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J Biomol Tech. 2010 September; 21(3 Suppl): S18.
PMCID: PMC2918105

Results of the PSRG 2010 Study: Edman and Mass Spectrometric Terminal Sequencing of a Monoclonal Antibody

B. Xiang,4 J. Walters,5 K. Mawuenyega,6 and J. Simpson7
1Genetech, Inc., South San Francisco, CA, United States;
2University of Texas Medical Branch, Galveston, TX, United States;
3University of Zurich, Zurich, Switzerland;
4Monsanto Company, St. Louis, MO, United States;
5Sigma-Aldrich, St. Louis, MO, United States;
6Washington University School of Medicine, St. Louis, MO, United States;
7National Cancer Institute, Frederick, MD, United States

Abstract

r9-1

N-terminal sequence analysis is an indispensable bioanalytical tool in the protein chemistry laboratory. N-terminal analysis is necessary for the quality control of protein biologics, for determining sites of biologically relevant proteolytic cleavage events, and is vital for the de novo characterization of monoclonal antibodies. Automated Edman degradation has historically been the method of choice for these analyses, though in recent years alternative mass spectrometric methods for determining N-terminal sequence have surfaced.The PSRG 2009 study made comparisons between the established Edman degradation techniques and alternative MS based techniques. Both methodologies were able to obtain N-terminal sequence information for the proteins provided however the study indicated reliance on protein databases to obtain N-terminal data. For the 2010 study, the PSRG distributed an intact monoclonal antibody. The variable regions for the heavy and light chains, which include the N-termini, are not recorded in the protein databases. The complexity of this sample challenged participants with a molecule containing two N-termini; one of which is N-terminally blocked. A total of 46 participants requested samples. Participants were provided with two vials containing 50ug each of a commercially available antibody. Participants were told that the sample was an intact antibody molecule. The participants were asked to determine as many amino acids of the N-terminus as possible from both termini by their method of choice, and were encouraged to try multiple methods for sequence elucidation. Study participants were directed to a website to anonymously upload data. The analysis of the results of the 2010 study focuses on the length and accuracy of the sequence calls reported by the participants, and a comparison of the results demonstrating some of the advantages and disadvantages of the classical Edman chemistry and alternative (MS based) technologies are noted. Information on the type of instruments and protocols are also reported.


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