(683c) Supramolecular Pegylation of Biopharmaceuticals

Introduction: The practice of medicine has been transformed by the use of protein drugs, but these also introduce an array of attendant complications arising from poor chemical and/or structural stability, thus requiring extensive effort in the development of stabilizing excipients and formulation methods. Direct chemical modification with poly(ethylene glycol) (PEG) has demonstrated improvements in protein stability and circulation half-life, a practice that has been used clinically for a number of protein therapies. Herein, we demonstrate for the first time a platform approach to PEGylation of biopharmaceuticals using only supramolecular interactions by leveraging high-affinity host-guest interactions in order to endow PEG-like functionality on proteins non-covalently.

Materials and Methods: A supramolecular macrocycle with high binding affinity was synthesized and functionalized with PEG chains of controlled molecular weight via â??clickâ? chemistry. Leveraging host-guest binding of this macrocycle with native residues on proteins, this PEGylated compound was added in formulation with insulin, glucagon, and a therapeutic antibody. Functional performance of all three biopharmaceuticals was assayed both in vitro and in vivo.

Results and Discussion: The supramolecular PEGylation (SM-PEG) of insulin extends in vitro stability and preserves potency when aged under agitating conditions, extending protein life from approximately 14 hours alone to over 100 days when in formulation with SM-PEG. Moreover, in a diabetic mouse model the duration of insulin activity can be tuned by selection of PEG molecular weight. Extending this approach to glucagon and a therapeutic anti-CD20 antibody, the SM-PEG lends significant stability and preservation of activity to these proteins as well. This approach to supramolecular modification of therapeutic proteins provides an excipient-only strategy to modulate protein activity and stability without the need for covalent modification of the protein drug molecule, and has broad possible use as a platform for biopharmaceutical formulation.

Conclusions: In biopharmaceutical practice, assuring stability and modulating pharmacokinetics are two primary areas of interest, and covalent PEGylation has been among the more successful strategies evaluated in this regard. Using high binding affinities afforded by specific supramolecular host-guest interactions, proteins can be modified non-covalently with PEG chains of various molecular weights. This supramolecular PEGylation strategy dramatically extended the stability of three therapeutic proteins, and also prolonged the activity of insulin in a modular fashion dependent on molecular weight. This approach may be broadly applicable as a platform of â??smartâ? excipients for formulation and shelf-life extension of biopharmaceuticals.