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Determination of the Primary Sequence/ Structure



Catherine A. Mouchahoir, Mellisa Ly, Michaella Levy, Lisa E. Kilpatrick, Scott C. Lute, Karen W. Phinney, Lisa Marzilli, Kurt A. Brorson, Michael T. Boyne, Darryl Davis, John E. Schiel


The primary sequence of a protein, including therapeutic monoclonal antibodies (mAbs), is a critical quality attribute that determines a great deal of its functionality and stability. Significant effort is devoted to determining the complete amino acid sequence of recombinant proteins of potential therapeutic benefit. Several methodologies may be employed to determine the fidelity of transcription and translation of the predicted protein. The focus of this chapter is to demonstrate primary structure confirmation using the recombinant human IgG1ҝ NIST mAb. Mass spectrometry based primary structure determination is covered in this chapter. It also will address the question of what necessary orthogonal characterization (additional enzymes, intact, and/or middle down) should be performed. A historical review of the methods employed to determine the sequence of proteins is included for completeness.
Current State-of-the-Art and Emerging Technologies for the Analysis of Monoclonal Antibodies
Publisher Info
ACS Publications, Washington, DC


NIST mAb, biotherapeutic, monoclonal antibody, primary structure, primary sequence, peptide mapping, top-down, middle-down, bottom-up, intact mass analysis


Mouchahoir, C. , Ly, M. , Levy, M. , Kilpatrick, L. , Lute, S. , Phinney, K. , Marzilli, L. , Brorson, K. , Boyne, M. , Davis, D. and Schiel, J. (2015), Determination of the Primary Sequence/ Structure, ACS Publications, Washington, DC, [online], (Accessed June 24, 2024)


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Created October 15, 2015, Updated November 10, 2018