Shrenik Mehta1, Heather Flores1, Benjamin Walters2, Alavattam Sreedhara3. 1. Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, California, 94080, USA. 2. Genentech Research and Early Development, 1 DNA Way, South San Francisco, California, 94080, USA. 3. Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, California, 94080, USA. sreedhaa@gene.com.
Abstract
PURPOSE: To evaluate the physical and chemical degradation of monoclonal antibodies in the presence of Zn2+. METHODS: A full length IgG1 monoclonal antibody (mAb1) was formulated with various amounts of Zn2+. The resulting mixture was incubated for several weeks at room temperature and analyzed using a variety of biochemical techniques to look for various physical (e.g. aggregation) and chemical (e.g. fragmentation) degradation pathways. RESULTS: mAb1 of the IgG1 subclass undergoes aggregation in the presence of Zn2+ in a concentration dependent manner. Up to hexamers were characterized using SEC-MALS. No fragmentation was noticed in the presence of Zn2+ as opposed to that found in our previous report when IgG1 mAbs were incubated in the presence of Cu2+ ions. Site directed mutagenesis indicated the involvement of Fc histidine (His 310) in Zn2+ mediated aggregation. CONCLUSIONS: A novel metal ion mediated isodesmic aggregation mechanism was found in IgG1 class of monoclonal antibodies. Histidine residues in the Fc region were determined to be the binding site and implicated in Zn2+ mediated aggregation.
PURPOSE: To evaluate the physical and chemical degradation of monoclonal antibodies in the presence of Zn2+. METHODS: A full length IgG1 monoclonal antibody (mAb1) was formulated with various amounts of Zn2+. The resulting mixture was incubated for several weeks at room temperature and analyzed using a variety of biochemical techniques to look for various physical (e.g. aggregation) and chemical (e.g. fragmentation) degradation pathways. RESULTS: mAb1 of the IgG1 subclass undergoes aggregation in the presence of Zn2+ in a concentration dependent manner. Up to hexamers were characterized using SEC-MALS. No fragmentation was noticed in the presence of Zn2+ as opposed to that found in our previous report when IgG1 mAbs were incubated in the presence of Cu2+ ions. Site directed mutagenesis indicated the involvement of Fc histidine (His 310) in Zn2+ mediated aggregation. CONCLUSIONS: A novel metal ion mediated isodesmic aggregation mechanism was found in IgG1 class of monoclonal antibodies. Histidine residues in the Fc region were determined to be the binding site and implicated in Zn2+ mediated aggregation.
Authors: R J Harris; B Kabakoff; F D Macchi; F J Shen; M Kwong; J D Andya; S J Shire; N Bjork; K Totpal; A B Chen Journal: J Chromatogr B Biomed Sci Appl Date: 2001-03-10
Authors: Vikas K Sharma; Thomas W Patapoff; Bruce Kabakoff; Satyan Pai; Eric Hilario; Boyan Zhang; Charlene Li; Oleg Borisov; Robert F Kelley; Ilya Chorny; Joe Z Zhou; Ken A Dill; Trevor E Swartz Journal: Proc Natl Acad Sci U S A Date: 2014-12-15 Impact factor: 11.205