Effect of Antimicrobial Preservatives on Partial Protein Unfolding and Aggregation

Abstract

One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation “hot-spot” reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol. All these enhanced the partial protein unfolding that includes a local region which was predicted to be the aggregation “hot-spot”. The extent of destabilization correlated with the extent of aggregation. Further, we show that stabilizing the “hot-spot” reduces aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas chlorobutanol causes the least protein aggregation. The same protein region acts as the “hot-spot” for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others.

Antimicrobial preservatives induce aggregation of interferon alpha-2a: The order in which preservatives induce protein aggregation is independent of the protein

“…No IFNA2 aggregation was observed in the absence of APs. This order in which APs induce IFNA2 aggregation is identical to what we observed earlier in the case of the model protein Cyt c: CR > PH > BA > PE (Hutchings et al 2013). …”

“…However, it is unknown whether APs cause IFNA2 aggregation. Here, we demonstrate that IFNA2 aggregation is enhanced in the presence of APs and that the extent and order of these effects match exactly to what we observed earlier in the case of the model protein Cyt c (Hutchings et al 2013). …”

“…Table 1 lists the concentrations of these APs used in formulations. Figure 2A shows the antimicrobial efficacy of these APs, measured using a simplified test (Sutton and Porter 2002, Hutchings et al 2013). We tested their effect on the growth of Escherichia coli bacteria.…”

“…To confirm the antimicrobial activity of APs, a simplified preservative efficacy test was performed (Sutton and Porter 2002, Hutchings et al 2013). A primary culture of Escherichia coli BL21(DE3) cells was incubated overnight at 37°C in a shaker.…”

“…Previous work from our laboratory has demonstrated that a specific order exists in which individual APs induce the aggregation of a model protein cytochrome c (Cyt c) (Hutchings et al 2013). The aim of the present study is to examine the effects of various APs on a pharmaceutically relevant protein and validate the results obtained with the model protein.…”