ALTERNATIVES TO PEG
Possible alternatives to PEGylation include, for example, HESylation, XTENylation, HSAylation, acylation, PASylation, and
glutamylation. The conjugation of peptides to hydroxyethyl starch (HES), XTEN (a polypeptide), human serum albumin (HSA),
lipids (acylation), poly-Pro-Ala-Ser (PAS), or polyglutamic acid (glutamylation) avoids the toxic issue of PEG because they
all can be biologically degraded and excreted. Like PEGs, most of these reagents can be customized to exhibit different release
HES has been used as a plasma expander for many years and is considered to have an exceptional safety profile. Acylation usually
involves the conjugation of a peptide to a naturally occurring fatty acid (e.g., palmitic acid) and does not seem to present
any toxicological issues. Liraglutide (Victoza), a palmitated peptide, was approved in January 2010 for the treatment of type-2
Despite the advantages of some of these other conjugates, they have a number of challenges. The conjugates based on polypeptides
(XTEN, HSA, PAS, polyGlu) are potentially immunogenic, but there is substantial evidence that such immugenicity is not realized
in vivo. A number of the alternative conjugate molecules face similar economic challenges to PEG. Companies involved in the development
of these alternative conjugates need to offer them at substantially lower costs than PEG to make them viable alternatives.
The availability of identical, activated polymers from multiple sources would be beneficial to mitigate vendor risk and improve
economic viability; however, as long as the respective polymers and linkers are patented, most innovators will remain exposed
to the well-known risks of single sourcing of raw materials. Contingency plans for secondary supply should not only benefit
innovators, but vendors as well by providing a secure supply of activated polymer.
Both HESylation and PEGylation lead to polydisperse bioconjugates that present unique analytical challenges. Polydisperse
conjugates have broad peaks and, in the case of PEG and HES, they tend to have low UV adsorption making it difficult to detect
peptide impurities generated in the manufacture of the peptide or during conjugation (5). From an analytical stand point,
the ability to link a conjugate to a peptide with a reversible linker would be attractive, although a reversible linker may
compromise the pharmacokinetics of the bioconjugate.
Currently, PEGylation for peptide and proteins involves two main families: lysine-active PEGs and sulfhydryl-selective PEG
reagents. Examples of lysine-active PEGS include NHS esters. The rate of coupling of a lysine-active PEG increases as the
pH is raised; however, peptides are not stable at high pH and therefore a balance between peptide stability and rate of coupling
has to be met. Coupling involves the formation of a peptide bond between the side chain NH2 functional group of lysine and the carbonyl portion of the succinimide. All of the lysine-active derivatives, except aldehydes
and ketones, can possibly react with other amino acids, such as imidazole groups of histidine and hydroxyl groups of tyrosine,
and therefore in the case of site specific PEGylation, a differential protection strategy may be necessary. Aldehyde- and
ketone-based lysine-active PEGs are selective for primary amines.
Examples of sulfhydryl-selective PEG reagents involve maleimides, vinyl sulfones, and thioethers. Sulfhydryl-selective PEG
reagents attach to the thiol group of a cysteine. Because of the lower abundance of cysteine amino acids (i.e., the second
least common amino acid) in peptides, more selective PEGylation can be achieved.
Both types of bonds are fairly strong and difficult to reverse. The ultimate reversible linkage would involve conjugation
that can be removed in vitro but is stable enough in vivo. Reversible, disulfide linkages are also selective to thiols; however, they are susceptible to reduction by biological reducing
agents such as glutathione. Although disulfide linkages could be reduced chemically to enable analysis of the peptide after
conjugation, the possibility of the bioconjugate being reduced in vivo presents a major challenge. The use of a conjugate-maleic-anhydride for conjugation to peptide would allow for the later
removal of the conjugate by treatment with mild acid at room temperature (6). Bentley et al. and Greenwald et al. have shown
that conjugation with PEG NHS esters can be reversed by hydrolysis under mild acid conditions (6). Zalipsky et al. showed
the release of the PEG from a PEG bioconjugate using mild reducing conditions (6).
In the case of PEGylated bioconjugates, use of analytical techniques such as enzymatic digestion and Edman degradation may
enable selective cleavage of the PEG-peptide bond. This technique may only be applicable for smaller bioconjugates. The PEG
conjugate bound to the peptide creates hindrance to the proteolytic enzymes and, thereby, prevents specific cleavage of the
PEG-peptide bond. On the contrary, Edman degradation typically results in cleavage of a peptide bond at adjacent amino acids
to the PEG, resulting in a missing amino acid. Veronese (2001) has stated these difficulties could be reduced by the use of
a PEG conjugate with a methionine in the side arm that is bound to an amine on the peptide. Cyanogen bromide treatment can
be used to break the peptide-methionine linkage and allow independent analysis of the peptide (5).
An alternative would be the formation of a bioconjugate linkage that can be enzymatically digested using nonmammalian enzymes.
This mechanism would enable the removal of the polymer in vitro in order to perform the desired analytical testing on the peptide component. Moreover, the selectivity of degradation of
the conjugate-peptide bond by nonmammalian enzymes would not affect the in vivo stability of the bioconjugate.
For conjugates that are currently manufactured recombinantly as fusion proteins with XTEN, HES, and other polypeptides, there
is the possibility for developing chemical technology that would create a fusion peptide (i.e., linkage through peptide bond)
that could potentially be manufactured by both chemical conjugation and by direct recombinant expression. The chemical synthesis
of a fusion peptide would involve chemical ligation technologies, which may include click chemistry, native chemical ligation,
and Staudinger ligation. This mechanism would create substantial economies in developing and clinically testing pre-proof-of-concept.