Description
Sessions: Poster Forum 2 - Monday - 12:00 pm
Date: November 13 - Monday
Time: 12:00 pm - 01:00 pm

Sub Topic | Secondary Topic: Biotherapeutics and Biotechnology - Biophysical and Mass Spec | Characterization


Authors: Yue Hu, University of Kansas (Main Author, Presenting Author); Jayant Arora, University of Kansas; Reza Esfandiary, MedImmune Inc.; Russ Middaugh, University of Kansas; Sangeeta Joshi, University of Kansas; David Weis, University of Kansas; David Volkin, University of Kansas

Presenting Author: Yue Hu

Purpose: Aim 1: To identify specific excipients (e.g. amino acids, hydrophobic salts, solvents of varying polarity, Hofmeister salts) that disrupt reversible self-association (RSA) of mAb-J (IgG1) in solution at high concentrations. Aim 2: To apply a series of biophysical techniques for determining different solution properties (e.g., viscosity, solubility, phase separation, aggregation propensity, etc.) of mAb-J in the presence of the selected excipients. Aim 3: To perform hydrogen-deuterium exchange mass spectrometry (HX-MS) experiments on mAb-J in the presence of different excipients, obtaining site-specific information to help identify the regions on the protein that are primarily affected by the selected excipients in solutions. Aim 4: To correlate HX-MS data with other information obtained from biophysical techniques, providing a more comprehensive understanding of the effects of various excipients on promotion or disruption of protein-protein interactions at high concentrations for mAb-J.

Methods: Multiple biophysical methods and hydrogen-deuterium exchange mass spectrometry were used for characterizing mAb solutions with and without excipients, including determining solution appearance, measureing viscosity, turbidity, solubility, and determing hydrodynamic diameter and oligomer fractions using dynamic light scattering (DLS) and component gradient multi-angle light scattering (CG-MALS).

Results: Several excipients (NaCl, ArgHCl, LysHCl, GluNa, and AspNa) were selected after excipient screening for researching on mAb-J molecule. At high mAb-J concentrations, strong protein-protein interactions (PPI) were found and solution was shown phase separation. At relatively low protein concentrations, although there was no phase separation, but it still showed strong opalescence (turbidity) for mAb-J solutions. However, the extent of RSA was reduced in the present of excipients. Specifically, the excipients could decrease solution viscosity and hydrodynamic diameter of mAb-J, and ArgHCl and LysHCl had the biggest effects. Furthermore, CG-MALS studies indicated that the excipients could increase percentage of monomer, preventing mAb-J forming oligomers. PEG-10,000 precipitation studies gave the solubility of mAb-J in the presence of selected excipients. The excipients increased solubility of mAb-J to different extents, and the highest apparent solubility was obtained by adding ArgHCl. Therefore, all selected excipients had the ability to stabilize mAb-J at high concentrations by decreasing hydrodynamic diameter, reducing viscosity, keeping mAb-J as a monomer, and increasing solubility. The HX-MS observations suggested that significant Fab-Fc interaction is the major driving force for mAb-J to undergo RSA at high protein concentrations. Interaction with charged amino acids weakened mAb-J PPI and thus disrupts RSA at high protein concentrations.

Conclusion: All selected excipients have ability to disrupt RSA for mAb-J. The negatively-charged amino acids (GluNa and AspNa) have more interactions with mAb-J due to net positive charge property of mAb-J. Several regions of mAb-J were demonstrated increased or decreased flexibility in the presence of GluNa and AspNa at high protein concentrations. The breaking of PPI and stabilization of mAb-J by GluNa and AspNa were mainly due to charge shielding effect. However, the poor RSA-disrupting efficiency of them may be caused by preferential hydration as well as the outcomes of increased flexibility in some regions. Thus, weak Fab-Fab interaction in CDR2L probably exists and new interactions among proteins may also play important part based on the excipient-induced high flexible regions. Therefore, a combination of different interactions and effects on mAb-J is existing for negatively-charged amino acids, giving the comprehensive biophysical and HX-MS results.

See attached abstract pdf for images.

Abstract Link: http://abstracts.aaps.org/Verify/AAPS2017/PosterSubmissions/M4020.pdf

Agenda