Several strategies for site-specific PEGylation have been successfully exploited to conjugate poly(ethylene glycol) (PEG) to pharmaceutical proteins. The advantages sought are those of improving efficacy and increasing the half-life of conjugated proteins while achieving a higher degree of homogeneity. Recombinant human growth hormone (hGH) was thus PEGylated exploiting two site-specific strategies: N-terminal PEGylation using the PEG20 kDa-aldehyde polymer and microbial transglutaminase (mTGase) mediated enzymatic PEGylation using PEG20 kDa-NH2. N-Terminal PEGylation of hGH was carried out by covalent attachment of PEG to the α-amine residue of Phe1 that yielded the monoconjugate PEG-Nter-hGH with a mass of 44152.2 Da, as measured by MALDI-TOF mass spectrometry. The mTGase mediated PEGylation, performed in a water/ethanol solution mixture, allowed a PEG coupling reaction only at the level of hGH Gln141, yielding the single monoconjugate PEG-Gln141-hGH with a mass of 44064.9 Da. Circular dichroism studies showed that both conjugation strategies preserved the native-like secondary structures of hGH. It is vital to maintain the structural integrity of hGH if PEGylated hGH is to be used in therapeutic applications. As expected, the pharmacokinetic profile in rats of PEG-Nter-hGH and PEG-Gln141-hGH revealed a significant increase in systemic exposure with respect to unmodified hGH. The conjugates showed a half-life increase of 4.5-fold with respect to hGH. These results demonstrate that both chemical and enzymatic site-selective PEGylation of hGH generates conjugates with a prolonged half-life.
PEGylation is a strategy that has been used to improve the biochemical properties of proteins and their physical and thermal stabilities. In this study, hen egg-white lysozyme (EC 3.2.1.17; LZ) was modified with methoxypolyethylene glycol-p-nitrophenyl carbonate (mPEG-pNP, MW 5000). This PEGylation of LZ produced conjugates that retained full enzyme activity with glycol chitosan, independent of degree of enzyme modification; its biological activity with the substrate Micrococcus lysodeikticus was altered according to its degree of modification. The conjugate obtained with a low degree of mPEG-pNP/NH(2) modification was studied by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), demonstrating a spectral peak at m/z 19,988 Da with 77% of its original enzymatic activity. Spectroscopic studies of Fourier transform infrared (FTIR) and circular dichroism (CD) did not show any relevant differences in protein structure between the native and conjugate LZ. Studies of the effects of pH and temperature on PEGylated LZ indicated that the conjugate was active over a broad pH range, stable at 50 degrees C, and demonstrated resistance to proteolytic degradation.
PEGylation is a successful strategy for improving the biochemical and biopharmaceutical properties of proteins and peptides through the covalent attachment of polyethylene glycol chains. In this work, purified recombinant uricase from Candida sp. (UC-r) was modified by PEGylation with metoxypolyethilenoglycol-p-nitrophenyl-carbonate (mPEG-pNP) and metoxypolyethyleneglycol-4,6-dichloro-s-triazine (mPEG-CN). The UC-r-mPEG-pNP and UC-r-mPEG-CN conjugates retained 87% and 75% enzyme activity respectively. The K(M) values obtained 2.7x10(-5) M (mPEG-pNP) or 3.0x10(-5) M (mPEG-CN) for the conjugates as compared to 5.4x10(-5) M for the native UC-r, suggesting enhancement in the substrate affinity of the enzyme attached. The effects of pH and temperature on PEGylated UC-r indicated that the conjugates were more active at close physiological pH and were stable up to 70 degrees C. Spectroscopic study performed by circular dichroism at 20 degrees C and 50 degrees C did not show any relevant difference in protein structure between native and PEGylated UC-r. In rabbit and Balb/c mice, the native UC-r elicited an intense immune response being highly immunogenic. On the other hand, the PEGylated UC-r when injected chronically in mice did not induce any detectable antibody response. This indicates sufficient reduction of the immunogenicity this enzyme by mPEG-pNP or mPEG-CN conjugation, making it suitable for a possible therapeutical use.
PEGylation is considered a successful technique to enhance the therapeutic and biotechnological potentials of peptides, proteins, toxins and drugs. The conjugation of polyethylene glycol (PEG) increases the size and molecular weight of conjugated molecule and improves its pharmacokinetics and pharmacodinamics by increasing water solubility, protecting from enzymatic degradation, reducing renal clearance and limiting immunogenic and antigenic reactions. These features are very useful for therapeutic proteins, since PEGylated proteins exhibit high stability and very low immunogenicity, ensuring a sustained clinical response with minimal dose and less frequent administration. The modification of snake venom toxins by PEGylation is a promising strategy to increase the use of these biomolecules in clinical practice, which has been limited by side effects of immune reactions in patients. Thrombin-like serine protease from Crotalus durissus collilineatus (SPCdc) is able to convert fibrinogen into fibrin and presents potential therapeutic application in cases of myocardial infarction, ischemic stroke and other thrombotic and vascular disorders. In this study we modified the SPCdc by site-specific PEGylation, producing the unique conjugate of molecular mass around 35 kDa, named SPCdc-PEG. Unexpectedly, the Km of the PEGylated enzyme (Km = 0.447 mM ± 0.025) was smaller than that of the native enzyme (Km = 0.770 mM ± 0.020), indicating that PEG-SPCdc has a higher affinity for the substrate TAME than SPCdc. Additionally, the values of Kcat/Km (1163 mM.min-1, for SPCdc-PEG and 350 mM.min-1, for SPCdc) showed that PEGylated enzyme has higher catalytic efficiency than the native form. These results demonstrated the relevant biopharmaceutical potential of SPCdc-PEG.
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