Open AccessCharacterization of AEBSF-antibody modifications for a protease inhibitor supplementation strategy
Author(s) -
Cindy X. Cai,
Nicole A. Schneck,
Weidong Zhao,
Daniel Blackstock,
Jia-Yan Cai,
Doug Harris,
Vera B. Ivleva,
Deepika Gollapudi,
Joe Horwitz,
Frank Arnold,
Jonathan W. Cooper,
Q. Paula Lei
Publication year - 2019
Publication title -
analytical and bioanalytical chemistry/analytical and bioanalytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.86
H-Index - 166
eISSN - 1618-2650
pISSN - 1618-2642
DOI - 10.1007/s00216-019-01995-9
Subject(s) - protease , protease inhibitor (pharmacology) , chemistry , potency , in vitro , biochemistry , medicine , immunology , human immunodeficiency virus (hiv) , viral load , enzyme , antiretroviral therapy
Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.