Application Note

Rapid Size Variant Analysis of Monoclonal Antibodies Using SEC Columns


High-throughput and rapid size-exclusion chromatography (SEC) methods can be useful to support biotherapeutic process and formulation development. In addition, a rapid SEC method will be more effective for real-time purity analyses to support a production process. In these applications, SEC can be used to monitor self-associated or aggregated protein impurities or protein fragmentation that can impact product safety or efficacy.

In a previous application note, it was demonstrated that effective separations and extended column lifetimes could be achieved using mobile phases at physiological pH (7.4) and ionic strength using Waters™ MaxPeak Premier Protein SEC Columns. Consequently, the evaluation of the performance and column lifetime of both UPLC and HPLC versions of these recently introduced columns were evaluated while operating near their specified maximum pressures, resulting in analysis times from 2.1 to 3.0 minutes.

For this study, the lifetimes of a Waters ACQUITY™ Premier Protein SEC (250 Å, 1.7 µm, 4.6 x 150 mm) Column and two XBridge™ Premier Protein SEC (250 Å, 2.5 µm) Columns with dimensions of 4.6 x 150 mm and 7.8 x 150 mm were successfully evaluated over the course of 500 analyses. The separation performance was evaluated for both self-associated and selected fragmented size variants of four marketed monoclonal antibody biosimilars.


  • Rapid SEC analyses (2.1 to 3.0 minutes), SEC analyses of mAb size variants on UPLC and HPLC systems
  • Column lifetimes greater than 500 analyses near maximum specified column pressures
  • Reproducible determinations of both HMWS and LMWS impurity levels throughout lifetime study
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