Study Of N-terminal-selective PEGylation For IFNa-2b

In the study, The N-terminal amino-group of IFNa-2b was site-selectively PEGylated with derivatives of mPEG-ALD-20KD, because of its basic amino reactivity and the difference of dissociation constants of amino group (pKa of a-amino is7.8,10.1for e-amino) the reaction is conductive to a-amino modification by adjusting reactive conditions. The influence of pH, mole ratio of PEG and IFNa-2b,temperature and time were evaluated, also the Physical and Chemical Properties of IFNa-2b, the mono-PEGylation of IFNa-2b has the highest percentage under the condition of pH4.0,4℃,48hs for reaction time and a mole ratio of PEG and IFNa-2b equal to10:1.MonoPEGylated IFNa-2b was isolated by SP cation exchange chromatography, and analytical methods were established to evaluate the uniformity of isomer of monoPEGylation because of different modification site. monoPEGylation was characterized using TSK-GEL SP-5PW(10um1000A7.5mm*7.5cm), the loading buffer is3.7mM sodium acetate(pH4.5), using a pH gradient elution, The separation was performed with a linear gradient from0%to70%of buffer B(10mM pH7.50K2HPO4) in110min at a flow rate of0.6ml/min and detected at226nm and the oven temperature is25℃. The monoPEGylation and orginal protein were digested by using trypsin and analyzed by peptide map, the different peaks were collected and analyzed by MALDI-TOF to determine the modification sites, six or more monoPEGylation with different modification site were isolated and characterized. The N-terminal amino-group of IFNa-2b account for around70%of all monoPEGylation. Based on the above, We can draw a conclusion that mPEG-ALD-20KD is highly selective for N-terminal amino-group.