The accuracy of antibody sequencing determines the success or failure of biopharmaceutical projects. In antibody development, sequence errors may lead to loss of activity or invalidation of patents; while at the intellectual property level, unvalidated sequences may lead to infringement lawsuits or loss of assets. Therefore, choosing professional sequencing services is not only a technical requirement, but also a core strategy to avoid R&D risks and protect business value.
When choosing antibody sequencing services, it is important to consider whether the service provider's technology platform (e.g., NGS deep sequencing, mass spectrometry, or a combination thereof) can accurately analyze the complex sequences of the antibody, especially the highly variable complementary decision region (CDR). Their sequencing depth, coverage, error rate, and ability to handle difficult samples need to be evaluated.
Successful antibody sequencing needs to be examined to see if it has a professional technical support team that can provide customized solutions according to project needs (e.g., full-length sequencing, variable region sequencing, pairwise information acquisition), clear data reporting formats (e.g., FASTA, annotation files), and professional consulting on results interpretation.
The Importance of Antibody Sequencing
Promoting Biopharmaceutical Innovation
Antibody sequencing is an integral and central part of the antibody drug development pipeline. Its importance is reflected in
Dissecting the structure of lead antibodies
In the antibody drug discovery stage, the precise determination of the amino acid sequence of antibody candidates, whether obtained by hybridoma technology, phage display, single B-cell sorting or other high-throughput screening methods, is required.
Only by obtaining the complete sequence can we gain an in-depth understanding of its structure-function relationship and predict its key attributes such as affinity, specificity and immunogenicity. This is the basis for subsequent engineering modifications, large-scale production process development and clinical trials.
Ensuring the quality of biosimilars
For original antibody drugs with expiring patents, the development of biosimilars is an important way to reduce healthcare costs. Biosimilars must be highly similar to the original drug in terms of quality, safety and efficacy. Antibody sequencing services are a core step in reverse engineering, providing an accurate "blueprint" for cell line construction, manufacturing process development and comprehensive comparability studies by accurately determining the amino acid sequence of the original antibody.
Patent avoidance and intellectual property Layout
Accurate antibody sequence information is the key to patent layout and avoidance of existing patents. By clarifying the sequence characteristics of the original antibody through sequencing, it helps to design new molecular entities with differences (e.g., changing the key amino acids in the CDR region) but retaining or enhancing the therapeutic effect, thus forming independent intellectual property rights.
Promoting Basic Research
Antibody sequencing services greatly empower basic immunology research and translational medicine exploration
Resolving immune response mechanisms
Sequencing B cell receptor (BCR) libraries by high-throughput sequencing technologies (e.g., NGS) can provide a comprehensive understanding of the diversity of an individual's antibody libraries in the state of infection, vaccination, and autoimmune disease.
Combined with functional analysis, rare antibodies with specific functions can be identified, providing key clues for understanding immune protection mechanisms and vaccine design.
Driving the development of novel antibody therapeutics and tools
Antibody fragments with specific functions (e.g., nanobodies, bispecific antibody prototypes, and targeting portions of antibody-coupled drugs) require sequencing to determine their sequences, a necessary step in translating them from laboratory discovery to potential therapeutic tools.
Mining valuable natural antibodies
Natural antibodies with specific properties isolated from immunized animals or human libraries are valuable biological resources.
After obtaining their precise sequences through antibody sequencing, recombinant expression, functional validation, and engineering optimization can be carried out
Types of Antibody Sequencing
Classification by Sequencing Target
Sequencing of hybridoma cells is performed by extracting cellular RNA/DNA, amplifying antibody genes (e.g., heavy and light chains) by RT-PCR, and resolving the sequences by combining with Sanger or NGS technology, which is suitable for preserving precious hybridoma monoclonal antibodies, and has the advantage of directly obtaining highly accurate sequences.
Single B-cell sequencing, on the other hand, isolates single B-cells, amplifies the variable region by single-cell PCR or 5'RACE, and captures natural antibody sequences by high-throughput platforms (e.g., 10x Genomics), which is especially suitable for the development of human antibodies and screening of rare antibodies.
Direct Protein Sequencing (Mass Spectrometry) Breaking through the genetic limitation, analyzing enzymatically cleaved peptide fragments by high resolution mass spectrometry (e.g. Orbitrap), together with the software such as PEAKS, splicing the amino acid sequences from the beginning, which can accurately analyze the modified antibodies (e.g. glycosylation sites, disulfide bonds).
Categorized by Sequencing Technology
Sanger sequencing is used as the gold standard to amplify specific gene fragments through primers, with an error rate of less than 0.001%, which is suitable for full-length sequencing of monoclonal antibodies, but with low throughput and high cost.
High-throughput sequencing (NGS) Parallel processing of a large number of samples based on platforms such as Illumina, which can tap into the diversity of antibody libraries, suitable for large-scale screening of hundreds of B cells, and needs to rely on bioinformatics to weed out errors.
Mass spectrometry ab initio sequencing combined with algorithms such as DASS/BADS to directly resolve amino acid sequences, capable of distinguishing equivalent amino acids (e.g., leucine/isoleucine), is the preferred solution for non-traditional antibodies (IgM, nanoantibodies), but requires stringent requirements for sample purity (>90%).
Classification by Sequencing Scope
Variable region sequencing focuses on the core functional regions of the antibody (VH/VL and CDR regions) and is used for mechanistic studies such as affinity optimization, with lower cost and shorter cycle time.
Full-length sequencing covers all gene sequences of the light and heavy chains (including the constant region), which provides complete data support for recombinant expression and patent filing, and industrial-grade projects need to verify the modification sites by mass spectrometry to comply with regulatory requirements.
Classified by Application Scenarios
Basic scientific research service mostly adopts Sanger sequencing + variable region analysis to meet the needs of laboratory hybridoma preservation or thesis publication, typical cases such as mouse monoclonal antibody sequence acquisition.
Industrial applications require NGS coupled with mass spectrometry to ensure the reliability of therapeutic antibody humanization or diagnostic reagent production, and the entire process must comply with GMP standards.
Special sample type for modified antibodies or rare samples (e.g. camel nanoantibodies), combining single B-cell sequencing and mass spectrometry validation technology, typical examples include the precise analysis of fluorescently labeled antibodies.
Combining traditional antibody library screening with high-throughput sequencing (Figure from Parola, C., 2018)
Key Factors in Selecting Antibody Sequencing Services
Technology-Sample Match
Sample attributes determine technology pathway selection, and a wrong match will result in sequencing failure or distorted data.
Technology adaptation scenarios
- Hybridoma sequencing: only for live hybridoma cells (>10⁶ cells or high-quality RNA is required), if the cells have been lost or contaminated, then you need to switch to mass spectrometry.
- Ab initio sequencing by mass spectrometry: requires high purity protein (>95%, concentration ≥0.5 mg/mL), suitable for frozen antibodies, serum poly-antibodies or rare samples that cannot be regenerated.
- Single-cell sequencing: relies on fresh B cells (>90% activity), fixed or frozen cells will result in missing data due to RNA degradation.
Differences in sample pretreatment
- Hybridoma sequencing requires inhibition of nuclease activity at the RNA extraction stage
- Mass spectrometry sequencing requires avoidance of protein solutions containing detergents (e.g., SDS), which can inhibit enzymatic efficiency
- Single-cell sequencing requires library construction to be completed within 24 hours of sampling
- Non-humanized antibodies (e.g., alpaca-derived single-domain antibodies) need to be confirmed for recognition of species-specific framework regions by the sequencing platform to ensure accurate alignment and analysis.
Data Reliability Validation
Data accuracy is the cornerstone; specify data quality assessment criteria (e.g., sequence coverage, error rate, splice accuracy report) and review example reports or validation data to ensure that the results are authentic and trustworthy.
Accuracy guarantee
- Hybridoma Sequencing: The main risk of error faced by this technology stems from preferential amplification during PCR. The industry validation protocol is to perform three reverse transcription-polymerase chain reaction (RT-PCR) cloning procedures and validate the final clones with bidirectional Sanger sequencing.
- Mass Spectrometry Sequencing: The limitation of this method is the difficulty in distinguishing between structurally similar leucines and isoleucines. Industry validation is performed by combining high-energy collisional dissociation fragmentation ion spectroscopy (HCD), electron transfer dissociation fragmentation ion spectroscopy (ETD), and mass spectrometry comparison of the synthesized target peptides.
- NGS High-Throughput Sequencing: The sequencing error rate inherent in next-generation sequencing technologies (usually around 0.1%) is their main source of error. It is common for the industry to introduce molecule-specific barcoding during the library construction process, and to correct errors by clustering and comparing sequencing reads that originate from the same molecular source.
In-depth validation requirements
- Coverage report: heat map of peptide coverage required for mass spectrometry (target: heavy chain > 98%, light chain > 95%)
- Modification analysis: glycosylation sites need to be verified by PNGase F digestion, oxidative modifications need to be reduced alkylation control
- Functional confirmation: final delivered sequences must be accompanied by recombinant expression validation (affinity ± 15% protoanatomics)
Application Scenario Synergy
Customize the depth of service based on end-use objectives: patent filing requires full-length sequence and CDR coordinates and screening for global patent conflicts; biosimilar development requires glycosylation site and charge variant analyses; diagnostic reagent production requires stability mutation prediction and expression system optimization solutions.
Cost Considerations
Budgets are important constraints. Conduct cost comparisons of different services to understand the pricing differences and services included in basic sequencing, deep sequencing, and biosignature analysis.
How to Choose Different Service Providers
Data Analysis and Interpretation Support
Antibody sequencing is not only about sequence determination, but also about subsequent data analysis and interpretation. Service providers should have strong data analysis capabilities and be able to analyze antibody sequences in depth through professional bioinformatics tools.
We adopt DASS technology to generate a large amount of sequence information through the use of mass spectrometers and data processing algorithms, and then apply computational programs to sequence MS/MS spectra from scratch to ensure sequence accuracy and comprehensive sequence coverage, so as to realize precise interpretation of antibody amino acid sequences.
Personalization and Customization of Services
Antibody sequencing needs vary depending on the purpose of the study, experimental design, and budget. Companies that choose to offer personalized and customized services are able to provide solutions that are tailored to specific research needs.
We can tailor our solutions to meet the needs of our clients. Whether it is the integrated analysis of single cell data or the option to measure only the light chain, heavy chain or variable region of an antibody, we can customize it.
Our process can be adapted every step of the way, from the choice of sequencing platform to the optimization of sequencing depth, from the format of data delivery to the implementation of strict confidentiality agreements.
Technical Support and Experience of the Service Provider
Our professional technical team can provide detailed program design before sequencing to ensure the accuracy of the sequencing process and provide timely technical support during data analysis.
Our core team consists of experienced proteomics and antibody engineering experts who have been specialized in antibody sequencing for many years. We not only have a large number of successfully delivered complex antibody sequencing cases (including monoclonal antibody, bis-antibody, nanoantibody, polyclonal serum, etc.), but also published relevant technical methods and application studies in international peer-reviewed journals, which have gained the recognition of the academic community for our technical strength.
Our team can provide you with advice on experimental design optimization (e.g., sample pretreatment, enzymatic digestion strategy), and even assist in the design or optimization of sequencing primers when needed. Leave your challenges to our specialized antibody sequencing experts, and you will receive reliable technical assurance and professional security.
Selected recent approaches aiming toward MS-based de novo sequencing of serum antibodies (Figure from Sebastiaan C. de Graaf, 2022)
References
- de Graaf, S. C., Hoek, M., Tamara, S., & Heck, A. J. R. (2022). A perspective toward mass spectrometry-based de novo sequencing of endogenous antibodies. mAbs, 14(1). https://doi.org/10.1080/19420862.2022.2079449
- Parola, C., Neumeier, D. and Reddy, S.T. (2018), Integrating high-throughput screening and sequencing for monoclonal antibody discovery and engineering. Immunology, 153: 31-41. https://doi.org/10.1111/imm.12838
For research use only, not intended for any clinical use.
