PXD047634

PXD047634 is an original dataset announced via ProteomeXchange.

Title	Identification of structural origins of complex charge heterogeneity in ACE2Fc fusion protein facilitated by free-flow isoelectric focusing
Description	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 fight against the widespread SARS-CoV-2 virus, as this coronavirus must bind to ACE2 to allow for its entry into host cells. The huge amount of charge isoforms and complicated charge heterogeneity have posed a challenge to charge variant investigation in pharmaceutical development. To address this, we employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed analysis of enriched ACE2Fc fractions using electrophoretic, chromatographic, mass spectrometric, and biolayer interferometric techniques, for unveiling the structural origins of ACE2Fc charge heterogeneity. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, utilizing the highest allowable voltage to maximize the FF-IEF separation window and pI resolution. Additionally, we preserved the structural integrity of ACE2Fc fractions by implementing a mild elution method for fraction recovery. Based on peptide mapping and other characterizations, we revealed that highly sialylated multi-antenna N-glycosylation significantly contributes to the complex profiles of ACE2Fc charge heterogeneity. In addition, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards SARS-CoV-2 S protein based on the correlation between modifications and the observed activity shift. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a clear sialylation-activity relationship in ACE2Fc enabled by FF-IEF fractionation.
HostingRepository	iProX
AnnounceDate	2023-12-08
AnnouncementXML	Submission_2024-05-14_19:14:07.681.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Lingyu Wang
SpeciesList	scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive Plus

Revision	Datetime	Status	ChangeLog Entry
0	2023-12-07 22:32:55	ID requested
1	2023-12-07 22:33:12	announced
⏵ 2	2024-05-14 19:14:08	announced	2024-05-15: Update information.

Wang L, Wu H, Cao T, Li H, Shen P, Lu L, Zhang Z, Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing. Eur J Pharm Biopharm, 198():114248(2024) [pubmed]

submitter keyword: free-flow electrophoresis (FFE), isoelectric focusing (IEF), Angiotensin-converting enzyme 2, fusion protein, charge heterogeneity, glycosylation, sialylation

Zhongli Zhang
contact affiliation	Shanghai Henlius Biologics Co., Ltd.
contact email	Zhongli_Zhang@henlius.com
lab head
Lingyu Wang
contact affiliation	Shanghai Henlius Biologics Co., Ltd.
contact email	lingyu_wang@henlius.com
dataset submitter

iProX dataset URI