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The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

Gentamicin is an aminoglycoside antibiotic, which induces renal tubular necrosis in rats. In the context of the European InnoMed PredTox project, transcriptomic and proteomic studies were performed to provide new insights into the molecular mechanisms of gentamicin-induced nephrotoxicity. Male Wistar rats were treated with 25 and 75 mg/kg/day subcutaneously for 1, 3 and 14 days. Histopathology observations showed mild tubular degeneration/necrosis and regeneration and moderate mononuclear cell infiltrate after long-term treatment. Transcriptomic data indicated a strong treatment-related gene expression modulation in kidney and blood cells at the high dose after 14 days of treatment, with the regulation of 463 and 3241 genes, respectively. Of note, the induction of NF-kappa B pathway via the p38 MAPK cascade in the kidney, together with the activation of T-cell receptor signaling in blood cells were suggestive of inflammatory processes in relation with the recruitment of mononuclear cells in the kidney. Proteomic results showed a regulation of 163 proteins in kidney at the high dose after 14 days of treatment. These protein modulations were suggestive of a mitochondrial dysfunction with impairment of cellular energy production, induction of oxidative stress, an effect on protein biosynthesis and on cellular assembly and organization. Proteomic results also provided clues for potential nephrotoxicity biomarkers such as AGAT and PRBP4 which were strongly modulated in the kidney. Transcriptomic and proteomic data turned out to be complementary and their integration gave a more comprehensive insight into the putative mode of nephrotoxicity of gentamicin which was in accordance with histopathological findings.

The main goal of the present work was to better understand the molecular mechanisms underlying liver hypertrophy (LH), a recurrent finding observed following acute or repeated drug administration to animals, using transcriptomic technologies together with the results from conventional toxicology methods. Administration of 5 terminated proprietary drug candidates from participating companies involved in the EU Innomed PredTox Project or the reference hepatotoxicant troglitazone to rats for up to a 14-day duration induced LH as the main liver phenotypic toxicity outcome. The integrated analysis of transcriptomic liver expression data across studies turned out to be the most informative approach for the generation of mechanistic models of LH. In response to a xenobiotic stimulus, a marked increase in the expression of xenobiotic metabolizing enzymes (XME) was observed in a subset of 4 studies. Accumulation of these newly-synthesized proteins within the smooth endoplasmic reticulum (SER) would suggest proliferation of this organelle, which most likely is the main molecular process underlying the LH observed in XME studies. In another subset of 2 studies (including troglitazone), a marked up-regulation of genes involved in peroxisomal fatty acid β-oxidation was noted, associated with induction of genes involved in peroxisome proliferation. Therefore, an increase in peroxisome abundance would be the main mechanism underlying LH noted in this second study subset. Together, the use of transcript profiling provides a means to generate putative mechanistic models underlying the pathogenesis of liver hypertrophy, to distinguish between subtle variations in subcellular organelle proliferation and creates opportunities for improved mechanism-based risk assessment.

A serious bottleneck in the drug development pipeline is the inability of current pre-clinical toxicology evaluation methods to predict early on, and with good accuracy, that a drug candidate will have to be removed from development due to toxicology/safety issues. The InnoMed PredTox consortium attempted to address this issue by assessing the value of using molecular profiling techniques (proteomics, transcriptomics, and metabonomics), in combination with conventional toxicology measurements, on decision making earlier in pre-clinical safety evaluation. In this study, we report on the SELDI-TOF-MS proteomics component of the InnoMed PredTox project. In this large scale, multi-site, multi-compound study, tissue and plasma samples from 14-day in vivo rat experiments conducted for 16 hepato- and nephro-toxicants with known toxicology endpoints (including 14 proprietary compounds and 2 reference compounds) were analyzed by SELDI-TOF-MS. We have identified seven plasma proteins and four liver proteins which were shown to be modulated by treatment, and correlated with histopathological evaluations and can be considered potential biomarker candidates for the given toxicology endpoints. In addition, we report on the intra- and inter-site variations observed based on measurements from a reference sample, and steps that can be taken to minimize this variation.

We consider QCD instanton-induced contributions to lepton pair production in hadron-hadron collisions. We relate these contributions to those known from deep inelastic scattering and demonstrate that they can be calculated reliably for sufficiently large momentum transfer. We observe that the instanton contribution to the angular distribution of the lepton pairs at finite momentum transfer strongly violates the Lam-Tung relation - a relation between coefficient functions of the angular distribution which is valid within the framework of ordinary perturbation theory. The drastic violation of this relation, as seen in experimental data, might be related to such instanton-induced effects., Comment: 24 pages, 9 figures, some comments added; to appear in Nuclear Physics B

The Alberta Complex is the most prominent member of a mafic to ultramafic igneous suite within the Rehoboth Basement Inlier/Namibia, being intrusive into sediments and mafic volcanics of the Paleoproterozoic Elim Formations. Subsequent amphibolite grade metamorphism of pre-Kibaran age obscured the original mineral assemblage. Two main units have been distinguished in the past within the Alberta Complex. (1) A thick pile of various amphibolites represents original norite, hyperite, gabbro and anorthosite and constitutes the Layered Sequence. It has been subdivided into four zones, thought to represent individual magmatic pulses. (2) Later stage magmatism comprises pegmatite veins as well as pyroxenite, harzburgite and dunite, emplaced in plugs and a cone sheet. In contrast to field observations, geochemical profiles of compatible elements across the complex indicate more complex layering and subdivision of the Layered Sequence into at least seven units. Incompatible trace elements are largely decoupled from compatible elements, which probably is the result of variable amount of interstitial liquid within cumulate phase. However, the shape of REE patterns has been controlled chiefly by fractionation of clinopyroxene and plagioclase. Finally, decoupling of hydrophile LILE from both compatible and incompatible elements may result from hydrothermal overprint or from redistribution during metamorphism. Gabbroic rocks from the Marginal Zone of the Alberta Compex display affinity to mafic volcanic rocks of the Elim Fm. Modeling based on in situ fractional crystallisation or, alternatively, mixing of evolved liquid with cumulate supports this hypothesis. In contrast, for samples within the magma chamber, only REE could be matched closely by these models, while modeled incompatible trace elements commonly deviate from the measured data. Additional complexity of local magma chamber probably best explains these deviations.

The production of top quark pairs accompanied by a hard gluon in $e^+e^-$ annihilation is studied including next-to-leading order corrections in the strong coupling. At leading order, the fraction r of $t\bar{t}g$ events with respect to all $t\bar{t}$ events is computed analytically as a function of the minimal gluon energy. Next-to-leading order results for r are given for center-of-mass energies of 0.5 and 1 TeV. We further calculate the differential distribution of r with respect to several variables, including the top quark energy and the $t\bar{t}$ invariant mass. We then investigate how our results depend on the choice of the renormalization scheme for the top quark mass by comparing results expressed in terms of either the pole mass or the $\msbar$ mass. Finally we estimate the sensitivity of the fraction r on the value of the running top quark mass at a scale of 1 TeV., Comment: 21 LaTeX pages, 12 figures, 1 reference added, 1 reference corrected, minor changes in the text

The reaction $e^+e^-$ annihilation into three jets was recently computed for massive quarks at next-to-leading order in perturbative QCD. We discuss some results of our calculation for b jets produced at the Z resonance., 4 pages, LaTeX, 2 postscript figures, uses espcrc2.sty. Talk given by W. Bernreuther at the workshop on Standard Model Physics, San Miniato, April 1997

We discuss rescattering effects that can be measured in {ital e}{sup +}{ital e}{sup {minus}} annihilation to three jets through a single gauge boson, by using triple product ({open_quote}{open_quote}event handedness{close_quote}{close_quote}) correlations of the {ital Z} ({gamma}{sup {asterisk}}) polarization with jet momenta. The gauge boson polarization may be produced either by polarized beams or through the natural polarization (left-right asymmetry) of the {ital Z}. QCD rescattering does not generate triple product correlations at one loop for massless quarks. We therefore calculate the QCD contribution for massive quarks, as well as the contribution of {ital W} and {ital Z} exchange loops for massless quarks. Because of the various cancellations, the standard model predictions for triple-product correlations at the {ital Z} are very small, making such measurements potentially sensitive to physics beyond the standard model. For example, the effects of a new gauge boson that couples only to baryon number may be larger than the standard model contributions; however, the effects would probably still be too small to effectively constrain it. {copyright} {ital 1996 The American Physical Society.}

We investigate for two Higgs-doublet models with intrinsic {ital CP} violation in the scalar potential {ital CP}-nonconserving effects in unpolarized {gamma}{gamma}{r_arrow}{ital t{bar t}} for a range of neutral Higgs boson masses which includes resonant {ital cphi} production and the subsequent decay of {ital cphi}{r_arrow}{ital t{bar t}}. The importance of taking into account, even in the resonant case, the interference with the nonresonant background is shown. Further, we propose and calculate three asymmetries which efficiently trace {ital CP}-violating effects in {gamma}{gamma}{r_arrow}{ital t{bar t}} using semileptonic {ital t{bar t}} decays.

The InnoMed PredTox consortium was formed to evaluate whether conventional preclinical safety assessment can be significantly enhanced by incorporation of molecular profiling (" omics" ) technologies. In short-term toxicological studies in rats, transcriptomics, proteomics and metabolomics data were collected and analyzed in relation to routine clinical chemistry and histopathology. Four of the sixteen hepato- and/or nephrotoxicants given to rats for 1, 3, or 14. days at two dose levels induced similar histopathological effects. These were characterized by bile duct necrosis and hyperplasia and/or increased bilirubin and cholestasis, in addition to hepatocyte necrosis and regeneration, hepatocyte hypertrophy, and hepatic inflammation. Combined analysis of liver transcriptomics data from these studies revealed common gene expression changes which allowed the development of a potential sequence of events on a mechanistic level in accordance with classical endpoint observations. This included genes implicated in early stress responses, regenerative processes, inflammation with inflammatory cell immigration, fibrotic processes, and cholestasis encompassing deregulation of certain membrane transporters. Furthermore, a preliminary classification analysis using transcriptomics data suggested that prediction of cholestasis may be possible based on gene expression changes seen at earlier time-points. Targeted bile acid analysis, based on LC-MS metabonomics data demonstrating increased levels of conjugated or unconjugated bile acids in response to individual compounds, did not provide earlier detection of toxicity as compared to conventional parameters, but may allow distinction of different types of hepatobiliary toxicity. Overall, liver transcriptomics data delivered mechanistic and molecular details in addition to the classical endpoint observations which were further enhanced by targeted bile acid analysis using LC/MS metabonomics. © 2010 Elsevier Inc.

The kidney is one of the main targets of drug toxicity, but early detection of renal damage is often difficult. As part of the InnoMed PredTox project, a collaborative effort aimed at assessing the value of combining omics technologies with conventional toxicology methods for improved preclinical safety assessment, we evaluated the performance of a panel of novel kidney biomarkers in preclinical toxicity studies. Rats were treated with a reference nephrotoxin or one of several proprietary compounds that were dropped from drug development in part due to renal toxicity. Animals were dosed at two dose levels for 1, 3, and 14 days. Putative kidney markers, including kidney injury molecule-1 (Kim-1), lipocalin-2 (Lcn2), clusterin, and tissue inhibitor of metalloproteinases-1, were analyzed in kidney and urine using quantitative real-time PCR, ELISA, and immunohistochemistry. Changes in gene/protein expression generally correlated well with renal histopathological alterations and were frequently detected at earlier time points or at lower doses than the traditional clinical parameters blood urea nitrogen and serum creatinine. Urinary Kim-1 and clusterin reflected changes in gene/protein expression and histopathological alterations in the target organ in the absence of functional changes. This confirms clusterin and Kim-1 as early and sensitive, noninvasive markers of renal injury. Although Lcn2 did not appear to be specific for kidney toxicity, its rapid response to inflammation and tissue damage in general may suggest its utility in routine toxicity testing.

We compute the thermal axino production rate in supersymmetric QCD to leading order in the gauge coupling. Using hard thermal loop resummation and the Braaten-Yuan prescription, we obtain a finite result in a gauge-invariant way, which takes into account Debye screening in the hot quark-gluon-squark-gluino plasma. The relic axino density from thermal reactions in the early Universe is evaluated assuming the axino is the lightest supersymmetric particle and stable due to R-parity conservation. From the comparison with the WMAP results, we find that axinos could provide the dominant part of cold dark matter, for example, for an axino mass of 100 keV and a reheating temperature of 10^6 GeV., 5 pages, 2 figures. Talk given at Strong and Electroweak Matter 2004, Helsinki, Finland, June 16-19, 2004

We present in detail a calculation of the next-to-leading order QCD corrections to the process $e^+e^-\to 3$ jets with massive quarks. To isolate the soft and collinear divergencies of the four parton matrix elements, we modify the phase space slicing method to account for masses. Our computation allows for the prediction of oriented three jet events involving heavy quarks, both on and off the Z resonance, and of any event shape variable which is dominated by three jet configurations. We show next-to-leading order results for the three jet fraction, the differential two jet rate, and for the thrust distribution at various c.m. energies., Comment: 51 pages, LaTeX, 16 postscript figures

We investigate the prospects to observe effects of transverse polarization of top quarks in $pp,p\bar{p}\to t\bar{t}X$. QCD absorptive parts generate a polarization of top quarks and antiquarks transverse to the production plane in the partonic processes $q\bar{q}\to t\bar{t}$ and $gg\to t\bar{t}$, which reaches values of a few percent. These perturbative QCD effects are decreased at the hadronic level. A measurement through momentum correlations among the $t$ and $\bar{t}$ decay products will be difficult both at an upgraded Tevatron and at the LHC., Comment: 14 pages, LaTeX; 8 uuencoded, tar-compressed figures added separately

We investigate the possibilities of searching for non-standard CP violation in $pp\to t\bar{t}X$ at multiple TeV collision energies. A general kinematic analysis of the underlying partonic production processes $gg\to t\bar{t}$ and $q\bar{q}\to t\bar{t}$ in terms of their density matrices is given. We evaluate the CP-violating parts of these matrices in two-Higgs doublet extensions of the standard model (SM) and give results for CP asymmetries at the parton level. We show that these asymmetries can be traced by measuring suitable observables constructed from energies and momenta of the decay products of $t$ and $\bar{t} $. We find CP-violating effects to be of the order of $10^{-3}$ and show that possible contaminations induced by SM interactions are savely below the expected signals., Comment: 24 pages, SLAC-PUB-6403, PITHA 93/43, 9 Figs. available upon request. Written in LaTeX