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Online CE-LIF-MS for the characterization of antibody glycosylation
By Tobias Preckel
Agilent Product Manager, OFFGEL Fractionator
Analytical methods for characterizing carbohydrate structures on therapeutic proteins are of critical importance due to the role of carbohydrates in clearance and mechanism of action. The most common techniques for carbohydrate profiling involve high performance liquid chromatography (HPLC), mass spectrometry (MS), and capillary electrophoresis (CE). For many users in biopharmaceutical and biotechnology applications, CE has become the method of choice due to high peak efficiencies and speed of analysis. [1] To overcome the potential difficulties associated with characterizing minor peaks in CE analysis, CE-MS is emerging as a powerful solution with continuous advances in separation capability, MS sensitivity, and assay robustness.
Unique setup allows correlation of LIF and MS traces
A typical CE-MS system allows direct coupling of the CE capillary with the MS sprayer inlet. In this mode, the capillary inlet is placed in the inlet buffer, while the capillary outlet exits the instrument and is placed within an electrospray MS source. In many cases, it is advantageous to not only rely on an ultraviolet (UV) signal from a diode array detector (DAD), but to also obtain an optical signal of the separated components just before they enter the MS. For online laser induced fluorescence (LIF) detection, Picometrics’ technology offers the advantage of flexibility of detection location. In this case, the small LIF detection cell is coupled to the capillary upstream of the electrospray tip, and is held in place by an adjustable arm. Figure 1 shows a schematic of this instrumentation.
In this arrangement, you can obtain both LIF and ion intensity signals within the same analysis, enabling accurate comparisons of the two data sets. Several researchers have successfully used CE-MS technology to elucidate structures of complex carbohydrates. In an industrial setting, CE-MS is used to identify peaks in routine assays involving UV or laser induced fluorescence detection. However, comparing CE-LIF and CE-MS data can often be challenging due to possible differences in peak resolution and sensitivity. To overcome this obstacle, you can use a Picometrics ZETALIF Discovery system to allow online LIF detection at the same time as CE-MS analysis.
Identification of minor glycan species
This online CE-LIF-MS technology has been used successfully for the direct identification of minor carbohydrate species. [2] An example is shown in Figure 2, where carbohydrates from a therapeutic monoclonal antibody (mAb) are analyzed by online CE-LIF-MS and minor species can be accurately identified and quantified.
Online CE-LIF-MS is a very useful tool for analysis of therapeutic glycoproteins. The Picometrics LIF detector integrates easily with the Agilent CE system, which can also be coupled with any of the Agilent 6000 Series LC/MS systems for identification of unexpected minor peaks during routine CE-LIF analysis. For more information, visit the Agilent CE-MS product page, or the Picometrics Web site.
Acknowledgment
We are extremely grateful to Lynn A. Gennaro, Protein Analytical Chemistry, Genentech Inc., South San Francisco, California, for implementing the present method, and for sharing the data with Picometrics and Agilent Technologies.
References
- Ma, S.; Nashabeh, W. “Carbohydrate Analysis of a Chimeric Recombinant Monoclonal Antibody by Capillary Electrophoresis with Laser-Induced Fluorescence Detection,” Anal. Chem. 1999, 71(22), 5185-5192.
- Gennaro, L.; Salas-Solano, O. “On-line CE-LIF-MS Technology for the Direct Characterization of N-Linked Glycans from Therapeutic Antibodies,” Anal. Chem. 2008, 80(10), 3838-3845.
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