Primary structure biopharmaceutical

Applications in Biotechnology. Biopharmaceutical products must be well characterized by means of analytical techniques. RP-HPLC with UV detection is suitable for the content assay and impurity profiling because of its high resolution. However, the bioactivity of protein-based pharmaceuticals not only relies on the primary structure but also on the higher order structure, i.e., the molecular confirmation, which is not revealed by UV absorbance. Circular dichroism and intrinsic fluorescence detection have been combined with UV detection in RP-HPLC to monitor the conformational properties of active compounds and impurities in biopharmaceutical products [57]. 

Once a peptide map is characterized using online MS, the chromatographic profile alone can serve as a routine analytical tool to monitor the protein s primary structure and covalent modifications, and is often used for batch release or stability testing of biopharmaceuticals. However, whenever in-depth characterization of a protein is needed, such as that required for comparability studies or reference material characterization, the peptide map should be coupled with MS to ensure a thorough examination of all peptides in the map. 

As discussed, biopharmaceuticals are characterized by large molecular sizes and various levels of structural complexity (i.e., primary, secondary, tertiary, or... 

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