This note describes a rapid IgM quantification method for cell culture product and purification process monitoring using the Agilent Bio-Monolith QA column.
This document details an analytical method for measuring IgM levels in cell culture supernatants and monitoring their distribution in chromatography fractions. It highlights the use of the Agilent Bio-Monolith OA column as an alternative to high-performance protein A columns, which can denature IgM antibodies under harsh elution conditions. The method utilizes ion exchange chromatography, specifically strong anion exchange, to effectively separate and quantify IgM.
| Column Part Number | 5069-3635 |
|---|---|
| Column Dimensions | 5.2 mm id x 4.95 mm |
| Mobile Phase A | 10 mM sodium phosphate, pH 7.0 |
| Mobile Phase B | 250 mM sodium phosphate, pH 7.0 |
| Gradient | Linear gradient from A to B within 10 min |
| Detection Wavelength | UV at 280 nm |
| Flow Rate | 2 mL/min |
The part number for the Agilent Bio-Monolith OA column is 5069-3635.
It is used because IgMs are easily denatured by the harsh conditions required for elution of most affinity ligands used with protein A columns.
Ion exchange chromatography is a strong candidate, as IgMs often exhibit strong retention on cation or anion exchangers.