Effect of pH, temperature, and salt on the stability of Escherichia coli- and Chinese hamster ovary cell-derived IgG1 Fc.
The circulation half-life of a potential therapeutic can be increased by fusing the molecule of interest (an active peptide, the extracellular domain of a receptor, an enzyme, etc.) to the Fc fragment of a monoclonal antibody. For the fusion protein to be a successful therapeutic, it must be stable to process and long-term storage conditions, as well as to physiological conditions. The stability of the Fc used is critical for obtaining a successful therapeutic protein. The effects of pH, temperature, and salt on the stabilities of Escherichia coli- and Chinese hamster ovary cell (CHO)-derived IgG1 Fc high-order structure were probed using a variety of biophysical techniques. Fc molecules derived from both E. coli and CHO were compared. The IgG1 Fc molecules from both sources (glycosylated and aglycosylated) are folded at neutral pH and behave similarly upon heat- and low pH-induced unfolding. The unfolding of both IgG1 Fc molecules occurs via a multistep unfolding process, with the tertiary structure and C(H)2 domain unfolding first, followed by changes in the secondary structure and C(H)3 domain. The acid-induced unfolding of IgG1 Fc molecules is only partially reversible, with the formation of high-molecular weight species. The CHO-derived Fc protein (glycosylated) is more compact (smaller hydrodynamic radius) than the E. coli-derived protein (aglycosylated) at neutral pH. Unfolding is dependent on pH and salt concentration. The glycosylated C(H)2 domain melts at a temperature 4-5 °C higher than that of the aglycosylated domain, and the low-pH-induced unfolding of the glycosylated Fc molecule occurs at a pH ~0.5 pH unit lower than that of the aglycosylated protein. The difference observed between E. coli- and CHO-derived Fc molecules primarily involves the C(H)2 domain, where the glycosylation of the Fc resides.
Product Attribute Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. cynthial@amgen.com
Cynthia H
CH
Li
Linda O
LO
Narhi
Jie
J
Wen
Mariana
M
Dimitrova
Zai-qing
ZQ
Wen
Jenny
J
Li
Joseph
J
Pollastrini
Xichdao
X
Nguyen
Trace
T
Tsuruda
Yijia
Y
Jiang
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000276:immunology; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D011487:Protein Conformation / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012965:Sodium Chloride / Q000494:pharmacology*; D013696:Temperature
D029968:Escherichia coli Proteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride