Ligand-Binding Mass Spectrometry to Study Biotransformation of Fusion Protein Drugs and Guide Immunoassay Development: Strategic Approach and Application to Peptibodies Targeting the Thrombopoietin Receptor
Journal Title: The AAPS Journal - Year 2010, Vol 12, Issue 4
Abstract
The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc (“peptibodies”) using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins.
Authors and Affiliations
Michael P. Hall, Colin Gegg, Kenneth Walker, Christopher Spahr, Robert Ortiz, Vimal Patel, Steven Yu, Liana Zhang, Hsieng Lu, Binodh DeSilva, Jean W. Lee
Keywords
Related Articles
- EP ID EP681421
- DOI 10.1208/s12248-010-9218-9
- Views 76
- Downloads 0
Design and Characterisation of a Polyethylene Oxide Matrix with the Potential Use as a Teat Insert for Prevention/Treatment of Bovine Mastitis
This manuscript reports (for the first time) on antibiotic-free polymeric inserts for the prevention and/or treatment of bovine mastitis. Polyethylene oxide (PEO)-based inserts were prepared using different concentration...
Reference Datasets for Bioequivalence Trials in a Two-Group Parallel Design
The online version of this article (doi:10.1208/s12248-014-9704-6) contains supplementary material, which is available to authorized users.
Ocular Drug Delivery
Ocular drug delivery has been a major challenge to pharmacologists and drug delivery scientists due to its unique anatomy and physiology. Static barriers (different layers of cornea, sclera, and retina including blood aq...
Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells
The online version of this article (doi:10.1208/s12248-015-9827-4) contains supplementary material, which is available to authorized users.
Prediction of Human Glomerular Filtration Rate from Preterm Neonates to Adults: Evaluation of Predictive Performance of Several Empirical Models
The objective of this study was to evaluate the predictive performance of several allometric empirical models (body weight dependent, age dependent, fixed exponent 0.75, a data-dependent single exponent, and maturation m...