Monoclonal Antibody Aggregation Associated with Free Radical Induced Oxidation.

Oxidation is an important degradation pathway of protein drugs. The susceptibility to oxidation is a common concern for therapeutic proteins as it may impact product efficacy and patient safety. In this work, we used 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) as an oxidative stress reagent to evaluate the oxidation of therapeutic antibodies. In addition to the oxidation of methionine (Met) and tryptophan (Trp) residues, we also observed an increase of protein aggregation. Size-exclusion chromatography and multi-angle light scattering showed that the soluble aggregates induced by AAPH consist of dimer, tetramer, and higher-order aggregate species. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that inter-molecular disulfide bonds contributed to the protein aggregation. Furthermore, intrinsic fluorescence spectra suggested that dimerization of tyrosine (Tyr) residues could account for the non-reducible cross-links. An excipient screening study demonstrated that Trp, pyridoxine, or Tyr could effectively reduce protein aggregation due to oxidative stress. This work provides valuable insight into the mechanisms of oxidative-stress induced protein aggregation, as well as strategies to minimize such aggregate formation during the development and storage of therapeutic proteins.

Structural characterization of H3K56Q nucleosomes and nucleosomal arrays.

The post-translational modification of histones is a key mechanism for the modulation of DNA accessibility. Acetylated lysine 56 in histone H3 is associated with nucleosome assembly during replication and DNA repair, and is thus likely to predominate in regions of chromatin containing nucleosome-free regions. Here we show by X-ray crystallography that mutation of H3 lysine 56 to glutamine (to mimic acetylation) or glutamate (to cause a charge reversal) has no detectable effects on the structure of the nucleosome. At the level of higher order chromatin structure, the K to Q substitution has no effect on the folding of model nucleosomal arrays in cis, regardless of the degree of nucleosome density. In contrast, defects in array-array interactions in trans ('oligomerization') are selectively observed for mutant H3 lysine 56 arrays that contain nucleosome-free regions. Our data suggests that H3K56 acetylation is one of the molecular mechanisms employed to keep chromatin with nucleosome-free regions accessible to the DNA replication and repair machinery.

Revealing a positive charge patch on a recombinant monoclonal antibody by chemical labeling and mass spectrometry.

During purification process development and analytical characterization, a recombinant human monoclonal antibody, referred to as rmAb1, showed an anomalous charge heterogeneity profile by cation-exchange chromatography (CIEC), characterized by extremely high retention and poor resolution between charge variants. Mass spectrometry-based footprinting methodologies that include selective labeling of lysine with sulfosuccinimidyl acetate and arginie with p-hydroxyphenylglyoxal were developed to map the positive charges on the rmAb1 surface. On the basis of the average percentages of labeling obtained for the lysine and arginine residues by peptide mapping analysis, the positive charges were more distributed on the surface in the Fab region than in the Fc region of rmAb1. By a comparative study of in-solution and on-resin labeling reaction dynamics, seven positively charged residues were identified to bind to the cation-exchange resin and they were located in the variable domains. Among them, three lysine and one arginine residues appeared to cluster together on the surface to form a positive charge patch. When the charge patch residues were neutralized by chemical labeling, rmAb1 exhibited a more typical CIEC retention time, confirming that the charge patch was responsible for the atypical CIEC profile of rmAb1. To our knowledge, this work is the first report revealing the amino acid composition of a surface charge patch on therapeutic monoclonal antibodies.

Cluster Size and Quinary Structure Determine the Rheological Effects of Antibody Self-Association at High Concentrations.

The question of how nonspecific reversible intermolecular protein interactions affect solution rheology at high concentrations is fundamentally rooted in the translation of nanometer-scale interactions into macroscopic properties. Well-defined solutions of purified monoclonal antibodies (mAbs) provide a useful system with which to investigate the manifold intricacies of weak protein interactions at high concentrations. Recently, characterization of self-associating IgG1 antibody (mAb2) solutions has established the direct role of protein clusters on concentrated mAb rheology. Expanding on our earlier work with three additional mAbs (mAb1, mAb3, and mAb4), the observed concentration-dependent static light scattering and rheological data present a substantially more complex relationship between protein interactions and solution viscosity at high concentrations. The four mAb systems exhibited divergent correlations between cluster formation (size) and concentrated solution viscosities dependent on mAb primary sequence and solution conditions. To address this challenge, well-established features of colloidal cluster phenomena could be applied as a framework for interpreting our observations. The initial stages of mAb cluster formation were investigated with small-angle X-ray scattering (SAXS) and ensemble-optimized fit methods, to uncover shifts in the dimer structure populations which are produced by changes in mAb interaction modes and association valence under the different solution conditions. Analysis of mAb average cluster number and effective hydrodynamic radii at high concentrations revealed cluster architectures can have a wide range of fractal dimensions. Collectively, the static light scattering, SAXS, and rheological characterization demonstrate that nonspecific and anisotropic attractive intermolecular interactions produce antibody clusters with different quinary structures to regulate the rheological properties of concentrated mAb solutions.

Monoclonal antibody self-association, cluster formation, and rheology at high concentrations.

The rheological properties of macromolecular and colloidal suspensions are dependent on the thermodynamic and kinetic parameters that define viscous flow, and remain an active field of study with broad implications in cellular biophysics, soft-matter theory, and biopharmaceutical technology. Here we use static light scattering, small-angle X-ray scattering, and viscosity measurements as a function of protein concentration to semiquantitatively correlate the oligomeric state of an IgG1 antibody (mAb1) with its rheological behavior at solution pH 6.0 and varying ionic strength (modified by 0.01-0.1 M Na2SO4). Solution SAXS characterization of 100 mM Na2SO4 solutions confirmed that mAb1 forms reversible dimers with extended structures in dilute solutions. Light-scattering measurements over a wide range of concentrations (1-175 mg/mL) provide detailed information on the equilibrium thermodynamic mAb1 interactions and their modulation by modest increases of Na2SO4. Through the use of interacting hard sphere models to fit light-scattering data, we establish that protein cluster formations consisting of 2-9 mAb1 molecules also increase the viscosity of 175 mg/mL IgG solutions from 52 up to 450 cP. The analysis of dilute and semidilute mAb1 solution rheology correlates linearly with the thermodynamic equilibrium cluster size, consistent with the viscosity behavior of elongated oligomeric structures that are not significantly dendrimeric or in a state of globular collapse. Furthermore, SAXS- and rheology-based structural modeling illustrate that only a small set of anisotropic interactions between complementary surfaces are required to nucleate and propagate protein clusters.

Influence of the cosolute environment on IgG solution structure analyzed by small-angle X-ray scattering.

Small-angle X-ray scattering experiments of two monoclonal antibodies (mAbs) were performed as a function of Hofmeister salt type and concentration including 100 mM Na(2)SO(4), 100-600 mM of NaSCN, or 100-600 mM arginine chloride at pH 6.0 to yield information on the effects of cosolutes on mAb solution conformation and flexibility. Minimal selected ensemble (MSE) procedures used to reconstruct the SAXS form factors revealed that both IgG1 mAbs exist in a conformational equilibrium with two subpopulations that vary in overall shape and size. The "closed" mAb conformation is characterized by a maximum dimension of ∼155 Šand shorter distances between Fab-Fab and Fab-FC domains. The "open" mAb conformation has a maximum dimension of ∼175 Šand an increase in the interdomain distances with concomitant increases in overall mAb flexibility. Analysis of the distribution of shapes and sizes of mAb structures within the conformational equilibrium indicates that they remain essentially unchanged under conditions with a broad range of chaotropic and kosmotropic salts including 100-600 mM NaSCN and 100 mM Na(2)SO(4). Analysis of the conformations within each MSE population under various conditions reveals a striking similarity between many of the MSE structures, IgG crystal structures, and single-molecule imaging studies; MSE analysis of mAb form factors also identified an overall relaxation of the mAb structure unique to solution conditions containing arginine chloride, characterized by an increased maximum dimension and a shift toward the population of the "open" mAb conformation. Our results provide the first comprehensive characterization of mAb conformational diversity in solution and are of direct relevance to understanding the effects of solution conditions on protein structural dynamics and stability.

Structural and biophysical studies of human PARP-1 in complex with damaged DNA.

The enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is a global monitor of chromatin structure and DNA damage repair. PARP-1 binds to nucleosomes and poly(ADP-ribosylates) histones and several chromatin-associated factors to expose specific DNA sequences to the cellular machinery involved in gene transcription and/or DNA damage repair. While these processes are critical to genomic stability, the molecular mechanisms of how DNA damage induces PARP-1 activation are poorly understood. We have used biochemical and thermodynamic measurements in conjunction with small-angle X-ray scattering to determine the stoichiometry, affinity, and overall structure of a human PARP-1 construct containing the entire DNA binding region, the zinc ribbon domain, and automodification domains (residues 1-486). The interaction of this PARP-1 protein construct with three different DNA damage models (DNA constructs containing a nick, a blunt end, or a 3' extension) was evaluated. Our data indicate that PARP-1 binds each DNA damage model as a monomer and with similar affinity, in all cases resulting in robust activation of the catalytic domain. Using small-angle X-ray scattering, we determined that the N-terminal half of PARP-1 behaves as an extended and flexible arrangement of individually folded domains in the absence of DNA. Upon binding DNA, PARP-1 undergoes a conformational change in the area surrounding the zinc ribbon domain. These data support a model in which PARP-1, upon binding DNA, undergoes a conformational change to become an active nuclear enzyme.

Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor.

The transformation potential of Simian Virus 40 depends on the activities of large T-antigen (LTag), which interacts with several cellular tumor suppressors including the important "guardian" of the genome, p53. Inhibition of p53 function by LTag is necessary for both efficient viral replication and cellular transformation. We determined the crystal structure of LTag in complex with p53. The structure reveals an unexpected hexameric complex of LTag binding six p53 monomers. Structure-guided mutagenesis of LTag and p53 residues supported the p53-LTag interface defined by the complex structure. The structure also shows that LTag binding induces dramatic conformational changes at the DNA-binding area of p53, which is achieved partially through an unusual "methionine switch" within p53. In the complex structure, LTag occupies the whole p53 DNA-binding surface and likely interferes with formation of a functional p53 tetramer. In addition, we showed that p53 inhibited LTag helicase function through direct complex formation.

Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen.

The oncoprotein large tumour antigen (LTag) is encoded by the DNA tumour virus simian virus 40. LTag transforms cells and induces tumours in animals by altering the functions of tumour suppressors (including pRB and p53) and other key cellular proteins. LTag is also a molecular machine that distorts/melts the replication origin of the viral genome and unwinds duplex DNA. LTag therefore seems to be a functional homologue of the eukaryotic minichromosome maintenance (MCM) complex. Here we present the X-ray structure of a hexameric LTag with DNA helicase activity. The structure identifies the p53-binding surface and reveals the structural basis of hexamerization. The hexamer contains a long, positively charged channel with an unusually large central chamber that binds both single-stranded and double-stranded DNA. The hexamer organizes into two tiers that can potentially rotate relative to each other through connecting alpha-helices to expand/constrict the channel, producing an 'iris' effect that could be used for distorting or melting the origin and unwinding DNA at the replication fork.