Antibody de novo Sequencing
peer-reviewed · Analytical Characterization of Biotherapeutics · 2017
| Date | 2017-08-08 |
| Type | peer-reviewed |
| Venue | Analytical Characterization of Biotherapeutics |
| Publisher | Wiley |
| Contribution | review |
| DOI | 10.1002/9781119384434.ch6 |
| Citations (OpenAlex) | 0 |
Abstract
Determining the amino acid sequence of a monoclonal antibody (mAb) is an early step in the long road to developing a therapeutic drug candidate. Historically this has been done by sequencing the DNA of the source hybridoma cell line. Unfortunately, source cells may not always be available and DNA sequencing efforts often fail by either returning multiple incorrect sequences or yielding a single mAb sequence that, once expressed in mammalian cells, is not as active as the hybridoma product. In these cases, proteomic-based sequencing of the hybridoma product may be the only viable option. We discuss the current techniques for mAb sequencing using tandem mass spectrometry (MS/MS). Besides giving basic advice on sample handling and preparation, we focus on methods that utilize spectral networks, or sets of MS/MS spectra from overlapping peptides, generated from multi-enzyme digests of the mAb. We also discuss how databases of known homologous mAbs can aid and how the latest advances in MS/MS instrumentation, particularly electron-transfer disassociation (ETD) and high-resolution mass accuracy, improve de novo sequencing accuracy.
Methods and tools
- Antibody de novo sequencing chapter: Book chapter on proteomic de novo sequencing of monoclonal antibodies when the hybridoma or its DNA sequence is unavailable or unreliable.