Validation of a shielded-hydrophobic-phase high-performance liquid chromatography method for the determination of residual methotrexate in recombinant protein biopharmaceuticals

A shielded-hydrophobic-phase (SHP) HPLC method for the determination of residual methotrexate in recombinant protein biopharmaceuticals was validated. The method requires no removal of protein or other prior sample “clean-up” and detects quantities of methotrexate as low as 2.5 ng in the presence of...

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Bibliographic Details
Published in:Journal of Chromatography A 1998-12, Vol.828 (1), p.303-309
Main Authors: Huang, Maggie M, Penn, Lara, Bongers, Jacob, Burman, Sudhir
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biotechnology
Fundamental and applied biological sciences. Psychology
Health. Pharmaceutical industry
Industrial applications and implications. Economical aspects
Methotrexate
Monoclonal antibodies
Production of active biomolecules
Proteins
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Summary:A shielded-hydrophobic-phase (SHP) HPLC method for the determination of residual methotrexate in recombinant protein biopharmaceuticals was validated. The method requires no removal of protein or other prior sample “clean-up” and detects quantities of methotrexate as low as 2.5 ng in the presence of up to 25 mg/ml of protein. Methotrexate was fully resolved from a recombinant IgG1 monoclonal antibody and associated matrix components. Accuracy was demonstrated by measuring “spiked” recoveries at the limit of quantitation (found 90–120% recovery with R.S.D.s ≤10%). Other validation parameters studied included range, precision, ruggedness, robustness and stability of “spiked” samples.
ISSN:0021-9673
DOI:10.1016/S0021-9673(98)00683-9