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: 3.13). 05/2012; 101(9):3062-77. DOI: 10.1002/jps.23187
Source: PubMed

ABSTRACT 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.

  • Source
    [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 · 3.99 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Arginine is widely used in biotechnology as a folding enhancer and aggregation suppressor. However, its action on the stability of complexly organized oligomeric proteins, on the one hand, and its role in the formation of supramolecular structures, on the other hand, are poorly known. The investigation is concerned with the effects of arginine on protein-protein interactions using phosphorylase kinase (PhK) as an example. PhK, a 1.3 MDa (αβγδ)4 hexadecameric complex, is a Ca(2+)-dependent regulatory enzyme that catalyzes phosphorylation and activation of glycogen phosphorylase b. On the basis of light scattering measurements it was shown that arginine induced aggregation of Ca(2+)-free PhK. On the contrary, when studying Ca(2+), Mg(2+)-induced aggregation of PhK at 37°C, the protective effect of arginine was demonstrated. The data on analytical ultracentrifugation are indicative of disruption of PhK hexadecameric structure under the action of arginine. Though HspB6 and HspB5 suppress aggregation of PhK they do not block the disruption effect of arginine with respect to both forms of PhK (Ca(2+)-free and Ca(2+), Mg(2+)-bound conformers). The dual effect of arginine has been interpreted from view-point of dual behaviour of arginine, functioning both like an osmolyte and a protein denaturant.
    International journal of biological macromolecules 05/2014; 68. DOI:10.1016/j.ijbiomac.2014.04.056 · 2.37 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Screening for pharmaceutically viable stability from measurements of thermally-induced protein unfolding and short-term accelerated stress underpins much molecule design, selection and formulation in the pharmaceutical biotechnology industry. However, the interrelationship between intrinsic protein conformational stability, thermal denaturation and pharmaceutical stability is complex. There are few publications in which predictions from thermal-unfolding-based screening methods are examined together with pharmaceutically relevant long-term storage stability performance. We have studied eight therapeutic IgG molecules under solution conditions optimized for large-scale commercial production and delivery. Thermal unfolding profiles were characterized by differential scanning calorimetry (DSC) and intrinsic fluorescence recorded simultaneously with static light scattering (SLS). These molecules exhibit a variety of thermal unfolding profiles under a common reference buffer condition and under individually optimized formulation conditions. Aggregation profiles by SE-HPLC and bioactivity upon long-term storage at 5(o)C, 25(o)C and 40(o)C establish that IgG molecules possessing a relatively wide range of conformational stabilities and thermal unfolding profiles can be formulated to achieve pharmaceutically stable drug products. We present evidence that a formulation design strategy that increases the thermal unfolding temperature of the Fab transition is likely to be a better general approach to improving pharmaceutical storage stability than one focused on increasing Tonset or Tm of the first unfolding transition.
    Molecular Pharmaceutics 02/2015; DOI:10.1021/mp400666b · 4.57 Impact Factor

Full-text (2 Sources)

Available from
Oct 1, 2014