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Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry.

Hagman, Charlotte and Ricke, Darrell and Ewert, Stefan and Bek, Stephan and Falchetto, Rocco Angelo and Bitsch, Francis (2008) Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry. Analytical Chemistry, 80 (4). pp. 1290-1296. ISSN 0003-2700

Abstract

The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.

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Date Deposited: 14 Dec 2009 14:04
Last Modified: 31 Jan 2013 01:25
URI: https://oak.novartis.com/id/eprint/193

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