Dr. Rodney Banwort walks through LC-MS peptide mapping for biologics, covering denaturation, digestion with trypsin, and mass spec analysis. A test on the NIST reference standard achieved 93% sequence coverage on both heavy and light chains, identifying oxidation, deamidation, and glycosylation.
Questions this video answers
Approximately 20 years.
QTFs and Orbitraps (high-field Orbitrap UPLC–MS system).
37 degrees Celsius.
93% for both heavy and light chains.
Oxidation, deamidation, glycosylation, and disulfide scrambling.
Chapters
Hello, My name is Dr. Rodney Banwort and I am a principal scientist here at Emory Pharma. Today I'm here to talk about LCMS peptide mapping of biologics. Prior to Emory, I was at ALCON and Novartis for 10 years where we studied biologics molecules by LC Ms. These molecules included antibodies, antibody fragments and therapeutic proteins. Before that I studied isolation and identification of small molecules at Pfizer in La Jolla. I have approximately 20 years total experience on LC Ms. Analysis working with instruments such as QTFs, Orbitraps. I have worked on biologic compounds from early development stage all the way through to commercialization. The peptide mapping aspect of this work characterizes sequence coverage, post translational modifications, disulfide scrambling, GMP release and sequence variants. Biologics such as monoclonal antibodies have molecular weights of more than 148,000 and and require several different analytical methods to characterize the chemical properties. By comparison, small molecule pharmaceuticals have molecular masses close to 500 and typically have one definitive HPLC method. This is not the case for biologic molecules however. The gold standard method for confirming the primary sequence of biologics is LC Ms. Peptide mapping. In this method, the protein sample is digested enzymatically and then injected into Emery Pharma's High field orbitrap UPLC Ms. System. The peptides are identified based on the observed versus expected molecular masses. In the peptide digest workflow, the protein sample is denatured followed by reduction and alkylation. The denaturation and reduction opens up the body of the protein by to allow the enzyme to access and digest the inner areas of the protein. Alkylation then prevents the cysteines from recombining. Trypsin or other enzyme is added and the sample is digested at 37 degrees. Trypsin hydrolyzes a sequence on the carboxyl side of the lysine and the arginine residues, generating peptides of a smaller size than that can be chromatographed. In the UPLCMS system, 100% coverage is not often obtained and so a frequent strategy uses a subsequent digest with an enzyme that has the complementary hydrolytic properties. Enzymes such as GLUSI can be used for this purpose. The peptide mapping chromatogram of the NIST reference material obtained here at Emery Pharma is presented on this slide. As can be seen, the result is complex with hundreds of peptides of varying intensities found, but with modern software and sufficient time, each of these peptides can be identified. This is how information on sequence coverage, post translational modifications and sequence variants is obtained at Emory Pharma. The sequence coverage found for The Nismab was 93% for both the heavy and light chains and is clearly illustrated on this slide. On the next slide, common modifications found in monoclonal antibodies and other proteins are illustrated. The detailed modifications include oxidation, deamidation, glycosylation and disulfide scrambling. In conclusion, LC Ms. Peptide mapping provides information on sequence verification, post translational modifications and sequence variants. The method is useful for early stage ID comparability and characterization studies and GMP release. Thank you and I hope this was useful information for you. If you have any more questions, please contact us@emorypharma.com for any further inquiries.
