Ranking mAb-excipient interactions in biologics formulations by NMR spectroscopy and computational approaches.

Excipients are added to biopharmaceutical formulations to enhance protein stability and enable the development of robust formulations with acceptable physicochemical properties, but the mechanism by which they confer stability is not fully understood. Here, we aimed to elucidate the mechanism through direct experimental evidence of the binding affinity of an excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. We ranked a series of excipients with respect to their dissociation constant (KD) and nonspecific binding constants (Ns). In parallel, molecular dynamic and site identification by ligand competitive saturation (SILCS)-Monte Carlo simulations were done to rank the excipient proximity to the proteins, thereby corroborating the ranking by STD NMR. Finally, the excipient ranking by NMR was correlated with mAb conformational and colloidal stability. Our approach can aid excipient selection in biologic formulations by providing insights into mAb-excipient affinities before conventional and time-consuming excipient screening studies are conducted.

Differential Surface Adsorption Phenomena for Conventional and Novel Surfactants Correlates with Changes in Interfacial mAb Stabilization.

Protein adsorption on surfaces can result in loss of drug product stability and efficacy during the production, storage, and administration of protein-based therapeutics. Surface-active agents (excipients) are typically added in protein formulations to prevent undesired interactions of proteins on surfaces and protein particle formation/aggregation in solution. The objective of this work is to understand the molecular-level competitive adsorption mechanism between the monoclonal antibody (mAb) and a commercially used excipient, polysorbate 80 (PS80), and a novel excipient, N-myristoyl phenylalanine-N-polyetheramine diamide (FM1000). The relative rate of adsorption of PS80 and FM1000 was studied by pendant bubble tensiometry. We find that FM1000 saturates the interface faster than PS80. Additionally, the surface-adsorbed amounts from X-ray reflectivity (XRR) measurements show that FM1000 blocks a larger percentage of interfacial area than PS80, indicating that a lower bulk FM1000 surface concentration is sufficient to prevent protein adsorption onto the air/water interface. XRR models reveal that with an increase in mAb concentration (0.5-2.5 mg/mL: IV based formulations), an increased amount of PS80 concentration (below critical micelle concentration, CMC) is required, whereas a fixed value of FM1000 concentration (above its relatively lower CMC) is sufficient to inhibit mAb adsorption, preventing mAb from co-existing with surfactants on the surface layer. With this observation, we show that the CMC of the surfactant is not the critical factor to indicate its ability to inhibit protein adsorption, especially for chemically different surfactants, PS80 and FM1000. Additionally, interface-induced aggregation studies indicate that at minimum surfactant concentration levels in protein formulations, fewer protein particles form in the presence of FM1000. Our results provide a mechanistic link between the adsorption of mAbs at the air/water interface and the aggregation induced by agitation in the presence of surfactants.

No ordinary proteins: Adsorption and molecular orientation of monoclonal antibodies.

The interaction of monoclonal antibodies (mAbs) with air/water interfaces plays a crucial role in their overall stability in solution. We aim to understand this behavior using pendant bubble measurements to track the dynamic tension reduction and x-ray reflectivity to obtain the electron density profiles (EDPs) at the surface. Native immunoglobulin G mAb is a rigid molecule with a flat, "Y" shape, and simulated EDPs are obtained by rotating a homology construct at the surface. Comparing simulations with experimental EDPs, we obtain surface orientation probability maps showing mAbs transition from flat-on Y-shape configurations to side-on or end-on configurations with increasing concentration. The modeling also shows the presence of ß sheets at the surface. Overall, the experiments and the homology modeling elucidate the orientational phase space during different stages of adsorption of mAbs at the air/water interface. These finding will help define new strategies for the manufacture and storage of antibody-based therapeutics.

Armoring the Interface with Surfactants to Prevent the Adsorption of Monoclonal Antibodies.

The pharmaceutical industry uses surface-active agents (excipients) in protein drug formulations to prevent the aggregation, denaturation, and unwanted immunological response of therapeutic drugs in solution as well as at the air/water interface. However, the mechanism of adsorption, desorption, and aggregation of proteins at the interface in the presence of excipients remains poorly understood. The objective of this work is to explore the molecular-scale competitive adsorption process between surfactant-based excipients and two monoclonal antibody (mAb) proteins, mAb-1 and mAb-2. We use pendant bubble tensiometry to measure the ensemble average adsorption dynamics of mAbs with and without the excipient. The surface tension measurements allow us to quantify the rate at which the molecules "race" to the interface in single-component and mixed systems. These results define the phase space, where coadsorption of both mAbs and excipients occurs onto the air/water interface. In parallel, we use X-ray reflectivity (XR) measurements to understand the molecular-scale dynamics of competitive adsorption, revealing the surface-adsorbed amounts of the antibody and excipient. XR has revealed that at a sufficiently high surface concentration of the excipient, mAb adsorption to the surface and subsurface domains was inhibited. In addition, despite the fact that both mAbs adsorb via a similar mechanistic pathway and with similar dynamics, a key finding is that the competition for the interface directly correlates with the surface activity of the two mAbs, resulting in a fivefold difference in the concentration of the excipient needed to displace the antibody.

'Reverse' Hofmeister effects on the sol-gel transition rates for an α-helical peptide-PEG bioconjugate.

We examine the dynamics of the sol-gel transition for end-functionalized linear- and 4-arm-peptides bioconjugated to poly-ethylene glycol (PEG) in aqueous environments with increasingly chaotropic (Cl- < Br- < I-) anions. A 23-amino acid peptide sequence is rationally designed to self-assemble upon folding into the ordered α-helical conformation due to the hydrophobic effect. We use Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) to quantify the ensemble average reversible secondary structure transitions as a function of electrolyte concentration and specific ion effects along the Hofmeister series. Subsequently, microrheology is used to quantify the kinetics of the gelation process, as it relates to folding and specific ion interactions. Our key findings were non-intuitive. We observe the faster evolution of the gel transitions in systems with more chaotropic anions. For our peptides in aqueous solution, "water-structuring" ions yield faster assembly behavior with a viscoelastic exponent, n, closer to unity representing self-assemblies that are Rouse-like. In contrast, ions that are "water-breaking" resulted in smaller viscoelastic exponents where self-assembly dynamics result in a viscoelastic exponent that suggests polymer entanglements.