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

Abstract

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.

In-situ biophysical characterization of high-concentration protein formulations using w NMR

“… 47 , 49 , 52 , 78 , 79 NMR is increasingly utilized as a sophisticated biophysical tool for exploring drug delivery and the stability of peptide and protein drugs across various formulations, including sterile solutions, suspensions, lyophilized, and frozen solutions. 39 , 80–85 In our study, the noninvasive nature of w NMR makes it a promising method for obtaining crucial information about protein stability in its original container, facilitating drug development and quality assessment processes.…”

Industrial Horizons in Pharmaceutical Science

“…In recent years, these tools have significantly advanced our understanding by pushing the boundaries in various areas. Examples include molecular or atomic level elucidation of key structures and interactions in amorphous solid dispersions within solid dosages, − critical (de)stabilizing interactions in parenteral peptide or protein formulations, − microenvironmental properties in lyophilized , and frozen biologics, and the encapsulation mechanisms of mRNA within LNPs for vaccine delivery. − These new applications exemplify the evolving mission of pharmaceutical sciences: to pose new questions to scientific and technical communities, to enhance collaboration, and to bring back innovative ideas and solutions. This dynamic approach helps drive forward the collective mission of delivering effective drugs to patients.…”

Study on the evaluation method of cigarette astringency in the simulated oral environment

“…Cigarette smoke is known to contain more than 50 phenolic compounds, with phenol, catechol, resorcinol, hydroquinone, o-cresol, m-cresol, and p-cresol being among the most prevalent (Wu et al, 2018). These phenolic compounds contribute to the aggregation and precipitation of proteins in saliva, leading to changes in the lubricating properties of saliva (Bis & Mallela, 2014;Heljo et al, 2015;Li et al, 2022;Singh et al, 2011). To quantify the primary phenolic compounds in MS, standard solutions of phenol, catechol, resorcinol, hydroquinone, o-cresol, m-cresol, and p-cresol were prepared at concentrations of 1000 mg/L and stored at a temperature of 4 C. The concentrations of these phenols in MS were determined using highperformance liquid chromatography (HPLC, Agilent Technologies, Inc., USA).…”

“…(Maa & Hsu, 1996; Singh et al, 2011; Heljo et al, 2015). However, phenolic compounds are not the sole contributor to oral astringency; a significant correlation exists between the pH of the MS and oral astringency (Li et al, 2017; Shi et al, 2019; Li, Qiu, et al, 2019; Li et al, 2022). This is due to the fact that it is close to or far from the isoelectric point of the protein solution by adjusting its pH.…”