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 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.

中文翻译：

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自由流等电聚焦促进治疗性 ACE2Fc 融合蛋白复杂电荷异质性结构起源的鉴定

Fc 融合蛋白代表了一种多功能分子平台，作为蛋白质治疗剂具有相当大的潜力，其电荷异质性应根据监管指南得到很好的表征。血管紧张素转换酶 2 Fc 融合蛋白 (ACE2Fc) 已被研究作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。复宏汉霖在研新药ACE2Fc已通过I期临床，但其电荷异构体数量巨大、电荷异质性复杂，给药物研发中的电荷异构体研究带来了挑战。我们采用离线自由流等电聚焦 (FF-IEF) 分级分离，然后对富集的 ACE2Fc 级分进行详细表征，以揭示重组 CHO 细胞表达的 ACE2Fc 中电荷异质性的结构起源。我们采用了经过精心调整的 3 组分分离介质进行 ACE2Fc 分级分离，采用最高允许电压来最大化 FF-IEF 分离窗口，并采用温和的 Protein A 洗脱方法来保存蛋白质结构完整性。通过肽图谱和其他表征，我们揭示了 ACE2Fc 电荷异质性的复杂特征主要是由高度唾液酸化的多天线 N-糖基化引起的。此外，基于组分表征和糖蛋白模型分析，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。我们的研究例证了 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。