Analytical Protein A columns are used to determine the titer of monoclonal antibodies for the optimal time for harvest of
the monoclonal antibody product. In this application note, Agilent Bio-Monolith Protein A columns are used to illustrate the
quick capture of monoclonal antibody titer from cell supernatant.
Methods and Materials
Sodium phosphate monobasic monohydrate (Sigma p/n S3522), sodium phosphate dibasic anhydrous (Sigma p/n RES20908-A7), citric
acid monohydrate (Sigma-Aldrich [p/n C1909]), and an Escherichia coli cell-lysis kit were purchased from Sigma-Aldrich Corp. (p/n CB0500). Humanized CHO-cell derived monoclonal antibody (IgG1)
was purchased from Bio-Creative Labs.
Eluent A is used for equilibration, binding, and re-equilibration. This buffer contained 20 mM sodium phosphate buffer, pH
7.4. To make 1 L of 20 mM sodium phosphate buffer, pH 7.4, dissolve 3.1 g of NaH2PO4•H2O and 10.9 g of Na2HPO4 (anhydrous) in distilled H2O to make a volume of 1 L. This is 0.1 M (100 mM) sodium phosphate buffer with pH 7.4 at 25 °C. This buffer can be stored
for up to 1 month at 4 °C. This stock solution is then diluted 1:5 with deionized water (take 200 mL of stock solution and
add 800 mL deionized water) to obtain 20 mM sodium phosphate buffer, pH 7.4. Eluent B is used for protein elution and contains
0.1 M citric acid, pH 2.8. To make 1 L of citric acid buffer, weigh out 21 g of citric acid monohydrate, dissolve it in approximately
600 mL of water with gentle stirring, and adjust the pH with 1 M HCl until the pH is 2.8. Finally, dilute the solution with
deionized water to the 1 L mark in a volumetric flask.
The manufacturer's recommended protocol was followed to obtain E. coli supernatant. The estimated concentration of protein was 40 mg/mL. The supernatant was then spiked with IgG1 as described;
40 mg/mL E. coli supernatant spiked with 2.5 mg/mL purified humanized IgG1. After mixing, the mixture was diluted further with mobile phase
A (20 mM sodium phosphate buffer, pH 7.4) with a 1:1 ratio to a final concentration of 1.25 mg/mL of IgG1 and 20 mg/mL E. coli supernatant.