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.
摘要：
Fc融合蛋白代表具有作为蛋白质治疗剂的相当大潜力的通用分子平台,其电荷异质性应根据监管指南良好地表征。血管紧张素转换酶2Fc融合蛋白（ACE2Fc）已被研究为各种冠状病毒的潜在中和剂，包括挥之不去的SARS-CoV-2，因为这种冠状病毒必须与ACE2结合才能进入宿主细胞。ACE2Fc,由Henlius（中国上海）开发的研究性新药，已经通过了I期临床试验,但是其大量的电荷同种型和复杂的电荷异质性对药物开发中的电荷变体研究提出了挑战。我们采用离线自由流动等电聚焦(FF-IEF)分馏，随后对富集的ACE2Fc级分进行详细表征，揭示重组CHO细胞表达的ACE2Fc中电荷异质性的结构起源。我们采用了经过良好调整的3组分分离介质进行ACE2Fc分馏，使FF-IEF分离窗口最大化的最高允许电压和用于保持蛋白质结构完整性的温和蛋白A洗脱方法。通过肽图谱和其他表征，我们揭示了ACE2Fc电荷异质性的复杂谱主要是由高度唾液酸化的多天线N-糖基化引起的。此外,基于级分表征和硅糖蛋白模型分析，我们发现ACE2Fc的N36，N73和N305处的大酸性聚糖能够降低对SARS-CoV-2的Spike（S）蛋白的结合活性。我们的研究举例说明了FF-IEF在高度复杂的融合蛋白表征中的价值，并揭示了ACE2Fc中的定量唾液酸化-活性关系。
