7 research outputs found
Using Empirical Phase Diagrams to Understand the Role of Intramolecular Dynamics in Immunoglobulin G Stability
Understanding the relationship between protein dynamics and stability is of paramount importance to the fields of biology and pharmaceutics. Clarifying this relationship is complicated by the large amount of experimental data that must be generated and analyzed if motions that exist over the wide range of timescales are to be included. To address this issue, we propose an approach that utilizes a multidimensional vector-based empirical phase diagram (EPD) to analyze a set of dynamic results acquired across a temperature-pH perturbation plane. This approach is applied to a humanized immunoglobulin G1 (IgG1), a protein of major biological and pharmaceutical importance whose dynamic nature is linked to its multiple biological roles. Static and dynamic measurements are used to characterize the IgG and to construct both static and dynamic empirical phase diagrams. Between pH 5 and 8, a single, pH-dependent transition is observed that corresponds to thermal unfolding of the IgG. Under more acidic conditions, evidence exists for the formation of a more compact, aggregation resistant state of the immunoglobulin, known as A-form. The dynamics-based EPD presents a considerably more detailed pattern of apparent phase transitions over the temperature-pH plane. The utility and potential applications of this approach are discussed
Coarse-Grained Modeling of the Self-Association of Therapeutic Monoclonal Antibodies
Coarse-grained computational models of two therapeutic
monoclonal
antibodies are constructed to understand the effect of domain-level
charge–charge electrostatics on the self-association phenomena
at high protein concentrations. The coarse-grained representations
of the individual antibodies are constructed using an elastic network
normal-mode analysis. Two different models are constructed for each
antibody for a compact Y-shaped and an extended Y-shaped configuration.
The resulting simulations of these coarse-grained antibodies that
interact through screened electrostatics are done at six different
concentrations. It is observed that a particular monoclonal antibody
(hereafter referred to as MAb1) forms three-dimensional heterogeneous
structures with dense regions or clusters compared to a different
monoclonal antibody (hereafter referred to as MAb2) that forms more
homogeneous structures (no clusters). These structures, together with
the potential mean force (PMF) and radial distribution functions (RDF)
between pairs of coarse-grained regions on the MAbs, are qualitatively
consistent with the experimental observation that MAb1 has a significantly
higher viscosity compared to MAb2, especially at concentrations >50
mg/mL, even though the only difference between the MAbs lies with a few amino acids at the antigen-binding loops (CDRs). It is also observed that the structures in MAb1 are formed
due to stronger Fab–Fab interactions in corroboration with
experimental observations. Evidence is also shown that Fab–Fc
interactions can be equally important in addition to Fab–Fab
interactions. The coarse-grained representations are effective in
picking up differences based on local charge distributions of domains
and make predictions on the self-association characteristics of these
protein solutions. This is the first computational study of its kind
to show that there are differences in structures formed by two different
monoclonal antibodies at high concentrations