Studies On The Application Of Affinity Chromatography Coupled With Bio-Mass Spectrometry In The Analysis Of Protein Drugs In Vivo

Recombinant human endostatin (rh-ET) specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. As the first I grade new recombinant anti-tumor medicine in China, now it had been approved by Chinese State Drug Administration to enter into clinical study.Regarding rh-ET with N-terminal 6 × His affinity tag as a model drug, we developed an analytical method for the analysis of microscale protein drugs, metabolites and complexes in serum. With the method, microscale protein drugs, metabolites and complexes in serum were extracted and enriched with immobilized affinity chromatography; protein drugs were analyzed and identified by bio-mass spectrometry.The structure of rh-ET was confirmed with amino acid composition analysis, N-terminal sequencing, molecular weight analysis by mass spectrometry and peptide mass fingerprinting (PMF). Rh-ET for this research is composed of two forms, one is same as design, and another is truncated with the N-terminal Met residue missing.Animal tests with the concentration distribution of radioactivity of ¹²⁵I labeled rh-ET on mice revealed that rh-ET was metabolized and excreted from excretory system mainly. The concentration of radioactivity in urine is the highest, and that in plasma is the-fourth, which is lower than that in urine, kidney, lung and liver. On the other hand, the main concentration ofradioactivity in urine comes from hydrophilic derivatives, such as metabolites, and the concentration of original protein drug in serum is higher.Original protein drugs, metabolites and complexes in serum were extracted and enriched with immobilized irnmunoaffinity chromatography and immobilized metal ion affinity chromatography. Molecular weights of the extraction were determined by directed MALDI-TOF-MS analysis on the affinity gel. Rh-ET in affinity gel was identified with PMF method by MALDI-TOF-MS and PST methods by two kinds of ESI-MS/MS combining with database searching.The experimental results showed that affinity extraction with irnmunoaffinity and metal ion affinity combined with biological mass spectrometry analysis by MALDI-TOF-MS and ESI-MS-MS is a very powerful technique to the recombinant protein drug analysis in vivo. Its high specificity, high sensitivity and easy to use make it a potential technique in the pharmacokinetics research of recombinant protein drug.The further research of the method developed by this study in the application of the pharmacokinetics research of recombinant protein drug, especially in the metabolites and complexes of the drug with the internal proteins is needed.