Antibody Sequencing for Monoclonals, Hybridomas, Biosimilars
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Antibody sequencing plays a foundational role in biopharmaceutical innovation by enabling accurate analysis of antibody structure at the molecular level. As monoclonal antibody development, hybridoma platforms, and biosimilar research continue to expand, sequencing technologies have evolved from basic tools into essential components of R&D workflows.
Traditional methods such as Edman degradation and Sanger sequencing have given way to advanced platforms like next-generation sequencing (NGS) and high-resolution mass spectrometry (MS), which offer higher throughput and more complete sequence coverage. In recent years, the integration of single-cell technologies and AI-driven analysis has further shortened discovery timelines and enhanced sequencing precision.
Precise antibody sequencing is foundational to every stage of biologics discovery and therapeutic development. The amino acid sequence of an antibody defines its three-dimensional structure, antigen-binding specificity, and overall functional integrity. Errors in sequence data can compromise expression, reduce therapeutic efficacy, or even introduce unexpected immunogenic responses.
By using specialized, high-accuracy sequencing platforms, researchers can avoid such risks and ensure that antibody candidates are both functional and safe. Reliable sequence data is essential not just for product performance but also for regulatory compliance and intellectual property protection.
Unlike genetic sequencing methods such as NGS, which infer protein structure from nucleic acid templates, mass spectrometry (MS) directly analyzes the physical antibody protein itself. This allows for label-free, reference-independent (ab initio) sequencing, providing a clear picture of the actual protein produced—especially critical when post-translational modifications (PTMs) are involved.
Advanced tandem MS/MS techniques, paired with expert bioinformatics analysis, allow for confident identification of:
These insights are crucial for functional validation, comparability studies, and downstream engineering.
1. Monoclonal Antibody (mAb) Validation
MS-based sequencing confirms that expressed antibody products match the intended design and helps detect cloning or expression-derived mutations early, ensuring product fidelity.
2. Hybridoma Rescue & Intellectual Property Protection
Sequencing antibodies directly from hybridoma supernatants enables preservation of sequence data even if cell lines are lost, securing both research value and patent rights.
3. Biosimilar Development & Analytical Similarity
For biosimilar programs, MS sequencing ensures that candidate molecules match originator antibodies not only in primary structure but also in PTM profiles (especially glycosylation), supporting regulatory comparability.
In monoclonal antibodies (mAb), accurate validation of their amino acid sequences is critical, but three major challenges are often faced: sequence drift (accidental mutations during cell culture), loss of original records, and batch-to-batch variation. These issues can disrupt antibody function and delay the development process.
High-fidelity sequencing provides a verified reference for downstream engineering. Since antibody engineering depends on the precision of the input sequence, any error at this stage can result in design failure or deviation from intended product characteristics.
In early-stage development, sequence confirmation serves as a critical quality control (QC) checkpoint. It ensures molecular consistency across cell line construction, process development, and early GMP manufacturing—forming a reliable foundation for subsequent characterization and comparability studies.
Mass spectrometry plays an irreplaceable role in this segment - it directly physically analyzes the expressed protein product. Through high-precision LC-MS/MS peptide mapping and de novo sequencing, we are able to:
Herceptin (trastuzumab) is a marketed humanized therapeutic monoclonal antibody targeting the HER2 receptor. In this study, it was used as a benchmark sample to validate the accuracy and reliability of mass spectrometry-based ab initio antibody sequencing methods. The goal was to establish a standardized, reproducible workflow for sequencing antibodies with unknown structures.
A bottom-up LC-MS/MS strategy was employed, integrating two key innovations:
1. Parallel Protease Digestion
Nine complementary proteases (e.g., trypsin, coagulation protease, elastase) were used in parallel under unified buffer conditions. This generated overlapping peptide fragments, enhancing overall sequence coverage and reducing blind spots in difficult regions.
2. Dual Fragmentation Strategy
Simultaneous use of:
This approach maximized sequence data by generating complementary ion types and leveraging different fragmentation mechanisms across all peptide precursors.
3. Data Processing
Raw MS/MS data were processed using Supernovo software. The software assembled sequences de novo, then iteratively refined them against antibody germline sequence templates to correct for somatic mutations.
Mass spectrometry-based de novo sequencing of the monoclonal antibody herceptin. (Figure from Weiwei Peng, 2021)
Common challenges in hybridoma technology: accidental loss of cell lines, low antibody secretion capacity, or missing sequence information due to genetic instability of cell clones
Directly analyze secreted antibody proteins in hybridoma cell culture supernatants. Even if a cell line fails to survive or expand, the presence of trace amounts of secreted antibodies in the supernatant can be resolved using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Enzymatic cleavage, peptide isolation and mass spectrometry acquisition, combined with de novo de novo sequencing algorithms, allow reconstruction of the complete antibody sequence directly from the physical molecules of the protein.
It does not rely on intact cells or stable genetic material (RNA/DNA), making it especially valuable in cases where viable cells are unavailable, nucleic acids are degraded, or antibody secretion is minimal.
Recombinant Antibody Production: Successful resolution of the sequence enables cloning into mammalian expression vectors (e.g., CHO cells) for stable, high-titer, scaleable recombinant antibody production.
Preservation of valuable traditional antibodies: For hybridomas with important research value but at risk of being lost, this method is an effective way to salvage their sequences, preserve their intellectual property and promote their further development.
The hybridoma-derived monoclonal antibody to the plasma membrane of human breast cancer, 139H2, has been a valuable research tool for Western blotting, ELISA, IHC, and IF, but the lack of sequence information has hindered its widespread use, necessitating the sequencing of this functional antibody to enable recombinant production.
LC-MS/MS-based bottom-up proteomics is the core analytical technique.
De novo sequencing of the hybridoma 139H2 based on bottom-up proteomics. (Figure from Weiwei Peng, 2024)
In biosimilar development, precise structural analysis of the original antibody (reference drug) is a core requirement to ensure similarity.
While traditional methods are limited by patent information barriers or reverse engineering uncertainties, mass spectrometry provides a direct and objective means of analysis.
Combination of bottom-up strategy through high-resolution LC-MS/MS:
The expiration of the original patent on recombinant human erythropoietin (rHuEPO) has led to the entry of a large number of biosimilars into the market, and these complex glycoproteins exhibit significant heterogeneity in glycosylation patterns due to different production systems and processes. This heterogeneity poses a challenge for drug quality control. Reliable methods to characterize, differentiate and identify these closely related rHuEPO products are therefore urgently needed.
The core analytical technique was performed using nanoLC-ESI-MS/MS (nano-liquid chromatography-electrospray ionization-tandem mass spectrometry) on the LTQ-Orbitrap Velos Pro platform.
Erythropoietin Biosimilar Profiling Flowchart (Figure from Mohd Afiq Hazlami Habib, 2019
We use LC-MS/MS as a core strategy for in-depth analysis of intact antibody molecules or complex peptide mixtures after their enzymatic digestion. The experimental procedure typically includes: sample reductive alkylation, enzymatic cleavage, peptide F separation, and finally the collection of precise mass-to-charge ratio data of the peptide parent ion and its fragment ions. This strategy is capable of comprehensively capturing key information about the primary sequence of the antibody and its post-translational modifications.
Based on the raw mass spectrometry data, we first performed Peptide Mapping, which is a preliminary method to confirm the known sequence regions by comparing the obtained peptide masses with the theoretical database.
For unknown sequences or regions with variations, we use De novo Sequencing: based on high quality MS/MS spectra, we directly analyze the b/y ion series generated by peptide breaks, and combined with bioinformatics algorithms, we obtain the order of amino acids, and realize the sequence assembly of the complete variable and constant regions.
Protein molecules themselves are directly characterized without relying on host cell lines, DNA templates or RNA. This feature makes it irreplaceably valuable in scenarios where traditional methods fail, such as when hybridoma cell lines are unstable, lost, or cannot be cultured, or when there is a need to directly characterize a final protein product that has undergone complex engineering modifications or post-translational modifications. Sequence resolution can be performed as long as trace amounts of the target protein are available.
Highly purified monoclonal antibodies are the ideal sample type. In addition, partially purified antibodies, hybridoma cell culture supernatants, and serum samples of polyclonal antibodies can be analyzed as effective starting materials.
References
For research use only, not intended for any clinical use.