Unlike small molecule drugs, biologics are produced in living systems and have, not only primary, but secondary, tertiary and possibly quaternary structures. Determination of a biologic drug's higher order structure is critical to ensure efficacy, stability, and safety.
NMR is perfectly suited to evaluate protein structures and to assess whether critical changes have occurred in a biotherapeutic drug during development and manufacturing. Because of its intrinsically high information content, NMR is proving to be a uniquely valuable tool for the evaluation of HOS, reducing the number of techniques needed to characterize biotherapeutic drugs. Recent advances in data acquisition and analysis have ensured that it is now possible to study intact materials (including monoclonal antibodies) at natural abundance, under conditions that are physiologically relevant. The new MbioHOS software offers a tool box of 1D and 2D method to evaluate higher order structure of biotherapeutic drugs. Additionally, the assessment of similarity between a biosimilar drug and its reference material is now fast and easy, allowing rapid pass / fail evaluations to be made.
FT-IR contributes to the portfolio of orthogonal techniques for HOS analysis by giving insight into the secondary structure of proteins. The analysis is performed very quickly (~1 min/sample) and is characterized by a high tolerance against varying sample properties such as protein concentration, buffer components and excipients. These properties make the method an optimal tool to monitor the conformation of biopharmaceutical proteins during the development phase and for routine quality control.
This example application shows the IR spectra of an antibody at different temperatures (25-95° C). As indicated by the unchanged amide I band shape the protein maintains its native-sheet structure up to ~60° C. Further heating causes a decrease in intensity indicative of intramolecular-β-sheets unfolding
The tertiary structure of therapeutic proteins is key to their activity and stability. Due to the complexity of these proteins and the fact that they will contain multiple disulfide bonds, analysis is a challenge, often requiring several LC/MS runs with the tryptic digests from reduced and non-reduced protein and a manual comparison of these two analyses.
The DisulfideDetect workflow allows the fully automated analysis of disulfide bonds in biopharmaceuticals, based on a single digest of the unreduced protein and without prior knowledge of enzyme specificity or native disulfide bonds. The workflow also provides a semi-quantitative scrambling analysis as well.
DisulfideDetect software sets a new standard in ease-of-use for detecting and evaluating disulfide bonds within therapeutic proteins, such as monoclonal antibodies. It provides an easy to understand, visually-based, workspace for disulfide bond analysis for both native and non-native bonds as well as inter- and intra-chain disulfide bonds within the protein/antibody.
DisulfideDetect software uses established methods from existing LC-MALDI instruments like the UltrafleXtremeTM, so if your lab is utilizing LC-MALDI you're all set! Just add the DisulfideDetect Module to begin analyzing disulfide bonds in your biotherapeutics. No additional hardware is necessary.
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