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32 BioPharm International eBook July 2021 www.biopharminternational.com PROTEIN CHARACTERIZATION FOR BIOPHARMACEUTICALS The therapeutic efficacy of a protein is directly inf luenced by its three- dimensional structure that is derived from interactions between the amino acids on constituent peptide chains. For biologic therapeutics, form fits function, making structural characterization and identification of changes in structure key requirements to understanding protein function. Candidates advancing through the drug pipeline are subject to increasingly rigorous structural e luc id at ion to de v e lop a robu st understanding of the correlation between structure and function, and each level or type of structure is studied. An important focus for the bio- pharmaceutical industry is identifying efficient analytical techniques to track each type of structure, with orthogonal techniques routinely deployed to gener- ate complementary information. Much of this focus centers on techniques for the elucidation of higher order struc- ture (HOS) at the secondary, tertiary, and quaternary levels. The primary structure of a protein is relatively fixed in the absence of intentional chemical modification, but HOS at all levels can change in response to a protein's localized environment. Extrinsic stresses such as changes in temperature or vibration can trigger conformational change within secondary structure motifs. Structural change at this level is often identified as a precursor for many processes that result in the formation of protein aggregates. Examples of respected analytical techniques valued for structural char- acterization include mass spectrometry for primary structure, and techniques including size-exclusion chromatogra- phy (SEC) and nuclear magnetic res- onance (NMR) to obser ve tertiar y structure. SEC is also used regularly in conjunction with dynamic light scatter- ing (DLS) for characterizing properties of quaternary structure. The two histor- ical spectroscopic techniques employed for measurements of secondary structure are Fourier-transform infrared (FTIR) spectroscopy and far-ultraviolet circular dichroism (far-UV CD). APPLYING IR SPECTROSCOPY TO PROTEIN ANALYSIS The secondary structure of a protein directly inf luences the strength and stretch vibration of carbonyl (C=O) bonds along the peptide backbone (1). F TIR is a vibrational spectroscopy technique that probes the A mide I band associated with absorbance by these bonds. Robust correlations have been established between absorbance at specific wavelengths and the defining motifs of secondary structure (2,3). Shifts in absorbance in the Amide I band are inherently sensitive to changes in hydrogen bonding, dipole– dipole interactions, and geometric orientations associated with α-helices, β-sheets, turns, and less prevalent forms of secondary structure. Sensitivity to changes in β-sheet structure, a motif prevalent in monoclonal antibodies (m Abs), is especia l ly notewor thy. Due to its ability to measure changes in intermolecular β-sheet structure, IR spectroscopy is one of very few techniques that can be used to directly monitor aggregate formation (4). Measurements of secondary structure have considerable utility because they help to elucidate the mechanisms that underpin drug eff icacy and stability, binding, and more, and provide funda- mental information for many biophys- ical investigations. The concept being that almost any change in conformation or association experienced by a protein, from ligand binding studies to aggre- gation processes to formation of oligo- meric complexes, includes a fundamental shift in secondary structure that can be observed using IR spectroscopy. In formulation development activ- ities, the aim is to preserve the ther- apeutically eff icacious form of the protein while at the same time enhance stability. Here, comparative measure- ments of secondary structure can be used to identify successful formulation strategies and accelerate investigations Biopharmaceutical Analysis Protein Characterization Figure 1. Schematic showing the core features of a Microfluidic Modulation Spectroscopy (MMS) flow cell where a sample is modulated across the laser path with a matching buffer stream to produce real-time differential scans of the Amide I band. FIGURES ARE COURTESY OF THE AUTHORS.