The antibody drug market has continued to expand in recent years and will continue to do so for the foreseeable future. With this expansion, more antibodies will be designed and produced for targeting specific diseases. Size exclusion chromatography (SEC) is widely used to quantitate monomers, dimers, aggregates, and fragments in antibody analysis. Due to a high demand for better resolution and faster analysis time, more well-designed SEC columns have been introduced. These are 2 µm and sub-2 µm particle size SEC columns with the appropriate pore size for analyzing antibodies with optimized particle chemistry and column packing methods. Despite this improvement, nonspecific absorption of antibodies onto the column gel matrix poses a challenge, with some newly engineered antibodies possessing a high degree of hydrophobicity. The use of organic solvents such as isopropyl alcohol (IPA) or salts can decrease this interaction as reported by many scientists. However, the additives may alter the diffusion of these molecules, which results in retention time shift and poor peak resolution that did not occur in a typical aqueous buffer system, such as sodium phosphate buffer at neutral pH.
In this application note, a TSKgel(r) UP-SW3000, 2 µm SEC column was used for analyzing monoclonal antibodies (mAbs) with the addition of 15% IPA in sodium phosphate buffer, pH 6.7. As demonstrated, peak resolution and retention time shift were not impacted.
Column: TSKgel UP-SW3000, 2 µm, 4.6 mm ID ? 30 cm
Instrument: UltiMate(r) 3000 UHPLC system run by
Chromeleon(r) (version 7.2)
Mobile phase: 15% IPA in 100 mmol/L KH2PO4 / Na2HPO4,
pH 6.7, 100 mmol/L Na2SO4, 0.05% NaN3
Flow rate: 0.30 mL/min
Detection: UV @ 280 nm
Temperature: 30 °C
Pressure: 22 MPa (maximum column pressure is 34 MPa)
Sample: USP mAb standard
The excellent reproducibility of injection to injection of the USP mAb standard onto the TSKgel UP-SW3000, 4.6 mm ID × 30 cm column, with a typical sodium phosphate buffer, pH 6.7, is shown in Figure 1. This figure is an overlay of 14 consecutive injections of the USP mAb standard sample at the flow rate of 0.3 mL/min. The retention times of monomer, dimer, aggregate, and fragment peaks are nearly unchanged. Peak width and peak shape are very consistent from injection to injection.
Table I consolidates the recorded calculated data from the monomer and dimer peaks of the 14 injections from Figure 1 with the %RSD of retention time and percent relative area below the allowance from the USP monograph guidance.
Figure 2 shows the overlay of 15 injections of the USP mAb standard sample onto the TSKgel UP-SW3000 column with the addition of 15% IPA. These injections are performed after the column is subjected to 15 injections of the USP mAb standard sample with sodium phosphate buffer, pH 6.7, without IPA. The baseline of the first injection (as shown in blue) indicates that the column takes only one to two injections to be stabilized. After that all subsequent injections are overlaid perfectly.
Table II lists the calculated data from the monomer and dimer peaks of the 15 injections from Figure 2 with the %RSD of retention time and percent relative area. As shown, the %RSD is below the allowance from the USP monograph guidance.
At 0.3 mL/min, the pressure of the column is slightly higher when IPA is added to the mobile phase compared to when the column is operated without IPA. However, the pressure is only at 22 MPa with the IPA added. It is still far below the allowance of the maximum pressure of 34 MPa of the column's rating. With this low operating pressure, the TSKgel UP-SW3000 column can be operated with both HPLC and UHPLC systems. As the chromatograms indicate, all runs are completed within 15 min.
Figure 3 is an overlay of injections with and without IPA added to the mobile phase. The overlay indicates the similarities of peak retention times, peak width and peak height of dimer, monomer, aggregate, and fragment peaks between the two different conditions.
An appropriate percentage of organic solvent such as IPA does not alter the diffusion of mAb molecules using a TSKgel UP-SW3000 column. As demonstrated, this column can be successfully operated with the addition of 15% IPA. Data indicates that the column's particle chemistry and packing are optimized so that with the addition of an appropriate amount of selected organic solvents, there is no alteration of peak retention time or poor peak resolution.
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