PEG-hGH: A Case Study
Ambrx selected human growth hormone (hGH) to demonstrate the power of its ReCODE™ protein engineering platform. Growth hormone represents a significant, expanding market: worldwide sales were nearly $2.0 billion in 2004 and are estimated to be growing at 10-15% annually. Ambrx’s hGH product with optimized pharmacology and preserved potency represents a significant commercial opportunity.
The grounding hypothesis for using ReCODE™ to enhance hGH performance centered on two objectives:
- Site-directed placement of a chemically reactive amino acid to provide a single point of attachment for a polyethylene glycol (PEG) polymer through orthogonal chemistry that does not react with any of the 20 natural amino acids
- Optimize placement of the PEGylation site on the surface of the hGH protein to improve longevity of action while maintaining potency
The process for engineering hGH began with incorporation of a novel amino acid, para-acetyl-phenylalanine at sites specified by the amber codon. Para-acetyl-phenylalanine (pAcF) is a derivative of phenylalanine that has been modified to include a ketone functional group. The ketone is chemically inert but it reacts efficiently and selectively with a specially-derivatized PEG polymer. As a result, Ambrx can introduce a single, selective attachment point at any site on the protein's surface, enabling protein engineering in a manner not previously possible.
Through supplementation of pAcF into the growth media of E. coli reconstituted with plasmids that direct the synthesis of tRNAo and tRNAo synthetase, pAcF is incorporated into recombinant hGH molecules at different single sites (depicted in red in the composite below).
A key feature of pAcF is its selectivity of chemical reaction. In the figure below, reaction of a specially-derivatized 5 kDa PEG polymer shifts the molecular size of hGH substituted with pAcF (incorporated at position 134) but the PEG does not modify unsubstituted hGH. This selectivity of reaction contrasts with succinimidyl-activated PEG polymers that react with multiple surface lysines, creating a heterogeneous mixture of products that differ in the number of PEG's added and in the sites at which they are attached. Importantly, many lysine residues are present at key sites; if these residues are disrupted through non-specific conjugation, the activity, potency or safety of the molecule may be compromised.
Among molecules that differ only in the site of PEGylation, several display exceptional performance in the pharmacological responses seen in animals, with differences in half life, bioavailability and pharmacodynamic performance. Ambrx’s first clinical candidate is among this group of enhanced performance PEG-hGH variants.
