The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle‐down characterization of antibodies

• DOI: 10.1111/febs.15813
• Publication Date: 2021-03-13T16:49:53Z
• Authors: Tomislav Čaval; Elizabeth Sara Hecht; Wilfred Tang; Maelia Uy‐Gomez; Andrew Nichols; Yong J. Kil; Wendy Sandoval; Marshall Bern; Albert J. R. Heck
• Keywords: Cleavage (geology); Cathepsin E; Cathepsin L; Edman degradation

Mass spectrometry is gaining momentum as a method of choice to de novo sequence antibodies (Abs). Adequate coverage the hypervariable regions remains one toughest identification challenges by either bottom‐up or top‐down workflows. Methods that efficiently generate mid‐size Ab fragments would further facilitate MS and decrease data complexity. Here, we explore proteases Cathepsins L D for forming protein from three IgG1s, IgG2, bispecific, knob‐and‐hole IgG1. We demonstrate high‐resolution native provides sensitive detection clipping sites. Both produced multiple, albeit specific cleavages. The Abs were cleaved immediately after CDR3 region, yielding ~ 12 kDa fragments, is, ideal sequencing‐sized. Cathepsin D, but not L, also directly below hinge, releasing F(ab’)2. When constrained different disulfide bonds found in IgG2 subtype tertiary structure hole‐containing bispecific IgG1, hinge region digest product was produced. motifs related sequences neutral amino acids Ab. A single pot (L + D) digestion protocol optimized achieve 100% efficiency. Nine corresponding VL, VH, CL, CH1, CH2, CH3, CL F(ab')2, constituted 70% summed intensities all deconvolved proteolytic products. Cleavage sites confirmed Edman degradation validated with sequencing. described work offers complementary middle‐down analysis may be applied Enzymes L— EC 3.4.22.15 , D— 3.4.23.5 .