Quantitative analysis of derivatized proteins prepared with pyridyl disulfide-containing cross-linkers by high-performance liquid chromatography

Determination of the introduced moieties into derivatized proteins is an es sential step in the preparation and quality control of chemically defined i mmunoconjugates. For the derivatized proteins using pyridyl disulfide-conta ining cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (S PDP) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), the derivatization degree (ratio of pyridyl disulfide moieties to protein) has been traditionally determined by measuring the absorbance o f both the derivatized protein and 2-thiopyridone (2-TP) released from the dithiothreitol (DTT) treatment (spectrophotometric method). This method, ho wever, causes several problems including false high and low determinations of the protein and 2-TP, respectively, low selectivity, poor reproducibilit y, and relatively large amounts of sample consumption. A quantitative deter mination method of the derivatization ratios using bovine serum albumin der ivatized with SPDP and SMPT as the model system has been developed. The con centration of protein and 2-TP released from the DTT treatment of derivatiz ed proteins was determined directly without consideration of different reag ents used and their concentrations. The present HPLC method was proved to b e better in terms of accuracy, selectivity, and reproducibility with micro sample consumption. Moreover, this HPLC method can be directly applied to a ll derivatized proteins prepared with pyridyl disulfide-containing cross-li nkers.