Biotechnology of gene fusion facilitates the production of recombinant fusion proteins that encompass combined properties and functions derived from the parental proteins. Fc-fusion proteins are homodimers composed of the IgG antibody Fc region covalently linked to a peptide or protein with desired functions. The prolonged plasma half-life and structural diversity of the Fc-fusion proteins consisting of multiple domains make them popular as a therapeutic molecular modality second only to monoclonal antibodies [1]. Originating from parental glycoproteins, Fc-fusion proteins with multiple glycosylation sites usually demonstrate charge heterogeneity profiles with much higher complexity than well-characterized monoclonal antibodies. The angiotensin-converting enzyme 2 (ACE2) is a pivotal human membrane protein involved in the renin–angiotensin–aldosterone system, crucial for maintaining blood pressure and, notably, serving as the entry point of several coronaviruses into cells, including SARS-CoV-2 [2], [3]. To overcome limited in vitro stability, ACE2 fused with IgG1 Fc, instead of soluble ACE2, holds promise as an antigen for neutralizing antibody screening, or directly as a potential large molecule therapeutic with acceptable druggability in the fight against pathogenic coronaviruses. Engineered soluble ACE2Fc with abrogated enzymatic activity has been validated as a decoy antiviral targeting a wide range of coronaviruses [4], [5]. ACE2Fc an investigational new drug developed by Henlius (Shanghai, China) has passed the Phase I clinical trial.

From the perspective of pharmaceutical process development, ACE2Fc brings difficulties as its intact molecule is comprised of more than 1200 residues, among which 16 asparagines and at least four serines/threonines are glycosylated, [2], [3] conferring huge microheterogeneity to this antiviral candidate, as illustrated in Fig. 1. Elucidation of charge variants is crucial as per regulatory guidance for protein therapeutics, whether based on antibody or fusion protein platforms, because charge isoforms may possess different efficacy/safety risks and charge profiles of therapeutic proteins directly reflect the consistency of the manufacturing process. Though charge variation characterization of ACE2Fc is an important and inevitable step in process development, limited relevant information has been reported, as most reported ACE2Fc research focused on early-stage ACE2Fc stability, sequence mutation, in vitro affinity, and in vivo efficacy [4], [5], [6]. While glycosylation of both ACE2 and the SARS-CoV-2 Spike (S) proteins has been extensively investigated through glycoproteomic approaches, its experimental correlation to charge heterogeneity profiles remains undetermined [7], [8], [9].

Interrogation of ACE2Fc charge heterogeneity is a challenging analytical task. Online coupling of capillary isoelectric focusing (cIEF) [10], [11], [12], [13] and ion exchange chromatography (IEC) [14], [15], [16] with mass spectrometry have been developed based on several dedicated ion sources, enabling fast MS identification of major antibody charge variants eluted from front-end pI separations. Nevertheless, complex protein samples with multiple glycosylation sites pose challenges for online analytical approaches, as the detected charge envelopes for intact proteins may be too intricate to be deconvoluted. Preparative IEC demonstrated sufficient resolution for charge variants of many therapeutic antibodies, mostly IgG1 subtype [17], [18], [19]. As to biopharmaceuticals with complex charge variations and subtle modifications, the IEF-based approach is superior to IEC as it provides high pI resolutions which can reveal fine details in charge profiles [13], [20]. IEF serves as the gold standard for charge heterogeneity profiling because of a large cohort of widely trusted carrier ampholytes, which can all be employed in preparative FF-IEF separation of complex fusion proteins. The commonly utilized 2D gel electrophoresis provides tandem pI- and size-based separation, but the protein isoforms separated in gels cannot be easily recovered for activity measurement. Up to date, offline free-flow isoelectric focusing (FF-IEF) fractionation followed by electrophoretic, chromatographic, and mass spectrometric characterization represents the most capable and in-depth approach for charge variant separation and identification [21], [22].

In this study, the structural origins of complex charge heterogeneity of ACE2Fc were interrogated using FF-IEF fractionation followed by detailed characterization of isolated variants, and the correlation of ACE2Fc backbone modifications to S protein binding activity was assessed. The charge heterogeneity profile of ACE2Fc profiled by imaging capillary isoelectric focusing (icIEF) exhibited the most intricate pattern we have ever observed, with up to 19 partially separated peaks, making it a crucial benchmark for our development strategy of FF-IEF methods. To achieve optimal fractionation and activity preservation, we employed a multiple-component medium, the highest separation voltage, and mild Protein A elution for fraction recovery. N-glycan profiling and tryptic peptide mapping were employed to identify the major roots of ACE2Fc charge heterogeneity. Furthermore, the S protein binding activities of representative ACE2Fc fractions were measured to reveal the sialylation-activity correlation, which are in line with reported glycoengineering and glycosylation deletion studies [23], [24]. This study introduces a paradigmatic approach for the development strategy of FF-IEF for complex fusion proteins. Additionally, the identification of the structural origins of ACE2Fc charge heterogeneity and their impact on S protein binding activity may contribute to a deeper understanding of ACE2, the receptor for various coronaviruses, including SARS-Cov-2, the culprit behind the lingering pandemic.