Novel Multidimensional Liquid Chromatography Workflow with In-Loop Enzymatic Digests of Multiple Heart-Cuts for Fast and Flexible Characterization of Biotherapeutic Protein Variants.

Multidimensional liquid chromatography (mD-LC) is becoming a powerful tool for complete characterization of individual peaks and protein variants through separation methods such as nondenaturing ion exchange (IEC) or size-exclusion chromatography coupled to reversed-phase (RP) chromatography. The flexibility of commercially available and customized mD-LC systems is still limited in terms of enzymatic peak processing between chromatographic dimensions. In this regard, only a few column-immobilized proteases are available for detailed peak characterization by mD-LC coupled to mass spectrometry (mD-LC-MS). Here, we present a purpose-built and automated multiple heart-cutting mD-LC design with a novel analytical workflow involving in-loop enzymatic heart-cut digestion between the first-dimensional column and transfer to the second dimension before MS or MS/MS analyses. The setup facilitates the spike-in of any enzyme to multiple heart-cuts for multilevel analysis, for example, for peptide mapping, fragment generation, or deglycosylation, to reduce heterogeneity and provide maximum flexibility in terms of incubation time for optimal peak characterization. We demonstrate the application of IEC coupled to RP-LC-MS and automated in-loop deglycosylation and on-column reduction of an IgG antibody combined with upper hinge region cleavage for Fab generation. We further employ mD-LC-MS and mD-LC-MS/MS to assess post-translational modifications of a bispecific antibody and to support molecule selection by evaluating the best downstream purification strategy. The novel design and automated workflow of the mD-LC system described here offers enhanced flexibility for in-solution processing and real-time monitoring of multiple heart-cuts enabling streamlined characterization of unknown biotherapeutic charge and size variants.

Aryldiones incorporating a [1,4,5]oxadiazepane ring. Part 2: chemistry and biology of the cereal herbicide pinoxaden.

BACKGROUND: Pinoxaden is a new cereal herbicide that provides outstanding levels of post-emergence activity against a broad spectrum of grass weed species for worldwide selective use in both wheat and barley. RESULTS: Factors influencing activity and tolerance to pinoxaden were in part linked to distinct structural parts of the active ingredient. Three complementary contributions that decisively impact upon the herbicidal potency against grasses were identified: a preferred 2,6-diethyl-4-methyl aromatic substitution pattern, a dione area suitable for proherbicide formation and beneficial adjuvant effects. The uptake and translocation pattern of pinoxaden when coapplied with its tailored adjuvant were analysed by autoradiography, indicating extensive and rapid penetration, followed by effective distribution throughout the plant. Crop injury reduction on incorporation of the [1,4,5]oxadiazepane ring into the aryldione template was reinforced with safener technology. Comparative studies on the behaviour of pinoxaden applied either alone or in combination with the safener cloquintocet-mexyl demonstrated that addition of the safener resulted in significant enhancement of metabolic degradation in wheat and barley, providing excellent crop tolerance and a substantial selectivity margin without adverse effects on weed control. CONCLUSION: The biological potential of pinoxaden and its active principle pinoxaden dione in terms of grass weed control and tolerance in cereals was fully exploited by inclusion of the safener cloquintocet-mexyl in the formulation in combination with a specific and tailor-made tank-mix adjuvant based on methylated rape seed oil.