Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry.

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab.

NMIHBA results from hypomorphic PRUNE1 variants that lack short-chain exopolyphosphatase activity.

Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.

Coarse-Grained Modeling of Antibodies from Small-Angle Scattering Profiles.

Predicting the concentrated solution behavior for monoclonal antibodies requires developing and using minimal models to describe their shape and interaction potential. Toward this end, the small-angle X-ray scattering (SAXS) profiles for a monoclonal antibody (COE-03) have been measured under solution conditions chosen to produce weak self-association. The experiments are complemented with molecular simulations of a three-bead antibody model with and without interbead attraction. The scattering profile is extracted directly from the molecular simulation to avoid using the decoupling approximation. We examine the ability of the three-bead model to capture features of the scattering profile and the dependence of compressibilty on protein concentration. The three-bead model is able to reproduce generic features of the experimental structure factor as a function of wave vector S(k) including a well-defined shoulder, which is a consequence of the planar structure of the antibody, and a well-defined minimum in S(k) at k ∼ 0.025 Å-1. We also show the decoupling approximation is incapable of accounting for highly anisotropic shapes. The best-fit parameters obtained from matching spherical models to simulated scattering profiles are protein concentration dependent, which limits their applicability for predicting thermodynamic properties. Nevertheless, the experimental compressibility curves can be accurately reproduced by an appropriate parametrization of the Baxter adhesive model, indicating the model provides a semiempirical equation of state for the antibody. The results provide insights into how equations of state can be improved for antibodies by accounting for their anisotropic shapes.

Both Reversible Self-Association and Structural Changes Underpin Molecular Viscoelasticity of mAb Solutions.

The role of antibody structure (conformation) in solution rheology is probed. It is demonstrated here that pH-dependent changes in the tertiary structure of 2 mAb solutions lead to viscoelasticity and not merely a shear viscosity (η) increase. Steady shear flow curves on mAb solutions are reported over broad pH (3.0 ≤ pH ≤ 8.7) and concentration (2 mg/mL ≤ c ≤ 120 mg/mL) ranges to comprehensively characterize their rheology. Results are interpreted using size exclusion chromatography, differential scanning calorimetry, analytical ultracentrifugation, near-UV circular dichroism, and dynamic light scattering. Changes in tertiary structure with concentration lead to elastic yield stress and increased solution viscosity in solution of "mAb1." These findings are supported by dynamic light scattering and differential scanning calorimetry, which show increased hydrodynamic radius of mAb1 at low pH and a reduced melting temperature Tm, respectively. Conversely, another molecule at 120 mg/mL solution concentration is a strong viscoelastic gel due to perturbed tertiary structure (seen in circular dichroism) at pH 3.0, but the same molecule responds as a viscous liquid due to reversible self-association at pH 7.4 (verified by analytical ultracentrifugation). Both protein-protein interactions and structural perturbations govern pH-dependent viscoelasticity of mAb solutions.

Relationship between particle elasticity, glass fragility, and structural relaxation in dense microgel suspensions.

"Fragile" glassy materials, which include most polymeric materials and organic liquids, exhibit a steep and super-Arrhenius dependence of relaxation time with temperature upon the glass transition and have been extensively studied. Yet, a full understanding of strong glass formers that exhibit an Arrhenius dependence on temperature is still lacking. In this work, we have investigated the glassy dynamics of poly(N-isopropylacrylamide) (PNIPAM) microgel particles of varied elasticity in dense aqueous suspensions, giving rise to a full spectrum of strong to fragile glass-forming behaviors. We have observed the dependence of particle motions and structural relaxation on particle volume fraction can be weakened by decreasing particle elasticity, due to particle deformation and the resulting interparticle elastic interaction upon intimate particle contacts at high particle concentration. Both measured α-relaxation time scales and dynamic length scales for cooperative rearranging motions of microgels in suspensions show similarly dependence on particle volume fraction and elasticity, thereby quantifying the glass fragility of dense microgel suspension of varied particle elasticity.

Critical examination of the colloidal particle model of globular proteins.

Recent studies of globular protein solutions have uniformly adopted a colloidal view of proteins as particles, a perspective that neglects the polymeric primary structure of these biological macromolecules, their intrinsic flexibility, and their ability to sample a large configurational space. While the colloidal perspective often serves as a useful idealization in many cases, the macromolecular identity of proteins must reveal itself under thermodynamic conditions in which the native state is no longer stable, such as denaturing solvents and high protein concentrations where macromolecules tend to have screened excluded volume, charge, and hydrodynamic interactions. Under extreme pH conditions, charge repulsion interactions within the protein chain can overcome the attractive hydrogen-bonding interactions, holding it in its native globular state. Conformational changes can therefore be expected to have great significance on the shear viscosity and other rheological properties of protein solutions. These changes are not envisioned in conventional colloidal protein models and we have initiated an investigation of the scattering and rheological properties of model proteins. We initiate this effort by considering bovine serum albumin because it is a globular protein whose solution properties have also been extensively investigated as a function of pH, temperature, ionic strength, and concentration. As we anticipated, near-ultraviolet circular dichroism measurements and intrinsic viscosity measurements clearly indicate that the bovine serum albumin tertiary structure changes as protein concentration and pH are varied. Our findings point to limited validity of the colloidal protein model and to the need for further consideration and quantification of the effects of conformational changes on protein solution viscosity, protein association, and the phase behavior. Small-angle Neutron Scattering measurements have allowed us to assess how these conformational changes influence protein size, shape, and interprotein interaction strength.

A microliter capillary rheometer for characterization of protein solutions.

Rheometry is an important characterization tool for therapeutic protein solutions because it determines syringeability and relates indirectly to solution stability and thermodynamic interactions. Despite the maturity of rheometry, there remains a need for a rheometer that meets the following three needs of the biopharamaceutical industry: small volume; large dynamic range of shear rates; and no air-sample interface. Here, we report the development of a miniaturized capillary rheometer that meets these needs and is potentially scalable to a multiwell format. These measurements consume only a few microliters of sample and have an uncertainty of a few percent. We demonstrate its performance on monoclonal antibody solutions at different concentrations and temperatures. The instrument has a dynamic range of approximately three decades (in shear rate) and can measure Newtonian, shear thinning, and yielding behaviors, which are representative of the different solution behaviors typically encountered. We compare our microliter capillary rheometer with existing instruments to describe the range of parameter space covered by our device.

The limitations of an exclusively colloidal view of protein solution hydrodynamics and rheology.

Proteins are complex macromolecules with dynamic conformations. They are charged like colloids, but unlike colloids, charge is heterogeneously distributed on their surfaces. Here we overturn entrenched doctrine that uncritically treats bovine serum albumin (BSA) as a colloidal hard sphere by elucidating the complex pH and surface hydration-dependence of solution viscosity. We measure the infinite shear viscosity of buffered BSA solutions in a parameter space chosen to tune competing long-range repulsions and short-range attractions (2 mg/mL ≤ [BSA] ≤ 500 mg/mL and 3.0 ≤ pH ≤ 7.4). We account for surface hydration through partial specific volume to define volume fraction and determine that the pH-dependent BSA intrinsic viscosity never equals the classical hard sphere result (2.5). We attempt to fit our data to the colloidal rheology models of Russel, Saville, and Schowalter (RSS) and Krieger-Dougherty (KD), which are each routinely and successfully applied to uniformly charged suspensions and to hard-sphere suspensions, respectively. We discover that the RSS model accurately describes our data at pH 3.0, 4.0, and 5.0, but fails at pH 6.0 and 7.4, due to steeply rising solution viscosity at high concentration. When we implement the KD model with the maximum packing volume fraction as the sole floating parameter while holding the intrinsic viscosity constant, we conclude that the model only succeeds at pH 6.0 and 7.4. These findings lead us to define a minimal framework for models of crowded protein solution viscosity wherein critical protein-specific attributes (namely, conformation, surface hydration, and surface charge distribution) are addressed.

Direct experimental evidence of growing dynamic length scales in confined colloidal liquids.

The modification of the glass transition in confined domains, particularly the length scales associated with cooperative motion, remains a mystery. Hard-sphere suspensions are confined between two surfaces to progressively smaller dimensions to probe the confinement effect on the growth of dynamic heterogeneities via confocal microscopy. The confinement length scale is defined as the critical spacing where deviations from bulk behaviors begin and is observed to occur at progressively larger gap spacings as the volume fraction is increased. However, dynamic length scales extracted from the four-point correlation function are on average smaller than the confinement length scale.

How does sensitivity to dynamical heterogeneity in supercooled colloidal liquids depend on tracer size?

Few experimental investigations have examined the dependence of probe length scale on the evolution of dynamical heterogeneity in supercooled liquids. In this study, we use fluorescent tracer probes, which are smaller than that of the constituent unlabeled particles in the matrix suspension, to investigate the tracer size sensitivity to the onset of dynamical heterogeneity in model hard-sphere colloidal suspensions close to the glass transition. The dynamics of poly(methyl methacrylate) (PMMA) tracer particles of radii a(S) = 137 nm embedded into constituent PMMA particles of a(L) = 212 and 637 nm with bulk volume fraction phi varied from 0.50 to 0.57 is examined using fluorescence correlation spectroscopy (FCS). Upon increasing phi for the largest tracer size ratio explored, f (= a(S)/a(L)) = 0.646, the onset of dynamic heterogeneity is observed from the broad distribution of characteristic decay times of the tracers, which is extracted from the measured FCS autocorrelation functions. The strong coupling between tracer size and its sensitivity to dynamical heterogeneity is reflected in the dynamic susceptibility, where dynamic correlation lengths are immeasurable for the smallest size ratio and show a marked increase to five tracer particle diameters for the largest size ratio.

Dielectrophoresis and AC-induced assembly in binary colloidal suspensions.

Dielectrophoretic behaviors and assembly of a binary suspension in aqueous media are examined in the presence of nonuniform alternating current (AC) electric field. A peculiar low-frequency threshold and dielectrophoresis (DEP) crossover frequency determine the applicable frequency window for binary assembly under positive DEP, which can be effectively tuned by medium conductivity and particle size, suggesting that the dynamic double-layer effect is responsible for the interfacial polarization of micrometer to submicrometer-sized particles in aqueous suspensions. Strong effects of AC-field frequency, medium conductivity, and size ratio on binary assembly morphology have been observed. A frequency-medium conductivity phase diagram is obtained to illustrate the morphological transition of assembled colloidal aggregates from segregated, ordered assemblies to inverted segregation with the appearance of amorphous phases upon increasing frequency and/or medium conductivity, which is a direct consequence of the competition between DEP and hydrodynamic mobility. Significantly, our results demonstrate a rapid method to form hybrid nanostructured materials.

Direct visualization of colloidal gelation under confinement.

The physical mechanism of colloidal gelation remains inadequately understood, particularly for intermediate to high volume fractions. Experiments to directly probe the complex evolution of structural and viscoelastic properties of gels have been few despite their fundamental importance in elucidating the physical mechanisms responsible for gelation. In this study, we use a home-built micron-gap rheometer combined with confocal microscopy to directly investigate the coupled structural and dynamic properties of colloidal gelation transition by spatial confinement. We observe that confinement-induced gelation proceeds by a spinodal decomposition route where strongly confined colloidal suspensions evolve into "colloid-rich" and "colloid-poor" regions; the propagation of the "colloid-rich" region in three dimensions is responsible for structural arrest and strong viscoelastic enhancement when a critical film thickness approaches 16-25 particle layers.

Impeded structural relaxation of a hard-sphere colloidal suspension under confinement.

The phenomenon of glass transition, such as the anomalous divergence in viscosity without apparent structural change, remains inadequately understood. We employ spatial confinement to probe length scale dependence on structural relaxation and concomitant glassy dynamics of a hard-sphere poly-(methyl methacrylate) colloidal suspension via confocal microscopy. Remarkable film thickness dependent scaling behavior is observed, where the mobility and relaxation processes of a "fluid" suspension are found to be significantly impeded as film thickness is reduced below 15-20 particle layers.