Higher concentration of trehalose dihydrate stabilizes recombinant IgG1 under forced stress conditions

Abstract

Stability of complex biotherapeutics like monoclonal antibodies is paramount for their safe and efficacious use. Excipients are inactive ingredients that are added to the purified product so as to offer it a stable environment. Trehalose dihydrate is a non-reducing sugar that is commonly used as a stabilizing agent in biotherapeutic formulations under liquid and frozen states. The stabilizing effect of trehalose against aggregation in protein formulations is well known. The present study aims to offer insights into the stability effects of higher trehalose concentration (230 mM) on liquid trastuzumab under different forced stress conditions including thermal, light with and without hydrogen peroxide (H₂O₂), humidity and extraction stresses. Under thermal stress, while high molecular weight (HMW) accounted for 38.80% in the trastuzumab sample without trehalose, it was 4.89% at high trehalose concentration. Similarly, under light stress with H₂O₂, the trastuzumab sample without trehalose had >80% more HMW than at high trehalose concentration. Two other IgG1 mAbs (rituximab and bevacizumab) were also evaluated for stability at higher trehalose concentrations (230 mM). Similar to trastuzumab, stabilization was observed under thermal stress for rituximab and bevacizumab at higher trehalose concentration compared to samples without trehalose (21.90% and 29.90% HMW, respectively). Likewise, accelerated (under humidity stress) and extraction stress induced secondary and tertiary structure disruptions were reduced at higher trehalose concentration. An in-silico study between binding interactions of trehalose and trastuzumab Fab region at different concentrations depicted an increase in hydrogen bonding with trastuzumab Fab when the trehalose concentration is increased, thereby reducing aggregation. Overall, mAb stability under forced stress conditions improved significantly at higher trehalose concentrations. While higher trehalose concentration (>200 mM) is used in mAb formulations and is known to minimise aggregation under thermal stress, however, the current study aims to also explore the stability imparted under light (with H₂O₂), humidity and extraction stresses for three different mAbs and attempts to explain the underlying mechanisms via in-silico studies.

Keywords: Excipients; Forced stress; Higher order structure; Monoclonal antibodies; Trehalose dihydrate.