Title: Complementary Methodologies for Size Heterogeneity Analysis of Antibody Impurities
Abstract: Monoclonal antibodies (mAbs) are critical therapeutic agents, and ensuring their quality is paramount. One key Critical Quality Attribute (CQA) of mAbs is size heterogeneity, as both aggregation and degradation can significantly impact their safety and efficacy. Size-based impurities include low molecular weight (LMW) degradation products and high molecular weight (HMW) aggregates, both of which must be thoroughly characterized to ensure therapeutic integrity.
In this presentation, we will explore the complementary techniques of Size Exclusion Chromatography (SEC) and Capillary Electrophoresis-sodium dodecyl sulfate (CE-SDS) for analyzing mAb size heterogeneity. SEC, performed under native conditions, is ideal for detecting HMW aggregates, while CE-SDS provides high-resolution separations of LMW species. Together, these methods offer a comprehensive approach to assessing mAb monomeric purity.
Using the Agilent 1290 Infinity II Bio LC system for SEC and the Agilent ProteoAnalyzer system, a parallel CE platform, we will demonstrate how these technologies were employed to analyze the NISTmAb reference standard. This talk will highlight how SEC and CE-SDS complement each other to provide a more complete picture of mAb quality, ensuring robust characterization of size heterogeneity.
Attendees will gain insights into the importance of employing multiple analytical techniques for thorough mAb quality assessment, and learn how these methods can be leveraged in both research and biopharmaceutical manufacturing settings.
Author/Presenter: Graham Robinett, Agilent Biopharma Business Development Manager
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Title: Advanced LC Technologies for Increasingly Complex Molecules
Abstract: Recent advances in drug delivery modalities, such as LNPs, AAVs, and ADCs, have forced scientists to optimize their analytical methodology to keep up with ever-changing critical quality attributes (CQAs). Now, labs are inundated with instrumentation to get the answers they seek. With the ever-changing analyte complexity, Wyatt offers a suite of advanced detection methods and alternative separations to meet these demands and report multiple quality attributes (MQAs). This seminar will address potential solutions for characterization of proteins, peptides, gene therapy using SEC-MALS and FFF-MALS. Case studies for ADC, AOC, and proteins will be presented to illustrate the addition of MALS to standard chromatography.
Author/Presenter: Benjamin Pulminskas is a field application scientist at Wyatt Technology LLC. He obtained his BS in Chemistry from Norwich University in Northfield, VT and MS in Chemistry from Northeastern University in Boston, MA. His research and professional focusses have been in analytical separation methods for proteins and gene therapies. Benjamin joined Wyatt Technology in 2022 and is Wyatt’s subject matter expert on light scattering based out of Atlanta, GA.
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Title: Effective and simultaneous aggregate and HCP reduction via flow-through mixed-mode chromatography in Mab purification- Detailed HCP clearance analysis by UHPLC-HRAM MS/MS.
Abstract: Advances in the design of engineered monoclonal antibody (mAb)–based therapeutics are generating a wide range of novel, efficacious biomolecules, but their manufacturing can also form ispaired products, fragments, and high levels of aggregation. Traditional downstream process steps developed for standard impurity reduction, such as HCPs, struggle to remove these high molecular weight (HMW) impurities while maintaining high yield and sufficient process economics. New polishing strategies need to be developed. In this talk, high monomer recovery and impurity reduction results will be shared from the purification of a high aggregate mAb feedstock post affinity capture using Thermo Scientific™ POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography Resin in flow-through mode. A particular focus is given to the identification and quantitation of HCP impurities both pre and post mixed-mode polish using HPLC-MS/MS 2 . Results show excellent clearance of HMW species and total HCPs, and hard-to-remove and high-risk HCPs were specifically identified in a single chromatography unit operation.
Speaker: Kelly Flook, Sr. Manager, Product Management, Purification Products, Thermo Fisher Scientific
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Title: Digital Membrane Chromatography.
Abstract: Electric fields within chromatography membranes provide a third dimension for biomolecule capture, concentration and elution using ion-exchange and affinity membranes. Membranes with a conductive surface permit an electric potential to be applied enabling a reversal of biomolecular charge facilitating elution with multiple cycles. Instantaneous Digital Membrane Chromatography (DMC) reduces dilution and analytical or process time. Molecular degradation resulting from extreme pH is eliminated. Biomolecules with proximal pI’s can be separated.
Author/Presenter: Dr. Stephan Brinke-Seiferth
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Title: FcR Affinity Chromatography for Intact Characterization of Antibody Glycan Variants
Abstract: Antibody- and protein-based biotherapeutics require extensive characterization efforts to link structural quality attributes to overall drug efficacy. Extended structural characterization performed on purified mAbs utilizes multiple chromatographic modes to characterize various quality attributes. Various glycans covalently linked to mAb therapeutics during bioprocessing is one major attribute of interest, as the presence of fucosylated or sialylated glycans can affect target binding strength, unwanted allergenic immune responses, and drug product/substance stability. Herein we present an affinity column manufactured by Tosoh Bioscience specifically geared as an orthogonal tool for mAb glycan analysis, driven by interactions between glycan variants present in the mAb Fc region and a protein-based Fc receptor ligand covalently linked to the stationary phase. Topics covered include the structural basis for the affinity interaction and applications for implementing glycan-based separations for mAbs relevant to scientists involved with structural characterization, bioprocess development, and functional testing groups.
Author/Presenter: Dr. Corey W. Meadows, Analytical Product Specialist at Tosoh Bioscience
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