LC–MS/MS Quantification of Next-Generation Biotherapeutics: A Case Study for an IgE Binding Nanobody in Cynomolgus Monkey Plasma

Abstract

Background: Nanobodies^® are therapeutic proteins derived from the smallest functional fragments of heavy chain-only antibodies. The development and validation of an LC–MS/MS-based method for the quantification of an IgE binding Nanobody in cynomolgus monkey plasma is presented. Results: Nanobody quantification was performed making use of a proteotypic tryptic peptide chromatographically enriched prior to LC–MS/MS analysis. The validated LLOQ at 36 ng/ml was measured with an intra- and inter-assay precision and accuracy <20%. The required sensitivity could be obtained based on the selectivity of 2D LC combined with MS/MS. No analyte specific tools for affinity purification were used. Plasma samples originating from a PK/PD study were analyzed and compared with the results obtained with a traditional ligand-binding assay. Excellent correlations between the two techniques were obtained, and similar PK parameters were estimated. Conclusion: A 2D LC–MS/MS method was successfully developed and validated for the quantification of a next generation biotherapeutic.

Financial & competing interests disclosure

The authors also gratefully acknowledge financial support from the IWT (grant 120333). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate insti-tutional review board approval or have followed the princi-ples outlined in the Declaration of Helsinki for all human or animal experimental investigations.

Acknowledgements

The authors would like to thank the Ablynx IgE project team members for their support, and the Ablynx CMC department for sharing expertise and equipment during initial set-up of this work.