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Use of predicted versus measured CCS values from different instrument platforms, and isomer separation on the SELECT SERIES Cyclic IMS
No full textBiotransformation activities require the comparison of metabolites across species and studies. In general, chromatographic retention time, accurate mass measurement and mass spectral data are used to align metabolites. Isomeric metabolite comparison may be more challenging particularly when retention times may differ depending on the analytical conditions used. Additionally, the elemental formulae as well as MS/MS spectra can be identical which significantly increases the complexity of the data interpretation and localization of the biotransformation. The use of collision cross section (CCS) values to compare metabolites analyzed using the SELECT SERIES Cyclic IMS and the SYNAPT G2-Si Q-Tof instruments located in different facilities has been shown here and demonstrates the benefit of such analyte-specific physiochemical property to align metabolites across studies.Moreover, computational prediction of CCS values may provide an additional data asset, allowing the comparison of predicted with measured CCS values. This can further provide additional insights to differentiate between isomers. The prediction can also be used to suggest when additional cyclic ion mobility separation (cIMS) would be beneficial in the separation of isomers and increase confidence in any assignment with the use of higher ion mobility resolution. Examples are given here where cIMS has been used to separate oxygenated metabolites of ranitidine and imipramine. This alternative separation mechanism adds to the separating power of UPLC and is of benefit when isomers co-elute
Navigating Between Right, Wrong, and Relevant: The Use of Mathematical Modeling in Preclinical Decision Making.
No full textThe goal of this mini-review is to summarize the collective experience of the authors for how modeling and simulation approaches have been used to inform various decision points from discovery to First-In-Human clinical trials. The article is divided into a high-level overview of the types of problems that are being aided by modeling and simulation approaches, followed by detailed case studies around drug design (Nektar Therapeutics, Genentech), feasibility analysis (Novartis Pharmaceuticals), improvement of preclinical drug design (Pfizer), and preclinical to clinical extrapolation (Merck, Takeda, and Amgen)
Global intercompany assessment of ICIEF platform comparability for the characterization of therapeutic proteins
No full textAn international team spanning 19 sites across 18 biopharmaceutical and in vitro diagnostics companies in the United States, Europe, and China, along with one regulatory agency, was formed to compare the precision and robustness of imaged CIEF (ICIEF) for the charge heterogeneity analysis of the National Institute of Standards and Technology (NIST) mAb and a rhPD-L1-Fc fusion protein on the iCE3 and the Maurice instruments. This information has been requested to help companies better understand how these instruments compare and how to transition ICIEF methods from iCE3 to the Maurice instrument. The different laboratories performed ICIEF on the NIST mAb and rhPD-L1-Fc with both the iCE3 and Maurice using analytical methods specifically developed for each of the molecules. After processing the electropherograms, statistical evaluation of the data was performed to determine consistencies within and between laboratory and outlying information. The apparent isoelectric point (pI) data generated, based on two-point calibration, for the main isoform of the NIST mAb showed high precision between laboratories, with RSD values of less than 0.3% on both instruments. The SDs for the NIST mAb and the rhPD-L1-Fc charged variants percent peak area values for both instruments are less than 1.02% across different laboratories. These results validate the appropriate use of both the iCE3 and Maurice for ICIEF in the biopharmaceutical industry in support of process development and regulatory submissions of biotherapeutic molecules. Further, the data comparability between the iCE3 and Maurice illustrates that the Maurice platform is a next-generation replacement for the iCE3 that provides comparable data
Titrating the preferences of altered lighting against temperature in female CD-1 laboratory mice, Mus musculus
No full textAspects of the laboratory are aversive to mice, such as being housed under bright lights and at temperatures below their thermal comfort, causing stress and poor welfare. While murine thermal preferences are well understood, light preferences in mice are under studied. To address this gap, reduced light intensity was titrated against varying temperatures. We hypothesized that mice would choose the red-tinted cage when offered temperatures below 30 °C but would choose the clear-heated cage when around 30 °C. Seven pairs of female CD-1 mice were randomly allocated to two connected cages (a red-tinted cage and a clear cage with a heated hot spot) for eight days, each containing 8 g of nesting material. Every 48 h, the clear, hot spot cage temperature was changed to 20, 24, 28, or 32 °C, in a random order, without repetition. Over the 8-day study, all cages experienced the 4 treatments for 2 days each. After an initial 24 h of exposure, nest complexity was scored, the amount of nesting material was weighed in each cage, and inactive behavior was observed. Temperature did not significantly alter nest weights; however, it did impact nest scores, with more complex nests being built in the red cage when 20–28 °C were available. Further, mice spent more time in the red-tinted cage when they had access to 20 and 24 °C. These temperatures do not outweigh the preference for lighting conditions, thus aversion to typical laboratory lighting may be more important than previously assumed
Novel insights into bile acid detoxification via CYP, UGT and SULT enzymes
No full textBile acid (BA) homeostasis is a complex and precisely regulated process to prevent impaired BA flow and the development of cholestasis. Several reactions, namely hydroxylation, glucuronidation and sulfation are involved in BA detoxification. In the present study, we employed a comprehensive and quantitative approach using human recombinant enzymes, human liver microsomes (HLM) and human liver cytosol (HLC) to delineate the interplay of the enzymatic processes involved in hepatic BA metabolism. We showed that CYP3A4 was a crucial step for the metabolism of several BA and their taurine and glycine conjugated forms and quantitatively described their metabolites. Glucuronidation and sulfation were also identified as important drivers of the BA detoxification process in humans. Moreover, lithocholic acid (LCA), the most hydrophobic BA with the highest toxicity potential, was a substrate for all investigated processes, demonstrating the importance of hepatic metabolism for its clearance. Collectively, this study describes the major contributing (metabolic) processes in the BA detoxification network and suggests that the inhibition potential of candidate drugs on CYP3A4, UGT1A3, UGT2B7 and SULT2A1 need to be assessed, together with inhibition of the relevant hepatic transporters and potential toxic metabolite formation, in the preclinical investigations of drug-induced cholestasis in humans
Chemoinformatics and Artificial Intelligence Colloquium: Progress and Challenges to Develop Bioactive Compounds
No full textWe report the main conclusions of the first Chemoinformatics and Artificial Intelligence Colloquium, Mexico City (virtual), June 15-17, 2022. Fifteen lectures were presented during a virtual and public event with speakers from industry, academia, and non-for-profit organizations. 1,290 participants, mainly students, and academics from more than 60 countries had registered. During the meeting, applications, challenges, and opportunities in drug discovery, de novo drug design, ADME-Tox (Adsorption, Distribution, Metabolism, Excretion and Toxicity) property predictions, organic chemistry and peptides, and antibiotic resistance were discussed. The program along with the recordings of all sessions is freely available at https://www.difacquim.com/english/events/2022-colloquium/
Energy Intake Models for Intermittent Operation of Dead-End Microfiltration Filling Line
No full textIn filling lines equipped with membrane separation devices in form of filters energy con-
sumption is only one of important working parameters, the other being sustainable filter
performance in terms of separation eficiency. As the filling line is typically equipped with a
valve, intermittent operation of the filter is an important form of its use. Whereas the overall
energy consumption of the filtration process is governed by the continuous operation mode,
the intermittent mode, characterized by opening/closing of the valve, contributes most to
problems of filter failure i.e. breakthrough of filtered particles through the membrane. In
this work, a model for determination of the energy intake of a microfiltration membrane
during the opening and closing of a valve is presented. The model is based on computa-
tional analysis of the pressure wave signals recorded during opening/closing of the valve
using Fourier transform, and expressed in a nondimensional filter area specific energy intake
form. The model is applied to the case of constant pressure dead-end microfiltration case
with three filter types: single membrane filter, stacked filter and pleated filter with filtration
surface areas ranging from 17.7cm2 to 2000cm2. Both clean filter as well as partially clogged
filter cases are taken into account. Based on extensive analysis of experimental data second
order polynomial models of the energy intake are developed and evaluated. The analysis of
energy intake results show, that for the clean filter case the largest energy intake is observed.
When at the constant
ow rate values membrane fouling occurs it leads to larger energy
intake, however due to a decreasing specific
ow rate during fouling these values do not
exceed the clean filter case
Droplet Microfluidics for the Label-Free Extraction of Complete Phase Diagrams and Kinetics of Liquid-Liquid Phase Separation in Finite Volumes
No full textLiquid-liquid phase separation of polymer and protein solutions is central in many areas of biology and material sciences. Here, an experimental and theoretical framework is provided to investigate the thermodynamics and kinetics of liquid-liquid phase separation in volumes comparable to cells. The strategy leverages droplet microfluidics to accurately measure the volume of the dense phase generated by liquid-liquid phase separation of solutions confined in micro-sized compartments. It is shown that the measurement of the volume fraction of the dense phase at different temperatures allows the evaluation of the binodal lines that determine the coexistence region of the two phases in the temperature-concentration phase diagram. By applying a thermodynamic model of phase separation in finite volumes, it is further shown that the platform can predict and validate kinetic barriers associated with the formation of a dense droplet in a parent dilute phase, therefore connecting thermodynamics and kinetics of liquid-liquid phase separation
Systematic profiling of conditional degron tag technologies for target validation studies.
No full textConditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource