Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain.
IgG antibodies (Abs) and fragments of IgG Abs are becoming major biotherapeutics to treat an assortment of human diseases. Commonly prepared fragments of IgGs include Fc, Fab, and F(ab')2 fragments, all of which can be made using the sulfhydryl protease papain, although prolonged digestion times and/or excessive amounts of papain typically result in further cleavage of the Fc domain into smaller fragments. During our attempts to use papain to isolate Fc fragments from different IgG monoclonal Abs, it was observed that prior removal of Fc glycans resulted in a faster rate of papain-mediated degradation of the Fc domain. Subsequent time-course experiments comparing glycosylated and deglycosylated versions of IgG antibodies showed that the majority of molecules in a deglycosylated IgG sample were converted into Fab, Fc, and smaller Fc fragments in less than one hour, whereas the original glycosylated IgG required more than two hours to convert into a comparable amount of Fab and Fc fragments. Furthermore, whereas papain digestion converted almost all of a deglycosylated Fc fragment into smaller fragments of approximately 10 and approximately 12 kDa within 4 h, more than 40% of a glycosylated Fc fragment remained intact even after 24 h of digestion. These results indicate that the presence of CH(2) domain glycans in either IgGs or purified Fc fragments increases resistance to papain digestion. Increased sensitivity of non-glycosylated Fc domains to papain is consistent with the Fc domains lacking a defined structure, as exemplified by their inability to bind Fcgamma receptors, since misfolded proteins are often degraded by proteases because of increased accessibility of their proteolytic cleavage sites. Based on these observations it is possible to use papain sensitivity as a means of assessing proper Fc structure of IgG molecules.
Raju
T Shantha
TS
Discovery Research, Centocor R&D Inc., 145 King of Prussia Road, Radnor, PA 19087, USA. traju@cntus.jnj.com
Scallon
Bernard J
BJ
D000818:Animals; D002236:Carbohydrate Conformation; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D010206:Papain / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D007141:Immunoglobulin Fc Fragments; D010206:Papain