NHS ester-activated PEGylation polymer can react with primary amines at pH 7.2 to 9 to yield a stable (covalent) amide bond. The reaction releases N-hydroxysuccinimide (NHS). Nucleophilic reaction between amine and active ester is most commonly performed in phosphate, carbonate-bicarbonate, HEPES or borate buffers at pH 7.2 to 8.5 for 0.5 to 4 h at room temperature or 4°C.