Application Notes: General

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Labile post-translational modifications are better preserved using ECD than conventional MS fragmentation techniques such as collision-induced dissociation (CID). Common modifications, such as glycosylation and phosphorylation, can impact the safety, efficacy, and binding activity of monoclonal antibodies but are challenging to identify. Stephen Sciuto from Agilent Technologies and colleagues analyzed tryptic digests of NIST mAb and Infliximab using an 6545XT Q-TOF mass spectrometer in ExD or CID mode. ECD provided higher sequence coverage than CID, reaching 100% for glycosylated peptides in Infliximab. The ability to pinpoint glycosylation sites using fragment ions demonstrates the power of ECD for characterizing glycopeptides.

The first reported combination of microdroplet reactions with ECD fragmentation offers a quicker way to analyze monoclonal antibodies for disease treatments. Thomas Walker from Agilent Technologies and co-workers demonstrate the use of an Agilent Jet Stream electrospray ionization source to facilitate in-spray chemical reduction and protease digestion of monoclonal antibodies. Downstream fragmentation of the microdroplet reaction products with ECD provided rapid characterization of intact antibodies in minutes. Efficient ECD fragmentation yielded rich sequence information including evidence of disulfide bond connectivity and confirmation of key sequences called complementary determining regions. These results highlight this method’s potential for fast and cost-effective antibody characterization with minimal sample preparation.

Charge heterogeneity is present in most biopharmaceutical protein products. During the manufacturing process, charge heterogeneity of the protein therapeutics can occur due to enzymatic cleavage and chemical post-translational modifications (PTM). For therapeutics like ADCs, not only the antibody contributes to the heterogeneity but also the linker and payload, which add even more complexity to the charge variant profiles. Characterizing the charge heterogeneity of ADCs is essential for critical quality attribute (CQA) assessment to ensure drug safety, efficacy, and potency.

For many years, Solid Phase Extraction (SPE) has been performed using plates and cartridges packed with a bed of loose media. Extracting samples of interest while removing contamination from samples presented to the chromatography system results in lower costs associated with detector maintenance and extends column lifespan. Cleaner samples also improve specificity in analysis and increase sensitivity. Traditional products, however, have inherent limitations in reproducibility of flow and recovery. The innovative composite technology found in the Microlute® range greatly improves flow consistency providing a step change in the reproducibility of processing and analyte recover.

The Microlute® CSi products use a novel composite technology which uses a blend of porous plastic and chromatographic SPE resin. This technology has been designed to eliminate issues caused by inherent issues and inconsistent packing of loose packed SPE plates. The data in this application note compares SPE results from a C18 10 mg loose packed plate with a C18 10 mg composite plate (Microlute® CSi) which were both created using the same batch of C18 resin.

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