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:
- antibody
- biotherapeutic quantitation
- denatured and native protein conformation
- immunocapture
- intact top-down protein quantitation
- multidimensional LC–MS
- non-specific binding
- NSB
- top-down QTOF LC/MS