An integrated methodology for quality assessment of therapeutic antibodies with potential long circulation half-life in harvested cell culture fluid using FcRn immobilized hydrophilic magnetic graphene

• Published in: Talanta (Oxford) 2024-05, Vol.272, p.125781-125781, Article 125781
• Main Authors: Feng, Jianan, Cao, Hao, Xiang, Yangjiayi, Deng, Chunhui, Li, Yan
• Format: Article
• Language: English
• Subjects: adsorbents; cell culture; dissociation; FcRn; gel chromatography; graphene; half life; hydrophilicity; Magnetic graphene; Magnetic solid phase extraction; magnetism; Overall quality assessment; pharmacokinetics; solid phase extraction; Therapeutic antibodies; therapeutics
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2024.125781

Designing modified therapeutic antibodies with enhanced FcRn-binding affinity holds promise in the extension of circulation half-lives and potential refinement of pharmacokinetics. During the development of these new-generation therapeutic antibodies, FcRn binding affinity of IgGs is emphasized and monitored as a critical quality attribute (CQA), alongside other critical assessments including titer and aggregation level. However, the traditional workflow for assessing the overall quality of expressed IgGs in harvested cell culture fluid (HCCF) is blamed to be cumbersome and time-consuming. This study presents an integrated methodology for the rapid quality assessment of IgGs in HCCF by selectively extracting IgGs with favorable high FcRn affinity for subsequent analysis using size exclusion chromatography (SEC). The approach utilizes innovative adsorbents known as FcRn immobilized hydrophilic magnetic graphene (MG@PDA@PAMAM-FcRn) in a magnetic solid-phase extraction (MSPE) process. To simulate the in vivo binding dynamics, MSPE binding and dissociation was performed at pH 6.0 and 7.4, respectively. The composite have demonstrated enhanced extraction efficiency and impurity removal ability in comparison to commercially available magnetic beads. The SEC monomer peak area value provides the output of this method, the ranking of which enabled the facile identification of superior HCCF samples with high overall quality of IgG. Optimization of MSPE parameters was performed, and the method was validated for specificity, precision, sensitivity, and accuracy. The proposed method exhibited an analytical time of 0.6 h, which is 7–22 times shortened in comparison to the conventional workflow. [Display omitted] •FcRn immobilized magnetic graphene with polydopamine and hydrophilic dendrimer coating was designed and synthesized for the first time.•IgG with different FcRn-affinity levels were differentially extracted from harvested cell culture fluid using FcRn immobilized material as magnetic adsorbents.•By leveraging the advantage of stepwise operation in MSPE technology, binding and dissociation at particular pH values were achieved, thus to accurately simulate the intricate pH-dependent binding dynamics in vivo between IgG and FcRn.•Purification procedure and FcRn-affinity evaluation were combined, thus only desired IgG species with high FcRn-affinity were selectively purified for further quality analysis.•The superior clones with desired overall quality of IgG could