WHO informal consultation on revision of guidelines on evaluation of similar biotherapeutic products, virtual meeting, 30 June - 2 July 2021.

The WHO informal consultation was held to promote the revision of WHO guidelines on evaluation of similar biotherapeutic products (SBPs) adopted by the Expert Committee on Biological Standardization (ECBS) in 2009. It was agreed in the past consultations that the evaluation principles in the guidelines are still valid, but a review was recommended to provide more clarity and case-by-case flexibility. The opportunity was therefore taken to review the experience and identify areas where the current guidance could be more permissive without compromising its basic principles, and where additional explanation could be provided regarding the possibility of reducing the amount of data needed for regulatory approval. The meeting participants applauded the leading role taken by the WHO in providing a much-needed streamlined approach for development and evaluation of SBPs which will provide efficient and cost-effective product development and increase patient access to treatments. It was recognized that the principles as currently described in the draft WHO guidelines are based on sound science and experience gained over the last fifteen years of biosimilar approvals. However, since these guidelines when finalised will constitute the global standard for biosimilar evaluation and assist national regulatory authorities in establishing revised guidance and regulatory practice in this complex area, it was felt that further revision and clarity on certain perspectives in specific areas was necessary to dispel uncertainties arising in the current revised version. This report describes the principles in the draft guidelines, including topics discussed and consensus reached.

Magnesium isotopes of the bulk solar wind from Genesis diamond-like carbon films.

NASA's Genesis Mission returned solar wind (SW) to the Earth for analysis to derive the composition of the solar photosphere from solar material. SW analyses control the precision of the derived solar compositions, but their ultimate accuracy is limited by the theoretical or empirical models of fractionation due to SW formation. Mg isotopes are "ground truth" for these models since, except for CAIs, planetary materials have a uniform Mg isotopic composition (within ≤1‰) so any significant isotopic fractionation of SW Mg is primarily that of SW formation and subsequent acceleration through the corona. This study analyzed Mg isotopes in a bulk SW diamond-like carbon (DLC) film on silicon collector returned by the Genesis Mission. A novel data reduction technique was required to account for variable ion yield and instrumental mass fractionation (IMF) in the DLC. The resulting SW Mg fractionation relative to the DSM-3 laboratory standard was (-14.4‰, -30.2‰) ± (4.1‰, 5.5‰), where the uncertainty is 2Æ¡ SE of the data combined with a 2.5‰ (total) error in the IMF determination. Two of the SW fractionation models considered generally agreed with our data. Their possible ramifications are discussed for O isotopes based on the CAI nebular composition of McKeegan et al. (2011).

Herbal feed additives containing essential oil: 1. Impact on the nutritional worth of complete feed in vitro.

This study was taken up to assess the impact of supplementing herbal feed additives [HFAs; fruit of Myristica fragrans (Jayphall), seeds of Anethum sowa (Suva), fruit of Apium graveolens (Ajmo), fruit of Cuminum cyminum (Jeera), bark of Cinnamonum zeylanicum (Dalchini), or whole plant of Eclipta alba (Bhangro)] containing essential oils as active component on the nutrient utilization and methane production using wheat straw-based total mixed ration (TMR) as a substrate by in vitro gas production technique. The essential oil content was the highest (P < 0.01) in M. fragrans followed by E. alba and A. sowa. In addition to essential oils, these HFAs also contained saponins, tannins, and antioxidants. The HFAs were supplemented at 1-3% of substrate dry matter (DM). The data were analyzed by 6 × 4 factorial design. Irrespective of level of HFA, the net gas production (NGP) and metabolizable energy (ME) availability was the highest (P < 0.01) in TMR supplemented with C. zeylanicum comparable with E. alba, but higher than TMR supplemented with other HFAs. Supplementation of TMR with different HFAs did not affect the digestibility of neutral detergent fiber (NDF) and true organic matter (TOM) and partitioning factor (PF). The total volatile fatty acids (VFAs), acetate, propionate (P < 0.01), and butyrate (P < 0.05) production was the highest in TMR supplemented with A. sowa, and the lowest was observed in TMR supplemented with C. cyminum. The isobutyrate and valerate production was also the highest (P < 0.01) in diet supplemented with A. sowa, but isovalerate production was the highest (P < 0.01) in diet supplemented with C. zeylanicum. The A:P ratio was the best in TMR supplemented with A. sowa. The efficiency of rumen fermentation was the highest, and efficiency of conversion of hexose to methane was the lowest in diet supplemented with A. sowa as compared to all other supplements. The in vitro methane production expressed as either percent of NGP, ml/100 mg DM of substrate/24 h, or as ml/100 mg of digestible OM/24 h was the lowest in TMR supplemented with A. sowa. The ammonia nitrogen production from TMR supplemented with M. fragrans and A. sowa was comparable, but significantly (P < 0.01) lower than TMR supplemented with other HFAs. Irrespective of the nature of HFA, the NGP and ME availability were significantly (P < 0.01) higher in TMR supplemented with HFAs at all levels as compared to un-supplemented TMR. As compared to control, the digestibility of NDF and that of TOM was depressed slightly in all the HFA-supplemented TMRs. The supplementation of HFAs at 2% of substrate DM improved (P < 0.01) the production of total VFAs, acetate, and propionate, and that of isovalerate in comparison to the un-supplemented TMR. The acetate to propionate ratio increased (P < 0.01) with the increase in the level of supplementation of HFAs containing essential oils. The methane and ammonia productions were depressed significantly when TMR was supplemented at 2% level of HFAs as compared to control TMR. It was concluded that supplementation of TMR with A. sowa at 2% of substrate was fermented better as indicated by the production of total and individual VFA, methane, and ammonia as compared to TMR supplemented with other HFA or un-supplemented TMR.

Collaborative study for the establishment of Etanercept Biological Reference Preparation Batch 1.

Three preparations of the human tumour necrosis factor (TNF) receptor II Fc fusion protein (TNFR II-Fc) Etanercept were formulated and lyophilised at the National Institute for Biological Standards & Control (NIBSC) prior to evaluation in a collaborative study for their suitability to serve as a World Health Organization (WHO) International Standard (IS)/European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the potency assay of Etanercept. Seven laboratories tested the preparations using an in vitro cell-based bioassay (TNF-α neutralisation) prescribed by the Ph. Eur. monograph on Etanercept (2895). The results of this study indicated that the candidate preparation, coded 13/204, established as the first IS for Etanercept with an assigned potency for TNF neutralisation activity of 10 000 IU per ampoule was also suitable to serve as Ph. Eur. BRP batch 1. The results were compared to those obtained with different cell-based neutralisation assays that were used by further laboratories in the context of establishing the 1st WHO IS for Etanercept. Based on these analyses, preparation 13/204 was adopted by the Ph. Eur. Commission as Etanercept BRP batch 1 with an assigned potency of 10 000 IU per ampoule.

Collaborative study for the establishment of Infliximab Biological Reference Preparation Batch 1.

Two preparations of the chimeric anti-Tumour Necrosis Factor (TNF) monoclonal antibody Infliximab were formulated and lyophilised at the National Institute for Biological Standards & Control (NIBSC) prior to evaluation in a collaborative study for their suitability to serve as a World Health Organization (WHO) International Standard (IS)/European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the potency assay of Infliximab. Twenty-six laboratories tested the preparations using different in vitro cell-based bioassays (TNF-α neutralisation, antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and binding assays. Amongst them, 19 laboratories performed cell-based bioassays. The results of this study indicated that the candidate preparation coded 16/170 was suitable to serve as an International Standard for Infliximab based on the data obtained for biological activity. This candidate standard was established in 2017 as the first International Standard for Infliximab with an assigned potency for TNF neutralisation activity of 500 IU per ampoule. In the same study, the suitability of preparation 16/170 of Infliximab to serve as the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the Infliximab potency assay as described in the Ph. Eur. monograph on Infliximab concentrated solution (2928) was also evaluated. The corresponding analysis, based on the measurement of the inhibitory action of anti-human TNF (Infliximab) on the cytotoxic activity of TNF-alpha, was performed using data from a subset of 9 laboratories using the TNF-alpha-sensitive fibrosarcoma cell line WEHI-164. The results obtained were compared to those obtained from different cell-based neutralisation assays that were used by other laboratories in the context of establishing the 1st World Health Organization (WHO) International Standard (IS) for Infliximab. Based on the analyses, preparation 16/170 was adopted by the Ph. Eur. Commission in June 2018 as Infliximab BRP batch 1 with an assigned potency of 500 IU per ampoule.

Redox state of Mars' upper mantle and crust from Eu anomalies in shergottite pyroxenes.

The oxidation state of basaltic martian meteorites is determined from the partitioning of europium (Eu) in their pyroxenes. The estimated redox conditions for these samples correlate with their initial neodymium and strontium isotopic compositions. This is interpreted to imply varying degrees of interaction between the basaltic parent melts, derived from a source in the martian mantle, and a crustal component. Thus, the mantle source of these martian basalts may have a redox state close to that of the iron-wüstite buffer, whereas the martian crust may be more oxidized (with a redox state higher than or equal to that of the quartz-fayalite-magnetite buffer). A difference in redox state of more than 3 log units between mantle and crustal reservoirs on Mars could result from oxidation of the crust by a process such as aqueous alteration, together with a subsequent lack of recycling of this oxidized crust through the reduced upper mantle.

Measurement of neutralising antibodies to type I interferons by gene expression assays specific for type 1 interferon-inducible 6-16 mRNA.

Human type I interferon products have been approved for the treatment of several diseases, though neutralising antibodies against them may develop and reduce therapeutic efficacy. Traditionally, potencies of human interferons (IFNs) and of neutralising antibodies (NAbs) against them are quantified by antiviral assays. These are being increasingly replaced by less cumbersome and faster bioassay methods. Since IFNs exert their biological effects by binding to receptors on target cells and stimulating the expression of IFN-inducible genes, measurement of transcribed mRNAs can form the basis of functional bioassays. In this study we have used two approaches, quantitative reverse transcription-polymerase chain reaction (qPCR) and branched DNA (bDNA), to develop efficient, sensitive and robust non-viral assays to quantify type I IFNs per se and NAbs in sera from patients treated with either IFNbeta or IFNalpha2a. We show the rapid (4h) induction of the type I IFN-inducible 6-16 mRNA in A549 lung carcinoma cells is sensitively and reproducibly concentration-dependent for both IFNbeta and IFNalpha2a stimulation, is quantifiable by either approach, and is readily adaptable for the detection and measurement of NAbs against type I IFNs. Quantitative neutralisation of IFN-stimulated 6-16 mRNA expression was achieved in both assays when sera from patients receiving IFNbeta or IFNalpha2a therapy known to contain NAbs against these IFNs were tested. Their rapid and potentially automatable performance strongly suggests these assays could be used in a clinical setting to monitor the development of neutralising antibodies in patients receiving IFN therapy.

Inherited interleukin 12 deficiency in a child with bacille Calmette-Guérin and Salmonella enteritidis disseminated infection.

Interferon-gamma receptor ligand-binding chain (IFN-gammaR1) or signaling chain (IFN-gammaR2) deficiency, like interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency, predispose to severe infections due to poorly virulent mycobacteria and salmonella. A child with bacille Calmette-Guérin and Salmonella enteritidis infection was investigated. Mutations in the genes for IFN-gammaR1, IFN-gammaR2, IL-12Rbeta1, and other molecules implicated in IL-12- or IFN-gamma-mediated immunity were sought. A large homozygous deletion within the IL-12 p40 subunit gene was found, precluding expression of functional IL-12 p70 cytokine by activated dendritic cells and phagocytes. As a result, IFN-gamma production by lymphocytes was markedly impaired. This is the first discovered human disease resulting from a cytokine gene defect. It suggests that IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella.

A genetic screen for increasing metabolic flux in the isoprenoid pathway of Saccharomyces cerevisiae: Isolation of SPT15 mutants using the screen.

A genetic screen to identify mutants that can increase flux in the isoprenoid pathway of yeast has been lacking. We describe a carotenoid-based visual screen built with the core carotenogenic enzymes from the red yeast Rhodosporidium toruloides. Enzymes from this yeast displayed the required, higher capacity in the carotenoid pathway. The development also included the identification of the metabolic bottlenecks, primarily phytoene dehydrogenase, that was subjected to a directed evolution strategy to yield more active mutants. To further limit phytoene pools, a less efficient version of GGPP synthase was employed. The screen was validated with a known flux increasing gene, tHMG1. New mutants in the TATA binding protein SPT15 were isolated using this screen that increased the yield of carotenoids, and an alternate isoprenoid, α-Farnesene confirming increase in overall flux. The findings indicate the presence of previously unknown links to the isoprenoid pathway that can be uncovered using this screen.

Adenosine A1 receptor antagonist mitigates deleterious effects of sleep deprivation on adult neurogenesis and spatial reference memory in rats.

Sleep deprivation (SD) upsurges intracellular levels of adenosine, impairs adult neuronal cell proliferation (NCP) and cognition while caffeine, a non-selective adenosine A1 receptor (A1R) antagonist improves cognition and adult NCP during SD. We examined the selective antagonistic effects of adenosine A1R using 8-cyclopentyl-1,3-dimethylxanthine (8-CPT) on impairment of spatial reference memory and adult NCP during 48h SD. Adult male Sprague Dawley rats were sleep deprived for 48h, using an automatic cage vibrating stimulus based on animal activity. Spatial reference memory was tested as a measure of cognitive performance employing Morris Water Maze. Rats were given 8-CPT dissolved in 50% dimethyl sulfoxide (DMSO), twice daily (10mg/kg, i.p.) along with 5-bromo-2-deoxyuridine (BrdU) (50mg/kg/day, i.p.). The rats treated with 8-CPT showed significantly short mean latency and path-length to reach the platform compared to the SD rats. Consistent with these findings, 8-CPT-treated group was found to have significantly increased the number of BrdU, Ki-67 and doublecortin (DCX) positive cells. However, no significant difference was seen in NeuN expression in the Dentate Gyrus (DG). Brain-derived neurotropic factor (BDNF) expression in the DG and CA1 region was observed to decrease significantly after SD and be rescued by 8-CPT treatment. Furthermore, latency to reach platform showed a negative correlation with number of BrdU, DCX type-1 cells and BDNF expression in DG. Thus, it may be concluded that treatment with 8-CPT, an adenosine A1R antagonist during SD mitigates SD induced decline in spatial reference memory and adult NCP possibly via up regulation of BDNF levels in DG and CA1 regions.

Electrophysiological and antiarrhythmic effects of the atrial selective 5-HT(4) receptor antagonist RS-100302 in experimental atrial flutter and fibrillation.

BACKGROUND: Stimulation of 5-HT(4) receptors increases atrial chronotropic and inotropic responses. Whether other electrophysiological effects are produced is unknown. In humans and swine, 5-HT(4) receptors are present only in atrium. Therefore, the effects of a novel 5-HT(4) receptor antagonist, RS-100302, and the partial agonist cisapride on atrial flutter and fibrillation induced in swine were studied to delineate the role of the 5-HT(4) receptor in modulating atrial electrophysiological properties and the antiarrhythmic potential of RS-100302. METHODS AND RESULTS: In 17 anesthetized, open-chest, juvenile pigs, atrial flutter or fibrillation was induced by rapid right atrial pacing with or without a right atrial free wall crush injury, respectively. Atrial effective refractory period (ERP), conduction velocity, wavelength, and dispersion of refractoriness were determined during programmed stimulation via a 56-electrode mapping plaque sutured to the right atrial free wall. Ventricular electrophysiological parameters were also measured. All electrophysiological parameters were measured at baseline and after infusion of RS-100302 and cisapride. In the atrium, RS-100302 prolonged mean ERP (115+/-8 versus 146+/-7 ms, P<0.01) and wavelength (8.3+/-0.9 versus 9.9+/-0.8 cm, P<0.01), reduced dispersion of ERP (15+/-5 versus 8+/-1 ms, P<0.01), and minimally slowed conduction velocity (72+/-4 versus 67+/-5 cm/s, P<0.01). These effects were all partially reversed by cisapride. RS-100302 produced no ventricular electrophysiological effects. RS-100302 terminated atrial flutter in 6 of 8 animals and atrial fibrillation in 8 of 9 animals and prevented reinduction of sustained tachycardia in all animals. CONCLUSIONS: The electrophysiological profile of RS-100302 suggests that it may have atrial antiarrhythmic potential without producing ventricular proarrhythmic effects.

Immunogenicity of granulocyte-macrophage colony-stimulating factor (GM-CSF) products in patients undergoing combination therapy with GM-CSF.

In this study, we have assessed the development of neutralizing and nonneutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in two groups of patients with metastatic colorectal carcinoma receiving two different GM-CSF products. Three clinical trials were carried out, and a combination of GM-CSF and a colon carcinoma-reactive antibody was used in the absence of any concomitant chemotherapy. Two different GM-CSF products, both rDNA-derived and produced in Escherichia coli, were used. Patients in Trial 1 received product X, and those in Trials 2 and 3 received product Y. Patients in Trial 2 also received interleukin 2 in an attempt to potentiate immune responses. After the first cycle of treatment, no GM-CSF antibodies were detected, but on subsequent therapy, 28 of the 38 patients tested receiving product Y (Trials 2 and 3) developed antibodies that bound to the GM-CSF product used for therapy. However, none of the patients developed antibodies that neutralized the biological activity of GM-CSF, as assessed using an in vitro bioassay. Furthermore, there was no in vivo impairment in GM-CSF-induced expansion of leukocytes, neutrophils, and eosinophils in the patients. In contrast, 19 of the 20 patients given product X (Trial 1) developed GM-CSF binding antibodies, and 9 of these patients were shown to develop antibodies that neutralized the biological activity of GM-CSF. The presence of the latter was associated with a significant reduction in GM-CSF-induced expansion of leukocytes, neutrophils, and eosinophils in patients. Therefore, product X appears to be more immunogenic than product Y. Immunochemical characterization confirmed that the specificity of the antibody responses varied depending on the product used for therapy. Whereas sera from Trial 1 patients treated with product X showed the presence of antibodies with strong recognition of GM-CSF proteins, sera from patients treated with product Y showed varied recognition of GM-CSF ranging from fairly strong to very weak but bound predominantly to two E. coli-derived, non-GM-CSF-related proteins of Mr approximately 20,000 and Mr approximately 30,000. Therefore, in sera from patients receiving product Y, the antibody specificity appeared to be directed not only against GM-CSF but also against non-product-related host cell contaminants. This study shows that GM-CSF products used for therapy are potentially immunogenic and generate antibodies to GM-CSF and/or other non-product-related contaminants. However, only antibodies that neutralize the biological activity of GM-CSF compromise therapeutic efficacy of the cytokine.

Alteration of interleukin 2 (IL-2) pharmacokinetics and function by IL-2 antibodies induced after treatment of colorectal carcinoma patients with a combination of monoclonal antibody 17-1A, granulocyte macrophage colony-stimulating factor, and IL-2.

In this study, we have assessed the development of neutralizing and non-neutralizing interleukin 2 (IL-2) antibodies in metastatic colorectal carcinoma patients receiving a colon carcinoma reactive monoclonal antibody (17-1A) in combination with granulocyte macrophage colony-stimulating factor and IL-2 therapy. Before treatment, no IL-2 antibodies were detected in any of the patients. After therapy, 10 of the 19 patients tested developed antibodies that bound to the IL-2 product used for therapy, but only one developed antibodies that neutralized the biological activity of IL-2 as assessed using an in vitro bioassay. We found that the induction of IL-2 antibodies in some patients irrespective of their neutralizing potential had a significant impact on IL-2 pharmacokinetics. A significant reduction of the area under the concentration-time curve and maximum concentration (C(max)) and increased IL-2 distribution and clearance were observed in IL-2 antibody-positive patients in comparison with IL-2 antibody-negative patients. A significant decrease in IL-2-mediated expansion of lymphocytes was also evident in patients positive for IL-2 antibodies in comparison with those negative for these antibodies. Further characterization of sera from patients with antibodies showed that, in most cases, the antibodies recognized different IL-2 preparations. Results also showed that serum IL-2 concentration at initiation of therapy in patients was significantly higher relative to healthy control donors. The endogenous production of IL-2 gradually increased during the treatment cycles. To conclude, induction of neutralizing and non-neutralizing antibodies in cytokine-treated patients should be carefully monitored in terms of their clinical significance.

Detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals.

Assessment of the unwanted immunogenicity of therapeutic biologicals in recipients is an important consideration in the evaluation of these medicinal products. Proper planning of immunogenicity studies with appropriately devised strategies is critical if valid and meaningful conclusions concerning the unwanted immunogenicity are to be derived. An essential requisite for such studies is the need for conducting carefully selected and validated procedures. Several techniques are available for detection, characterization and measurement of antibodies elicited in an immune response. These include various formats of immunoassays, surface plasmon resonance and biological assays. None of these assays alone can provide sufficient information on the characteristics of the induced antibodies. A combination of methods is therefore usually necessary for a detailed understanding of the quantity and type(s) of antibodies generated against a therapeutic product. This manuscript considers the benefits and limitations of the various techniques available for antibody detection and outlines a brief strategy for the assessment of unwanted immunogenicity of therapeutic products.

Caffeine and modafinil given during 48 h sleep deprivation modulate object recognition memory and synaptic proteins in the hippocampus of the rat.

We aimed to evaluate the effect of caffeine/modafinil on sleep deprivation (SD) induced alterations in recognition memory and synaptic proteins. The data revealed a beneficial effect of caffeine/modafinil against deficit in the familiar object retrieval performance and object exploration ratio after 48 h SD. Caffeine treatment prevented the SD induced down-regulation of synaptophysin and synapsin I proteins with no change in PSD-95 protein in hippocampus. However, modafinil administration improved the down-regulation of synaptophysin, synapsin I and PSD-95 proteins in hippocampus. Hence, caffeine/modafinil can serve as counter measures in amelioration of SD induced consequences at behavioural and protein levels.

Biodistribution and gene expression of lipid/plasmid complexes after systemic administration.

The objectives of this study were to investigate the influence of physicochemical properties of lipid/plasmid complexes on in vivo gene transfer and biodistribution characteristics. Formulations based on 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and novel biodegradable cationic lipids, such as ethyl dioleoyl phosphatidylcholine (EDOPC), ethyl palmitoyl myristyl phosphatidylcholine (EPMPC), myristyl myristoyl carnitine ester (MMCE), and oleyl oleoyl L-carnitine ester (DOLCE), were assessed for gene expression after tail vein injection of lipid/plasmid complexes in mice. Gene expression was influenced by cationic lipid structure, cationic lipid-to-colipid molar ratios, plasmid-to-lipid charge ratios, and precondensation liposome size. Detectable levels of human growth hormone (hGH) in serum, human factor IX (hFIX) in plasma, and chloramphenicol acetyltransferase (CAT) in the lung and liver were observed with positively charged lipid/plasmid complexes prepared from 400-nm extruded liposomes with a cationic lipid-to-colipid ratio of 4:1 (mol/mol). Intravenous administration of lipid/CAT plasmid complexes resulted in distribution of plasmid DNA mainly to the lung at 15 min after injection. Plasmid DNA accumulation in the liver increased with time up to 24 hr postinjection. There was a 10-fold decrease in the amount of plasmid DNA in the lung at 15 min after injection, when the lipid/plasmid complex charge ratio was decreased from 3:1 to 0.5:1 (+/-). Bright fluorescent aggregates were evident in in vivo-transfected lung with the positively charged pCMV-CAT/DOLCE:dioleyl phosphatidylethanolamine (DOPE) (1:1, mol/mol) complexes, while more discrete punctate fluorescence was observed with a 4:1 molar ratio of cationic lipid:colipid formulations. Preinjection of polyanions such as plasmid, dextran sulfate, polycytidic acid, and polyinosinic acid decreased hGH expression, whereas the preinjection of both positively charged and neutral liposomes had no effect on hGH serum levels. Of the cationic lipids tested, DOLCE was found to be the most effective potentially biodegradable cationic lipid. A correlation between gene expression and cationic lipid:colipid ratios and lipid-to-plasmid charge ratio was also observed for DOTMA- and DOLCE-based formulations.

Detection and measurement of cytokines.

Accurate and sensitive methods for the measurement and detection of cytokines are an obvious pre-requisite for the study of cytokine biology, biochemistry and the possible involvement of these molecules in pathology. In this review, the various methods available for cytokine measurement and detection (bioassays, immunoassays and other procedures) are described and compared. A critical appraisal of the potential advantages and limitations of the techniques is included.

Cytokine immunoassays: recommendations for standardisation, calibration and validation.

Accurate and sensitive methods for measurement and detection of cytokines are important for the understanding of cytokine biology and biochemistry and the assessment of cytokine involvement in pathology and physiological processes. Various types of assays are available for estimation of cytokine levels in biological fluids. Often, immunoassays are used for this purpose but in many cases inappropriate assay choice, design and standardisation have led to confusing or erroneous data and conclusions. Only if well characterized, validated and standardized methods are used and results carefully analysed can useful unambiguous information be obtained. This article contributes significantly towards the standardisation, calibration and validation aspects of immunoassays for cytokines.

Strategies for detection, measurement and characterization of unwanted antibodies induced by therapeutic biologicals.

An important aspect of evaluating the safety of therapeutic biologicals is the assessment of the unwanted immunogenicity of such biologicals in recipients. Properly planned immunogenicity studies with appropriately devised strategies are critical if valid conclusions concerning the unwanted immunogenicity are to be derived. Such studies need to be conducted using carefully selected and validated procedures. Several techniques are available for detection and measurement of immunogenicity including immunoassays, radioimmunoprecipitation assays (RIPAs), surface plasmon resonance (SPR) and bioassays. A combination of methods for characterization of the induced antibodies is usually necessary for a detailed understanding of the type(s) of antibodies generated against a therapeutic product. This review considers the benefits and limitations of the various techniques available for antibody detection and outlines a strategy for the assessment of unwanted immunogenicity of therapeutic products.

A novel, sensitive bioassay for transforming growth factor beta.

We have developed a simple, sensitive bioassay for transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) based on the ability of these cytokines to inhibit the interleukin-5 induced proliferation of the erythroleukaemia cell line, TF-1. This assay is rapid, reproducible and sensitive to less than 500 fg/ml of TGF-beta 1, and 5-10 pg/ml TGF-beta 2. The assay is 100-1000-fold less sensitive to other inhibitory molecules such as interferon-beta, interferon-gamma and TNF-alpha. The assay can be made specific for TGF-beta 1 or TGF-beta 2 by including specific neutralising antibodies for TGF-beta 1 or TGF-beta 2. The assay can recognise all the readily available recombinant molecular species of these molecules as well as the natural proteins produced from human and bovine platelets and detects TGF-beta in serum samples.