Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing

Abstract

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.

Keywords: Angiotensin-converting enzyme 2 (ACE2); Binding activity; Charge heterogeneity; Free-flow electrophoresis (FFE); Fusion protein; Glycosylation; Isoelectric focusing (IEF); Sialylation.