High-Throughput Analysis of Antibody Subunits Using a BioResolve RP mAb Polyphenyl Column

The objective of this application note is to demonstrate how the BioResolve RP mAb Polyphenyl Column can be leveraged to improve productivity through the development of a highly-efficient and robust method for the analysis of mAb subunits.

Benefits

• Solid-core particle technology improves performance for the separation of biomacromolecules
• Accelerated characterization and method development of therapeutic proteins

The clinical use of therapeutic monoclonal antibodies (mAbs) has grown exponentially over the past 30 years due to their effectiveness in treating diseases while minimizing the risk of adverse effects in comparison to conventional drug-based treatments.¹ However, as a result of their manufacturing process, mAb-based products usually contain various  post-translational modifications and product/process related impurities  that can impact their safety and efficacy.² Comprehensive characterization is therefore critical for drug development and quality control. To characterize and monitor the critical quality attributes (CQAs) of mAbs, reversed-phase liquid chromatography (RPLC) is extensively used in process development and stability studies at several molecular levels, as encountered with intact analysis, middle down/subunit profiling, and peptide mapping.³ Methods that incorporate conventional fully-porous RPLC-based chemistries often sacrifice throughput for separation performance in the analysis of mAbs at the subunit or intact level. This, in part, is due to a non-ideal pairing between stationary phase attributes (e.g. morphology, pore size, and  surface chemistry) and analyte physicochemical properties (e.g., size). For that reason, there is promise in using purposefully-designed column technology to increase throughput and reduce the time to develop a new therapeutic mAb. 

The Waters BioResolve RP mAb Polyphenyl Column was designed to improve the efficiency of large biomolecule separations via the specialization of its particle technology.^4,5 Unlike reversed-phase columns containing fully-porous particles, BioResolve RP mAb Polyphenyl Columns contain the solid core, silica based particles with a defined porous layer (i.e., SCP) that allows for high-resolution separations at high flow rates, and rapid column equilibration, leading to increased throughput for the analysis of large biomolecules, such as mAbs, in various environments.

The objective of this application note is to demonstrate how the BioResolve RP mAb Polyphenyl Column can be leveraged to improve productivity through the development of a highly-efficient and robust method for the analysis of mAb subunits.

Chemical and reagents

Waters mAb Subunit Standard (P/N: 186008927) that contains 25 µg of reduced, IdeS-digested NIST mAb (Reference Material 8671) was dissolved in 100 µL water to obtain a solution at a concentration of 0.25 µg/µL. HPLC grade water, acetonitrile, and  TFA were purchased from Fisher Scientific and used as received. 

The BioResolve RP mAb Polyphenyl Column is comprised of wide-pore solid core particles to enhance its kinetic properties in application to resolving biomacromolecules, such as proteins, under aggressive conditions that incorporate short runtimes and/or high flow rates. To evaluate such conditions, an appropriate sample and chromatographic separation must be selected for comparison. With this in mind, NIST mAb (Reference Material 8671) was selected as a representative sample commonly encountered in the biopharmaceutical industry. This material was subjected to limited proteolysis to facilitate a subunit-level analysis, an emerging technique for the profiling of mAb impurities that is adaptable to high-throughput methods. To this end, the NIST mAb was enzymatically treated with IdeS⁶ and thereafter reduced to generate the three main sub-units (Fc/2, LC, and Fd') and used as a case study. To establish a baseline comparison, a 15 minute RPLC platform method was performed using an industry leading, fully-porous C₄ column on an ACQUITY UPLC H-Class Bio System with a Tunable Ultra-Violet (TUV) Detector  and compared against the BioResolve RP mAb Polypehenyl Column. As shown in Figure 1, using a 10 minute gradient with a  0.3 mL/min flow rate, it was possible to generate a chromatogram that is representative of conventional protein RPLC conditions.  With the employed 0.1% TFA mobile phases, both columns were able to resolve the three mAb subunits sufficiently, as evidenced by the observation of comparable peak widths. Nonetheless, the unique selectivity imparted by the novel polyphenyl bonded  phase led to the improved resolution of impurities, as with the separation of several putative, Fd' oxidation variants (insets). This increase in the resolution of subunit impurities typifies the superior performance of the BioResolve RP mAb Polyphenyl Column, which can lead to improved quantitation of product related impurities.

With baseline performance established using a conventional method, the BioResolve RP mAb Polyphenyl Column was further tested to fully utilize its kinetic advantages and facilitate a higher-throughput separation. To explore column performance with shorter analysis times, the 15 minute method (10 minute gradient) was scaled to 10 minutes (5 minute gradient) and 3 minutes (1 minute gradient), as shown in Figure 2. Even with the 1 minute gradient (red trace), the three main subunits of the IdeS-digested NIST mAb could be baseline resolved, demonstrating the BioResolve column’s unique stationary phase is well suited for high-throughput separations of biomacromolecules. 

To probe the amenability of column technologies to high-throughput analyses, the 1 minute gradient (3 minute method) was optimized at the flow rate of 0.5 mL/min for the BioResolve RP mAb Polyphenyl Column, the previously mentioned C₄ column, and a polymeric column. As shown in Figure 3, baseline resolution of each subunit was obtained using all three columns, though the BioResolve RP mAb Polyphenyl Column showed the narrowest peak widths and highest resolution. Increased intensities were observed in the separation using the BioResolve RP mAb Polyphenyl Column for the three main subunits and the low abundance peaks associated with the Fd' fragment. This demonstrates the higher sensitivity provided by the BioResolve RP mAb Polyphenyl Column, which could allow for more accurate analysis of trace impurities during process development and quality control. Calculated results showed the BioResolve RP mAb Polyphenyl Column to produce a higher peak capacity (Pc=32) versus both the C4 (Pc=28), and polymeric column (Pc=24), indicating the advantage of the enhanced kinetics of BioResolve RP mAb Polyphenyl Column. Another benefit of using the BioResolve RP mAb Polyphenyl Column is the low backpressure (1800 psi at 0.5 mL/min) afforded by its low permittivity, offering compatibility with HPLC and UPLC instrumentation for increased flexibility in method deployment. In addition, the BioResolve RP mAb Polyphenyl Column had the shortest column re-equilibration time, which resulted in a shorter cycle time (3 minutes) in comparison to the C₄ (4 minutes) and polymeric column (4 minutes). This of particular interest for high-throughput methods in the context of productivity as even a difference of 1 minute per cycle (30%) can add up to 100 plus additional injections over 24 hours.

To further validate the utility of the BioResolve RP mAb Polyphenyl Column for developing high-throughput separations of proteins, a repeatability study was performed. To test the reproducibility of the 1 minute gradient method, variation in peak areas and retention times from three consecutive injections of the mAb Subunit Standard was evaluated. As shown in Figure 4, highly reproducible retention times (RSD <0.05%) and peak areas (RSD <0.8%) were observed, confirming that the BioResolve RP mAb Polyphenyl Column can consistently and efficiently resolve proteins even when used with short analysis times. Together, this study demonstrates that the BioResolve RP mAb Polyphenyl Column can be used to develop high-throughput methods in the analysis of antibody subunits.

Through this work, it has been demonstrated that a high-throughput method for antibody subunit analysis can be readily obtained using a BioResolve RP mAb Polyphenyl Column and ACQUITY UPLC H-Class Bio System. With methods based on several different lengths of time, it was the BioResolve RP mAb Polyphenyl Column, as compared to a leading C₄ and polymeric column, that produced the highest resolution separations of a sample comprised of Ides-digested, NIST mAb subunits. With a total analysis time of only 3 minutes, high resolution and robust separations were obtained using the BioResolve RP mAb Polyphenyl Column. The ability of the column to deliver efficient and robust separations of mAb subunits makes it well suited for the characterization and monitoring of mAb product throughout the discovery, development, and manufacturing processes.

1. Liu, J.K.H. The history of monoclonal antibody development-progress, remaining challenges, and future innovations. Annals of Medicine and Surgery. 2014, 3, 113–116.
2. ICH Q8. Pharmaceutical Development. 2009.
3. Dong, M., et al. HPLC for characterization and quality control of therapeutic monoclonal antibodies. LCGC North America. 32, 10, 796–808.
4. Nguyen, J., et al. Designing a New Particle Technology for Reversed Phase Protein Separations Through the Optimization of Diffusion Properties, Waters Application Note, 720006168EN, Jan 2018.
5. Nguyen, J., et al. A Novel Phenyl Bonded Phase for Improved Protein Reversed Phase Separation, Waters Application Note, 720006169EN, Jan 2018.
6. An, Y., et al. A new tool for monoclonal antibody analysis: application of IdeS proteolysis in IgG domain specific characterization. Mabs. 2014, 6:4, 879–893.