Biophysical characterization and molecular simulation of electrostatically driven self‐association of a single‐chain antibody

Abstract

Colloidal protein-protein interactions (PPI) are often expected to impact key behaviors of proteins in solution, such as aggregation rates and mechanisms, aggregate structure, protein solubility, and solution viscosity. PPI of an anti-fluorescein single chain antibody variable fragment (scFv) were characterized experimentally at low to intermediate ionic strength using a combination of static light scattering and sedimentation equilibrium ultracentrifugation. Surprisingly, the results indicated that interactions were strongly net-attractive and electrostatics promoted self-association. Only repulsive interactions were expected based on prior work and calculations based a homology model of a related scFv crystal structure. However, the crystal structure lacks the charged, net-neutral linker sequence. PyRosetta was used to generate a set of scFv structures with different linker conformations, and coarse-grained Monte Carlo simulations were used to evaluate the effect of different linker configurations via second osmotic virial coefficient (B ) simulations. The results show that the configuration of the linker has a significant effect on the calculated B values, and can result in strong electrostatic attractions between oppositely charged residues on the protein surface. This is particularly relevant for development of non-natural antibody products, where charged linkers and other loop regions may be prevalent. The results also provide a preliminary computational framework to evaluate the effect of unstructured linkers on experimental protein-protein interaction parameters such as B .

Electrostatically Mediated Protein-Protein Interactions for Monoclonal Antibodies: A Combined Experimental and Coarse-Grained Molecular Modeling Approach

“…This corresponds to a lower effective molecular charge, such as has been observed for MAbs in prior work. 14,23,53 For MAb B, the simulations show the same characteristic nonmonotonic pH 5 behavior that was observed experimentally. In some cases, the simulations used j > 1. j values greater than 1 indicates the simulation assigns more charge to the molecule than found in the sequence.…”

“…However, initial calculations with simpler systems that have flexible linkers indicate that the computational burden increases by orders of magnitude. 23 The update to the 1bAA force field proposed and implemented in previous work was employed here. 12,14 This force field allows 1 to evaluate the combination of the effects of short-ranged nonelectrostatic attractions (e.g., hydration effects and van der Waals attractions), screened electrostatic interactions, and excluded volume effects at the scale of individual amino acids with high computational efficiency.…”

“…14,38 j is used to account for deviations from theoretical net charges due to effects such as preferential counterion binding as shown in previous work. 23 j ¼ q effective .…”

“…14,[20][21][22] In the present work, the reference system sampling was treated analytically by choosing the reference potential to be that of a steric-only 1bAA system. 23 The simulations returned B 22 /B 22,steric where B 22,steric denotes be the B 22 value if only steric interactions were present. B 22,steric is the value of B 22 based on the 3-dimensional structures of the 2 protein molecules (with each amino acid described in the 1bAA model), in the absence of all nonsteric interactions (electrostatics, van der Waals attractions, hydrophobic attractions, etc.).…”

“…14,[20][21][22] Prior work in this vein focused primarily on globular proteins. 23 The present report examines the PPI of a series of MAbs that show qualitatively and quantitatively different experimental PPI across a range of TIS (~5-300 mM) and pH (5, 6.5, 8) conditions that are commonly used in protein formulations. The 3 MAbs are referred to as MAb A, MAb B, and MAb C in what follows below.…”