2008
DOI: 10.1021/ac7021234
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Immunopurification and Mass Spectrometric Quantification of the Active Form of a Chimeric Therapeutic Antibody in Human Serum

Abstract: In this study, we show that liquid chromatography coupled with tandem mass spectrometry provides a sensitive, specific, and accurate absolute quantification of Erbitux, a human:murine chimeric mAb used for the treatment of colorectal cancer. Micrometric magnetized beads, functionalized with soluble epidermal growth factor receptor (sEGFR), the pharmacological target of Erbitux, were used for specific immunocapture of Erbitux allowing assessment of the antibody's biological potency and sample purification. Foll… Show more

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Cited by 137 publications
(84 citation statements)
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“…When no protein or peptide purification step is employed, LC/MS sensitivity is significantly compromised due to matrix interference from the peptide-rich digest [24]. For high-sensitivity LC/MS applications, immunopurification is the most effective way to improve assay sensitivity and robustness by isolating target protein from matrix [25,26]. Further immunopurification of surrogate peptide from the digest could be carried out using an anti-peptide Ab [27].…”
Section: Resultsmentioning
confidence: 99%
“…When no protein or peptide purification step is employed, LC/MS sensitivity is significantly compromised due to matrix interference from the peptide-rich digest [24]. For high-sensitivity LC/MS applications, immunopurification is the most effective way to improve assay sensitivity and robustness by isolating target protein from matrix [25,26]. Further immunopurification of surrogate peptide from the digest could be carried out using an anti-peptide Ab [27].…”
Section: Resultsmentioning
confidence: 99%
“…There were a few publications using tandem high-performance chromatography mass spectrometry (LC-MS/MS) methods to quantify mAb (10)(11)(12)(13)(14)(15). The method approach involves enzyme digestion to reduce mAb into small peptides to be within the mass range of the mass spectrometry.…”
Section: Discussionmentioning
confidence: 99%
“…Biotherapeutics or associated surrogate peptides as well as any important catabolites could be specifically quantified by MS. In fact, several researchers have published studies with this ultimate goal in mind (Dubois et al, 2007(Dubois et al, , 2008Heudi et al, 2008;Ezan et al, 2009;Liu et al, 2011b;Bronsema et al, 2012;Li et al, 2012). Historically, there have been several drawbacks associated with quantitative MS methods compared with LBA, including the following: 1) MS has been generally less sensitive, 2) large protein analytes have suffered from low throughput, and 3) quantitative MS methods for macromolecules are much more nascent compared with established antibody-based methodologies.…”
Section: Impact Of Protein Biotherapeutic Biotransformation On Bioanamentioning
confidence: 99%