Liyun Zhang1, Lisa A Vasicek2, SuChun Hsieh3, Shuli Zhang3, Kevin P Bateman2, Jack Henion1. 1. Q2 Solutions, 19 Brown Rd, Ithaca, NY 14850, USA. 2. Department of Pharmacokinetics, Pharmacodynamics & Drug Metabolism, Merck & Co., Inc., West Point, PA, 19486, USA. 3. Department of Pharmacokinetics, Pharmacodynamics & Drug Metabolism, Merck & Co., Inc., Palo Alto, CA, 94304, USA.
Abstract
AIM: The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line multidimensional LC-quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while maintaining the intact native biologically active conformation of the antibody. RESULTS: Following method optimization for a model antibody, an incurred biotherapeutic in cynomologus monkey was quantified in its intact top-down native conformation. A partial method validation demonstrated acceptable precision and accuracy although improved sensitivity requires further studies. CONCLUSION: An on-line multidimensional LC-MS approach presents a proof-of-principle example for quantifying an intact, native antibody isolated from an incurred biological sample via immunoaffinity techniques coupled with top-down QTOF LC-MS bioanalysis.
AIM: The requirements for developing antibody biotherapeutics benefit from understanding the nature and relevant aspects of the entire molecule. The method presented herein employs on-line multidimensional LC-quadrupole time-of-flight (QTOF)-MS for the quantitative determination of an antibody isolated from biological samples while maintaining the intact native biologically active conformation of the antibody. RESULTS: Following method optimization for a model antibody, an incurred biotherapeutic in cynomologus monkey was quantified in its intact top-down native conformation. A partial method validation demonstrated acceptable precision and accuracy although improved sensitivity requires further studies. CONCLUSION: An on-line multidimensional LC-MS approach presents a proof-of-principle example for quantifying an intact, native antibody isolated from an incurred biological sample via immunoaffinity techniques coupled with top-down QTOF LC-MS bioanalysis.
Keywords:
NSB; antibody; biotherapeutic quantitation; denatured and native protein conformation; immunocapture; intact top-down protein quantitation; multidimensional LC–MS; non-specific binding; top-down QTOF LC/MS
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