Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 1

Abstract

Many challenges limit the formulation of antibodies as high-concentration liquid dosage forms including elevated solution viscosity, decreased physical stability, and in some cases, liquid-liquid phase separation. In this work, an IgG1 monoclonal antibody (mAb-J), which undergoes concentration-dependent reversible self-association (RSA), is characterized in the presence of 4 amino acids (Arg, Lys, Asp, Glu) and NaCl using biophysical techniques and hydrogen exchange-mass spectrometry. The 5 additives disrupt RSA, prevent phase separation, and reduce solution viscosity to varying extents. These excipients also cause decreased turbidity, reduced average hydrodynamic diameter, and increased relative solubility of mAb-J in solution. The RSA disrupting efficacy of the positively charged amino acids is greater than either negatively charged amino acids or NaCl. As measured by hydrogen exchange-mass spectrometry, anionic excipients induced more alterations of mAb-J backbone dynamics at pH 6.0, and weak Fab-Fab interactions likely remained with the addition of either cationic or anionic excipients at high protein concentrations. Along with a companion paper examining a different mAb with a different molecular mechanism of RSA, these results are discussed in the context of various excipient strategies to disrupt protein-protein interactions to formulate mAbs at high protein concentrations with good stability profiles and favorable pharmaceutical properties for subcutaneous administration.

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 2

“…This can be pursued by either an excipient screening approach to semiempirically identify stabilizers 33 or by a more rational additive selection process based on a mechanistic understanding of RSA interactions for a particular mAb (as outlined in the companion paper in this issue with mAb-J). 4 In this work, we continue to examine the latter, more targeted approach to excipient selection by using HX-MS to identify regions of mAb-C (and associated molecular mechanisms), resulting in a more rational selection of additives to disrupt specific RSA interactions within a mAb.…”

“…Other weak intermolecular forces, such as dipole-dipole coupling, hydrogen bonding, or even electrostatic interactions, could also contribute to mAb-C RSA to some extent. 21 Similar to the HX-MS analysis described in the companion paper with mAb-J, 4 potential chemical exchange rate differences in mAb-C formulated with the various additives prevent direct comparisons between HX kinetics in the different formulations. 36 Thus, we focused on HX difference between high and low protein concentrations of mAb-C in the presence of each additive separately.…”

“…In this paper (Part 2) with mAb-C, along with a companion paper (Part 1) in this issue with mAb-J, 4 we examine the role of additives and excipients in disrupting and enhancing specific PPIs that lead to the RSA of 2 different IgG1 mAbs at high protein concentrations. In this work, the solution properties, molecular features, and backbone flexibility (at specific sites known to facilitate RSA) were evaluated for a human monoclonal IgG1 antibody (mAb-C), previously demonstrated to undergo RSA via Fab-Fab interactions (at specific peptide segments within the CDRs of mAb-C rich in hydrophobic amino acids).…”

“…1 The latter route offers the potential of reducing costs, saving time, and increasing patient compliance by having patients self-administer. [1][2][3] As outlined in detail in the Part 1 companion paper, 4 successful formulation of mAbs as highconcentration protein dosage forms is a challenge in terms of both protein instability (e.g., aggregation and reversible selfassociation [RSA]) and pharmaceutical solution properties (e.g., high viscosity).…”

Strategies for high‐concentration drug substance manufacturing to facilitate subcutaneous administration: A review

“…As discussed above, excipients can be added to the formulation to provide stabilizing effects (Chang et al, 1996; Kerwin, 2008; Mahler et al, 2005; Randolph & Jones, 2002). These include detergents, protein‐stabilizing agents, and other additives that can reduce protein–protein interactions to enable high protein concentration formulations with good stability profiles (Hu, Arora et al, 2019; Hu, Toth et al, 2019). Polysorbates are often added to stabilize proteins against interface‐induced aggregation and to minimize surface adsorption of proteins (Bam et al, 1998; Carpenter, Kendrick, Chang, Manning, & Randolph, 1999).…”