Molecular computations of preferential interactions of proline, arginine.HCl, and NaCl with IgG1 antibodies and their impact on aggregation and viscosity.
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ABSTRACT:
SUBMITTER: Cloutier TK
PROVIDER: S-EPMC7531574 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Preferential interactions of weakly interacting formulation excipients govern their effect on the equilibrium and kinetics of several reactions of protein molecules in solution. Using vapor pressure osmometry, we characterized the preferential interactions of commonly used excipients trehalose, L-arginine.HCl and NaCl with three therapeutically-relevant, IgG1 monoclonal antibodies that have similar size and shape, but differ in their surface hydrophobicity and net charge. We further characterized the effect of these excipients on the reversible self-association, aggregation and viscosity behavior of these antibody molecules. We report that trehalose, L-arginine.HCl and NaCl are all excluded from the surface of the three IgG1 monoclonal antibodies, and that the exclusion behavior is linearly related to the excipient molality in the case of trehalose and NaCl, whereas a non-linear behavior is observed for L-arginine.HCl. Interestingly, we find that the magnitude of trehalose exclusion depends upon the nature of the protein surface. Such behavior is not observed in case of NaCl and L-arginine.HCl as they are excluded to the same extent from the surface of all three antibody molecules tested in this study. Analysis of data presented in this study provides further insight into the mechanisms governing excipient-mediated stabilization of mAb formulations.
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Project description:Human/humanized IgG4 antibodies have reduced effector function relative to IgG1 antibodies, which is desirable for certain therapeutic purposes. However, the developability and biophysical properties for IgG4 antibodies are not well understood. This work focuses on the head-to-head comparison of key biophysical properties, such as self-interaction and viscosity, for 14 human/humanized, and chimeric IgG1 and IgG4 S228P monoclonal antibody pairs that contain the identical variable regions. Experimental measurements showed that the IgG4 S228P antibodies have similar or higher self-interaction and viscosity than that of IgG1 antibodies in 20 mM sodium acetate, pH 5.5. We report sequence and structural drivers for the increased viscosity and self-interaction detected in IgG4 S228P antibodies through a combination of experimental data and computational models. Further, we applied and extended a previously established computational model for IgG1 antibodies to predict the self-interaction and viscosity behavior for each antibody pair, providing insight into the structural characteristics and differences of these two isotypes. Interestingly, we observed that the IgG4 S228P swapped variants, where the CH3 domain was swapped for that of an IgG1, showed reduced self-interaction behavior. These domain swapped IgG4 S228P molecules also showed reduced viscosity from experiment and coarse-grained simulations. We also observed that experimental diffusion interaction parameter (kD) values have a high correlation with computational diffusivity prediction for both IgG1 and IgG4 S228P isotypes.Abbreviations: AHc, constant region Hamaker constant; AHv, variable region Hamaker constant; CDRs, Complementarity-determining regions; CG, Coarse-grained model; CH1, Constant heavy chain 1; CH2 Constant heavy chain 2; CH3 Constant heavy chain 3; chgCH3 Effective charge on the CH3 region; CL Constant light chain; cP, Centipoise; DLS, Dynamic light scattering; Fab, Fragment antigen-binding; Fc, Fragment crystallizable; Fv, Variable domaing; (r) Radial distribution function; H1 CDR1 of Heavy Chain; H2 CDR2 of Heavy Chain; H3 CDR3 of Heavy Chain; HVI, High viscosity index; IgG1 human immunoglobulin of IgG1 subclass; IgG4 human immunoglobulin of IgG4 subclass; kD, Diffusion interaction parameter; L1 CDR1 of Light Chain; L2 CDR2 of Light Chain; L3 CDR3 of Light Chain; mAb, Monoclonal antibody; MD, Molecular dynamics; PPI Protein-protein interactions; SCM, Spatial charge map; UP-SEC, Ultra-high-performance size-exclusion chromatography; VH, Variable domain of Heavy Chain; VL, Variable domain of Light Chain.
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Project description:Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.
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Project description:Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in CH3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.
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Project description:The viscosity of household care products plays an important role in pleasant delivery using consumer experience at home. A novel solution to mitigate the sharp rising of viscosities at low temperatures of detergents was proposed. By designing the formulation of the surfactant blend, formulators can achieve acceptable viscosity profiles in the temperature range encountered in daily life. The verification and modulation of formulas bearing parabolic viscosity-temperature behavior were systematically studied, including in single, binary, and ternary systems, based on the modulation of sodium ethoxylated alkyl sulfate (AES) by other anions, zwitterions, and nonions. The R ratio theory was used to have a better understanding of the molecular assembly of surfactants behind the parabolic behavior exhibited in rheology analyses. One of the key findings is that the parabolic viscosity-temperature phenomenon could be easily observed in the highly hydrated ethoxylated anionic systems like AES-based systems. For those anions lacking ethoxylation, especially sodium linear alkylbenzene sulfonate (LAS), the monotonic variation of hydration affinity with temperature led to the disappearance of parabola in the observed temperature window (>0 °C). Moreover, salinity played an important role in the hydration affinity of the polar group and the interaction between the hydrophilic headgroups. A balanced salinity should be optimized to modulate the hydration affinity in a desired range so that the parabola could be easily tuned within the target temperature region. These findings provide opportunities for the formulators in the household care industry to design products with better pourability through carefully selecting a combination of surfactants and fine-tuning their ratios to improve consumer use experience, especially in winter.
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Project description:MotivationCodon models are widely used to identify the signature of selection at the molecular level and to test for changes in selective pressure during the evolution of genes encoding proteins. The large size of the state space of the Markov processes used to model codon evolution makes it difficult to use these models with large biological datasets. We propose here to use state aggregation to reduce the state space of codon models and, thus, improve the computational performance of likelihood estimation on these models.ResultsWe show that this heuristic speeds up the computations of the M0 and branch-site models up to 6.8 times. We also show through simulations that state aggregation does not introduce a detectable bias. We analyzed a real dataset and show that aggregation provides highly correlated predictions compared to the full likelihood computations. Finally, state aggregation is a very general approach and can be applied to any continuous-time Markov process-based model with large state space, such as amino acid and coevolution models. We therefore discuss different ways to apply state aggregation to Markov models used in phylogenetics.Availability and implementationThe heuristic is implemented in the godon package (https://bitbucket.org/Davydov/godon) and in a version of FastCodeML (https://gitlab.isb-sib.ch/phylo/fastcodeml).Contactnicolas.salamin@unil.chSupplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Aggregation of monoclonal antibodies is often a multi-step process involving structural alterations in monomeric proteins and subsequent formation of soluble or insoluble oligomers. The role of local conformational stability and dynamics of native and/or partially altered structures in determining the aggregation propensity of monoclonal antibodies, however, is not well understood. Here, we investigate the role of conformational stability and dynamics of regions with distinct solvent exposure in determining the aggregation propensity of an IgG1 and IgG2 monoclonal antibody. The temperatures employed span the pre-unfolding range (10-40°C) and the onset temperatures (T onset ) for exposure of apolar residues (≈ 50°C), alterations in secondary structures (≈ 60°C) and initiation of visible aggregate formation (≈ 60°C). Solvent-exposed regions were found to precede solvent-shielded regions in an initiation of aggregation for both proteins. Such a process was observed upon alterations in overall tertiary structure while retaining the secondary structures in both the proteins. In addition, a greater dynamic nature of solvent-shielded regions in potential intermediates of IgG1 and the improved conformational stability increased its resistance to aggregation when compared to IgG2. These results suggest that local conformational stability and fluctuations of partially altered structures can influence the aggregation propensity of immunoglobulins.
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Project description:Only around 80% of patients with generalized myasthenia gravis (MG) have serum antibodies to acetylcholine receptor [AChR; acetylcholine receptor antibody positive myasthenia gravis (AChR-MG)] by the radioimmunoprecipitation assay used worldwide. Antibodies to muscle specific kinase [MuSK; MuSK antibody positive myasthenia gravis (MuSK-MG)] make up a variable proportion of the remaining 20%. The patients with neither AChR nor MuSK antibodies are often called seronegative (seronegative MG, SNMG). There is accumulating evidence that SNMG patients are similar to AChR-MG in clinical features and thymic pathology. We hypothesized that SNMG patients have low-affinity antibodies to AChR that cannot be detected in solution phase assays, but would be detected by binding to the AChRs on the cell membrane, particularly if they were clustered at the high density that is found at the neuromuscular junction. We expressed recombinant AChR subunits with the clustering protein, rapsyn, in human embryonic kidney cells and tested for binding of antibodies by immunofluorescence. To identify AChRs, we tagged either AChR or rapsyn with enhanced green fluorescence protein, and visualized human antibodies with Alexa Fluor-labelled secondary or tertiary antibodies, or by fluorescence-activated cell sorter (FACS). We correlated the results with the thymic pathology where available. We detected AChR antibodies to rapsyn-clustered AChR in 66% (25/38) of sera previously negative for binding to AChR in solution and confirmed the results with FACS. The antibodies were mainly IgG1 subclass and showed ability to activate complement. In addition, there was a correlation between serum binding to clustered AChR and complement deposition on myoid cells in patients' thymus tissue. A similar approach was used to demonstrate that MuSK antibodies, although mainly IgG4, were partially IgG1 subclass and capable of activating complement when bound to MuSK on the cell surface. These observations throw new light on different forms of MG paving the way for improved diagnosis and management, and the approaches used have applicability to other antibody-mediated conditions.
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Project description:Proline residues have unique roles in protein folding, structure, and function. Proline and the aromatic amino acids comprise the encoded cyclic protein residues. Aromatic protein side chains are defined by their negatively charged ? faces, while the faces of the proline ring are partially positively charged. This polarity results from their two-point connection of the side chain to the electron-withdrawing protein backbone, and the lower electronegativity of hydrogen compared to carbon, nitrogen, and oxygen. The hydrogens adjacent to the carbonyl and amide nitrogen, H? and H?, respectively, are the most partially positive. Proline's side chain is also conformationally restricted, allowing for interaction with aromatic residues with minimal entropic or steric penalty. Proline and aromatic residues can interact favorably with each other, due to both the hydrophobic effect and the interaction between the ? aromatic face and the polarized C-H bonds, called a CH/? interaction. Aromatic-proline interactions can occur locally, for example, to stabilize cis-amide bonds, and over larger distances, in the tertiary structures of proteins, and intermolecularly in protein-protein interactions. In peptides and proteins, aromatic-proline sequences more readily adopt cis-prolyl amide bonds, where the aromatic ring interacts with the proline ring in the cis conformation. In aromatic-proline sequences, Trp and Tyr are more likely to induce cis-amide bonds than Phe, suggesting an aromatic electronic effect. This result would be expected for a CH/? interaction, in which a more electron-rich aromatic would have a stronger (more cis-stabilizing) interaction with partial positive charges on prolyl hydrogens. In this Account, we describe our investigations into the nature of local aromatic-proline interactions, using peptide models. We synthesized a series of 26 peptides, TXPN, varying X from electron-rich to electron poor aromatic amino acids, and found that the population of cis-amide bond (Ktrans/cis) is tunable by aromatic electronics. With 4-substituted phenylalanines, we observed a Hammett correlation between aromatic electronics and Ktrans/cis, with cis-trans isomerism electronically controllable by 1.0 kcal/mol. All aromatic residues exhibit a higher cis population than Ala or cyclohexylalanine, with Trp showing the strongest aromatic-proline interaction. In addition, proline stereoelectronic effects can modulate cis-trans isomerism by an additional 1.0 kcal/mol. The aromatic-proline interaction is enthalpic, consistent with its description as a CH/? interaction. Proline-aromatic sequences can also promote cis-prolyl bonds, either through interactions of the aromatic ring with the preceding cis-proline or with the H? prior to cis-proline. Within proline-rich peptides, sequences commonly found in natively disordered proteins, aromatic residues promote multiple cis-amide bonds due to multiple favorable aromatic-proline interactions. Collectively, we found aromatic-proline interactions to be significantly CH/? in nature, tunable by aromatic electronics. We discuss these data in the context of aromatic-proline and aromatic-glycine interactions in local structure, in tertiary structure, in protein-protein interactions, and in protein assemblies.
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Project description:The immunoglobulin (Ig) CH2 domain is a promising scaffold for the development of candidate therapeutics. We have previously shown that the stability of isolated CH2 could be increased by the introduction of an additional disulfide bond and removal of seven N-terminal residues (m01s). However, both isolated CH2 and m01s aggregate, likely due to the existence of aggregation-prone regions (APRs) that we identified by using computational methods. This knowledge was used to generate a phage display library of mutants. The library was incubated at high temperature to remove aggregating CH2 domains, and then panned against a mouse anti-human CH2 monoclonal antibody targeting a conformational epitope to remove misfolded CH2s. After two rounds of panning, one clone, m01s5, with smaller APRs, was identified. After additional mutagenesis one clone, m01s5.4, which aggregated much less than m01s as measured by a turbidity assay and dynamic light scattering, was identified. m01s5.4 also exhibited much lower nonspecific binding than m01s. Engineering of a previously identified m01s-based tumor antigen-specific binder led to a dramatic reduction of its aggregation without affecting its binding. In summary, we describe a new approach for reducing aggregation based on a combination of computational and phage display methodologies, and show that aggregation of CH2-based scaffolds can be significantly reduced by the newly identified mutants, which can improve the developability of potential CH2-based therapeutics.
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