Antibody de novo Sequencing

peer-reviewed · Analytical Characterization of Biotherapeutics · 2017

peer-reviewed · Analytical Characterization of Biotherapeutics · 2017. Natalie Castellana et al. Determining the amino acid sequence of a monoclonal antibody (mAb) is an early step in the long road to…
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.

Authors

  1. Natalie Castellana · Abterra Biosciences (United States), Mapp Biopharmaceutical (United States), University of California San Diego
  2. Adrian Guthals · Abterra Biosciences (United States), Mapp Biopharmaceutical (United States), Mapp Biopharmaceutical, Inc., University of California San Diego, University of California System

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.

Cites (15)

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