De Novo Antibody Sequencing

De novo antibody sequencing service offered by Biologics International Corp (BIC) shows unparalleled accuracy, which can significantly advances your research in diagnostic and therapeutic fields. Combining our proprietary analysis method, bioinformatic assisted de novo sequencing, with well-established antibody database, BIC guarantees that the antibodies encoded by final sequences show the same activity as the antibodies you provide.

Want to know the how does BIC work or benefits of sequenced antibodies?

Service Highlights

• The same antibody activity guaranteed! Only few company in the world is able to make the same guarantee.
• Professional team. The BIC team, consisted of experienced MS experts, bioinformatic scientists, and high skilled stuffs, has perfectly performed dozens of de novo sequencing projects.
• High-throughput. It allows more than 500 antibody sequencing projects to be performed simultaneously, and BIC is glad to take on more of your projects, even the difficult ones.
• One-stop solution. From monoclonal antibody, antibody sequencing, to recombinant antibody, our comprehensive service can definitely save your time and money.

De Novo Antibody Sequencing Service

Customer Provides	Deliverables	Timeline
0.05 - 0.1 mg antibody sample with purity ≥ 95%	Sequencing report Mass spectrum report Experimental report	3-4 weeks

Please note: If your sample has limited quantity, lower purity or sample mixtures, contact us first. We would try our best to solve these problems.

De Novo Antibody Sequencing at BIC

Orthogonal Enzymes Digestion

5 - 8 types of proteases are chosen according to our experience, digesting antibody protein to overlapping peptides. At least 5× coverage is reached, resulting in 100% coverage.

Mass Spectrometry Equipment

At BIC, we use an advanced Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) with a 0.02 Da difference in the molecular weight. This high-tech equipment gives us many advantages, such as flexibility of fragmentation (CID, HCD and ETD are available), novel ETD HD (high dynamic range ETD provides significantly increased fragment ion coverage), and advanced peak determination (great identification of unique peptides and experimental throughput).

Peptides Analysis by LC-MS/MS

De novo antibody sequencing based on LC-MS/MS requires CID/HCD/ETD or other fragmentation to increase the coverage. In CID, peptide bonds are randomly cleaved, primarily forming b (the ions from N terminal) and y (the ions from C terminal) series ions. And HCD/ETD form c and z type ions through cleavage of the peptide bond between the amino group and alpha carbon.

For b, c, y, z or other series ions, some characteristic fragments are frequently observed which can be used for the identification of peptides. The combination of both CID and HCD in our Orbitrap Fusion MS allows that the same sample using two fragmentation techniques generates complementary series of fragment ions, resulting in a more reliable and accurate spectra result.

De Novo Sequencing

Based on our in-house algorithm, we can directly interpret spectrum obtained in high throughput MS/MS. That is very different from some other analysis softwares which generally convert the spectrum to a graph.

The basic principles:

1) Using a dynamic programming algorithm to compute thousands of peptide candidates from raw MS/MS data.

2) Combining antibody database with inputted basic search parameters, it can rebuild the peptide ion series, generating a peptide sequence with the highest score from above candidates.

3) A strict mass error tolerance (the +/- 1.5 Da range for variable regions) and a local confidence score are used to calibrate the sequence data.

4) In rare cases, if there are still some unclear residues in peptides, BIC offers a high-throughput expression method to screen for the exactly sequence.

Differentiation of Peptides With Identical Mass

Some amino acid residues, like Isoleucine (Ile) residue and leucine (Leu) residue, share exactly the same molecular weight, which are difficult to distinguished them in traditional MS. Some other cases, such as GG=N, GE=W, and Q=K, would show vague result. BIC can differentiate these amino acid residues by high quality and accurate MS. Our antibody database and enzyme diagnosis methods can also distinguish them.

Confirmation & Report Generation

After a series of well-designed sequencing process described above, BIC would provide a verification programme: an antibody-antigen binding method is employed to confirm the final sequence. This added confirmation step can make sure that the activity of antibody encoded by final sequence are the same with the antibody you provide.

At last, our scientists would confirm the result and generate the report manually.

Why Do You Need Antibody Sequencing?

• Safety. Hybridoma cell lines are easily lost due to gene loss, gene mutation or contamination. De novo antibody sequencing can avoid these problems.
• Patent applications. To get protection of your unique antibody through patent applications, sequence information is one of the first steps.
• Antibody engineering. A 100% sure sequence provides valuable data for further engineering modifications, such as antibody humanization, bi-specific antibodies, and antibody-drug conjugates.
• Recombinant antibody expression. Diagnostic or therapeutic antibody development always requires a stable cell line for producing a high amount of stable antibodies without contaminants. Accurate sequences ensure batch to batch consistency throughout research, development and manufacturing programs.

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