Role of Benzyl Alcohol in the Unfolding and Aggregation of Interferon α-2a

Abstract

Benzyl alcohol (BA) is the most widely used antimicrobial preservative in multi-dose protein formulations, and has been shown to cause protein aggregation. Our previous work on a model protein cytochrome c demonstrated that this phenomenon occurs via partial unfolding. Here, we examine the validity of these results by investigating the effect of BA on a pharmaceutically relevant protein, interferon α-2a (IFNA2). IFNA2 therapeutic formulations available on the pharmaceutical market contain BA as a preservative. Isothermal aggregation kinetics and temperature scanning demonstrated that BA induced IFNA2 aggregation in a concentration dependent manner. With increasing concentration of BA, the apparent aggregation temperature of IFNA2 linearly decreased. Denaturant melts measured using intrinsic protein fluorescence and that of the ANS dye indicated that IFNA2 stability decreased with increasing BA concentration populating a partially unfolded intermediate. Changes in NMR chemical shifts and hydrogen exchange rates identified the structural nature of this intermediate, which correlated with an aggregation “hot-spot” predicted by computational methods. These results indicate that BA induces IFNA2 aggregation by partial unfolding rather than global unfolding of the entire protein, and is consistent with our earlier conclusions from model protein studies.

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy

“…Compared to Fc, lesser stable Fab will have increased population of partially unfolded states with hydrophobic residues exposed to solvent, which can interact with polysorbates. Similar observations were made in our earlier studies where the least stable region of a protein has been shown to be affected most by the excipients used in protein formulations 37–39,41 . The lesser stability of Fab is due to its variable domain (Fig.…”

“…Possible explanation underlying this differential effect of polysorbates is in the intrinsic thermodynamic stabilities of Fab and Fc regions. Thermodynamically lesser stable domain is more vulnerable to structural perturbations, because of its increased vulnerability to partial unfolding, compared to a more stable domain 37–39,41,42 . When the stabilities of the Fab and Fc regions were measured using chemical denaturant melts with guanidinium chloride (GdmCl) as the denaturant (Fig.…”

Pentanol and Benzyl Alcohol Attack Bacterial Surface Structures Differently

“…In a Bacillus subtilis strain, the lethal concentration of BzA increased the membrane fluidity (43). For protein structures, BzA induced the aggregation of proteins, such as human interleukin (44) and interleukin-1 receptor antagonist (45), by partially unfolding these proteins (46,47). Unfolding actions were also reported for short-chain alcohols (48).…”

“…The reason for this remains obscure, but we speculated that the low specificities of the alcohols and variation in the transportation paths for the dyes might have contributed to these differences. BzA was reported to partially unfold a pharmaceutically relevant protein, interferon ␣-2a (46). This implied that BzA might unfold multiple proteins, suggesting that PeA and BzA might affect the functions of other surface proteins, including porins responsible for the transportation of fluorescent probes in addition to AcrABTolC.…”

Not changes in membrane fluidity but proteotoxic stress triggers heat shock protein expression in Chlamydomonas reinhardtii

“…Whether these aggregates consist of unfolded membrane proteins that form as a consequence of the increased membrane fluidity or by a denaturing effect of BA on soluble proteins is unclear. In any case, a denaturing effect of BA on purified, soluble recombinant proteins has been reported, including cytochrome c (Hutchings, Singh, Cabello‐Villegas, & Mallela, ; Singh, Cabello‐Villegas, Hutchings, & Mallela, ), interferon‐γ (Tobler, Holmes, Cromwell, & Fernandez, ), interferon α‐2a (Bis, Singh, Cabello‐Villegas, & Mallela, ), interleukin‐1 receptor antagonist (Roy, Jung, Kerwin, Randolph, & Carpenter, ), and human granulocyte colony‐stimulating factor (Thirumangalathu, Krishnan, Brems, Randolph, & Carpenter, ), albeit at BA concentration exceeding those used in our in vivo studies by at least 2.5‐fold. Whatever the origin of the proteins in BA‐induced aggregates is, they are indicative for a BA‐induced disturbance of cellular protein homeostasis, which in turn may trigger HSP expression by the classical cytosolic UPR (Figure ).…”