Protein A Affinity Capture followed By AdvanceBio SEC Aggregation Analysis using the Agilent InfinityLab Series 2D-LC System

Protein biopharmaceuticals such as recombinant monoclonal antibodies are currently in widespread use for the treatment of various life-threatening diseases including cancer and autoimmune diseases 1,2,3. In contrast to small molecule drugs, mAbs are large (150 kDa) and heterogeneous. As a result of the biosynthetic process and subsequent manufacturing and storage, hundreds of different variants may co-exist, differing in aspects such as N-glycosylation, N- and C-terminal processing, deamidation, oxidation, amino acid sequence, and disulfide bridges. Fragmentation and aggregation further add to the complexity. Due to the structural complexity of mAbs, a variety of tests are required to monitor Critical Quality Attributes (CQAs) throughout the development and manufacturing process, hence, unraveling this complexity represents an analytical challenge. In recent years, two-dimensional liquid chromatography (2D-LC) has been shown to be highly promising for the detailed characterization of monoclonal antibodies (mAbs) 4.

Protein A is an immunoglobulin-Fc (IgG) receptor found in the cell wall of Staphylococcus aureus. It has strong affinity for polyclonal and monoclonal IgGs like human IgG 1, IgG 2, and IgG 4, in addition to IgGs from some other species such as rabbits and some mouse IgGs. Immobilized Protein A is commonly used for preparative and process scale purifications of IgG. At the analytical scale, the Agilent Bio-Monolith Protein A HPLC Column can be used for fast quantification and small scale purification of IgGs in complex mixtures or pure samples.5 Size Exclusion Chromatography is the method of choice for quantification of aggregates in Bio therapeutic samples. Protein A Capture followed by size exclusion chromatography for aggregation analysis yields more accurate characterization as purer sample is analyzed. With the Agilent 2D LC System, this analysis can be done with one injection. Protein A capture is performed in the 1st dimension. Eluted IgG is trapped in the multiple heart cutting loops and eluted in the 2nd dimension onto an Agilent AdvanceBio SEC sizing column for accurate aggregate analysis 6.

1. Sandra, K.; Vandenheede, I.; Sandra, P. Modern Chromatographic and Mass Spectrometric Techniques for Protein Biopharmaceutical Characterization. J. Chromatogr. A 2014, 1335, 81–103.

2. Fekete, S.; et al. Chromatographic, Electrophoretic and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals. Anal. Chem. 2016, 88, 480–507.

3. Walsh, G. Biopharmaceutical benchmarks 2018. Nat. Biotechnol. 2018, 36, 1136–1145.

4. Vanhoenacker, G.; et al. Multi-Attribute Analysis of Monoclonal Antibodies Using the Agilent InfinityLab 2D-LC Solution and Q-TOF MS 2019,2020. Agilent Technologies, Inc. Application Note, publication number 5994-0947EN.

5. Duong, P. T. Agilent Bio-Monolith Protein A Monitors Monoclonal Antibody Titer from Cell Cultures. Agilent Technologies Application Note, publication number 5991-2990EN, 2014.

6. Cronan, P.; et al. Protein A Affinity Capture followed By AdvanceBio SEC Aggregation Analysis using the Agilent InfinityLab Series 2D-LC System, 2016. Agilent Technologies, Inc. Application Note, publication number 5991-7223EN

Presenter: Patrick Cronan (LC Applications Scientist, Agilent Technologies, Inc.)

Patrick Cronan is an LC applications Scientist with Agilent Technologies. He is an accomplished analytical chemist with over 20 years of experience in the pharmaceutical and Biopharma industries; has facilitated method transfer from HPLC to UHPLC while using LCMS to characterize large and small molecule drug-like compounds. Patrick has also worked extensively with Agilent’s 2D Chromatography system developing methods to increase throughput of biologic separations by coupling multiple techniques like SEC x IEX.

Presenter: Regina Black (LC Applications Scientist, Agilent Technologies Inc.)

Gina joined Agilent over 2 years ago and brought with her a wealth of experience in the pharmaceutical industry. She began her career at Merck as a synthetic chemist and became a subject matter expert in preparative HPLC, eventually joining the Separations COE, supporting both discovery and process development efforts. After twenty-two years at Merck, Gina moved to Bristol-Myers Squibb where she served as a site lead for their Single Compound Purification group, using a highly automated workflow to analyze, purify and deliver final products for biological testing. Gina then moved back into large scale separations and enabling technologies supporting Chemical Development at BMS. Over the years, Gina has developed an extensive background in both reverse phase and normal phase HPLC, SFC and IEX methodology.