De Novo Monoclonal Antibody Sequencing: Technical Analysis, Advantages, and Applications
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Purification and quality assessment of antibody protein samples;
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Multi-enzyme digestion (e.g., trypsin, chymotrypsin) to generate high-coverage peptide fragments;
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Acquisition of peptide fragmentation spectra using high-resolution mass spectrometry;
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Sequence inference using De Novo sequencing algorithms (such as PEAKS or Novor);
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Peptide assembly and full-length antibody sequence reconstruction;
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Manual curation, post-translational modification identification, structural alignment, and optional expression validation.
What Is De Novo Monoclonal Antibody Sequencing?
De Novo antibody sequencing is a technique that reconstructs complete monoclonal antibody sequences directly from protein samples, without relying on any reference gene or sequence databases. Using high-resolution liquid chromatography–tandem mass spectrometry (LC-MS/MS), enzymatically digested antibody peptides are analyzed, and algorithms are applied to interpret MS/MS fragment spectra and progressively deduce the corresponding amino acid sequences.
In contrast to traditional sequencing approaches that depend on database matching, De Novo sequencing starts from protein-level data and enables full structural reconstruction. This is particularly valuable for antibodies, whose most functionally critical elements—the variable regions, especially the complementarity-determining regions (CDRs)—exhibit extreme sequence variability and are often absent or underrepresented in public databases. Consequently, De Novo sequencing offers a uniquely effective method for characterizing such highly diverse structures.
A comprehensive De Novo monoclonal antibody sequencing workflow typically involves the following steps:
Technical Advantages: Why Choose De Novo Monoclonal Antibody Sequencing?
1. Independent of Genetic Material and Expression Constraints
De Novo sequencing is particularly advantageous for antibody samples derived from clinical sources, immunized animals, or commercial providers, where the original B cells or expression constructs are inaccessible. Unlike gene-based sequencing approaches, this method requires no DNA or RNA information and is the only viable solution for sequence determination in such contexts.
2. High Compatibility with Sequence Variability, Including CDR Regions
The antibody variable region—especially CDR1 to CDR3—comprises the primary functional sites but exhibits extensive sequence diversity that makes database matching ineffective. By analyzing actual fragment ion patterns, De Novo sequencing enables accurate identification of novel mutations and unique structural variants, making it ideal for antibody engineering, humanization, and affinity optimization.
3. Simultaneous Detection of Post-Translational Modifications
Antibodies often undergo critical post-translational modifications (PTMs), such as glycosylation, deamidation, and oxidation, which influence their stability and function. De Novo sequencing not only reveals the linear amino acid sequence but also detects the location and type of PTMs, thereby providing insights into the antibody's native conformation as expressed in the production system.
4. Direct Utility in Recombinant Expression and Structural Reconstitution
The peptide sequences obtained through De Novo analysis can be reverse-engineered into cDNA templates for recombinant expression. This makes the technique particularly valuable for reproducing legacy antibodies, validating functional properties, modeling three-dimensional structures, and designing patent-evading variants.
Practical Application Scenarios: From Scientific Research to Industrial Translation
1. Sequence Recovery of Legacy Antibodies
In cases where a functional antibody is already available in the laboratory but its original source is unknown or no longer accessible, De Novo sequencing enables full sequence reconstruction. This allows for recombinant expression or patent filing by effectively re-establishing the functional antibody structure.
2. Cross-Vendor Antibody Comparison and Validation
De Novo sequencing can be employed to evaluate whether antibodies with the same catalog number from different manufacturers are structurally identical. It helps detect potential mutations, post-translational modifications, or batch-to-batch variability that may affect product consistency.
3. Consistency Assessment in Therapeutic Antibody Development
This technique supports the structural comparison and consistency evaluation of antibody products expressed in different host systems (such as CHO and HEK293 cells) or under varying process conditions. The resulting data can be used to satisfy regulatory requirements for IND or BLA submissions.
4. Rational Patent Strategy and Antibody Design
By characterizing the structural features of patented therapeutic antibodies through De Novo sequencing, researchers can design sequence-divergent yet functionally similar alternatives. This facilitates strategic planning in intellectual property (IP) management during novel antibody drug development.
5. Antibody Discovery in Vaccine and Immunological Research
De Novo monoclonal antibody sequencing allows for the identification of high-affinity antibody candidates directly from immunized animal serum. Key CDR sequences can be recovered for recombinant expression, high-throughput screening, and subsequent affinity maturation.
MtoZ Biolabs’ De Novo Antibody Sequencing Solution
At MtoZ Biolabs, we have developed a dedicated De Novo sequencing platform optimized for antibody structural analysis. By integrating experimental design, mass spectrometry, and bioinformatics, we deliver high-coverage, high-accuracy, and highly reproducible antibody sequence data for every project.
1. Parallel Multi-Enzyme Digestion: Enhances sequence coverage in structurally complex or variable regions such as complementarity-determining regions (CDRs).
2. Dual Mass Spectrometry Platforms (Orbitrap and timsTOF): Combines high resolution and high throughput to ensure data quality.
3. Proprietary Assembly Algorithms with Manual Curation: Ensures precise and confident sequence reconstruction.
4. Extended Analytical Modules: Includes post-translational modification identification, structural modeling, and sequence comparison to support preclinical studies and IND-enabling research.
5. Optional CHO-Based Expression Validation: Enables closed-loop verification from sequence to expressed functional antibody.
To date, we have successfully completed hundreds of antibody sequencing and expression projects for pharmaceutical companies, academic institutions, hospitals, and vaccine developers. Applications include legacy antibody sequence retrieval, therapeutic antibody comparability assessment, structural differentiation analysis, and immune repertoire profiling.
In scenarios where antibody functionality is known but sequence information is unavailable, De Novo monoclonal antibody sequencing offers a robust and scientifically validated pathway for sequence reconstruction. Beyond making the sequence accessible, it enables structural control, reproducible expression, and strategic patent positioning. If you are working with antibody samples of unknown origin or are engaged in expression, re-engineering, or comparability studies, De Novo sequencing should be considered a core enabling technology. Please contact the MtoZ Biolabs technical team for sample evaluation, project consultation, and reference reports.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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