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Creative Biolabs is gladly to show a series of unrivalled de novo antibody sequencing services for research, diagnostic and therapeutic industries. To overcome the current drawback of sequencing based on traditional methods, Creative Biolabs has developed the proprietary Database Assisted Shotgun Sequencing (DASS) technology, which based on our next generation antibody sequencing platform. Our services consist of variable region, variable plus leader region and full length heavy- and light-chain antibody sequencing for all species, isotypes and allotypes. Purified monoclonal antibodies in multivalent forms can be sequenced with 100% coverage of the desired regions as well as excellent accuracy. Numerous successful cases from Creative Biolabs have confirmed our qualification to provide antibody sequencing with 100% accuracy and satisfaction guarantee to meet our customers’ needs.
Our world-leading DASS system provides reliable supporting proof for:
• Confirmation & validation of produced mAb
• QC analysis
• IND/NDA application
Fig. 1 De Novo Antibody Sequencing Workflow
Sequencing of the V and J and C segments by DASS
The V and J and C gene segments of antibodies are available in public databases. However, during the maturation of an antibody, the B cell introduces hypermutations into the sequence to optimize the affinity. Our mapping algorithm is error tolerant and can match the “mutated” peptides to the corresponding germline, reliably.
Due to the high number of peptides, we get sequence information for EVERY peptide bond in the antibody. Typically, 20-70 different MS/MS spectra are generated for each amino acid (AA) position. Hence, even the hardest sequences of proline and arginine rich peptides can be resolved. As the order of all amino acids is clear, there is no need for time consuming techniques like edman sequencing. If we do not reach 100 % coverage, you will not be charged.
De Novo sequencing of the CDR3 region
While the CDR3 of the light chain is mostly encoded by the germline sequences, the CDR3 of the heavy chain is usually not available in databases. It is encoded by the so called D-segments but these are modified by nucleases and terminal transferases. Typically, only 1-4 AA of a D-segment remain in the matured antibody. The rest of the D-segment is “artificial” and has to be sequenced de novo.
Our method generates many overlapping peptides during the fragmentation process, enabling us to sequence very long stretches of unknown amino acids. The high quality of MS/MS spectra in combination with intelligent data mining, allows us to read the CDR3 like a book. The technique is so powerful, that we were able to sequence a 20 kDa protein, which had no homologue in the database.
Isobaric amino acids
In contrast to other MS based methods we can discriminate most isobaric amino acid combinations. E.g.:
• W can be distinguished from GE, AD and SV (by mass difference) .
• R can be distinguished from GV (by mass difference) .
• Q can be distinguished from GA and K (by fragment spectra and mass difference) .
• N can be distinguished from GG (by derivatization and fragment spectra) .
• Leucine and isoleucine cannot be distinguished. However, most of these positions can be determined using the corresponding germline sequence (see Fig. 1). As antigen binding is mostly mediated by salt bridges and hydrogen bonds the impact of Leu/Ile is usually negligible.
Sequencing of fluorochrome mAbs, IgMs and other non-standard antibodies
• Our method is usually not affected by small ligands (FITC, Biotin, Alexa) coupled to antibodies. Larger protein ligands make sequencing slightly more difficult. However, this can be compensated by using a higher protein concentration, because sequencing quality is dependent on sample amount.
• IgMs can usually be sequenced like normal IgGs. Slightly more sample may be required. As the constant region is modified by several glycans, which cannot be cleaved by PNGase F, we can only guarantee complete coverage for the variable part of the antibody.
• Our method works best with mouse and rat antibodies. However, ~50 antibodies from rabbit, hamster and lama have been sequenced successfully.
• Some mAbs are coupled to matrices. This is compatible with our sequencing method but more sample may be required.
• Many older hybridoma produce 2 light chains (one is a Kappa-chain of myelom MOPC 21). These mixtures can usually be sequenced, even when the MOPC 21 kappa chain is in twofold molar excess.
Fig. 2 Sequencing result from PEAKS mass spectrometry software. Grey/Black: antibody sequence. Blue: perfect peptide matches with a mass deviation of < 4 ppm.
Validation of your sequence
Before we start sequencing, the masses of the reduced light- and heavy-chain will be determined. The masses of the intact chains serve to validate the determined sequences. The light chain sequence must match within a +/- 1.2 Da range. For the heavy chain +/- 1.8 Da are allowed. If the determined sequences do not match with the protein mass, you will not be charged.
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