Excipients differentially influence the conformational stability and pretransition dynamics of two IgG1 monoclonal antibodies

Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047, USA.
Journal of Pharmaceutical Sciences (Impact Factor: 2.59). 09/2012; 101(9):3062-77. DOI: 10.1002/jps.23187
Source: PubMed


Since immunoglobulins are conformationally dynamic molecules in solution, we studied the effect of stabilizing and destabilizing excipients on the conformational stability and dynamics of two IgG1 monoclonal antibodies (mAbs; mAb-A and mAb-B) using a variety of biophysical approaches. Even though the two mAbs are of the same IgG1 subtype, the unfolding patterns, aggregation behavior, and pretransition dynamics of these two antibodies were strikingly different in response to external perturbations such as pH, temperature, and presence of excipients. Sucrose and arginine were identified as stabilizers and destabilizers, respectively, on the basis of their influence on conformational stability for both the IgG1 mAbs. The two excipients, however, had distinct effective concentrations and different effects on the conformational stability and pretransition dynamics of the two mAbs as measured by a combination of differential scanning calorimetry, high-resolution ultrasonic spectroscopy, and red-edge excitation shift fluorescence studies. Stabilizing concentrations of sucrose were found to decrease the internal motions of mAb-B, whereas arginine marginally increased its adiabatic compressibility in the pretransition region. Both sucrose and arginine did not influence the pretransition dynamics of mAb-A. The potential reasons for such differences in excipient effects between two IgG1 mAbs are discussed.

Download full-text


Available from: Charles Russell Middaugh, Oct 01, 2014
  • Source
    • "When considering approaches to choosing mAbs formulation for the best stability, the correct selection of buffer, pH and excipient(s) is essential. The solution pH can have profound effects on protein structure, stability and biological activity (Kopec and Schneider, 2011; Thakkar et al., 2012). In the context of formulation, pH is optimized to minimize physical and chemical degradation pathways (Cromwell et al., 2006; Gokarn et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Finding excipients which mitigate protein self-association and aggregation is an important task during formulation. Here, the effect of an equimolar mixture of l-Arg and l-Glu (Arg·Glu) on colloidal and conformational stability of four monoclonal antibodies (mAb1–mAb4) at different pH is explored, with the temperatures of the on-set of aggregation (Tagg) and unfolding (Tm1) measured by static light scattering and intrinsic fluorescence, respectively. Arg·Glu increased the Tagg of all four mAbs in concentration-dependent manner, especially as pH increased to neutral. Arg·Glu also increased Tm1 of the least thermally stable mAb3, but without similar direct effect on the Tm1 of other mAbs. Raising pH itself from 5 to 7 increased Tm1 for all four mAbs. Selected mAb formulations were assessed under accelerated stability conditions for the monomer fraction remaining in solution after storage. The aggregation of mAb3 was suppressed to a greater extent by Arg·Glu than by Arg·HCl. Furthermore, Arg·Glu suppressed the aggregation of mAb1 at neutral pH such that the fraction monomer was near to that at the more typical formulation pH of 5.5. We conclude that Arg·Glu can suppress mAb aggregation with increasing temperature/pH and, importantly, under accelerated stability conditions at weakly acidic to neutral pH.
    International Journal of Pharmaceutics 10/2014; 473(1-2):126-133. DOI:10.1016/j.ijpharm.2014.06.053 · 3.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Differential scanning calorimetry measures the heat capacity of states and the excess heat associated with transitions that can be induced by temperature change. The integral of the excess heat capacity is the enthalpy for this process. Despite this potentially intimidating sounding physical chemistry background, DSC has found almost universal application in studying biological macromolecules. In the case of proteins, DSC can be used to determine equilibrium thermodynamic stability and folding mechanism but can also be used in a more qualitative manner screening for thermal stability as an indicator for, ligand binding, pharmaceutical formulation or conditions conducive to crystal growth. DSC usually forms part of a wider biophysical characterisation of the biological system of interest and so the literature is diverse and difficult to categorise for the technique in isolation. This review therefore describes the potential uses of DSC in studying protein folding and stability, giving brief examples of applications from the recent literature. There have also been some interesting developments in the use of DSC to determine barrier heights for fast folding proteins and in studying complex protein mixtures such as human plasma that are considered in more detail.
    Archives of Biochemistry and Biophysics 09/2012; 531(1). DOI:10.1016/j.abb.2012.09.008 · 3.02 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A molecular understanding of excipient effects on the interrelationship(s) between dynamics and conformational stability of proteins, such as monoclonal antibodies (mAbs), can be important for their pharmaceutical development. The current study examines stabilizing and destabilizing effects of excipients on the conformational stability and local dynamics of distinct solvent-exposed regions within an IgG1 monoclonal antibody (mAb-B). The principles of site-selective photoselection upon red-edge excitation, accompanied by acrylamide quenching of tryptophan fluorescence were employed in this study. The initiation of mAb-B thermal unfolding occurs by structural alterations in the more solvent-exposed regions of the antibody, which subsequently leads to a cascade of structural alterations in its relatively more solvent-shielded regions. In addition, an increase in internal dynamics of solvent-shielded regions made mAb-B more susceptible to thermally induced structural perturbations resulting in its global destabilization. Sucrose and arginine exert their stabilizing and destabilizing effects by predominantly influencing the conformational stability of solvent-exposed regions in mAb-B. The complex molecular effects of sucrose and arginine on local dynamics of different regions in mAb-B and their correlation with the protein's conformational stability are described within the pretransition range, at the onset temperature (T(onset) ) and at the thermal melting temperature (T(M) ). © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.
    Journal of Pharmaceutical Sciences 12/2012; 101(12). DOI:10.1002/jps.23332 · 2.59 Impact Factor
Show more