Arnold McAuley

Molecular origins of surfactant- mediated stabilization of protein

Loss of activity through aggregation and surface-induced denaturation is a significant problem in the production, formulation and administration of therapeutic proteins. Surfactants are commonly used in upstream and downstream processing and drug formulation. However, the effectiveness of a surfactant strongly depends on its mechanism(s) of action and properties of the protein and interfaces. Surfactants can modulate adsorption loss and aggregation by coating interfaces and/or participating in protein-surfactant associations. Minimizing protein loss from colloidal and interfacial interaction requires a fundamental understanding of the molecular factors underlying surfactant effectiveness and mechanism. These concepts provide direction for improvements in the manufacture and finishing of therapeutic proteins. We summarize the roles of surfactants, proteins, and surfactant-protein complexes in modulating interfacial behavior and aggregation. These events depend on surfactant properties that may be quantified using a thermodynamic model, to provide physical/chemical direction for surfactant selection or design, and to effectively reduce aggregation and adsorption loss.

Isomerization of a Single Aspartyl Residue of Anti-Epidermal Growth Factor Receptor Immunoglobulin γ2 Antibody Highlights the Role Avidity Plays in Antibody Activity

A new isoform of the light chain of a fully human monoclonal immunoglobulin gamma2 (IgG2) antibody panitumumab against human epidermal growth factor receptor (EGFR) was generated by in vitro aging. The isoform was attributed to the isomerization of aspartate 92 located between phenylalanine 91 and histidine 93 residues in the antigen-binding region. The isomerization rate increased with increased temperature and decreased pH. A size-exclusion chromatography binding assay was used to show that one antibody molecule was able to bind two soluble extracellular EGFR molecules in solution, and isomerization of one or both Asp-92 residues deactivated one or both antigen-binding regions, respectively. In addition, isomerization of Asp-92 showed a decrease in in vitro potency as measured by a cell proliferation assay with a 32D cell line that expressed the full-length human EGFR. The data indicate that antibodies containing either one or two isomerized residues were not effective in inhibiting EGFR-mediated cell proliferation, and that two unmodified antigen binding regions were needed to achieve full efficacy. For comparison, the potency of an intact IgG1 antibody cetuximab against the same receptor was correlated with the bioactivity of its individual antigen-binding fragments. The intact IgG1 antibody with two antigen-binding fragments was also much more active in suppressing cell proliferation than the individual fragments, similar to the IgG2 results. These results indicated that avidity played a key role in the inhibition of cell proliferation by these antibodies against the human EGFR, suggesting that their mechanisms of action are similar.

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain

Recombinant human monoclonal antibodies have become important protein‐based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of β‐sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the CH3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human CH3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized CH3 dimer were similar, but differences were observed. The reduced CH3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer–dimer equilibrium. Interestingly, the dimer–monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human CH3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer–dimer equilibrium of the human CH3 domain. Hence, these results may have implications for the stability of the intact antibody.

Small-angle neutron scattering study of a monoclonal antibody using free-energy constraints.

Monoclonal antibodies (mAbs) contain hinge-like regions that enable structural flexibility of globular domains that have a direct effect on biological function. A subclass of mAbs, IgG2, have several interchain disulfide bonds in the hinge region that could potentially limit structural flexibility of the globular domains and affect the overall configuration space available to the mAb. We have characterized human IgG2 mAb in solution via small-angle neutron scattering (SANS) and interpreted the scattering data using atomistic models. Molecular Monte Carlo combined with molecular dynamics simulations of a model mAb indicate that a wide range of structural configurations are plausible, spanning radius of gyration values from ∼39 to ∼55 Å. Structural ensembles and representative single structure solutions were derived by comparison of theoretical SANS profiles of mAb models to experimental SANS data. Additionally, molecular mechanical and solvation free-energy calculations were carried out on the ensemble of best-fitting mAb structures. The results of this study indicate that low-resolution techniques like small-angle scattering combined with atomistic molecular simulations with free-energy analysis may be helpful to determine the types of intramolecular interactions that influence function and could lead to deleterious changes to mAb structure. This methodology will be useful to analyze small-angle scattering data of many macromolecular systems.

Small-Angle Neutron Scattering Study of Protein Crowding in Liquid and Solid Phases: Lysozyme in Aqueous Solution, Frozen Solution, and Carbohydrate Powders

The structure, interactions, and interprotein configurations of the protein lysozyme were studied in a variety of phases. These properties have been studied under a variety of solution conditions before, during, and after freezing and after freeze-drying in the presence of glucose and trehalose. Contrast variation experiments have also been performed to determine which features of the scattering in the frozen solutions are from the protein and which are from the ice structure. Data from lysozyme at concentrations ranging from 1 to 100 mg/mL in solution and water ice with NaCl concentrations ranging from 0 to 0.4 mol/L are fit to model small-angle neutron scattering (SANS) intensity functions consisting of an ellipsoidal form factor and either a screened-Coulomb or hard-sphere structure factor. Parameters such as protein volume fraction and long dimension are followed as a function of temperature and salt concentration. The SANS results are compared to real space models of concentrated lysozyme solutions at the same volume fractions obtained from Monte Carlo simulations. A cartoon representation of the frozen lysozyme solution in 0 mol/L NaCl is presented based on the SANS and Monte Carlo results, along with those obtained from other complementary methods.

Modulation of Protein Adsorption by Poloxamer 188 in Relation to Polysorbates 80 and 20 at Solid Surfaces

Poloxamer 188 (BASF Pluronic® F68) is widely used as a shear-protective excipient to enhance cell yield in agitated cultures and reduce cell adhesion in stationary cultures. However, little is known in any quantitative sense of its effect on protein adsorption and aggregation. Optical waveguide lightmode spectroscopy was used here to compare the adsorption kinetics exhibited by poloxamer 188, and polysorbates 80 and 20, in the presence and absence of a model protein (chicken egg white lysozyme) and in separate experiments, a recombinant protein (human granulocyte colony-stimulating factor) at hydrophilic, silica-titania surfaces. Experiments were performed in sequential and competitive adsorption modes, enabling the adsorption kinetic patterns to be interpreted in a fashion revealing the dominant mode of surfactant-mediated stabilization of protein in each case. Kinetic results showed that polysorbates 80 and 20 are able to inhibit protein adsorption only by their preferential location at an interface to which they show sufficient affinity, and not by formation of less surface active, protein-surfactant complexes. On the other hand, poloxamer 188 is able to inhibit protein adsorption by entering into formation of protein-surfactant complexes of low adsorption affinity (i.e., high colloidal stability), and not by its preferential location at the interface.

Protein structure and interactions in the solid state studied by small-angle neutron scattering.

Small-angle neutron scattering (SANS) is uniquely qualified to study the structure of proteins in liquid and solid phases that are relevant to food science and biotechnological applications. We have used SANS to study a model protein, lysozyme, in both the liquid and water ice phases to determine its gross-structure, interparticle interactions and other properties. These properties have been examined under a variety of solution conditions before, during, and after freezing. Results for lysozyme at concentrations of 50 mg mL(-1) and 100 mg mL(-1), with NaCl concentrations of 0.4 M and 0 M, respectively, both in the liquid and frozen states, are presented and implications for food science are discussed.

Investigating Structure and Dynamics of Proteins in Amorphous Phases Using Neutron Scattering

In order to increase shelf life and minimize aggregation during storage, many biotherapeutic drugs are formulated and stored as either frozen solutions or lyophilized powders. However, characterizing amorphous solids can be challenging with the commonly available set of biophysical measurements used for proteins in liquid solutions. Therefore, some questions remain regarding the structure of the active pharmaceutical ingredient during freezing and drying of the drug product and the molecular role of excipients. Neutron scattering is a powerful technique to study structure and dynamics of a variety of systems in both solid and liquid phases. Moreover, neutron scattering experiments can generally be correlated with theory and molecular simulations to analyze experimental data. In this article, we focus on the use of neutron techniques to address problems of biotechnological interest. We describe the use of small-angle neutron scattering to study the solution structure of biological molecules and the packing arrangement in amorphous phases, that is, frozen glasses and freeze-dried protein powders. In addition, we discuss the use of neutron spectroscopy to measure the dynamics of glassy systems at different time and length scales. Overall, we expect that the present article will guide and prompt the use of neutron scattering to provide unique insights on many of the outstanding questions in biotechnology.

Characterization of Monoclonal Antibody–Protein Antigen Complexes Using Small-Angle Scattering and Molecular Modeling

The determination of monoclonal antibody interactions with protein antigens in solution can lead to important insights guiding physical characterization and molecular engineering of therapeutic targets. We used small-angle scattering (SAS) combined with size-exclusion multi-angle light scattering high-performance liquid chromatography to obtain monodisperse samples with defined stoichiometry to study an anti-streptavidin monoclonal antibody interacting with tetrameric streptavidin. Ensembles of structures with both monodentate and bidentate antibody–antigen complexes were generated using molecular docking protocols and molecular simulations. By comparing theoretical SAS profiles to the experimental data it was determined that the primary component(s) were compact monodentate and/or bidentate complexes. SAS profiles of extended monodentate complexes were not consistent with the experimental data. These results highlight the capability for determining the shape of monoclonal antibody–antigen complexes in solution using SAS data and physics-based molecular modeling.