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

Abstract

Antimicrobial preservatives (APs) are included in liquid multi-dose protein formulations to combat the growth of microbes and bacteria. These compounds have been shown to cause protein aggregation, which leads to serious immunogenic and toxic side-effects in patients. Our earlier work on a model protein cytochrome c (Cyt c) demonstrated that APs cause protein aggregation in a specific manner. The aim of this study is to validate the conclusions obtained from our model protein studies on a pharmaceutical protein. Interferon α-2a (IFNA2) is available as a therapeutic treatment for numerous immune-compromised disorders including leukemia and hepatitis c, and APs have been used in its multi-dose formulation. Similar to Cyt c, APs induced IFNA2 aggregation, demonstrated by the loss of soluble monomer and increase in solution turbidity. The extent of IFNA2 aggregation increased with the increase in AP concentration. IFNA2 aggregation also depended on the nature of AP, and followed the order m-cresol > phenol > benzyl alcohol > phenoxyethanol. This specific order exactly matched with that observed for the model protein Cyt c. These and previously published results on antibodies and other recombinant proteins suggest that the general mechanism by which APs induce protein aggregation may be independent of the protein.

Interactions Between Peptide and Preservatives: Effects on Peptide Self-Interactions and Antimicrobial Efficiency In Aqueous Multi-Dose Formulations

“…Most significant changes in antimicrobial efficacy as a function of peptide concentration were seen with m-cresol, followed by phenol, while least changes were seen when benzyl alcohol was used. This is the same ranking order with which these preservatives have been shown to induce the aggregation of several different proteins (7,9,10). However, our study is the first one to propose that the interaction may also lead to the reduction of their antimicrobial efficacy with increasing concentrations of peptide in the same ranking order.…”

“…Several examples can be found in literature where commonly used aromatic preservatives exhibit a negative effect on the physicochemical stability of proteins (e.g., 4-7). These preservative-protein interactions can lead to changes in protein conformation and increased aggregation tendency (5), decreased stability at higher temperatures (6,7) and/or chemical modifications (8). Several studies comparing the effects of phenol, m-cresol and benzyl alcohol on protein stability have suggested that benzyl alcohol generally appears to cause fewer instability issues than phenol or mcresol (6,7,9,10).…”

Effect of Aluminum Adjuvant and Preservatives on Structural Integrity and Physicochemical Stability Profiles of Three Recombinant Subunit Rotavirus Vaccine Antigens

“…2-PE is also known to destabilize a model protein cytochrome c and therapeutic proteins including an IgG1 mAb and interferon a-2a by causing structural perturbations and aggregation. [54][55][56] Ongoing and Future Work…”

Antimicrobial Excipient-Induced Reversible Association of Therapeutic Peptides in Parenteral Formulations

“…22 The findings of Maa and Hsu were extended by Gupta and Kaisheva, who reported on the incompatibility of a 150 kDa therapeutic antibody with phenol and meta-cresol, with optimal stability and preservative effectiveness were achieved at intermediate concentrations of benzyl alcohol. 23 Additional investigations into the mechanism of aggregation induced by antimicrobial preservatives for proteins, including Cytochrome C (12 kDa), 24e26 recombinant human interleukin-1 receptor agonist (17.3 kDa), 27,28 interferon alpha-2a (19.2 kDa), 24 and recombinant human granulocyte colony stimulating factor (18.8 kDa), 29 have provided evidence suggesting that these excipients induce partial unfolding which drives irreversible aggregation, rather than complete unfolding. While benzyl alcohol has been reported to decrease the temperature of thermal denaturation of lysozyme (14.3 kDa), it was also found to stabilize a partially unfolded state to inhibit heat-induced aggregation that occurred extensively without benzyl alcohol.…”