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The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.

Aggregation is often the major issue during formulation and manufacturing development of therapeutic proteins, in particular human monoclonal antibody. Currently, there is a lack of structural information of aggregates of such large protein as human antibodies, due to the large molecular sizes of the aggregates. In this article, we shall discuss the application of vibrational spectroscopies including FT-IR, Raman and Raman Optical Activity (ROA), to characterize the structures of various types of monoclonal antibody aggregates formed under different stresses. Two different classes of human monoclonal antibodies, namely IgG1 and IgG2, have been subjected to this structural investigation. The common stresses leading to antibody aggregation, mis-folding or unfolding during manufacturing and formulation include exposure to acidic pHs, heat and shear stress. The effect of different types of stresses on the structure and aggregate formation of human monoclonal antibodies has been investigated by employing vibrational spectroscopy. While data present only monoclonal antibody, the same technology can be used for any protein aggregates.

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