Achieving site-specific PEGylation

PEGylation has been around for 30 years and it is surprising that it is still widely used given the significant advances that have been made in biopharmaceutical manufacture since then. So why is this the case?

PEGylation is the only established half‑life modification technology that has been clinically proven in at least eight marketed products — total sales of which have exceeded $5 billion. Optimised half‑life for a peptide or protein-based medicine is required to ensure the medicine is safe and remains in the body long enough to be as efficacious as possible. According to estimates, more than 5000 peptides and proteins with therapeutic potential require half-life optimisation, and at least 15 such products are currently going through clinical trials in PEGylated form. However, there are known challenges with existing PEGylated products, ranging from the cost of product development through to transient side effects from sustained exposure to polymer. Several of these issues can be addressed either directly or indirectly with a more precise and targeted approach to PEGylation.

Current PEGylation methods are inefficient because they yield heterogeneous products of varying potency. Maximising the commercial potential of the next generation of biopharmaceuticals will depend on well‑defined systems for PEG conjugation to deliver homogeneous products as cost effectively as possible. It is now possible to selectively design and control protein half-life using a technology that applies precision chemistry to site‑specific PEGylation. The technology, TheraPEG, originated from the concept of attaching a PEG molecule across a natural disulphide bond within a protein. Most classes of therapeutic protein contain disulphide bonds that can be site specifically modified in this way, PEGylating the protein while retaining biological activity. Once reformed across a PEG molecule, the disulphide bond only increases in size by about 1 nm and the resulting TheraPEG conjugate has greater stability, owing to protection of sulphur atoms from disulphide cleavage or exchange reactions. In addition, the protein shows greater resistance to degradation and aggregation, which are often significant problems in the manufacture of biopharmaceuticals.

Site-specific PEGylation chemistry can also be applied to polyhistidine tags used for affinity purification of recombinant proteins. The so-called HiPEG approach enables PEG conjugation at two consecutive histidine residues without losing the ability to purify the protein. This can be thought of as an adaptor socket, allowing PEG attachment at will. For instance, HiPEG PEGylation can increase protein molecular weight (MW) site-specifically: a MW increase of 60000 can be achieved using a single 60000 MW linear PEG, three 20000 MW linear PEGs or two 15000 MW branched PEGs. Using these alternative constructs, it is possible to maximise options in optimisation of a biologic rather than be limited to a prescribed PEGylation strategy.

A diverse range of proteins, including cytokines, antibody fragments, enzymes, peptides and blood proteins, is amenable to site‑specific PEGylation by either TheraPEG or HiPEG. Their efficiency and predictability in terms of retained activity in PEGylated products make both technologies attractive and cost effective in extending protein half‑life for therapeutic application.