Modern Size-Exclusion Chromatography Separations of Biosimilar Antibodies at Physiological pH and Ionic Strength

Significance of the Topic

The accurate quantification of protein aggregates and fragments is critical for the quality control of therapeutic monoclonal antibodies. Conventional SEC mobile phases often require elevated ionic strength or non-physiological pH to suppress secondary interactions, but these conditions can dissociate transient aggregates and misrepresent critical quality attributes.

Objectives and Study Overview

This application note investigates the use of physiological buffer conditions (Dulbecco’s PBS at pH 7.4, 150 mM ionic strength) as an SEC eluent to characterize four FDA-approved biosimilar antibodies. The performance of three modern SEC columns—with different hardware surface chemistries and particle designs—is compared to a dextran-agarose reference to assess aggregate recovery, fragment resolution, and analysis speed.

Methodology and Experimental Design

Biosimilar samples of bevacizumab, infliximab, rituximab, and trastuzumab were analyzed neat after freeze–thaw. Mobile phase compositions ranged from 0.8X to 2X DPBS. Columns tested included a diol-bonded BEH 200 Å, 2.5 µm BioResolve SEC mAb; a PEO-bonded BEH 250 Å, 2.5 µm Waters XBridge Premier; and an 8.6 µm dextran-agarose Cytiva SuperDex 200 Increase. A 1.7 µm UPLC ACQUITY Premier version of XBridge was also evaluated. Flow rates varied to optimize resolution (0.25–0.5 mL/min). UV detection at 214 nm captured low-abundance size variants.

Used Instrumentation

• ACQUITY UPLC H-Class Bio system with CH-30A column heater
• ACQUITY UPLC Tunable UV detector (titanium flow cell)
• Columns: Waters XBridge Premier Protein SEC 250 Å, BioResolve SEC mAb 200 Å, Cytiva SuperDex 200 Increase, ACQUITY Premier Protein SEC 250 Å
• Empower 3 chromatography data system

Key Results and Discussion

• XBridge Premier maintained consistent recovery of high-molecular-weight species (HMWS) and fragment resolution from 0.8X–2X DPBS, while BioResolve required ≥1.2X DPBS to avoid loss of basic mAbs (pI ≥9.1).
• The SuperDex reference demonstrated minimal nonspecific interactions across DPBS concentrations but at the expense of a four- to eight-fold longer runtime.
• The ACQUITY Premier column achieved similar aggregate quantification to XBridge Premier at twice the linear velocity, reducing analysis time to 12.5 min, though fragment resolution was slightly decreased due to system dispersion.

Benefits and Practical Applications of the Method

• True physiological conditions improve relevance to in vivo aggregate behavior
• Broader buffer tolerance on Premier columns enables streamlined platform methods
• High throughput separations—up to eight-fold faster than dextran-agarose columns
• Simplified method transfer using pre-made DPBS minimizes development time

Future Trends and Potential Applications

Expanding physiological SEC to other biotherapeutics may enhance predictive aggregation profiles. Integration with online light scattering or mass spectrometry could further characterize aggregate species without buffer exchange. Continued optimization of column surface chemistries will likely broaden pH and ionic strength compatibility for diverse protein classes.

Conclusion

MaxPeak Premier HPS and BEH-PEO bonded SEC columns enable robust analysis of antibody size variants using physiological DPBS mobile phases. They match the inertness of traditional dextran-agarose media while delivering significantly higher throughput and platform flexibility for therapeutic protein characterization.

References

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2. Goyon A, Excoffier M, Janin-Bussat MC, et al. Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies. J Chromatogr B. 2017;1065–1066:119–128.
3. Wen J, Arakawa T, Philo JS. Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal Biochem. 1996;240(2):155–166.
4. Koza SM, Reed C, Chen W. Impact of LC system dispersion on the size-exclusion chromatography analysis of monoclonal IgG antibody aggregates and fragments. Waters Application Note; 2019.