The development and successful commercialization of protein-based biopharmaceuticals and diagnostic reagents frequently depends on the ability to adequately characterize these complex biomolecules. Waters ACQUITY UPLC^® BEH300, C4 and C18 RP, Protein-Pak^TM Hi Res IEX, ACQUITY UPLC BEH125, BEH200 1.7 µm and BEH450 2.5 µm SEC columns and associated methods can help improve your protein separation and characterization challenges.

Waters ACQUITY UPLC SEC System Solution
ACQUITY^® BEH125, BEH200 1.7 µm and BEH450 2.5 µm SEC columns are recommended for use on ACQUITY UPLC Systems, and are ideally suited for the separation and analysis of peptides and proteins including monoclonal antibody monomers from aggregates. Customers report that they can frequently provide higher sample throughput with improved component Rs compared to use of traditional HPLC-based SEC offerings. In addition, our BEH125, BEH200 1.7 µm and BEH450 2.5 µm SEC chemistries are quality controlled with relevant proteins to help ensure consistent and predictable batch-to-batch performance in validated methods.

Protein-Pak Hi Res Columns
Non-porous, hydrophilic polymer-based, high-ligand density particles in cation (CM and SP) and anion-exchange (Q) chemistries for characterization of protein and protein variants. 

ACQUITY UPLC BEH300, C4 and C18 Reversed-Phase UPLC Columns
Wide pore, Ethylene Bridged Hybrid (BEH) based 1.7 µm particles with C4 or C18 ligands designed for UPLC-based protein and polypeptide characterization.

UPLC^® Technology allows analytical chemists to reach far beyond conventional LC separations and has proven itself to be a major asset in increasing the productivity of laboratories around the world.  The latest addition to this application driven portfolio, is the ACQUITY UPLC^® SEC System Solution, enabled by the unique Ethylene Bridged Hybrid (BEH) Diol coated 1.7 µm particle technology.

• Determines aggregation levels in therapeutic monoclonal antibodies up to 10 times faster than traditional HPLC based SEC methods
• Fully optimized column chemistry significantly reduces the requirement for high salt concentration mobile phases
• QC tested with relevant proteins, ensuring unmatched batch-to-batch consistency and increased confidence in validated QC release methods

Waters Protein-Pak™ Hi Res IEX family of columns were developed to assist in the UPLC^® characterization of recombinant proteins and monoclonal antibodies destined to be used as therapeutics.  The non-porous, high-ligand density particles in these columns give higher resolution for high molecular weight proteins compared to traditional porous IEX particles. In addition, the particles in Protein-Pak Hi Res Ion-Exchange columns are surface modified with a multi-layered network of ion-exchange groups (sulfopropyl, carboxymethyl or quaternary ammonium), which assists in the effective binding of charged proteins. This allows for high sample loading capacities and component resolution while minimizing column fouling. 

• Innovative particle and ligand bonding processes yield protein binding capacities typically seen with porous IEX materials while delivering component resolution of large proteins usually observed with non-porous IEX columns.
• Quality-control tested with protein samples to help ensure consistent batch-to-batch performance.
• Columns containing hydrophillic, polymer-based IEX particles can be effectively cleaned with aggressive wash protocols.
• Column history and performance optimized when used on an ACQUITY^® UPLC System.

Waters invests significant research and development time to deliver solutions that help organizations address today's separation challenges. As a primary manufacturer of separations media, our ability to consistently deliver reliable, quality products, and support is second to none in the industry. Discover how our BEH300 C₄ columns supported by our world-class R&D, applications, and distribution organizations, help make the "Science of What's Possible" a reality.

• Well characterized, state-of-the-art bonding procedures
• Low pH and high temperature stability
• Quality-control tested with diverse protein mixture
• Consistent batch-to-batch reproducibility
• Available 1.7 μm particles for UPLC and nano UPLC applications

Rigorous Manufacturing and Testing Procedures Help Ensure Consistent Batch-to-Batch Performance

In 2006, Waters introduced UPLC^® Technology that demonstrated outstanding performance enhancements in resolution and speed compared to use of traditional HPLC. Our BEH300 C₄ material is available in two particle sizes for easy transfer from an HPLC protein separation method using 3.5 µm particles to the benefits of ACQUITY UPLC^® Technology using 1.7 µm particles. In addition, BEH300 C₄ material can be effectively run with mass spectrometry compatible eluents for collection of the information rich data possible using this advanced detection technique.

• Easily scaled 3.5 μm HPLC to 1.7 μm UPLC for enhanced separations
• Effective with MS compatible eluents to address the need for advanced detection techniques

 1.7 µm Particle Technology Benefits to Complex Protein Separations

Use of 1.7 μm particle technology yields improved resolution within mAb heavy chain fragment. Note that the full performance benefits of the ACQUITY^® BEH300 C₄, 1.7 μm column will only be realized with the low system volume and low detector dispersion of an optimized ACQUITY UPLC system.

 Successful Application of BEH300 C₄ to UPLC/MS Protein Analyses

Compared to many 100% silica-based reversed-phase columns, BEH300 C₄ offerings can be successfully used with MS friendly eluents.