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Accurate quantification of soluble glypican-3 in clinical samples using immunoassays is challenging, because of the lack of appropriate antibody reagents to provide a full spectrum measurement of all potential soluble glypican-3 fragments in vivo. Glypican-3 SOMAmer (slow off-rate modified aptamer) is a novel reagent that binds, with high affinity, to a far distinct epitope of glypican-3, when compared to all available antibody reagents generated in-house. This paper describes an integrated analytical approach to rational selection of key reagents based on molecular characterization by epitope mapping, with the focus on our work using a SOMAmer as a new reagent to address development challenges with traditional antibody reagents for the soluble glypican-3 immunoassay. A qualified SOMAmer-based assay was developed and used for soluble glypican-3 quantification in hepatocellular carcinoma (HCC) patient samples. The assay demonstrated good sensitivity, accuracy, and precision. Data correlated with those obtained using the traditional antibody-based assay were used to confirm the clinically relevant soluble glypican-3 forms in vivo. This result was reinforced by a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay quantifying signature peptides generated from trypsin digestion. The work presented here offers an integrated strategy for qualifying aptamers as an alternative affinity platform for immunoassay reagents that can enable speedy assay development, especially when traditional antibody reagents cannot meet assay requirements.

Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. Graphical Abstract ᅟ.

Background: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. Results: For the quantitation of BMS-986089, both accuracy (99–103%) and precision (2.4–12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100–108%) and precision (0.7–18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94–103%) and precision (2.4–14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). Conclusion: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC–MS/MS-based methods to eventually assess in vivo drug–target engagement.

Clopidogrel is a prodrug anticoagulant with active metabolites that irreversibly inhibit the platelet surface GPCR P2Y12 and thus inhibit platelet activation. However, gaining an understanding of patient response has been limited due to imprecise understanding of metabolite activity and stereochemistry, and a lack of acceptable analytes for quantifying in vivo metabolite formation. Methods for the production of all bioactive metabolites of clopidogrel, their stereochemical assignment, and the development of stable analytes via three conceptually orthogonal routes are disclosed.

Interleukin-23 (IL-23) is an important therapeutic target for the treatment of inflammatory diseases. Adnectins are targeted protein therapeutics that are derived from domain III of human fibronectin, and have similar protein scaffold to antibodies. A specific adnectin (Adnectin 2) was identified to bind to IL-23 and compete with IL-23/IL-23R interaction, being a potential protein therapeutic. Hydrogen/deuterium exchange mass spectrometry (HDX MS) and computational methods were applied to probe the binding interactions between IL-23 and Adnectin2 and to determine the correlation between the two orthogonal methods. This review article summarizes the current structural knowledge about Il-23 and it focuses on the applicability of HDX MS to investigate the higher order structure of proteins, which plays an important role for the discovery of new and improved biotherapeutics.

Higher order structure (HOS) is a crucial determinant for the biological functions and quality attributes of protein therapeutics. Mass spectrometry (MS)-based protein footprinting approaches play an important role in elucidating the relationship between protein biophysical properties and structure. Here, we describe the use of a combined method including hydrogen-deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP) and site-specific carboxyl group footprinting to investigate the HOS of protein and protein complexes. The work focuses on implementing complementary solution-phase footprinting approaches that differ in time scale, specificity for protein residue side chains vs. backbone as well as selectivity for different residue types to map integratively the epitope of human interleukin-6 receptor (IL-6R) for two adnectins with distinct affinities (Kd, Adnectin1 ∼ 6.2 pM vs. Kd, Adenctin2 ∼ 46 nM), and evaluate the resultant conformation/dynamic change of IL-6R. The suggested epitope, which is conserved for adenctin1 and adenctin2 binding, is a flexible loop that connects two β-strands in the cytokine-binding domain (DII) of IL-6R. We also found that adnectin1, the more strongly binding ligand, induces structural perturbations on two unstructured loops that are distally located beyond the epitope. Those changes are either attenuated or not detected for the case of adnectin2 binding. In addition to providing credibility in epitope determination, utilization of those combined approaches reveals the structural effects that can differentiate protein therapeutics with similar apparent biophysical properties.

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.ᅟ

The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this article, HDX-MS based workflows designed for both protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein/drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS to protein therapeutics characterization are also described.

Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.Graphical Abstract.

Background: There is considerable interest in the pharmaceutical industry today in both development of therapeutic proteins as viable biopharmaceutical agents as well as the implementation of microsampling techniques, such as dried blood spots (DBS), as an alternative to current sample collection and handling procedures for biological samples generated in drug discovery and development studies. We have demonstrated that these two techniques can be integrated by developing bioanalytical methods that simultaneously determine the concentrations of unique therapeutic protein constructs, using LC–MS-based detection of multiple surrogate peptides following direct trypsin digestion of DBS. Results: Bioanalytical methods were developed for the simultaneous determination of two structurally different therapeutic proteins (PEGylated-Adnectin™-1, MW 11,144 amu and an Fc-fusion protein, MW 67,082 amu) in a single DBS sample using LC–MS-based detection of multiple peptides generated from different regions of the proteins following trypsin digestion. The same methodology was applied to the analysis of DBS samples collected following dosing of a third unique protein (PEGylated-Adnectin-2) to mice. Although these initial DBS methods were slightly less sensitive than those developed specifically for each individual protein in plasma or serum, the generic digestion procedure yielded sufficient accuracy, precision and an extended linear dynamic range to justify their further evaluation in pharmacokinetic, pharmacodynamic and toxicological studies of selected therapeutic proteins following dosing in preclinical discovery studies. Additionally, DBS samples may offer a convenient, generic platform approach for direct enzymatic digestion and sample preparation for LC–MS-based quantitation of proteins. DBS samples prepared for two of the therapeutic proteins were also stable for at least 2 weeks when stored at room temperature. Conclusion: Although the same clarification and interpretation of DBS results will be required (e.g., blood vs plasma levels, hematocrit effects on DBS determinations and red blood cell partitioning) as for small-molecules, there still remains the potential to further develop and expand this strategy with appropriate proteins of interest. While additional studies will be required to validate this approach in specific applications, we have demonstrated the feasibility of using DBS sampling to directly quantify structurally different types of therapeutic proteins in blood in discovery studies and present the potential to simultaneously measure other proteins, such as biomarkers, to augment and integrate data generated from in vivo studies.

It has recently been proposed that plasma levels of 4β-hydroxycholesterol (4βHC) may be indicative of cytochrome P450 3A4 (P450 3A) activity and therefore could be used to probe for P450 3A-mediated drug-drug interactions. With this in mind, we describe a highly sensitive and precise liquid chromatography-electrospray ionization-tandem mass spectrometry method for the measurement of 4βHC in human plasma with a lower limit of quantification established at 2 ng/mL using 50 μL of plasma. The entire sample preparation scheme including saponification and derivatization of 4βHC to the corresponding dipicolinyl ester (DPE) was completed in less than 8 h using an automated sample preparation scheme enabling higher-throughput capabilities. Chromatographic resolution of 4βHC from 4α-hydroxycholesterol and other endogenous isobaric species was achieved in 11-min using an isocratic gradient on a C18 column. Because of endogenous concentrations of 4βHC in plasma, a stable isotope labeled (SIL) analogue, d7-4βHC, was used as a surrogate analyte and measured in the standard curve and quality control samples prepared in plasma. A second SIL analogue, d4-4βHC, was used as the internal standard. The intraday and interday accuracy for the assay was within 6% of nominal concentrations, and the precision for these measurements was less than 5% relative standard deviation. Rigorous stability assessments demonstrated adequate stability of endogenous 4βHC in plasma and the corresponding DPE derivative for the analysis of clinical study samples. The results from clinical samples following treatment with a potent P450 3A inducer (rifampin) or inhibitor (ketoconazole) are reported and demonstrate the potential future application for this highly precise and robust analytical assay.

Mass spectrometry (MS) has become a powerful technology in the discovery and development of protein therapeutics in the biopharmaceutical industry. This review article describes recent developments and future trends in the characterization of protein therapeutics using MS. We discuss top-down MS for the characterization of protein modifications, hydrogen/deuterium exchange MS and ion mobility MS methods for higher order protein structure studies. Quantitative analysis of protein therapeutics (in vivo) by MS as an orthogonal approach to immunoassay for pharmacokinetics studies will also be illustrated.

The stereoselective determination of stereoisomers in biological samples provides vital information on stereospecific metabolism and pharmacokinetic profiles of the drugs. Despite the unique advantage and the great success of normal-phase (NP) HPLC for the separations of drug stereoisomers using polysaccharide-type chiral stationary phases (CSPs), the technique is rarely applied to quantitative HPLC-MS-MS bioanalysis. This is, at least in part, due to the incompatibility between the usual mobile phase (n-hexane or n-heptane) in normal-phase HPLC and the MS ionization sources which poses a potential detonation hazard. An environmentally friendly and nonflammable alternative solvent, ethoxynonafluorobutane (ENFB), was reported previously to potentially provide an ideal solution for combining the powers of stereoselective NP chromatographic separation and MS-MS detection. In this study, a stereoselective NP-HPLC-MS-MS method was developed using ENFB to quantify a pair of Bristol Myers Squibb (BMS) proprietary drug stereoisomers and their ketone metabolite for an in vitro study, which demonstrated, for the first time, the practical applicability and utility of ENFB for bioanalysis in pharmaceutical industry. The effects of different organic modifiers and temperature, as well as the comparison between ENFB and the usual solvent, heptane, for the separation, are discussed. The resolution of the stereoisomers was achieved using 63% of 3:1 mixture of ethanol and methanol with 37% ENFB on a Chiralpak AD-H column at 50 degrees C. High sensitivity was obtained using the MS-MS detection in the positive ion atmospheric pressure chemical ionization (APCI) mode. The lower limit of quantitation (LLOQ) for the first stereoisomer and the ketone metabolite was 5 ng/mL, and was 10 ng/mL for the second isomer in the human liver microsome-potassium phosphate buffer matrix. The linear dynamic range of 5-1000 ng/mL for both isomers and 10-1000 ng/mL for the metabolite were demonstrated with R2or =0.997. The precision of the analysis was5% R.S.D. at or above the nominal concentration of 80 ng/mL, and20% R.S.D. at 8 ng/mL. The mean bias was less than 15%. Extraction recovery and acceptable matrix interference were demonstrated using one isomer and the ketone, and better than 75% recovery and less than 25% ion suppression or interference were found. The method was successfully implemented for an in vitro intrinsic metabolic clearance study.

Metabolomics has roots in the pharmaceutical industry that go back nearly three decades. Initially focused on applications in toxicology and disease pathology, more recent academic and commercial efforts have helped advance metabolomics as a tool to reveal the molecular basis of biological processes and pharmacological responses to drugs. This article will discuss areas where metabolomic technologies and applications are poised to have the greatest impact in the discovery and development of pharmaceuticals.

Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin ((10)Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.

Lysophosphatidic acids (LPAs) are biologically active signaling molecules involved in the regulation of many cellular processes and have been implicated as potential mediators of fibroblast recruitment to the pulmonary airspace, pointing to possible involvement of LPA in the pathology of pulmonary fibrosis. LPAs have been measured in various biological matrices and many challenges involved with their analyses have been documented. However, little published information is available describing LPA levels in human bronchoalveolar lavage fluid (BALF). We therefore conducted detailed investigations into the effects of extensive sample handling and sample preparation conditions on LPA levels in human BALF. Further, targeted lipid profiling of human BALF and plasma identified the most abundant lysophospholipids likely to interfere with LPA measurements. We present the findings from these investigations, highlighting the importance of well-controlled sample handling for the accurate quantitation of LPA. Further, we show that chromatographic separation of individual LPA species from their corresponding lysophospholipid species is critical to avoid reporting artificially elevated levels. The optimized sample preparation and LC/MS/MS method was qualified using a stable isotope-labeled LPA as a surrogate calibrant and used to determine LPA levels in human BALF and plasma from a Phase 0 clinical study comparing idiopathic pulmonary fibrosis patients to healthy controls.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method.The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ).Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method.The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.

Background: There is a need for general and reliable LC–MS assays capable of supporting the bioanalysis of a variety of human monoclonal antibody-based therapeutic drug candidates in animal PK/TK studies. Results: We present herein improvements in our previously reported universal peptide approach to the bioanalysis of human monoclonal antibody protein drug candidates in animal studies. These improvements include incorporation of a second, light chain-based universal peptide into the assay, thus introducing the concept of a dual universal peptide assay; and incorporation of a universal stable isotope-labeled monoclonal antibody into the assay. Conclusion: Improvements reported herein to the universal peptide assay will enable more reliable quantification of human monoclonal antibody protein drug candidates in animal studies.

High-resolution MS (HRMS) is a central analytical technique for the study of biomolecules and is widely used in the biopharmaceutical industry. This paper reviews recent advances in commonly used HRMS instrumentation and experimental strategies for HRMS-based structural characterization of protein therapeutics. An overview of protein higher order structural characterization using HRMS-based technologies is presented, including the use of hydrogen/deuterium exchange and hydroxyl radical footprinting methods for probing protein conformational dynamics and interactions in solution. Future directions in application of HRMS for characterizing protein therapeutics are also described.

The specific, high affinity binding of plant-derived digitalis glycosides by the mammalian sodium and potassium transporting adenosine triphosphatase (Na,K-ATPase, or sodium pump), a plasma membrane enzyme with critical physiological importance in mammalian tissues, has raised the possibility that a mammalian analog of digitalis might exist. We previously isolated and structurally characterized from bovine hypothalamus a novel isomer of the plant glycoside, ouabain, which differs structurally only in the attachment site and/or the stereochemistry of the steroid moiety [Tymiak et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8189-8193]. Hamlyn and co-workers reported a molecule purified from human plasma which by mass spectrometry could not be distinguished from plant ouabain [Hamlyn et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6259-6263]. Since rhamnoside cardiotonic steroids are not known as natural products from mammalian sources, it became important to compare these two pure isolates to determine if the same or structurally distinct compounds has been found. Our results indicate that the human and bovine Na,K-ATPase-inhibitors are identical, but different from plant ouabain. This supports the notion that the human sodium pump may be under specific physiological regulation by a mammalian analog of the digitalis glycosides.

Recombinant human G granulocyte-colony stimulating factor (rhG-CSF) produced in Escherichia coli is a non-glycosylated polypeptide containing five cysteine residues. The reported major disulfide (S-S) linkages in mature human G-CSF are C36 -C42 and C64 -C74 , leaving C17 as a free cysteine, which could potentially result in S-S scrambling. The purpose of this work is to illustrate different mass spectrometry (MS) approaches for characterization of S-S linkages in therapeutic proteins including S-S scrambling using rhG-CSF as a model protein.Peptide mapping analysis of both non-reduced and reduced digests of rhG-CSF was performed to demonstrate the presence of S-S linked peptides and their corresponding reduced peptides. High mass accuracy measurements of these peptides provided the initial identifications of S-S linkages. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) were used to fragment these peptides in order to obtain further sequence information and identify S-S linkages.S-S linked peptides and their corresponding reduced peptides correlating with major S-S linkages were observed. Peptides that correlated with other S-S linkages as a result of S-S scrambling were also observed.Presence of the reported major S-S linkages in rhG-CSF was confirmed. S-S scrambling was also observed in which C18 was involved in S-S linkages and C37 , C65 or C75 were present as free cysteines. This study demonstrates the practical utility of combining different MS methods for characterization of S-S linkages in therapeutic proteins.

Mass spectrometry (MS) is one of the key techniques in protein characterization. In this article, the workflow for MS-based structural characterization of biologics in biopharmaceutical drug discovery is presented, including characterization of primary and higher order structures. Advances in MS techniques in protein characterization are illustrated, including electron transfer dissociation MS (ETD-MS) for primary structure analysis and hydrogen/deuterium exchange MS (HDX-MS) for probing protein higher order structures and mapping epitopes. Future trends in applications of MS to evaluate and optimize candidate molecules in biologics stability studies is also described.

A flexible two-step protocol has been developed for high-throughput assays of endothelin converting enzyme (ECE) activity. The method is illustrated using endothelial cell monolayers and crude cell extracts as enzyme sources for conversion of exogenous substrates (big endothelin isopeptides) to their respective vasoactive products (endothelin isopeptides). Cleavage conditions have been optimized to minimize substrate utilization (reducing cost) while maintaining sensitivity and selectivity for mature endothelin detection in receptor- and antibody-based assays. Endothelins are quantified in a separate step and comparable estimates of mature endothelin formation are obtained using both EIA and radioreceptor assays in 96-well formats. Human umbilical vein (HUVEC) and human aorta (HAEC) endothelial cells characterized by this method are found to preferentially convert the big endothelin-1 (big ET-1) isopeptide through a membrane-bound, thiorphan-insensitive, and phosphoramidon-sensitive zinc metalloendopeptidase. Both intact cells and membrane preparations used as the enzyme source predict similar IC50 values for phosphoramidon inhibition of ECE (ca 1 microM). The procedure described is simple, rapid, and suitable for high-volume screening.

Unstable drug candidates often lead to complexity for both sample collection and bioanalysis. Dried blood spot (DBS) technology is believed to be a viable solution to address this problem. However, it is currently a challenge to evaluate compound stability on DBS due to its solid format. The observed compound loss on a DBS card could be degradation and/or incomplete recovery. Therefore, a reliable bioanalytical method which can differentiate recovery loss from degradation is necessary for such stability evaluation. In this paper, the stability of an unstable drug candidate (KAI-9803) in human blood was evaluated using DBS. A reliable approach to evaluating analyte stability on DBS was developed with an appropriate time-zero sample, a consistent DBS sample processing method, and a suitable positive control. Commercially available DBS cards were evaluated, and it was found that KAI-9803 degraded during the drying process. An in-house modified DBS card was developed and demonstrated to be able to stabilize KAI-9803 during the drying process by rapidly lowering the pH of the spotted blood sample. The storage stability of KAI-9803 in human blood on this new card has been established for at least 48 days at room temperature. This in-house modified DBS card could provide a generic approach for other compounds which require stabilization at a low pH.

The role that metabonomics has in the evaluation of xenobiotic toxicity studies is presented here together with a brief summary of published studies. To provide a comprehensive assessment of this approach, the Consortium for Metabonomic Toxicology (COMET) has been formed between six pharmaceutical companies and Imperial College of Science, Technology and Medicine (IC), London, UK. The objective of this group is to define methodologies and to apply metabonomic data generated using H-1 NMR spectroscopy of urine and blood serum for preclinical toxicological screening of candidate drugs. This is being achieved by generating databases of results for a wide range of model toxins which serve as the raw material for computer-based expert systems for toxicity prediction. The project progress on the generation of comprehensive metabonomic databases and multivariate statistical models for prediction of toxicity, initially for Ever and kidney toxicity in the rat and mouse, is reported. Additionally, both the analytical and biological variation which might arise through the use of metabonomics has been evaluated. An evaluation of intersite NMR analytical reproducibility has revealed a high degree of robustness. Second, a detailed comparison has been made of the ability of the six companies to provide consistent urine and serum samples using a study of the toxicity of hydrazine at two doses in the male rat, this study showing a high degree of consistency between samples from the various companies in terms of spectral patterns and biochemical composition. Differences between samples from the various companies were small compared to the biochemical effects of the toxin. A metabonomic model has been constructed for urine from control rats, enabling identification of outlier samples and the metabolic reasons for the deviation. Building on this success, and with the completion of studies on approximately 80 model toxins, first expert systems for prediction of liver and kidney toxicity have been generated. (C) 2003 Elsevier Science (USA). All rights reserved.