TSKgel® UP-SW3000 Method Optimization
Optimizing mobile phase conditions ensures that the highest resolution separation is achieved. Tosoh Bioscience recommends starting with the following standard mobile phase conditions for mAbs and related product (proteins, bispecifics, ADC, etc.) analysis by size exclusion chromatography (SEC) using the TSKgel® UP-SW3000 (Figure 1):
Standard Mobile Phase Conditions
Column:
Mobile phase:
Gradient:
Flow rate:
Detection:
Temperature:
Instruments:
Samples: |
TSKgel® UP-SW3000
100 mmol/L sodium phosphate, 100 mmol/L Na2SO4, 0.05% NaN3, pH 6.7
isocratic
0.35 mL/min
UV @ 280 nm
25 °C
HPLC or UHPLC system
mAb or proteins |
Figure 1: UHPLC Separation of a mAb on TSKgel® UP-SW3000 (4.6 mm ID × 30 cm) Under Standard Mobile Phase Conditions
For mAbs or related products with more hydrophobic character or those that do not exhibit favorable separation under standard conditions, mobile phase additives can be added to the standard condition in order to reduce unwanted secondary interactions. This can result in a decrease in peak tailing and an increase in efficiency and resolution.
Tosoh Bioscience recommends the use of up to 20% organic or up to 500 mmol/L arginine to improve mAb or related product separations (Figures 2 and 3).
Figure 2: UHPLC Separation of a mAb on TSKgel® UP-SW3000 (4.6 mm ID × 15 cm) Under Standard Mobile Phase Conditions with IPA (as indicated)
Figure 3: UHPLC Separation of an ADC on TSKgel® UP-SW3000 (4.6 mm ID × 15 cm) Under Standard Mobile Phase Conditions with Arginine (as indicated)
If you would like additional support optimizing your analysis method, please reach out to our technical service team who are always willing to assist in any way (contact info to the left).