UHPLC size exclusion column for protein analysis by light scattering and mass spectrometry
The TSKgel UP-SW3000-LS columns are dedicated to UHPLC size-exclusion analyses of monoclonal antibodies and other biopharmaceuticals combined with advanced detection such as multi-angle light scattering (MALS) or mass spectrometry (MS). The 2 µm silica particles are diol bonded for a hydrophilic surface. In combination with the 25 nm pore size, the columns are optimally suited for the analysis of proteins ranging between 10 kDa – 500 kDa. The column is also available without improvements for advanced detection methods: TSKgel UP-SW3000.
Protein Calibration Curves for UP-SW3000-LS SEC column |
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| Column: TSKgel UP-SW3000-LS, 2 µm, 4.6 mm ID × 30 cm
TSKgel UP-SW3000, 2 µm, 4.6 mm ID × 30 cm
Competitor SEC, 1.7 µm, 4.6 mm ID × 30 cm
Mobile phase: 100 mmol/L phosphate buffer, pH 6.7 + 100 mmol/L Na2SO4
Flow rate: 0.35 mL/min
Detection: UV @ 280 nm
Temperature: 25 °C
Inj. vol.: 10 μL
Samples: 1. thyroglobulin, 640 kDa
1’. thyroglobulin dimer
2. gamma-globulin, 155 kDa
2’. gamma-globulin dimer
3. ovalbumin, 47 kDa
4. ribonuclease A, 13,700 Da
5. p-amino benzoic acid, 137 Da |
Noise of buffer injection in multi-angle light scattering detection |
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| Column: TSKgel UP-SW3000-LS, 2 µm, 4.6 mm ID × 30 cm
Competitor SEC, 1.7 µm, 4.6 mm ID × 30 cm
Mobile phase: 100 mmol/L phosphate buffer, pH 6.7 + 100 mmol/L Na2SO4
Flow rate: 0.35 mL/min
Detection: Low angle light scattering (LALS) detected with LenS3 MALS detector
Temperature: 25 °C
Inj. vol.: 10 μL
Samples: Mobile phase only |
Key feature of UP-SW3000-LS UHPLC columns is a highly reduced noise level in advanced detectors saving time for equilibration and improving data quality. For multi-angle light scattering (MALS) applications, a low noise level is achieved in minutes, leading to high signal-to-noise ratios and thus high sensitivity. For mass spectrometry applications, ionization efficiency by electrospray ionization is improved and increases overall MS performance and instrument uptime. This makes the UP-SW3000-LS column particularly suited for in-depth analyses of aggregates and fragments of mAbs, bi- and multispecifics.
TSKgel UP-SW3000-LS lot-to-lot reproducibility |
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| Column: TSKgel UP-SW3000-LS, 2 µm, 4.6 mm ID × 30 cm of three different production lots
Mobile phase: 100 mmol/L phosphate buffer, pH 6.7 + 100 mmol/L Na2SO4
Flow rate: 0.35 mL/min
Detection: UV @ 280 nm
Temperature: 25 °C
Inj. vol.: 10 μL
Samples: Protein standard containing thyroglobulin, gamma-globulin, ovalbumin, ribonuclease A, p-amino
benzoic acid, 137 Da |
In addition, the UP-SW3000-LS columns offer reproducible and reliable size exclusion protein separations. This is explained by robust separations over multiple injections as well as a high column lifetime which reduces the need for frequent column replacements. However, low variations between column production lots facilitate column exchange.