TY  - JOUR
PB  - AMER CHEMICAL SOC
A1  - Pandya, Akash
A1  - Zhang, Cheng
A1  - Barata, Teresa S
A1  - Brocchini, Steve
A1  - Howard, Mark J
A1  - Zloh, Mire
A1  - Dalby, Paul A
JF  - Molecular Pharmaceutics
KW  - Science & Technology
KW  -  Life Sciences & Biomedicine
KW  -  Medicine
KW  -  Research & Experimental
KW  -  Pharmacology & Pharmacy
KW  -  Research & Experimental Medicine
KW  -  Fab
KW  -  formulation
KW  -  stability
KW  -  aggregation
KW  -  melting temperature
KW  -  enthalpy change
KW  -  preferentialinteraction
KW  -  SELF-ASSOCIATION
KW  -  FAB FRAGMENT
KW  -  AGGREGATION
KW  -  PREDICTION
KW  -  STABILIZATION
KW  -  EXCIPIENTS
KW  -  DESIGN
KW  -  SERVER
KW  -  SERIES
N1  - This publication is licensed under

CC-BY 4.0 .
cc licence
by licence
Copyright © 2024 The Authors. Published by American Chemical Society
N2  - The design of stable formulations remains a major challenge for protein therapeutics, particularly the need to minimize aggregation. Experimental formulation screens are typically based on thermal transition midpoints (Tm), and forced degradation studies at elevated temperatures. Both approaches give limited predictions of long-term storage stability, particularly at low temperatures. Better understanding of the mechanisms of action for formulation of excipients and buffers could lead to improved strategies for formulation design. Here, we identified a complex impact of glycine concentration on the experimentally determined stability of an antibody Fab fragment and then used molecular dynamics simulations to reveal mechanisms that underpin these complex behaviors. Tm values increased monotonically with glycine concentration, but associated ?Svh measurements revealed more complex changes in the native ensemble dynamics, which reached a maximum at 30 mg/mL. The aggregation kinetics at 65 °C were similar at 0 and 20 mg/mL glycine, but then significantly slower at 50 mg/mL. These complex behaviors indicated changes in the dominant stabilizing mechanisms as the glycine concentration was increased. MD revealed a complex balance of glycine self-interaction, and differentially preferred interactions of glycine with the Fab as it displaced hydration-shell water, and surface-bound water and citrate buffer molecules. As a result, glycine binding to the Fab surface had different effects at different concentrations, and led from preferential interactions at low concentrations to preferential exclusion at higher concentrations. During preferential interaction, glycine displaced water from the Fab hydration shell, and a small number of water and citrate molecules from the Fab surface, which reduced the protein dynamics as measured by root-mean-square fluctuation (RMSF) on the short time scales of MD. By contrast, the native ensemble dynamics increased according to ?Svh, suggesting increased conformational changes on longer time scales. The aggregation kinetics did not change at low glycine concentrations, and so the opposing dynamics effects either canceled out or were not directly relevant to aggregation. During preferential exclusion at higher glycine concentrations, glycine could only bind to the Fab surface through the displacement of citrate buffer molecules already favorably bound on the Fab surface. Displacement of citrate increased the flexibility (RMSF) of the Fab, as glycine formed fewer bridging hydrogen bonds to the Fab surface. Overall, the slowing of aggregation kinetics coincided with reduced flexibility in the Fab ensemble at the very highest glycine concentrations, as determined by both RMSF and ?Svh, and occurred at a point where glycine binding displaced neither water nor citrate. These final interactions with the Fab surface were driven by mass action and were the least favorable, leading to a macromolecular crowding effect under the regime of preferential exclusion that stabilized the dynamics of Fab.
ID  - discovery10199720
UR  - http://dx.doi.org/10.1021/acs.molpharmaceut.4c00332
EP  - 13
SN  - 1543-8384
Y1  - 2024/01/01/
TI  - Molecular Dynamics Simulations Reveal How Competing Protein-Surface Interactions for Glycine, Citrate, and Water Modulate Stability in Antibody Fragment Formulations
AV  - public
ER  -