Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography–mass spectrometry
Section snippets
Materials
The recombinant monoclonal antibody was produced by transfected Chinese hamster ovary (CHO) cell lines and purified at Abbott Bioresearch Center (Worcester, MA). The same antibody was also produced by culturing the same CHO cell line in medium containing Arg and Lys, each with six carbon-13 isotopes and purified by protein A chromatography at Abbott Bioresearch Center. Acetonitrile and trifluoroacetic acid (TFA) were purchased from J.T. Baker (Phillipsburg, NJ). 13C-Arg and 13C-Lys were
Principle of the method
The concentration of the recombinant antibody decreased from approximately 130 to 4 μg/mL from several hours to 42 days after administration as measured by ELISA, as a result of protein degradation [34]. It was necessary to enrich the recombinant antibody and remove interfering serum proteins including endogenous monkey IgG, which is present at approximately 16 mg/mL [35]. Several approaches have been reported in the literature to reduce interference including the use of albumin depletion kits [5]
Conclusion
A method including protein A purification, limited Lys-C digestion, and LC–MS was developed to quantify a recombinant human monoclonal antibody in cynomolgus monkey serum for a pharmacokinetic study. The same recombinant monoclonal antibody with isotopic labeling was used as an internal standard, which was spiked into the serum samples. Interferences of serum proteins including endogenous IgG were sufficiently reduced using protein A purification and limited Lys-C digestion, which selectively
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