Biotherapeutics are generally larger molecules such as peptides, proteins and monoclonal antibodies with monomer molecular weight ranging from 3,000-150,000 Da. The drug must remain free from impurities such as fragment, dimer and other higher order aggregates as they may cause severe immunogenic response. This becomes even more important if the biotherapeutic protein is thermally susceptible. Historically, size exclusion is the preferred mode of chromatography used for separation and characterization of such applications. Here we report the online detection of absolute molecular weight of two recombinant protein samples using a UHPLC size exclusion chromatography (SEC) column directly connected to the LenS3TM Multi-Angle Light Scattering (MALS) detector.
Samples: BSA (Calibration standard)
Sample 1: Re. Protein (~90 kDa) at 1.72 mg/mL
Sample 2: Re. Protein (~90 kDa) at 3.64 mg/mL
The samples were stored at -20 °C and thawed to 8 °C just before analysis. The concentration was adjusted by diluting the sample in mobile phase pre-chilled at 8 °C.
Instrument: ThermoFisher Ultimate® 3000 UHPLC
Column: TSKgel UP-SW2000 (P/N 0023514),
2 μm, 4.6 mm ID × 30 cm L
Mobile BupH modified Dulbecco’s phosphate buffer
phase: prepared in light scattering grade water and
filtered through a 0.1 µm PES membrane
Flow rate: 0.20 mL/min
Detection: UV @ 280 nm and LenS3 MALS detector
(positioned in series: → UV → MALS)
Temperature: 25 °C
Injection vol.: Sample 1: 25 µL; ; Sample 2: 10 µL
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