Aggregation Kinetics of Polysorbate 80/<i>m</i>-Cresol Solutions: A Small-Angle Neutron Scattering Study

Gilbert, Peter H.; Zhang, Zhenhuan; Qian, Ken K.; Allen, David P.; Ford, Rachel R.; Wagner, Norman J.

doi:10.1021/acs.molpharmaceut.1c00803

Cited by 2 publications

(1 citation statement)

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“…Due to the diversity of protein and peptide properties, including sequence, structure, and chemical modifications, multiple types of interactions likely contribute to the mechanism of AP-induced aggregation. , In addition to the impact on the peptide stability, m -cresol has been reported to be incompatible with polysorbate 80 (PS80), a routinely utilized surfactant in sterile formulations . Most recently, very nice structural characterization has been carried out to elaborate the kinetic mechanism of the interplay between m -cresol and PS80 using small-angle neutron scattering. , Nevertheless, it remains unclear if there is an underlying mechanism of aromatic AP-induced peptide aggregation, e.g., a global conformational change or site-specific interactions.…”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanism of Antimicrobial Excipient-Induced Aggregation in Parenteral Formulations of Peptide Therapeutics

Falk

et al. 2022

Mol. Pharmaceutics

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Antimicrobial preservatives are used as functional excipients in multidose formulations of biological therapeutics to destroy or inhibit the growth of microbial contaminants, which may be introduced by repeatedly administering doses. Antimicrobial agents can also induce the biophysical instability of proteins and peptides, which presents a challenge in optimizing the drug product formulation. Elucidating the structural basis for aggregation aids in understanding the underlying mechanism and can offer valuable knowledge and rationale for designing drug substances and drug products; however, this remains largely unexplored due to the lack of high-resolution characterization. In this work, we utilize solution nuclear magnetic resonance (NMR) as an advanced biophysical tool to study an acylated 31-residue peptide, acyl-peptide A, and its interaction with commonly used antimicrobial agents, benzyl alcohol and m-cresol. Our results suggest that acyl-peptide A forms soluble octamers in the aqueous solution, which tumble slowly due to an increased molecular weight as measured by diffusion ordered spectroscopy and 1H relaxation measurement. The addition of benzyl alcohol does not induce aggregation of acyl-peptide A and has no chemical shift perturbation in 1H–1H NOESY spectra, suggesting no detectable interaction with the peptide. In contrast, the addition of 1% (w/v) m-cresol results in insoluble aggregates composed of 25% (w/w) peptides after a 24-hour incubation at room temperature as quantified by 1H NMR. Interestingly, 1H–13C heteronuclear single-quantum coherence and 1H–1H total correlation experiment spectroscopy have identified m-cresol and peptide interactions at specific residues, including Met, Lys, Glu, and Gln, suggesting that there may be a combination of hydrophobic, hydrogen bonding, and electrostatic interactions with m-cresol driving this phenomenon. These site-specific interactions have promoted the formation of higher-order oligomerization such as dimers and trimers of octamers, eventually resulting in insoluble aggregates. Our study has elucidated a structural basis of m-cresol-induced self-association that can inform the optimized design of drug substances and products. Moreover, it has demonstrated solution NMR as a high-resolution tool to investigate the structure and dynamics of biological drug products and provide an understanding of excipient-induced peptide and protein aggregation.

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