J Pharm Pharmacogn Res 2(Suppl. 1): S115, 2014

Special supplement with the abstract book of LATINFARMA 2013



Pasut G.

Department of Pharmaceutical and Pharmacological Sciences, University of Padova, F. Marzolo 5, 35131, Padova, Italy.

Biocatalysis represents a promising area of research in organic chemistry. The specificity and selectivity of enzymes can dramatically improve the yields of synthesis, especially for those involving very complex structures. In the field of polymer conjugation, enzymes have also been considered as tools for site-specific conjugation at the level of amino acids not usually addressed with chemical methods. Different enzymatic protocols have been developed and proposed for polymer conjugation to protein. The most advanced is GlycoPEGylation that, applied to coagulation factor IX, yielded a conjugate presently in clinical trials . In this case the method is based on two enzymes to mimic O-glycosylationor N-glycosylation and consequently it has the advantage to couple PEG at the site of naturally occurring glycosylation. Although innovative, this approach involves two enzymes and therefore its feasibility at industrial scale might be limited. Another approach exploits the enzyme Sortase A to couple a polymer at the C-terminus of proteins. Also in this case there are some limitations such as the requirement of a specific C-terminal consensus amino acid sequence as substrate for the enzyme.

A promising alternative is the transglutaminase (TGase) enzymes that catalyze the transfer of a primary amine (amino donor) to the gammacarboxamide group of a glutamine (acyl donor). Interestingly, these enzymes recognize other substrate than ε-amino group of a lysine as amino donor, such as for example a PEG-NH2. My lab focused in microbial TGase (mTGase) from Streptomyces mobaraense, already approved as food additive. Beside the fact that this approach can target an amino acid that otherwise cannot be modified by chemical method another advantage of mTGase is its selectively. Among the several glutamines present in a protein only those inserted in very flexible loops can be a substrate, therefore very homogeneous conjugates can be obtained. Furthermore, mTGase preserves a good level of activity in a wide range of pH and ionic strength values and also in the presence of organic co-solvents (up to 50% v/v). We exploited this feature to develop tailor-made reaction conditions for specific proteins with the aim to stabilize the protein or to reduce the number of glutamines substrate of mTGase, thus obtaining mono-PEGylated forms.

The potential of mTGase-mediated PEGylation has been compared in our lab to another known approach of site selective conjugation: Nterminal PEGylation. The methods were applied to hGH by using a 20kDa PEG, and the results were comparable in terms of yield, and pharmacokinetic/pharmacodynamic. Relevant was the efficacy of the conjugates able to produce a weight growth with a single weekly injection in hypophysectomized rats that was comparable to that obtained with 6 weekly injections of free hGH.

The present and future development of TGase as tool for PEGylation is the development of a immobilized mTGase on resin beads, which will offer the relevant advantage of a fast removal of the enzymes and the possibility of a fast screening of several reactions conditions. Furthermore, we discovered that immobilized mTGase present an increased selectivity towards the Glu inserted in the most flexible loops.

This new era of enzymatic polymer conjugation is at the beginning and it might possess greater potentials of development and feasibility for even more advanced protein conjugates.