Faster Separations Using Agilent Weak Cation-Exchange Columns Andrew Coffey, Agilent Technologies
Ion-exchange is a commonly used technique for the separation of complex protein mixtures. This application note demonstrates how analysis times can be significantly reduced, increasing throughput without compromising analytical performance by exploiting the benefits of small particle size, nonporous ion-exchange sorbents.
pH Gradient Elution for Improved Separation of Monoclonal Antibody Charge Variants Andrew Coffey, Agilent Technologies
Ion-exchange is used to separate protein mixtures under mild conditions usually by applying a shallow gradient of increasing salt concentration as eluent. pH gradient elution is used less because it can require complex buffer systems; however, modern quaternary HPLC systems are suited to generating pH gradient elution profiles from conventional buffer salts. This application note demonstrates the benefits of using pH gradient elution for separation of charge isoforms of monoclonal antibodies using Agilent Bio MAb columns.
Optimizing Protein Separations with Agilent Weak Cation-Exchange Columns Andrew Coffey, Agilent Technologies
Columns containing weak cation-exchange stationary phases offer a degree of versatility when used for separation of positively charged proteins. This application note explores the difference in selectivity that can be obtained by adjusting the pH of the mobile phase eluent, and how these can be changed to optimize the resolution of the compounds of interest using Agilent Bio WCX columns.
Analysis of Oxidized Insulin Chains Using Reversed-Phase Agilent ZORBAX RRHD 300SB-C18 Phu T. Duong and Linda Lloyd, Agilent Technologies
A new reversed-phase media, Agilent ZORBAX RRHD 300SB-C18 1.8 µm, was used for the separation of a typical protein biopharmaceutical, insulin. The column designed for UHPLC systems significantly reduced analysis time, which is critical for increasing the efficiency of QC for protein primary structure analysis. The 300 Å pore-size media is stable under acidic conditions, providing the robust reproducible separations required for protein QC.
Fast Separation of Recombinant Human Erythropoietin Using Reversed-Phase Agilent ZORBAX RRHD 300SB-C18, 1.8 µm Phu T. Duong and Linda Lloyd, Agilent Technologies
The Agilent ZORBAX 300SB-C18 1.8 µm RRHD column provides increased sensitivity, exceptional pH, and thermal stability. The use of a 1.8 µm column designed for UHPLC systems reduces analysis time, increasing the efficiency of QC for protein primary structure analysis. This application note focuses on the fast separation of various recombinant human erythropoietin isoforms. Methods are optimized for flow rates, gradient, and reproducibility under acidic conditions that contain trifluoroacetic acid.
Analyze MAb and BSA Digests by UHPLC with UV Detection and Agilent ZORBAX RRHD 300SB-C18 Phu T. Duong and Linda Lloyd, Agilent Technologies
A new reversed-phase media, Agilent ZORBAX RRHD 300SB-C18 1.8 µm, is used for the analysis of trypsin-digested monoclonal antibody and bovine serum albumen. The robustness of the media is demonstrated using different acidic eluents, temperatures, and flow rates. Good reproducibility is also evident.
Rapid UHPLC Analysis of Reduced Monoclonal Antibodies Using an Agilent ZORBAX Rapid Resolution High Definition (RRHD) 300SB-C8 Column James Martosella and Phu T. Duong, Agilent Technologies
An Agilent ZORBAX 1.8 µm RRHD 300SB-C8 reversed-phase column was used under optimized chromatographic conditions for delivering highly resolved ultra-fast separations of reduced and alkylated monoclonal antibodies. The column enabled separation of light chain and two heavy chain monoclonal antibody variants in less than 4 min. Alternate mobile phase compositions were also employed, making separations flexible for ultra-fast LC–MS analysis. At elevated operating pressures and temperature, the column delivered highly reproducible separations and excellent chromatographic run-to-run results.
Ultra High Speed and High Resolution Separations of Reduced and Intact Monoclonal Antibodies with Agilent ZORBAX RRHD Sub-2 µm 300 Å Diphenyl UHPLC Colum James Martosella and Phu T. Duong, Agilent Technologies
Rapid separations of intact and reduced MAbs were achieved with the use of an Agilent ZORBAX RRHD 300 Å 1.8 µm diphenyl reversed-phase column. MAbs expressed by both Chinese hamster ovary and CDH media cell lines were evaluated and compared with the goal of obtaining high resolution and high efficiency separations during rapid run times. The column also was evaluated at elevated operating pressures and temperature, and exhibited high operational tolerance during continuous investigations of reproducibility and lifetime.
Fast Separation of Monoclonal Antibody and Dimer by SEC with Agilent Bio SEC Tim Rice, Agilent Technologies
One of the main concerns with protein aggregates is that they may cause an adverse immune response in vivo. Size exclusion chromatography is the most widely used method to detect and separate protein aggregates from monomers. We present a method using Agilent Bio SEC-3, 300 Å pore size silica columns to achieve a separation of monoclonal antibody aggregates from monomer in less than 5 min, significantly less time than normally achieved by conventional HPLC.
Optimum Pore Size for Characterizing Biomolecules with Agilent Bio SEC Columns Keeley Mapp, Agilent Technologies
Size exclusion chromatography (SEC) is a technique for separating proteins, oligonucleotides, and other complex biopolymers in their native forms by size using aqueous eluents. For molecules of discreet molecular weight, such as proteins, SEC can be used to detect and quantitate monomers, dimers, aggregates, and fragments.
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