Purpose: The enzyme microbial transglutaminase (mTGase) has been used for site-specific protein derivatization at specific glutamines. mTGase-mediated can selectively conjugate PEG-NH2 to G-CSF at Gln135. Here we investigated if mTGase can catalyze the same reaction but with reversed substrates, namely a Lys in the protein and a Gln in the polymer. A specific acyl donor PEG derivative was used Z-QG-PEG. mTGase catalyzed the reaction generating a high-yield PEG-G-CSF conjugate in which the polymer was selective linked to Lys41 of the protein. The PEG-K41-G-CSF conjugate was compared with the PEG-Q135-G-CSF, obtained by mTGase catalysis in presence of PEG-NH2. Biophysical characterization showed that the two positional isomers have similar properties, and the studies in rats demonstrated that they have comparable pharmacokinetics. mTGase revealed to be a very useful tool for site-selective protein modification at either Lys or Gln residues.
Methods: mTGase-mediated Gln conjugation of G-CSF
PEG20kDa-NH2 (10-fold molar excess) was added to a solution of 2 mg/ml G-CSF in 0.1 M phosphate buffer pH 7.2. mTGase was added in order to reach an enzyme/substrate (E/S) ratio of 1:50. The reactions was incubated at 25°C, and analyzed by RP-HPLC. The reaction mixture was desalted by a HiTrap column. The conjugate (PEG-Q135-G-CSF) was purified by IEC-HPLC and buffer exchanged with 10 mM pH 4.6 sodium acetate buffer containing 5% sorbitol.
mTGase-mediated Lys conjugation of G-CSF
ZQG-PEG20kDa (10-fold molar excess) was added to a G-CSF solution as above reported; also mTGase was added as above reported. The reactions were incubated at 25°C and were quenched after 24 h of incubation upon addition of a stock solution of iodoacetamide at a molar ratio with mTGase of 30/1 (iodoacetamide/mTGase). Aliquots of the reaction mixtures were analyzed by RP-HPLC. The conjugate (PEG-K41-G-CSF) was purified as described for PEG-Q135-G-CSF
Conjugates were characterized by SDS-PAGE, MALDI-TOF mass spectrometry, circular dichroism, peptide mapping.
Pharmacokinetic investigation in rats
Pharmacokinetics of PEG-K41-G-CSF, PEG-Q135-G-CSF and G-CSF were studied in Sprague-Dawley rats (250-350 g, 3 animals per group). A dose corresponding to 100 μg/kg of G-CSF (equiv.) was i.v. administered to anesthetized rats (anesthesia 5% isoflurane gas mixed with O2) via tail vein. At predetermined times, blood samples were collected from the tail vein and centrifuged at 1500 × g for 15 min. The G-CSF (equiv.) content in the serum samples was quantified using a Human G-CSF ELISA Kit (Life Technologies).
Results: We have previously demonstrated that TGase recognizes as substrate the Gln resides that are located in flexible protein loops . In the case of Gln-selective coupling by TGase PEGylation the reaction yielded the formation of a single mono-conjugate in which the polymer was specifically attached only to Gln135 . We determined that TGase has a great specificity also in terms of Lys modification; in fact Lys-selective coupling by TGase PEGylation yielded a single mono-PEGylated isomer at the level of Lys41. Therefore TGase has great versatility because it can generates monoPEGylated conjugates at specific Lys or Gln residues on the basis of PEG reagent used, namely a PEG-NH2 for Gln modification or a ZQG-PEG for Lys modification. The specific Lys or Gln substrate of TGase must fulfill the basic requirement of being inserted in flexible regions (Fig. 1).
In Figure 1 it is shown that the Lys41 and Gln145 (Note that the numbers refer to the rhG-CSF while Figure 1 reports the sequence of hG-CSF) are located in non-structured regions with relatively high value of B-factor, an index of backbone flexibility.
For both conjugates the coupling reaction was quite fast, reaching approximately 90% of conversion in less than 4 hours. The purified conjugates were analyzed by SDS-PAGE (Fig. 2) and MALDI-TOF mass spectrometry confirming the purity and expected molecular mass.
The Circular dichroism analysis demonstrated that in both cases the PEG chain did not interfere with the G-CSF secondary structure.
The pharmacokinetic study confirmed that, as seen in other cases, the conjugates achieved a half-life prolongation that is correlated to the molecular weight of the PEG coupled. In particular the conjugates showed a half-life increase in the range of 3-3.5 fold with respect to native G-CSF.
Conclusion: This work demonstrated that TGase-mediated enzymatic PEGylation offers the unique feature to obtained site-selective polymer protein conjugates at specific Lys or Gln residues. The site of modification is determined by selecting the proper PEGylating reagent according to the desired site of modification. The high TGase selectivity ensured the obtainment of site-specific monoPEGylated isomers in high yield and under physiological conditions, relevant properties for conjugation of also unstable proteins.