Substituted Polyesters by Thiol–Ene Modification: Rapid Diversification for Therapeutic Protein Stabilization

Abstract

Many proteins, especially those used as therapeutics, are unstable to storage and shipping temperatures, leading to increased costs in research and industry. Therefore, the design and synthesis of novel stabilizers is an important area of investigation. Herein we report new degradable polymers that stabilize proteins to environmental stressors such as refrigeration and elevated temperature. Specifically, polycaprolactones with different pendant groups were synthesized and surveyed for their ability to stabilize an important therapeutic protein to storage and shipping conditions. Ring-opening polymerization (ROP) of an allyl-substituted caprolactone monomer was carried out using the organocatalyst 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) to yield a well-defined, alkene-substituted degradable polymer, which was used as a common backbone to control for degree of polymerization. Relevant side chains such as trehalose, lactose, glucose, carboxybetaine and oligo(polyethylene glycol) were installed via post-polymerization thiol-ene reactions. These degradable polymers were then employed as excipients for the stabilization of the therapeutic protein granulocyte colony-stimulating factor (G-CSF) against storage at 4 °C and shipping temperatures of 60 °C. The best stabilization was observed using the trehalose- and zwitterion-substituted polyesters. Both the trehalose- and carboxybetaine-substituted pCL were further investigated with regard to molecular weight dependence, and it was found that the molecular weight was minimally important for stabilization to refrigeration, but critical for G-CSF stabilization at elevated temperatures. Both high performing zwitterionic and trehalose polyesters were also degraded and the polymers and degradation products shown to be non-cytotoxic. This work provides potential biocompatible polymers for stabilization of the important therapeutic G-CSF, as well as a general platform for the future discovery of new polymeric protein stabilizers.

Synthesis and Biological Evaluation of a Degradable Trehalose Glycopolymer Prepared by RAFT Polymerization

“…[13] And our group has previously reported on glycopolymers with trehalose side chains for enhancing stability of a wide range of proteins against lyophilization, mechanical, and heat stress. [14][15][16][17] Yet, most of the reported stabilizers are not degradable.…”

“…[18,19] To address this need, we recently described stabilizing polymers with polyester backbones. [17] Specifically, polycaprolactone was prepared with zwitterionic and trehalose side chains. These polymers were highly effective protein stabilizers and also degraded through ester hydrolysis.…”

“…Additionally, synthetic carboxylated polyamidosaccharides have been shown to retain lysozyme activity after multiple lyophilization cycles . And our group has previously reported on glycopolymers with trehalose side chains for enhancing stability of a wide range of proteins against lyophilization, mechanical, and heat stress . Yet, most of the reported stabilizers are not degradable.…”

“…For instance, hydroxyethyl starch has been extensively used for the preparation of protein conjugates with increased half‐lives, but may accumulate in some organs increasing risk of death for critically ill patients . To address this need, we recently described stabilizing polymers with polyester backbones . Specifically, polycaprolactone was prepared with zwitterionic and trehalose side chains.…”

Polypeptides with High Zwitterion Density for Safe and Effective Therapeutics

“…[28] Other types of polymers,i ncluding biodegradable poly(amino acid)s and nonbiodegradable poly(glycerol), poly(2-oxazoline), poly(acrylamide), poly(vinylpyrrolidone),a nd poly(N-(2-hydroxypropyl)methacrylamide), have also been proposed as PEG alternatives,however their immunogenicity when conjugated to proteins has not been fully studied. [30] The PCL zwitterion was shown to preserve therapeutic protein activities.However,the hydrophobicity of the long caprolactone repeating unit and low zwitterion density might compromise its hydration capacity and its stealth properties. It would be interesting to develop ab iodegradable zwitterionic polymer.M aynard et al reported the development of ab iodegradable zwitterionic polyester by attaching carboxybetaine moieties to an allyl-substituted polycaprolactone (PCL) through thiol-ene chemistry.…”