Erik J. Fernandez

Distinct Aggregation Mechanisms of Monoclonal Antibody Under Thermal and Freeze-Thaw Stresses Revealed by Hydrogen Exchange

ABSTRACTPurposeAggregation of monoclonal antibodies (mAbs) is a common yet poorly understood issue in therapeutic development. There remains a need for high-resolution structural information about conformational changes and intermolecular contacts during antibody aggregation.MethodsWe used hydrogen exchange mass spectrometry (HX-MS) to compare the aggregation mechanism and resultant aggregate structures of the pharmaceutical antibody Bevacizumab under freeze-thaw (F/T) and thermal stresses.ResultsBevacizumab aggregation increased with number of F/T cycles and decreased with protein concentration. HX-MS showed native-like aggregates. Conversely, thermal stress triggered non-native aggregation at temperatures below melting point of the least stable CH2 domain. Under these conditions, HX was significantly enhanced in much of the Fab fragment while being decreased relative to native HX in CDRs. Analysis of intrinsic fluorescence Trp and extrinsic ANS dye binding supported structural differences between two antibody aggregates formed by F/T vs. thermal stresses.ConclusionsReduced hydrogen exchange in three CDRs suggests these residues may form strong intermolecular contacts in the antibody aggregates; regions of enhanced HX indicate unfolding. Residue level modeling methods with varying levels of atomistic detail were unable to identify aggregation patterns predictively.

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Aβ fibril formation pathway

β‐amyloid peptide (Aβ) is one of the main protein components of senile plaques associated with Alzheimers disease (AD). Aβ readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Aβ aggregates or oligomers is still under investigation. In this article, we show that different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Aβ aggregates faster than Aβ prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Aβ aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Aβ fibrils formed with agitation. In addition, Aβ fibrils formed under quiescent conditions were less toxic to differentiated SH‐SY5Y cells than the Aβ aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Aβ aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Aβ oligomers.

How does a protein unfold on a reversed-phase liquid chromatography surface? ☆

Nuclear magnetic resonance and isotope-exchange techniques were used to study unfolding of lysozyme adsorbed to reversed-phase liquid chromatography surfaces. All surfaces resulted in significant amide exchange, indicating solvent exposure and some loss of native structure. However, none of the surfaces resulted in complete exchange. The greatest amount of structure was preserved on the C4 silica, with the most protection in the α-helix domain. C18 silica and Source RPLC resulted in much greater solvent exposure. No simple correlation was found between chromatographic retention and degree of surface unfolding. Variations in residual conformation may explain the complex retention behavior of proteins vs. small molecules.

Structural Differences between Aβ(1-40) Intermediate Oligomers and Fibrils Elucidated by Proteolytic Fragmentation and Hydrogen/Deuterium Exchange

The aggregation of amyloid-beta protein (Abeta) in vivo is a critical pathological event in Alzheimers disease. Although more and more evidence shows that the intermediate oligomers are the primary neurotoxic species in Alzheimers disease, the particular structural features responsible for the toxicity of these intermediates are poorly understood. We measured the peptide level solvent accessibility of multiple Abeta(1-40) aggregated states using hydrogen exchange detected by mass spectrometry. A gradual reduction in solvent accessibility, spreading from the C-terminal region to the N-terminal region was observed with ever more aggregated states of Abeta peptide. The observed hydrogen exchange protection begins with reporter peptides 20-34 and 35-40 in low molecular weight oligomers found in fresh samples and culminates with increasing solvent protection of reporter peptide 1-16 in long time aged fibrillar species. The more solvent exposed structure of intermediate oligomers in the N-termini relative to well-developed fibrils provides a novel explanation for the structure-dependent neurotoxicity of soluble oligomers reported previously.

α-Lactalbumin tertiary structure changes on hydrophobic interaction chromatography surfaces

Hydrogen exchange (HX) detected by mass spectrometry (MS) was used to analyze the structure of calcium-free alpha-lactalbumin, a model protein with marginal stability. Two chromatographic peaks were observed from samples of pure protein eluted from SOURCE phenyl hydrophobic interaction chromatography (HIC) media. Whole-protein HX measurements showed that the less-retained peak had solvent exposure similar to native protein in the absence of the HIC surface while the retained protein was nearly, although not fully, solvent exposed. The formation of these two peaks was kinetically limited. The protein also refolded successfully following elution. In addition, proteolytic fragmentation was used to analyze HX at the peptide level. This approach revealed that helix C was the most stable region of alpha-lactalbumin under native conditions and in the flow-through peak. Helix C also formed the core of residual native structure in the partially unfolded protein in the retained peak. The results suggest that residues that are most solvent accessible under native conditions may be those most likely to unfold upon adsorption.

THE EFFECTS OF PERMEABILITY HETEROGENEITY ON MISCIBLE VISCOUS FINGERING : A THREE-DIMENSIONAL MAGNETIC RESONANCE IMAGING ANALYSIS

The three‐dimensional evolution of the viscous fingering instability has been visualized directly with magnetic resonance imaging (MRI). Miscible displacement of thin solute bands by aqueous solvent was investigated in packed beds of 30 μm chromatographic particles. Fingering behavior into samples of glycerol and a protein, bovine serum albumin (BSA), with viscosity ratios ranging from 1 to approximately 4, were compared. The three‐dimensional morphology and dynamics of fingers were monitored to approximately millimeter spatial resolution using MRI. Linear and nonlinear fingering behavior were observed. Permeability heterogeneities with length scales on the order of the finger wavelength induced complex three‐dimensional fingering patterns. Sample and column boundary effects on fingering dynamics were also noted. The differences in fingering behavior observed between albumin and glycerol samples are consistent with the wavelength predictions of linear stability analysis and the large differences in molecu...

Aggregation of anti-streptavidin immunoglobulin gamma-1 involves Fab unfolding and competing growth pathways mediated by pH and salt concentration.

Changes in non-native aggregation mechanisms of an anti-streptavidin (anti-SA) IgG1 antibody were determined over a wide range of pH and [NaCl] under accelerated (high temperature) conditions, using a combination of calorimetry, chromatography, static light scattering, dye binding, and spectroscopy (fluorescence, infra-red, and circular dichroism). Aggregation rates were strongly influenced by conformational stability of at least the Fab regions, but were only weakly affected by changes in electrostatic colloidal interactions. This was in contrast to the effects of electrostatic interactions on aggregate growth, as the dominant growth mechanism shifted dramatically with pH and [NaCl]. Pre-formed aggregates also displayed a reversible cloud-point boundary that quantitatively aligned with the overall pattern of aggregation mechanisms as a function of pH and [NaCl], suggesting an underlying thermodynamic transition may dictate whether molecular aggregates will coalesce into macroscopic particles. Structural changes upon unfolding and aggregation were also sensitive to pH and [NaCl]. Interestingly, Thioflavin T binding was essentially indistinguishable for aggregates formed in different pH and [NaCl] conditions, however, the other assays indicated notable differences across different solvent conditions. This suggests that the overall degree of conformational change during aggregation can be influenced by electrostatic interactions, but suggests caution in interpreting whether available techniques detect changes that are directly relevant to the mechanism(s) of aggregate formation and growth.

Viscous fingering in chromatography visualized via magnetic resonance imaging

Abstract Magnetic resonance imaging (MRI) and chromatographic peak shape analysis have been used to study the elution of bovine serum albumin (BSA) and glycerol in size-exclusion chromatography columns. Chromatograms of BSA samples show evidence of dramatic fingering at concentrations above 30–50 mg/ml. However, MRI of BSA samples reveals eluent fingers invading the sample band inside the column at concentrations as low as 10 mg/ml. Growth rates of fingers increase with protein concentration and the impact of fingering on chromatograms at lower concentrations is reduced because of dispersion. MRI analysis of glycerol samples demonstrates similar behavior, although finger widths are larger. Fingering in high concentration samples of glycerol causes portions of a sample to trail and separate from the main sample band. Nonuniform flow at the inlet and a self-correcting leading edge of the sample band were noted and attributed to viscous effects.

Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009.

Computational Design and Biophysical Characterization of Aggregation-Resistant Point Mutations for γD Crystallin Illustrate a Balance of Conformational Stability and Intrinsic Aggregation Propensity

γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have ΔΔG(un) ≫ 0, without significantly affecting IAP. S130P was predicted to have ΔΔG(un) ∼ 0 but with reduced IAP. The stability, conformation, and aggregation kinetics in acidic solution were experimentally characterized and compared for the variants and wild-type (WT) protein using circular dichroism and intrinsic fluorescence spectroscopy, calorimetric and chemical unfolding, thioflavin-T binding, chromatography, static laser light scattering, and kinetic modeling. Monomer secondary and tertiary structures of both variants were indistinguishable from WT, while ΔΔG(un) > 0 for M69Q and ΔΔG(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q.