Monoclonal antibody molecules play a crucial role in drug discovery and diagnostic reagent development. The primary structure of the antibody molecule itself, especially the amino acid sequence of its CDR region, is the core of its biological function, and accurate and rapid analysis of its complete sequence is of great significance. For example, directly analyzing the sequence of existing antibody products on the market is a shortcut for developing antibody drugs, antibody diagnostic reagents, and other antibody products. Many commercially available antibodies have neutralizing activity against drug targets. By determining the primary sequence of the monoclonal antibody, it can be used as a basis for affinity enhancement and humanization to develop antibody drugs. In the process of developing biosimilar antibody drugs, accurate primary structural information of the original drug is the foundation and premise for biosimilar antibody drug development.
Biotechnology company Biotye Park has established a new-generation antibody sequencing platform based on the advanced high-resolution mass spectrometer Obitrap Fusion Lumos, combined with extensive bioinformatics analysis experience, to achieve rapid and accurate analysis of the primary structure of antibodies. This platform is suitable for sequence analysis of different subtypes of antibodies (e.g., IgG and IgM) and different types of antibodies (fluorescent-labeled antibodies, immobilized antibodies, antibodies from different species).
Monoclonal Antibody De Novo Sequencing Service Process
Advantages of Monoclonal Antibody De Novo Sequencing Service:
1. Full Sequence Coverage of Monoclonal Antibody Light and Heavy Chains
During the antibody sequence determination process, Biotye Park selects five different proteases for enzymatic digestion, utilizing the complementarity between different enzymatic digestion peptides to achieve 100% full sequence assembly of the light and heavy chains of the antibody molecule. For the core CDR region of the antibody, the sequence analysis process requires identification at least 10 times to fully ensure the accuracy of the sequencing results (as shown in the figure below).
Monoclonal Antibody De Novo Sequencing 2
2. Advanced Data Processing Algorithms
Biotye Park has developed its own mass spectrometry raw data calculation method based on literature published in Nature Biotechnology and the Journal of Proteome Research (JPR), in combination with professional software for antibody sequence analysis, PEAKS Mass Spectrometry Software. Using a wide-to-narrow approach, it achieves accurate identification of antibody sequences without missing any valid data information.
3. High Precision and Accuracy
During the sequencing process, Biotye Park uses the currently high-resolution and high-sensitivity Orbitrap Fusion Lumos mass spectrometer. Compared with the previous generation of mass spectrometers (e.g., Obitrap Elite, Q Exactive, etc.), Lumos improves the speed of first- and second-level mass spectrometry scans while ensuring the accuracy of peptide molecular weights. The increase in first-level mass spectrometry scan speed helps identify the same peptide multiple times, improving the reliability of the results. The increase in second-level mass spectrometry scan speed allows for better fragmentation, ensuring result accuracy, and enables accurate determination of leucine and isoleucine through characteristic peaks of secondary fragment ions (as shown in the figure below).
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4. Sequencing Capability for Various Subtypes and Different Types of Antibodies
Our established technology platform is not affected by small groups (such as FITC, Biotin, Alexa), although larger protein groups pose challenges to sequencing. However, if a higher concentration of protein is provided, successful sequencing is still possible.
5. Rigorous Sequence Prediction and Strict Sequence Verification
We first conduct sequence prediction on the sequencing results, marking the CDR region of the antibody (as shown in the figure below). Meanwhile, we establish a new database with the determined sequence and re-analyze the collected mass spectrometry data using the Sequence Confirmation mode. We only provide results to clients that are completely matched.
Monoclonal Antibody De Novo Sequencing 4
Bilingual Project Report in Chinese/English
In the technical report, Biotye Park Biotechnology will provide you with a detailed technical report in both Chinese and English, including:
1. Experimental Steps (Chinese and English)
2. Relevant Mass Spectrometry Parameters (Chinese and English)
3. Mass Spectrometry Images
4. Raw Data
5. Peptide Sequence Information
6. Antibody Sequence Information
