Persistence and Fab-specific interaction of acyl-protein thioesterase-1 with monoclonal antibodies during downstream processing

Šprager, Ernest (author), Krajnc, Aleksander (author), Lunder, Mojca (author), Vašl, Jožica (author), Bratkovič, Tomaž (author)

Difficult-to-remove host cell proteins (HCPs) can compromise monoclonal antibody (mAb) product quality and stability. This study investigated the persistence of acyl-protein thioesterase-1, a polysorbate-degrading HCP, during downstream processing and its interaction with a set of mAbs. Acyl-protein thioesterase-1 remained in the eluates after reprocessing a specific mAb with protein A affinity liquid chromatography, even when various wash buffer additives were used, indicating a strong product:HCP interaction that resists conventional purification strategies. Upon cleaving the mAb and purifying its Fab and Fc fragments, we used biolayer interferometry to show that acyl-protein thioesterase-1 selectively and tightly binds to the Fab region. Domain-specific sensor-immobilization approach suggested that the binding site was located on the antibody's CH1 domain, and the interaction was influenced by preincubation with the mAb's cognate antigen. These findings highlight the importance of characterizing HCP:mAb interactions as a basis for improving HCP reduction strategies.

• Language: English
• Keywords: monoclonal antibodies, chromatography, host cell proteins, acyl-protein thioesterase-1, protein-protein interactions, biolayer interferometry
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: Str. 1-7
• Numbering: Vol. 241, art. 106920
• PID: 20.500.12556/RUL-181020
• UDC: 577
• ISSN on article: 1046-5928
• DOI: 10.1016/j.pep.2026.106920
• COBISS.SI-ID: 272547075
• Publication date in RUL: 23.03.2026

Record is a part of a journal

• Title: Protein expression and purification
• Shortened title: Protein expr. purif.
• Publisher: Academic Press
• ISSN: 1046-5928
• COBISS.SI-ID: 2627623

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: monoklonska protitelesa, kromatografija, proteini gostiteljskih celic, acil-protein tioesteraza-1, interakcije protein-protein, interferometrija biološke plasti

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P1-0420
• Name: Napredna imunološka zdravila in celični pristopi v farmaciji