PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins

Abstract

A major limitation of biopharmaceutical proteins is their fast clearance from circulation via kidney filtration, which strongly hampers efficacy both in animal studies and in human therapy. We have developed conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala and Ser (PAS). PAS sequences are hydrophilic, uncharged biological polymers with biophysical properties very similar to poly-ethylene glycol (PEG), whose chemical conjugation to drugs is an established method for plasma half-life extension. In contrast, PAS polypeptides offer fusion to a therapeutic protein on the genetic level, permitting Escherichia coli production of fully active proteins and obviating in vitro coupling or modification steps. Furthermore, they are biodegradable, thus avoiding organ accumulation, while showing stability in serum and lacking toxicity or immunogenicity in mice. We demonstrate that PASylation bestows typical biologics, such as interferon, growth hormone or Fab fragments, with considerably prolonged circulation and boosts bioactivity in vivo.

The polypeptide biophysics of proline/alanine‐rich sequences (PAS): Recombinant biopolymers with PEG‐like properties

“…Interestingly, both the isolated PAS polypeptides and their fusion proteins with IL‐1Ra and TrxA migrated at positions corresponding to much higher molecular weight than normally expected. While such a retarded electrophoretic mobility in comparison with conventional marker proteins was seen for other PASylated proteins before,9 it was most pronounced for the isolated polymers, which did not even leave the stacking gel (Figure 1B,C). This behavior is most likely explained by poor binding of SDS, whose negatively charged head groups provide the driving force in the electric field, probably due to the lack of any hydrophobic amino acid side chains.…”

“…Originally, PASylation technology was devised on the basis of a repetitive sequence comprising Pro, Ala and Ser residues 9. Motivated by the pronounced solubility of these natively disordered polypeptides in water—despite complete lack of charged side chains—we became interested in the role of the only polar side chain present, the Ser hydroxyl group.…”

“…Interestingly, the observation that PAS polypeptides, which do not bind the common protein dye Coomassie Brilliant Blue,9 can be stained using such a method typical for PEG21 illustrates certain chemical similarity of both polymers with regard to the complexation of Ba 2+ ions. However, uncharged PEG polymers of comparable molecular size moved much faster through the gel than the PAS polypeptides.…”

“…As an alternative, we have developed a novel class of biodegradable polymers based on the natural L ‐amino acids Pro, Ala, and Ser 9. These Pro/Ala‐rich sequences (PAS) show high solubility in aqueous buffers and exhibit natively disordered conformation, in particular absence of detectable secondary or tertiary structure, thus inherently mimicking the polymer behavior of PEG.…”

“…Indeed, several PASylated biopharmaceuticals have been generated up to now, for example growth hormone,9 leptin,10 antibody fragments,9, 11 interferons,9, 12, 13 erythropoietin,14 a complement inhibitor,15 and interleukin‐1 receptor antagonist (IL‐1Ra). All these PASylated proteins have demonstrated beneficial properties:8 (i) high solubility and stability, (ii) full biofunctionality both in vitro and in animal models, (iii) high bioavailability when injected subcutaneously, intraperitoneally or intramuscularly and (iv) drastically prolonged plasma half‐life—by one to two orders of magnitude—depending on the length of the PAS chain (typically, 600 residues).…”