Dr. Rodney Banward walks through intact-mass and subunit analyses of monoclonal antibodies using high-field Orbitrap UPLC-MS. The method achieves ±6 ppm mass accuracy on intact protein and identifies modifications like glycans, lysine clipping, and pyroglutamate. These workflows can transfer to cheaper instruments for routine quality control.
Chapters
Hello, my name is Dr. Rodney Banward and I am a principal scientist here at Emory Farmer. Today I'm talking about LC Ms. Intact and subunit analyses of biologics molecules. Prior to coming to emory, I had 10 years of LCMS analyses of biologic molecules at ALCON and Novartis. We analyze molecules such as monoclonal antibodies, antibody fragments and therapeutic proteins. Before that, I perform isolation and identification of small molecules at Pfizer. I have over 20 years experience of LC Ms. Working primarily with QTFs and Orbitrap instruments. I have worked on biologic compounds from the early development stage all the way through to commercialization. Biologics such as monoclonal antibodies have molecular weights of more than 148,000 Daltons and require several different analytical methods to characterize their chemical properties. By comparison, small molecule pharmaceuticals have molecular masses closer to 500 Daltons and typically have one definitive 8HPLC method. This is not the case for biologic molecules. However. LC MF's intact mass determination is a straightforward and easy to perform analysis that is useful throughout the whole product development cycle from early discovery up through and including commercialization. Indeed, the consistency of drug production is supported by the consistency of intact mass results throughout the project lifetime. The information obtained from the LC Ms. Intact mask analyses includes verification of the primary sequence, major glycosylations, N and C terminal modifications, glycation, GMP release characterization and comparability studies. In this method, the protein sample is diluted with buffer and then injected into Emery Pharma's High Field Orbitrap UPLC Ms. System. We used two different chromatographic methods, one using a C4 column and the other using a guard column. The methods produced equivalent results. The deconvoluted spectrum is shown here. The observed masses are consistent with extensive C terminal lysine clipping, N terminal pyroglutamate formation and the major glycans. The only sample preparation step was the dilution of the NISMAB with bond buffer. The mass accuracy attained on our Orbitrap Explorers 240 was plus or minus 6 ppm, an excellent result. The glycans of interest are shown on this slide. They consist of N acetylglucosamine, mannose, fucose, galactose and sialic acid. Subunit analyses of mabs can characterize more detailed modifications than are accessible in the simple intact LCMS experiment, but with a minimum of sample preparation. In a one hour digest, the MAB is treated with the I enzyme and reduced with TCEP, producing three subunits each with molecular masses of approximately 24 kilodaltons. The results on our case study of the NIST MAB are presented here. In addition to the modifications characterized in the intact study, glycation, lysine modifications and a glycosylation are observed. Again, the mass accuracy in our experiment was excellent at + or -3.1 ppm. As can be seen, the study demonstrated that hexose modification is located on the light chain. The reduction of interchange disulfide bonds separates the heavy and light chains chemically. The resulting chromatogram is presented here. Localized and defilled information A modification such as glycation of the light chain is obtained. The D glycosylated MAB was obtained after treatment with the enzyme PNGASF at 37 degrees Celsius. Some protocols call for this information because this answers the question what is the mass of the protein without any glycosylation? Here we summarize the type of information obtained through intact and subunit LC Ms. Analyses. At the top, the intact mass can be determined with either a reverse phase or size exclusion. LC Ms. Analysis information on major glycosylations, lysine clipping and pyroglutamate formation is obtained. The SAC method is also useful for analyzing the drug to antibody ratio. For antibody drug condensates with reduced or decalcosylated proteins, additional characterizations become available. Glycation and lysine additions and more in depth glycosylation information became available. The most detailed information came from samples which were reduced and digested with the eyes enzyme. Fourteen traits, seven minor and two major modifications were observed. In summary, the LC Ms. Analyses of the intact and subuno biologic molecules are simple analyses which characterize detailed modifications of complex molecules. This information rich study is useful throughout the product development cycle from research to commercialization including GMP product release comparability and characterization studies. While our work utilized a modern high field orbitrap, the method can be transferred to a low cost mass spectrometer such as a single quadrupole instrument. The low cost mass spectrometers can be affordable for QC labs. Thus, the intact LC Ms. Analyses can be utilized in ID tests and commercial release testing. Thank you and please contact us@emripharma.com with any further questions or inquiries. It.
