have also been several recent technological developments that could lead to more rapid and reproducible bioassays.     

Growth hormone secretagogues: recent advances and applications

The discovery of a new class of compounds that stimulate the release of growth hormone (GH) in a manner distinctly different from growth hormone-releasing hormone (GHRH) is advancing the understanding of the mechanisms that control GH secretion. These compounds, the GH secretagogues, act at both pituitary and hypothalamic levels, and might even elicit effects in the CNS and peripheral systems. A receptor with high affinity for the GH secretagogues has been identified and several observations suggest the presence of additional receptors. The existence of these specific endogenous receptors could indicate that the mechanism of GH release is not yet fully understood. Several potential indications have been explored clinically and, as some of these compounds are orally active, they could offer attractive alternatives to recombinant human growth hormone (hGH) in treating GH disorders such as growth hormone deficiency (GHD), age-related conditions, obesity and catabolic conditions.     

Detection of the cyanobacterial hepatotoxins microcystins

Concern regarding the presence of microcystins in drinking water and their possible contamination in food (e.g., salad vegetables, fish, shellfish) has resulted in the need for reliable methods for the detection and accurate quantification of this class of toxins. Currently, routine analysis of microcystins is most commonly carried out using high-performance liquid chromatography with photodiode array detection (HPLC-PDA), although more sensitive biological assays such as antibody-based ELISAs and protein phosphatase inhibition assays have also proven useful. However, many of these methods have been hindered by the availability of a wide range of purified microcystins. Although over 60 variants have now been reported, only a very small number are commercially available and calibrated standards are not yet obtainable. This has led to the common practice of reporting microcystin-LR equivalence regardless of which variant is present. The increased availability of HPLC with online mass spectral analysis (HPLC-MS) may facilitate more accurate detection of toxin variants but as several microcystins share the same molecular mass, definitive identification can be difficult. A further difficulty in analyzing microcystins is the requirement for sample processing before analysis. Solid phase extraction (SPE) is typically used to enrich environmental concentrations of microcystins, or to eliminate contaminants from complex samples such as animal and plant tissues. Recently, new technologies employing recombinant antibodies and molecularly imprinted polymers have been exploited to develop assays and biosensors for microcystins. These novel detection systems are highly sensitive, often do not require sample processing, and offer a simpler, less expensive alternative to analytical techniques. They have also been successfully employed in solid phase extraction formats for the concentration and clean up of environmental samples before HPLC analysis.     

蛋白多肽类药物体内动力学分析方法的研究新进展

蛋白多肽类药物由于其在生物体内含量低、半衰期短和易受内源性物质干扰等特点而使其体内动力学的研究面临很多挑战。生物样品分析方法是进行药物体内动力学研究的基础。综述了研究蛋白多肽类药物体内动力学的常规分析方法:免疫学分析法、同位素标记示踪法、生物检定法和理化分析技术。展望了荧光标记近红外检测技术在蛋白多肽类药物体内动力学无损实时在位分析上的应用前景。     

Detection of doping with recombinant human growth hormone

Detection of doping with recombinant human growth hormone is one of the challenges for antidoping analysis. This review focuses on the most important relevant publications that provide insight into the laboratory measurement of human growth hormone (hGH), antibodies and standards, the isoform approach and the biomarker approach. The isoform approach monitors the changes of hGH molecular isoform composition in serum and was applied at the Olympic Games in Athens in 2004, Turin in 2006 and Beijing in 2008. The markers approach detects a formula score, which reflects the changes in concentration of IGF-1 and P-III-P. All these methodologies measure the concentrations of growth hormone and its isoforms for isoform approach, or the concentrations of IGF-1 and P-III-P. All factors that affect these measurements should be taken into account for the development of methods to detect doping with recombinant hGH.     

重组人生长激素治疗肝硬化低蛋白血症

目的 :观察重组人生长激素治疗肝硬化引起的低蛋白血症疗效。方法 :病例选择为血清清蛋白含量低于 35g·L- 1的肝硬化病人 30例 ,男性 2 4例 ,女性 6例 ,年龄 ( 4 8±s11)a( 2 8～ 68a)。治疗方法为重组人生长激素 4IU ,sc ,qd ,10d为一个疗程。结果 :治疗后食欲增加为 70 % ( 2 1/30 )、腹胀减轻为 63 % ( 19/30 )、乏力减轻为 83 % ( 2 5/30 )、恶心减轻为 58% ( 11/19)。血清清蛋白由治疗前的 ( 2 8± 4 ) g·L- 1增加到 ( 32± 3) g·L- 1,P <0 .0 1;停药后 2wk血清清蛋白增加到 ( 36± 4 ) g·L- 1( 17例 )。Child积分由治疗前 ( 9.4± 2 .3)降低到 ( 8.5± 2 ) ,P<0 .0 1。结论 :重组人生长激素可以增加肝硬化病人的清蛋白水平 ,改善营养状况 ,无明显不良反应     

Regeneration of native structure and biological activity by air oxidation of the reduced bovine trypsin inhibitor (Kunitz)*

Derivatives of the tetraguanidinated bovine trypsin-kallikrein inhibitor lacking up to four amino-acid residues have been prepared by Edman degradation of the amino-terminal Arg-Pro-Asp-Phe sequence. Comparative studies on the native inhibitor, tetraguanidinated inhibitor, and truncated sequences demonstrated that none of the four N-terminal amino-acid residues are essential for the regeneration of native structure and biological activity by air oxidation of the reduced molecule. However residues 2, 3, and 4, although to a different extent, are important for the protein stability and significantly affect the conformation of the tetraguanidinated inhibitor and the rate of the reactivation process. Removal of the terminal positive charge from Arg 1 by diazotization gave a fully active deaminated, tetraguanidinated inhibitor but the lack of the salt bridge interaction between the amino and the carboxyl terminus of the protein prevented the correct folding of the reduced molecule by air oxidation as shown by activity measurements and conformational studies. The stability constants and the standard free energies of binding of the complexes between trypsin and the different inhibitor derivatives have been determined.     

Changes in Endothelial Dysfunction and Associated Cardiovascular Disease Morbidity Markers in GH-IGF Axis Pathology

Arterial endothelial dysfunction is an early event in the pathogenesis of atherosclerosis and predisposes individuals to the deposition of unstable atherosclerotic plaques. It can also lead to increased arterial stiffness, which is an accepted cause of increased arterial pulse wave velocity (APWV). Endothelial dysfunction is reversed by recombinant human growth hormone (rhGH) therapy in patients with growth hormone (GH) deficiency (GHD), favorably influencing the risk for atherogenesis. Endogenous human growth hormone (hGH), secreted by the anterior pituitary, and levels of insulin-like growth factor-I (IGF-I), produced in response to hGH stimulation of the liver, peak during early adulthood, but decline throughout adulthood. It is suspected that low-grade inflammatory cardiovascular pathophysiologic markers such as homocysteine, nitric oxide, C-reactive protein (CRP), and fibrinogen and plasminogen activator inhibitor along with changes in lipid and glucose metabolism may all contribute to GHD-associated metabolic and cardiovascular complications. These effects are associated with increased APWV, but are attenuated by rhGH therapy in GHD. GH replacement increases IGF-I levels and reduces CRP and large-artery stiffness. Reviews of rhGH in the somatopause have not been overtly favorable. Whereas reviews of rhGH/rhIGF-I combinations in GH resistance are more positive than those for rhGH alone, their combined use in the somatopause is limited. Senescent individuals may benefit from such a combination.     

Growth hormone for the treatment of growth failure in children

The etiology, diagnosis, and clinical features of growth failure in children are presented, with discussion of exogenous growth hormone (GH) replacement, its indications, efficacy, and adverse effects. Causes of growth delay include malnutrition, systemic illness, emotional deprivation, deficiency of endogenous growth hormone, thyroid hormone deficiency, and cortisol excess. Growth hormone (somatotropin) is secreted by the anterior pituitary gland in response to various stimuli, including exercise, hypoglycemia, and arginine. This hormone stimulates growth of skeletal muscle and connective tissue, increases rate of protein synthesis, and decreases rate of glucose use. Diagnosis of GH deficiency usually relies upon detection of adequate GH release in response to two stimuli. However, because patients with adequate endogenous GH release (determined by testing) may also grow when given exogenous GH, other methods are being evaluated for diagnosis of GH-dependent states. In many children, exogenous GH replacement produces increased rates of growth within 6 to 12 months; subsequently, growth rates decline. Distribution of the pituitary-derived GH product somatotropin was halted because of reports of Creutzfeldt-Jakob disease in some patients receiving it. Somatrem, a biosynthetic GH produced by recombinant DNA technology, has shown efficacy similar to somatotropin in clinical studies and is currently available to pediatric endocrinologists. Growth hormone replacement is beneficial in idiopathic and acquired GH deficiency, including partial GH deficiency. Further testing is needed to determine the usefulness and cost-benefit of somatrem therapy in GH-dependent and other types of growth failure.