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Understanding protein-protein interactions and formation of reversible oligomers (clusters) in concentrated monoclonal antibody (mAb) solutions is necessary for designing stable, low viscosity (η) concentrated formulations for processing and subcutaneous injection. Here we characterize the strength ( K ) of short-range anisotropic attractions (SRA) for 75-200 mg/mL mAb2 solutions at different pH and cosolute conditions by analyzing structure factors ( S eff ( q )) from small-angle X-ray scattering (SAXS) using coarse-grained molecular dynamics simulations. Best fit simulations additionally provide cluster size distributions, fractal dimensions, cluster occluded volume, and mAb coordination numbers. These equilibrium properties are utilized in a model to account for increases in viscosity caused by occluded volume in the clusters (packing effects) and dissipation of stress across lubricated fractal clusters. S eff ( q ) is highly sensitive to K at 75 mg/mL where mAbs can mutually align to form SRA contacts but becomes less sensitive at 200 mg/mL as steric repulsion due to packing becomes dominant. In contrast, η at 200 mg/mL is highly sensitive to SRA and the average cluster size from SAXS/simulation, which is observed to track the cluster relaxation time from shear thinning. By analyzing the distribution of sub-bead hot spots on the 3D mAb surface, we identify a strongly attractive hydrophobic patch in the complementarity determining region (CDR) at pH 4.5 that contributes to the high K and consequently large cluster sizes and high η. Adding NaCl screens electrostatic interactions and increases the impact of hydrophobic attraction on cluster size and raises η, whereas nonspecific binding of Arg attenuates all SRA, reducing η. The hydrophobic patch is absent at higher pH values, leading to smaller K , smaller clusters, and lower η. This work constitutes a first attempt to use SAXS and CG modeling to link both structural and rheological properties of concentrated mAb solutions to the energetics of specific hydrophobic patches on mAb surfaces. As such, our work opens an avenue for future research, including the possibility of designing coarse-grained models with physically meaningful interacting hot spots.

The effects of a subclass of monoclonal antibodies (mAbs) on protein-protein interactions, formation of reversible oligomers (clusters), and viscosity (η) are not well understood at high concentrations. Herein, we quantify a short-range anisotropic attraction between the complementarity-determining region (CDR) and CH3 domains (K CDR-CH3 ) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor S eff ( q ) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The K CDR-CH3 bead attraction strength was isolated from the strength of long-range electrostatic repulsion for the full mAb, which was determined from the theoretical net charge and a scaling parameter ψ to account for solvent accessibility and ion pairing. At low ionic strength (IS), the strongest short-range attraction (K CDR-CH3 ) and consequently the largest clusters and highest η were observed with IgG1, the subclass with the most positively charged CH3 domain. Furthermore, the trend in K CDR-CH3 with the subclass followed the electrostatic interaction energy between the CDR and CH3 regions calculated with the BioLuminate software using the 3D mAb structure and molecular interaction potentials. Whereas the equilibrium cluster size distributions and fractal dimensions were determined from fits of SAXS with the MD simulations, the degree of cluster rigidity under flow was estimated from the experimental η with a phenomenological model. For the systems with the largest clusters, especially IgG1, the inefficient packing of mAbs in the clusters played the largest role in increasing η, whereas for other systems, the relative contribution from stress produced by the clusters was more significant. The ability to relate η to short-range attraction from SAXS measurements at high concentrations and to theoretical characterization of electrostatic patches on the 3D surface is not only of fundamental interest but also of practical value for mAb discovery, processing, formulation, and subcutaneous delivery.

Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations. The goal of this distillation is to help harmonize aggregate quantitation approaches across the biopharmaceutical industry. We review key considerations for sample and instrument suitability, experimental design, and data analysis best practices and conversely, highlight potential pitfalls to accurate aggregate analysis. Our goal is to provide experienced users benchmarks against which they can standardize their analyses and to provide guidance for new AUC analysts that will aid them to become proficient in this fundamental technique., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

This work compares the conformational stability, backbone flexibility, and aggregation propensity of monomer and dimer fractions of an IgG1 monoclonal antibody (mAb) generated on UVA light exposure for up to 72 h collected by preparative size-exclusion chromatography, compared with unstressed control. UVA light exposure induced covalent aggregation, and fragmentation as measured by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and extensive oxidation of specific methionine residues (Met 257, Met 433, and Met 109) in both size fractions identified by reverse phase chromatography coupled to mass spectrometry. Compared with unstressed mAb, both the monomer and dimer fractionated from 72 h UVA light-exposed mAb had decreased thermal melting temperatures (T m1 ) by 1.4°C as measured by differential scanning calorimetry, minor changes in tertiary structure as measured by near-UV CD, increased monomer loss, and aggregation on accelerated storage at 35°C. Hydrogen/deuterium exchange mass spectrometry identified local segments with increased flexibility in C H 2 and C H 3 domains of both size fractions, and decreased flexibility in few segments of F ab and C H 1 domains in the dimer fraction. Segment 247-256 in heavy chain, an established aggregation hotspot in IgG1 mAbs had large increase in flexibility in both size fractions compared with unstressed mAb., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The dynamic nature of the structure of monoclonal antibodies (mAbs) can be probed at a resolution of 5-20 residues using hydrogen-deuterium exchange mass spectrometry (H/D-MS). Recent studies using H/D-MS have shown that distinct regions of IgG1 mAbs experience significant changes in backbone dynamics in response to specific physicochemical alterations, varying solution conditions, or exposure to different environmental stresses. Tracking such changes in local dynamics may therefore serve as a key analytical tool, not only to monitor stability changes, but also to design improved, and more stable formulations of therapeutic mAbs in pharmaceutical dosage forms. This review article describes the H/D-MS method as applied to the analysis of formulations containing mAbs and summarizes recent studies monitoring changes in mAb local dynamics in response to chemical modifications, physical degradation, and presence of stabilizing and destabilizing excipients. Furthermore, the nature of the local dynamics of a highly conserved peptide segment in the CH 2 domain of IgG1 mAbs is reviewed, and the results are correlated with decreased pharmaceutical stability, supporting the identification of a common aggregation hotspot sequence in the Fc region of human IgG1 mAbs. In addition, unresolved challenges (and opportunities) in applying H/D-MS technology for stabilization and formulation development of mAbs are discussed., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the CH2 and distant segments in the VH, CH1, and VL domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244-254 segment of the CH2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (Tonset) and unfolding (Tm1) temperatures of 7°C and 6.7°C, respectively, for the CH2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244-254 segment, providing a site-directed mutant example that this segment of the CH2 domain is an aggregation hot spot in IgG1 mAbs.

The structural integrity and conformational stability of an IgG1 monoclonal antibody (mAb), after partial or complete enzymatic removal of the N-linked Fc glycan, were compared with the untreated mAb over a wide range of temperature (10°C-90°C) and solution pH (3-8) using circular dichroism, fluorescence spectroscopy, and static light scattering combined with data visualization employing empirical phase diagrams. Subtle-to-larger stability differences between the different glycoforms were observed. Improved detection of physical stability differences was then demonstrated over narrower pH range (4.0-6.0) using smaller temperature increments, especially when combined with an alternative data visualization method (radar plots). Differential scanning calorimetry and differential scanning fluorimetry were then utilized and also showed an improved ability to detect differences in the physical stability of a mAb glycoform. On the basis of these results, a two-step methodology was used in which conformational stability of a mAb glycoform is first screened with a wide variety of instruments and environmental stresses, followed by a second evaluation with optimally sensitive experimental conditions, analytical techniques, and data visualization methods. With this approach, a high-throughput biophysical analysis to assess relatively subtle conformational stability differences in protein glycoforms is demonstrated., (© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.)

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (T(m) values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (C(H)2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the C(H)2 domain (notably heavy chain 241-251) and the mAb's overall physical stability., (© 2013 Wiley Periodicals, Inc.)

This work examines the effect of three anions from the Hofmeister series (sulfate, chloride, and thiocyanate) on the conformational stability and aggregation rate of an IgG1 monoclonal antibody (mAb) and corresponding changes in the mAb's backbone flexibility (at pH 6 and 25 °C). Compared to a 0.1 M NaCl control, thiocyanate (0.5 M) decreased the melting temperatures (Tm) for three observed conformational transitions within the mAb by 6-9 °C, as measured by differential scanning calorimetry. Thiocyanate also accelerated the rate of monomer loss at 40 °C over 12 months, as monitored by size exclusion chromatography. Backbone flexibility, as measured via H/D exchange mass spectrometry, increased in two segments in the CH2 domain with more subtle changes across several additional regions. Chloride (0.5 M) caused slight increases in the Tm values, small changes in aggregation rate, and minimal yet consistent decreases in flexibility across various domains with larger effects noted within the VL, CH1, and CH3 domains. In contrast, 0.5 M sulfate increased Tm values, had small stabilizing influences on aggregate formation over time, yet substantially increased the flexibility of two specific regions in the CH1 and VL domains. While thiocyanate-induced conformational destabilization of the mAb correlated with increased local flexibility of specific regions in the CH2 domain (especially residues 241-251 in the heavy chain), the stabilizing anion sulfate did not affect these CH2 regions.

Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30% of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5%)/isopropanol (5%)/acetic acid (20%) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.

The exoloops of glycoprotein hormone receptors (GpHRs) transduce the signal generated by the ligand-ectodomain interactions to the transmembrane helices either through direct hormonal contact and/or by modulating the interdomain interactions between the hinge region (HinR) and the transmembrane domain (TMD). The ligand-induced conformational alterations in the HinRs and the interhelical loops of luteinizing hormone receptor/follicle stimulating hormone receptor/thyroid stimulating hormone receptor were mapped using exoloop-specific antibodies generated against a mini-TMD protein designed to mimic the native exoloop conformations that were created by joining the thyroid stimulating hormone receptor exoloops constrained through helical tethers and library-derived linkers. The antibody against the mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone-stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones, suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the HinRs, exoloops, and TMDs such as those involved in precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, we demonstrate that changes in the HinR-exoloop interactions play an important role in receptor activation. Computational analysis suggests that the mini-TMD antibodies act by conformationally locking the transmembrane helices by means of restraining the exoloops and the juxta-membrane regions. Using GpHRs as a model, we describe a novel computational approach of generating soluble TMD mimics that can be used to explain the role of exoloops during receptor activation and their interplay with TMDs.

We report two antibodies, scFv 13B1 and MAb PD1.37, against the hinge regions of LHR and TSHR, respectively, which have similar epitopes but different effects on receptor function. While neither of them affected hormone binding, with marginal effects on hormone response, scFv 13B1 stimulated LHR in a dose-dependent manner, whereas MAb PD1.37 acted as an inverse agonist of TSHR. Moreover, PD1.37 could decrease the basal activity of hinge region CAMs, but had varied effects on those present in ECLs, whereas 13B1 was refractory to any CAMs in LHR. Using truncation mutants and peptide phage display, we compared the differential roles of the hinge region cysteine box-2/3 as well as the exoloops in the activation of these two homologus receptors., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)