Analysis of Monoclonal Antibodies Using De Novo Sequencing

Monoclonal antibodies (mAbs) are antibodies produced by a single B cell clone, known for their high specificity and consistency. They have become crucial in therapeutic, diagnostic, and research applications. 

 

De Novo sequencing refers to the process of determining the amino acid sequence of a protein without relying on a reference database. This method employs tandem mass spectrometry (MS/MS) to fragment the protein into peptides and then analyzes the resulting data to reconstruct the original sequence. Unlike traditional database-dependent methods, De Novo sequencing is essential when dealing with new or uncharacterized antibodies, providing detailed insights into the protein's sequence, including post-translational modifications (PTMs) and sequence variations.

 

Steps in Monoclonal Antibody Analysis Using De Novo Sequencing

1. Sample Preparation

The monoclonal antibody is purified from the culture supernatant or biological sample using techniques like affinity chromatography. The purified antibody is then subjected to enzymatic digestion, typically using proteases such as trypsin, to generate smaller peptide fragments.

 

2. Mass Spectrometry Analysis

The peptide fragments are analyzed using tandem mass spectrometry (LC-MS/MS). High-resolution MS/MS data is acquired to ensure accurate identification of the peptide fragments. The mass spectra of the peptides are recorded, capturing detailed information on the mass-to-charge ratio (m/z) of fragment ions.

 

3. De Novo Sequencing

Advanced algorithms are used to interpret the MS/MS data, reconstructing the amino acid sequence of each peptide. Special attention is given to identifying PTMs and detecting sequence variants, which are crucial for understanding the functional properties of the antibody.

 

4. Sequence Assembly

The individual peptide sequences are assembled to reconstruct the full-length monoclonal antibody sequence, covering both the heavy and light chains.

 

Applications

The De Novo sequencing of monoclonal antibodies is essential in various applications, including:

 

1. Characterization of Novel Antibodies

For newly developed therapeutic mAbs, De Novo sequencing ensures accurate sequence determination, which is critical for regulatory approval and intellectual property protection.

 

2. Biosimilar Development

De Novo sequencing is used to compare the sequence of biosimilars with that of the original reference product, ensuring structural equivalence.

 

3. Quality Control

Monitoring sequence integrity during production to detect any sequence variations or modifications that could affect efficacy or safety.

 

4. Post-Translational Modification Analysis

Identifying PTMs, such as glycosylation or phosphorylation, that can influence the mAb's therapeutic properties.