The influence of synthetic conditions on the stability of methotrexate-monoclonal antibody conjugates determined by reversed phase high performance liquid chromatography

✍ Scribed by F. Hudecz; M. C. Garnett; T. Khan; R. W. Baldwin

• Publisher: John Wiley and Sons
• Year: 1992
• Tongue: English
• Weight: 428 KB
• Volume: 6
• Category: Article
• ISSN: 0269-3879
• DOI: 10.1002/bmc.1130060306

✦ Synopsis

Methotrexate (MTX) has been convalently attached to an IgG-type monoclonal antibody (791TI36) directed to tumour-associated antigen gp72. Conjugates were synthesized by the active ester method using MTX N-succinimidyl ester at various pH values (7.5-10.5). Following purification by gel filtration, high performance liquid chromatography was used to assess the free drug or its derivatives in samples of MTX-791T/36 conjugates previously treated (or not) with hydroxylamine. Quantitative analysis, performed on a reversed phase column (pore size 300 A) with isocratic acetonitrile-sodium acetate buffer (pH 4.8) as mobile phase, indicated no detectable amount of free methotrexate in hydroxylamine-treated conjugates even six months after their preparation. Similar observations were made with conjugates, whose synthesis were performed at pH 3 10. In contrast, the presence of increasing amounts of drug/metabolite could be demonstrated in samples produced at lower pH values. Based on these findings, the pH-dependent kinetics of MTX release has been determined and used to design conditions under which stable MTX-791T/36 conjugates could be prepared without hydroxylamine reaction.