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

F. Hudecz, M. C. Garnett, T. Khan, R. W. Baldwin

Research output: Contribution to journalArticle

22 Citations (Scopus)


Methotrexate (MTX) has been convalently attached to an IgG‐type monoclonal antibody (791T/36) 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 Å) 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 ⩾ 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.

Original languageEnglish
Pages (from-to)128-132
Number of pages5
JournalBiomedical Chromatography
Issue number3
Publication statusPublished - Jan 1 1992


ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

Cite this