Elsevier

Analytical Biochemistry

Volume 414, Issue 1, 1 July 2011, Pages 147-153
Analytical Biochemistry

Quantitation of a recombinant monoclonal antibody in monkey serum by liquid chromatography–mass spectrometry

https://doi.org/10.1016/j.ab.2011.03.004Get rights and content

Abstract

A method including protein A purification, limited Lys-C digestion, and mass spectrometry analysis was used in the study to quantify a recombinant monoclonal antibody in cynomolgus monkey serum. The same antibody that was isotopically labeled was used as an internal standard. Interferences from serum proteins were first significantly reduced by protein A purification and then by limited Lys-C digestion of protein A bound IgG, including both monkey and the recombinant IgG. Fab fragment of the recombinant human IgG was analyzed directly by LC–MS, while monkey IgG and the Fc fragment of the recombinant human IgG remained bound to protein A resin. Quantitation was achieved by measuring the peak intensity of the Fab from the recombinant human IgG and comparing it to that of the Fab from the stable isotope-labeled internal standard. The results were in good agreement with the values from ELISA. LC–MS can therefore be used as a complementary approach to ELISA to quantify recombinant monoclonal antibodies in serum for pharmacokinetics studies and it can also be used where specific reagents such as antigens are not readily available for ELISA.

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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