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Background: Monoclonal antibodies and Fc fusion proteins contain an IgG Fc moiety, which is associated with various degradation processes, including aggregation. Results: Fc unfolding is triggered by the protonation of acidic residues and depends on the IgG subclass and C H 2 domain glycosylation. Conclusion: Fc aggregation in acidic conditions is determined by C H 2 stability. Significance: Understanding Fc aggregation is important for improving the quality of Fc-based therapeutics.

The prevention of aggregation in therapeutic antibodies is of great importance to the biopharmaceutical industry. In our investigation, acid-induced aggregation of monoclonal IgG1 and IgG2 antibodies was studied at pH 3.5 as a function of salt concentration and buffer type. The extent of aggregation was estimated using a native cation-exchange chromatography (CEX) method based on the loss of soluble monomer. This approach allowed quantitative analysis of antibody aggregation kinetics for individual and mixed protein solutions. Information regarding the aggregation mechanism was gained by assessing stabilities of intact antibodies relative to their Fc and Fab fragments. The role of protein thermodynamic stability in aggregation was deduced from differential scanning calorimetry (DSC). The rate of aggregation under conditions mimicking the viral inactivation step during monoclonal antibody (mAb) processing was found to be strongly dependent on the antibody subclass (IgG1 vs IgG2). At 25 °C, IgG1s were resistant to low pH aggregation, but IgG2s aggregated readily in the presence of salt. The observed distinction between IgG1 and IgG2 aggregation resulted from differential stability of the corresponding C(H)2 domains. This was further confirmed by experimenting with an IgG1 molecule containing an aglycosylated C(H)2 domain. Interestingly, comparative analysis of two buffer systems (based on acetic acid vs citric acid) revealed differences in mAb aggregation under identical pH conditions. Evidence is provided for the importance of the total acid concentration for antibody aggregation at low pH. The effects of C(H)2 instability and solution composition on aggregation are significant and deserve careful consideration during the development of mAb- or Fc-based therapeutics.

We report the observation of liquid-liquid phase separation in a solution of human monoclonal antibody, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective interprotein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable proetin condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.ntibodies are widely used in research and biotechnology, as well as in medical and pharmaceutical applications. In some cases, concentrated solutions of specific antibodies are required. In particular, monoclonal antibodies (MAb) have become a major category of drugs in the treatment of a variety of diseases (1). In some drug delivery routes e.g., subcutaneous administration, formulations with concentrated antibody solutions are required to achieve therapeutic dosing (2).The physiological functions of antibodies are mostly determined by antibody-antigen and antibody-receptor specific interactions. However, the nonspecific interactions between antibodies (i.e., self-association) in the concentrated antibody solutions can also affect their functions. Nonspecific attractive interactions can cause various forms of condensation, including liquid-liquid phase separation, aggregation, and crystallization. Upon such condensation, antibodies lose their solubility, and may lose their biological activity. Particularly, in pharmaceutical industry, these processes impact storage stability and safety of protein therapeutics thus impeding drug development (3). For example, immunogenicity of some biologics has been attributed to formation of protein aggregates (4). The mechanisms of protein condensation are complex and depend on protein concentration, buffer composition, temperature, etc. Clearly, the factors which affect protein condensation throughout the shelf-life must be understood and controlled to ensure biotherapeutic effectiveness.One important condensation process is liquid-liquid phase separation (LLPS). In LLPS, a homogeneous protein solution spontaneously separates into two coexisting phases with different protein concentrations. This phenomenon takes place upon changing the temperature or other solution conditions, and is reversible. In contrast to aggregation or crystallization, LLPS, while highly sensitive to the average "net" attractive interaction between proteins, are much less sensitive to the distribution pattern and the nature of the "local" interactions on the protein surface. As a result, LLPS exhibits universal features applicable to a variety...