Tractable Method for Rapid Quality Assessment of Therapeutic Antibodies in Harvested Cell Culture Fluid based on FcγRIIIa-Immobilized Magnetic Microspheres.

ABSTRACT

FcγRIIIa-binding affinity is one of the key factors to ensure the efficacy of many antitumor therapeutic antibodies, which should be monitored along with the titer, protein aggregation, and other critical quality attributes. The conventional workflow for the quality assessment of therapeutic antibodies in harvested cell culture fluid (HCCF) is time-consuming and costly nevertheless. In this study, a tractable method was established for rapid quality assessment of a HCCF sample through differentially extracting IgG with different FcγRIIIa affinity levels using FcγRIIIa-immobilized magnetic microspheres, followed by size exclusion chromatography (SEC) to determine the amount and monomer percentage of IgGs in the preceding eluate. FcγRIIIa-immobilized magnetic microspheres with polydopamine (PDA) and hydrophilic dendrimer (PAMAM) coating (denoted as Fe3O4@PDA@PAMAM-FcγRIIIa) were synthesized for the first time as magnetic adsorbents. The PDA cladding endowed the composites with good chemical stability in acidic elution buffer, and the PAMAM dendrimer empowered the composites of high ligand immobilization capacity and hydrophilic surface. The labile FcγRIIIa was immobilized under mild conditions. By directly applying a simple magnetic solid phase extraction procedure to treat HCCF, favored IgG species with high FcγRIIIa affinity would be selectively captured by Fe3O4@PDA@PAMAM-FcγRIIIa composites for subsequent SEC analysis. The monomer peak area value in SEC, which was set as the read-out of the proposed method, correlated directly with the theoretical overall quality of standard-spiked HCCF samples.