Introduction
Antibody therapeutics are enjoying high growth rates, the major areas of therapeutic application being cancer and immune/inflammation-related disorders including arthritis and multiple sclerosis. In 2013, six of the top ten best-selling global drug brands were monoclonal antibodies (mAbs) and more than 400 monoclonals were in clinical trials. The characterization of these complex biomolecules is a major challenge in process monitoring and quality control. The main product characteristics to be monitored are aggregate and fragment content, glycosylation pattern and charged isoforms.
The standard method used in biopharmaceutical QC for mAb aggregate and fragment analysis is size exclusion chromatography (SEC). A new series of 2 micron silica based UHPLC columns with 25 nm (250 Å) pore size can be applied to either increase speed or improve resolution of the separation of antibody fragments, monomers, and dimers.
Experimental
Columns: TSKgel UP-SW3000 (P/N 0023449), 2 μm
Competitor Protein SEC Column, 1.7 μm
Column size: 4.6 mm ID x 15 cm
Eluent: 100 mmol/L phosphate buffer (pH 6.7) +
100 mmol/L sodium sulfate + 0.05% NaN3
Flow rate: 0.35 mL/min
Temperature: 25 °C
Detection: UV @ 280 nm, micro flow cell
Sample (Calibration):
1. thyroglobulin, 640,000 Da
(1’ thyroglobulin dimer);
2. γ-globulin, 155,000 Da
(2’ γ-globulin dimer);
3. ovalbumin, 47,000 Da;
4. ribonuclease A, 13,700 Da;
5. p-aminobenzoic acid, 137 Da
Inj. Volume: 5 μL
Sample (mAb Analysis):
therapeutic mAb (mouse-human chimeric)
1: trimer; 2: dimer;
3: monomer ; 4: fragment
Inj. Volume: 10 μL
Results
Figure 1 shows the calibration curves of the new TSKgel UP-SW 3000 2 μm column and a commercially available 1.7 micron UHPLC column. The calibration of TSKgel UP-SW3000 shows a shallower slope in the region of the molecular weight of γ-globulin. These differences in the separation range and steepness of the curves are related to a slight difference in pore size (25 nm for TSKgel versus 20 nm for the 1.7 µm material).
Calibration curves
>> Download the full Application Note as PDF
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