High-Yield Production of PASylated Human Growth Hormone Using Secretory E. coli Technology

level: intermediate Since the 1985 approval of the first recombinant human growth hormone (hGH, such as Protropin/somatrem human growth hormone from Genentech, now Roche), the number of clinical indications for therapy with hGH has steadily increased (1). That led to a highly successful drug with more than US$3 billion sales in 2011 (2). Even so, hGH shares a common problem with most other firstgeneration protein therapeutics: a very short plasma half-life of just about two hours in humans. Because such biologics are relatively small molecules, they are rapidly eliminated by renal filtration (3). So they usually have to be injected daily to accomplish the desired therapeutic effect. The same holds true for antibody fragments (4) and for the growing class of alternative protein scaffolds (5). Thus, a technology is needed to prolong their plasma halflives to meet clinical demands. Indeed, resulting “biobetters” could promise lower dosing with longer time intervals, leading to in improved tolerability and enhanced patient compliance. PEGylation technology is an established strategy for extending plasma half-life of small-sized biopharmaceuticals. Through chemical conjugation to polyethylene glycol (PEG), the hydrodynamic radius of a protein becomes larger than the pore size of the glomerular filtration barrier in kidneys, so its circulation time in blood is extended to a therapeutically useful range (6). However, chemically activated GMP-grade PEG can be an expensive raw material, and PEGylated proteins have to be recovered from the reaction mixture by additional purification steps that ultimately lower yields and raise manufacturing costs. Furthermore, the activity of a PEGylated therapeutic protein can be impaired if amino-acid side chains in the vicinity of its active site become modified through PEG attachment (7). In response to those drawbacks of PEGylation, XL-protein GmbH has developed what we consider to be a competitive technology — called PASylation — that extends the plasma half-life of biopharmaceuticals by applying a natively disordered amino-acid chain as a biological alternative to PEG. A therapeutic protein is genetically fused with a polypeptide sequence comprising several hundred residues of the small amino acids proline, alanine, and/or serine (“PAS”). Like PEG, PAS sequences adopt a random coil structure in aqueous solution, so they generate a large hydrodynamic volume that retards renal filtration of a “PASylated” biologically active protein (8). Through such means, the typically short plasma half-life of small therapeutic proteins can be prolonged by several orders of magnitude (Figure 1), which allows their dosing frequency to be Figure 1: PASylation technology genetically fuses a therapeutic protein with a sequence composed of the amino acids Pro, Ala, and/or Ser (PAS, left) to produce a fusion protein in a single expression step (middle). The PAS sequence adopts a random coil formation, increasing the hydrodynamic volume of the resulting molecule larger than the kidney pore size. That strongly prolongs the protein’s plasma half-life (right).