Study Of PEGylation DhHP-6 With Activity PEG Derivative

Along with the continuous exaltation of the technique level of the research and the synthesis in biochemistry realm, more and more medicine of protein (peptide) is discovered or synthesized. According to a general statistics, more than 500 kinds of bio-product have already been placed in a clinical trial stage just in 2000. But when these medicines is really applied, many problems appeared, such as short half- life, having the immunogenicity, is easily hydrolysisd by protease, low solubility, etc. Researchers developed a lot of methods for overcoming these problems, among these methods, the PEGylation of protein (peptide) have drawn more and more concern. The common used PEG in protein PEGylation is Methoxy-PEG which have a structure like: CH3-(OCH2CH2)nOH. It's a hydrophilic polymer without any electric charge. A biocompatible interface or a protection layer can be gained when PEG is grafted onto the surface.This protection layer can reduce the immunogenicity of exogenous material. Consumedly lower the adsorbability of proteins, cells and bacteria. Because of the large volume of PEG, it also can lower the kidney clearance speed of PEGylated drugs. The PEG molecule will be quickly clearance without structure variety in vivo, the clearance speed is only related with the molecular weight of PEG . Even a high molecular weight PEG only has very weak immunogenicity. In practice, a PEGylation reaction usually needs a critical condition which may destroy the protein (peptide), therefore, researchers often use an activate PEG as modifier. Currently, there has been variety commercialize of activate PEG derivatives appearing on market.In this paper, we chose a common used activate PEG derivative (m-SC-PEG) as modifier and studied a few factor which will influence the yield. First of all, we investigate the influence of PH. As PH value increasing, yield of modify reaction become higher. But a higher PH may denature DhHP-6. Investigation of temperature show, a higher yield was obtained when reaction was carried out in higher temperature. The same problem to PH was appeared when a higher temperature used. A prolonged reaction time gave higher yield. After reaction for a certain time, yield reached a top value and further prolonged time didn't case an obvious change. Use more m-SC-PEG also got higher yield. But it's hard to separate PEG and PEG-DhHP-6 from the reaction solution. And more cost was cost as more reactant was used. So a proper reactant ratio was necessary. At last, considering for reduce hydrolysis reaction, m-SC-PEG was dissolved in organic solvent, then added into DhHP-6 solution to start modify reaction. Result showed yield increased. Comprehensive consider the above mentioned experiment result and restriction, chosen modify reaction condition is PH=7.0, T=25℃, reaction time is 2h, reactant molar ratio is m-SC-PEG:DhHP-6=10:1, solvent uses H2O or H2O and acetonitrile.After reaction condition is decided, we investigate the reaction dynamics of the PEGylation reaction. At first, the hydrolysis reaction of m-SC-PEG is studied. H2O is used as solvent and reactant at the same time. So the water is extremely over quantity. This reaction can be considered as a first order reaction. First order reaction dynamics is used to study the hydrolysis reaction. Among these, NHS is used to detect the extent of reaction. Because NHS have a ultraviolet absorbsion peak in 260nm. A series different concentration of NHS is used to get a standard absorbsion curve. Then certain concentration of reactant can be determined by NHS'absorbsion. And corresponding reaction dynamics function and reaction rate constant can be calculated. The function is ln( a - x)=-0.0208t-9.8829Reaction rate constant k1=0.0208min^-1After reaction rate constant of hydrolysis reaction is calculated. Reaction dynamics of PEGylation may be investigated. In practice, residual mass of m-SC-PEG in solution is a sum of a first order function and an exponential function about time. The differential dynamics function of PEGylation reaction is transformed to a first order linear inhomogeneity differential function. Its solution has a complex form. So it's discommodious for further discussion and study. A simplified model is founded which is composed with a hydrolysis reaction and a modification reaction which is independent to each other. From this model, a simple second order reaction dynamics function is got. And a approximate solution of reaction rate constant can be obtained. The result is k2'= 1.132×104mol-1Lmin-1. Following study is the relationship of k2'and real reaction rate constant k2. Research shows that k2'is a average value of a set of numerical values. Only when the residual concentration of two reactants is equality, k2'and k2 have the same value.At last, bioactivity of DhHP-6 and PEG-DhHP-6 is compared. If PEG-DhHP-6 have no bioactivity, the modification reaction is meaningless. Study of enzyme activity shows PEG-DhHP-6 remains 80% of enzyme activity of DhHP-6. In a word, the PEGylation reaction keeps most of DhHP-6's bioactivity. In another hand, study of enzyme stability of DhHP-6 and PEG-DhHP-6 show PEG-DhHP-6 is obviously more stable than DhHP-6. All this result show this PEGylation reaction is worthwell.