Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses.

The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 min and plasmid purification <60 min. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention.

Parallel sample processing using dispersive INtip micro-purification on programmable multichannel pipettes.

Automation gives researchers the ability to process and screen orders of magnitude higher numbers of samples than manual experimentation. Current biomacromolecule separation methodologies suffer from necessary manual intervention, making their translation to high-throughput automation difficult. Herein, we present the first characterization of biomacromolecule affinity purification via dispersive solid-phase extraction in a pipette tip (INtip). We use commercially available resin and compare efficiency with batch and spin column methodologies. Moreover, we measure the kinetics of binding and evaluate resin binding capacities. INtip technology is effective on, and scalable for, an automated platform (INTEGRA ASSIST). The results suggest that high-throughput biomolecular workflows will benefit from the integration of INtip separations.

Immune-modulating enzyme indoleamine 2,3-dioxygenase is effectively inhibited by targeting its apo-form.

For cancer cells to survive and proliferate, they must escape normal immune destruction. One mechanism by which this is accomplished is through immune suppression effected by up-regulation of indoleamine 2,3-dioxygenase (IDO1), a heme enzyme that catalyzes the oxidation of tryptophan to N-formylkynurenine. On deformylation, kynurenine and downstream metabolites suppress T cell function. The importance of this immunosuppressive mechanism has spurred intense interest in the development of clinical IDO1 inhibitors. Herein, we describe the mechanism by which a class of compounds effectively and specifically inhibits IDO1 by targeting its apo-form. We show that the in vitro kinetics of inhibition coincide with an unusually high rate of intrinsic enzyme-heme dissociation, especially in the ferric form. X-ray crystal structures of the inhibitor-enzyme complexes show that heme is displaced from the enzyme and blocked from rebinding by these compounds. The results reveal that apo-IDO1 serves as a unique target for inhibition and that heme lability plays an important role in posttranslational regulation.

Alkyl Isocyanates via Manganese-Catalyzed C-H Activation for the Preparation of Substituted Ureas.

Organic isocyanates are versatile intermediates that provide access to a wide range of functionalities. In this work, we have developed the first synthetic method for preparing aliphatic isocyanates via direct C-H activation. This method proceeds efficiently at room temperature and can be applied to functionalize secondary, tertiary, and benzylic C-H bonds with good yields and functional group compatibility. Moreover, the isocyanate products can be readily converted to substituted ureas without isolation, demonstrating the synthetic potential of the method. To study the reaction mechanism, we have synthesized and characterized a rare MnIV-NCO intermediate and demonstrated its ability to transfer the isocyanate moiety to alkyl radicals. Using EPR spectroscopy, we have directly observed a MnIV intermediate under catalytic conditions. Isocyanation of celestolide with a chiral manganese salen catalyst followed by trapping with aniline afforded the urea product in 51% enantiomeric excess. This represents the only example of an asymmetric synthesis of an organic urea via C-H activation. When combined with our DFT calculations, these results clearly demonstrate that the C-NCO bond was formed through capture of a substrate radical by a MnIV-NCO intermediate.