robin curtis

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Papers by robin curtis

Towards an improved prediction of concentrated antibody solution viscosity using the Huggins coefficient

Journal of Colloid and Interface Science

Arginine to lysine mutations increase the aggregation stability of a single chain variable fragment through unfolded state interactions

Biochemistry

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

The Journal of Physical Chemistry B

Predicting the concentrated solution behavior for monoclonal antibodies requires developing and u... more 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.

Hard Spheres with Purely Repulsive Interactions Have Positive Diffusion Interaction Parameter, kD

Biophysical journal, Jan 8, 2017

Sorret et al. (1) recently critically discussed our paper in Biophysical Journal (2,3) where we e... more Sorret et al. (1) recently critically discussed our paper in Biophysical Journal (2,3) where we emphasized the need to consider the macromolecular nature of proteins when considering their structure over a large range of protein concentration (c), pH, etc. In particular, they criticized our interpretation of pH-dependent measurements of Bovine Serum Albumin (BSA) Stokes radius using Dynamic Light Scattering (DLS) (4), and independent intrinsic viscosity [h] measurements (5), as implying a change in conformation when pH is varied. We specifically quote from their work (1): ''Sarangapani et al. (78) concluded that BSA increased in size by 20% from pH 7.2 to 5.0, and by 40% from pH 7.2 to 3.0, based on fluctuations in Ds,2 values in 20 mM ionic strength buffers. These findings are surprising considering that other studies (88,89) did not observe such significant changes in the size of BSA as a function of pH >2 under similar conditions. Furthermore, Tanford and Buzzell demonstrated that apparent size changes in BSA as a function of pH are due to electroviscous effects alone, and not due to deformation of the BSA molecule (90).''

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Evaluation of aggregate and silicone-oil counts in pre-filled siliconized syringes: An orthogonal study characterising the entire subvisible size range

International journal of pharmaceutics, Jan 15, 2017

Characterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routine... more Characterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routinely extended to the sub-visible size-range (0.1-10μm). Additionally, with the increased use of pre-filled syringes (PFS), particle differentiation is required between proteinaceous and non-proteinaceous particles such as silicone-oil droplets. Here, three orthogonal techniques: Raster Image Correlation Spectroscopy (RICS), Resonance Mass Measurements (RMM) and Micro-Flow Imaging (MFI), were evaluated with respect to their sub-visible particle measurement and characterisation capabilities. Particle formation in mAb PFS solutions was evaluated with increasing polysorbate-20 (PS-20) concentrations. All three techniques provided complementary but distinct information on protein aggregate and silicone-oil droplet presence. PS-20 limited the generation of mAb aggregates during agitation, while increasing the number of silicone-oil droplets (PS-20 concentration dependant). MFI and RMM revealed P...

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Reply to "Comment on 'Osmolyte Effects on Monoclonal Antibody Stability and Concentration-Dependent Protein Interactions with Water and Common Osmolytes

The journal of physical chemistry. B, Nov 3, 2016

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Proofreading of substrate structure by the Twin-Arginine Translocase is highly dependent on substrate conformational flexibility but surprisingly tolerant of surface charge and hydrophobicity changes

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2016

The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichi... more The Tat system transports folded proteins across the bacterial plasma membrane, and in Escherichia coli preferentially transports correctly-folded proteins. Little is known of the mechanism by which Tat proofreads a substrate&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s conformational state, and in this study we have addressed this question using a heterologous single-chain variable fragment (scFv) with a defined structure. We introduced mutations to surface residues while leaving the folded structure intact, and also tested the importance of conformational flexibility. We show that while the scFv is stably folded and active in the reduced form, formation of the 2 intra-domain disulphide bonds enhances Tat-dependent export 10-fold, indicating Tat senses the conformational flexibility and preferentially exports the more rigid structure. We further show that a 26-residue unstructured tail at the C-terminus blocks export, suggesting that even this short sequence can be sensed by the proofreading system. In contrast, the Tat system can tolerate significant changes in charge or hydrophobicity on the scFv surface; substitution of uncharged residues by up to 3 Lys-Glu pairs has little effect, as has the introduction of up to 5 Lys or Glu residues in a confined domain, or the introduction of a patch of 4 to 6 Leu residues in a hydrophilic region. We propose that the proofreading system has evolved to sense conformational flexibility and detect even very transiently-exposed internal regions, or the presence of unfolded peptide sections. In contrast, it tolerates major changes in surface charge or hydrophobicity.

Protein-Protein and Protein-Salt Interactions. A Study of Specific ION Effects on Homopolypeptides

Poly-L-lysine provides a good model compound for understanding protein interactions as the peptid... more

A molecular approach to bioseparations: Protein–protein and protein–salt interactions

Chemical Engineering Science 2006 61 907 923, 2006

... accessible protein mass concentration, c p , (1) View the MathML source Here, R is the ideal ... more

Osmolyte Effects on Monoclonal Antibody Stability and Concentration-Dependent Protein Interactions with Water and Common Osmolytes

The journal of physical chemistry. B, Jan 7, 2016

Preferential interactions of proteins with water and osmolytes play a major role in controlling t... more Preferential interactions of proteins with water and osmolytes play a major role in controlling the thermodynamics of protein solutions. While changes in protein stability and shifts in phase behavior are often reported with the addition of osmolytes, the underlying protein interactions with water and/or osmolytes are typically inferred rather than measured directly. In this work, Kirkwood-Buff integrals for protein-water interactions (G12) and protein-osmolyte interactions (G23) were determined as a function of osmolyte concentration from density measurements of antistreptavidin immunoglobulin gamma-1 (AS-IgG1) in ternary aqueous solutions for a set of common neutral osmolytes: sucrose, trehalose, sorbitol, and poly(ethylene glycol) (PEG). For sucrose and PEG solutions, both protein-water and protein-osmolyte interactions depend strongly on osmolyte concentrations (c3). Strikingly, both osmolytes change from being preferentially excluded to preferentially accumulated with increasin...