Phase separation in solutions of monoclonal antibodies and the effect of human serum albumin

Abstract

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...

Liquid–Liquid Phase Separation in a Dual Variable Domain Immunoglobulin Protein Solution: Effect of Formulation Factors and Protein–Protein Interactions

“…Critical concentrations for crystallins and lysozyme are 240 ± 10 mg/ mL 21 and 230 ± 10 mg/mL, 22 respectively, while for the mAb the critical concentration is a range of 50−100 mg/mL. 29,30,33,38 From Figure 1, DVD-Ig protein with a molecular weight of around 200 kDa has a coexistence curve wider (C c is around 20−100 mg/mL) than that reported for mAbs. This is consistent with the hypothesis that the width of the coexistence curve (or concentration range in which solution exhibits opalescence and LLPS) increases as the size and the asymmetric shape of the molecule increase.…”

Coarse-Grained Model for Colloidal Protein Interactions, B22, and Protein Cluster Formation

“…Protein–protein interactions in solution are important mediators of protein phase behavior, 1,2 non-native aggregation, 3,4 increases in viscosity at high concentrations, 5,6 and in vivo self-assembly in biological systems. 7,8 In dilute solution in vitro , most soluble proteins fall into one of two categories: (i) they form stable dimers or oligomers, with equilibrium dissociation constants ( K d ) on the order of μ M or smaller values; 9,10 (ii) they are natively monomeric, and their protein–protein interactions are instead described in terms of the second osmotic virial coefficient B 22 .…”

Impact of surface interactions on the phase behavior of Y-shaped molecules

“…Of particular interest to this paper are the experimental studies of liquid-liquid phase transitions by Benedek et al [6,11,17]. They have reported several important results in a recent study of liquid-liquid phase separation in eight human myeloma IgGs and two recombinant pharmaceutical human IgGs.…”

“…Although IgGs are typically quite soluble at physiological conditions, sometimes they can become insoluble. In fact, recent studies of protein condensation have been published both for recombinant pharmaceutical IgGs and monoclonal IgGs [10][11][12][13][14][15][16][17]. A detailed discussion of the importance of IgGs in physiological and pharmaceutical situations is given by Wang et al [6], as well as Nezlin [18].…”