Increasing Speed for Biocharacterizations: Peptide Mapping ofMonoclonal Antibodies with an Agilent AdvanceBio Peptide Mapping Column - - Chromatography Online
Increasing Speed for Biocharacterizations: Peptide Mapping ofMonoclonal Antibodies with an Agilent AdvanceBio Peptide Mapping Column


The Application Notebook
pp. 706-708

Peptide mapping by reversed-phase chromatography is the mainstay technique in biotherapeutic analysis, delivering the comprehensive characterization of biopharmaceutical products. When interfaced with a mass spectrometer (MS), it can deliver the identification of proteins and its variants, determine post-translational modifications (PTMs) and locations, and confirm protein sequences.

However, peptide mapping represents a significant chromatographic challenge because of the inherent complexity of protein digests. As a result, many organizations struggle with developing robust and reliable peptide maps. In general, their peptide maps have suffered from low sensitivity, poor peak shapes, and very long separation times to achieve the desired resolution.


Figure 1: Fast HPLC optimizations of mAb tryptic digest. Mobile phase A: water (0.1% FA) Mobile phase B: 90% acetonitrile (0.1% FA), Injection: 15 L, Temperature: 40 C, DAD: 215 nm (a): 75 min. separation on a 2.1 150 mm AdvanceBio Peptide Mapping column generating 59 peptide peaks. (b): 14 min. separation on a 2.1 100 mm AdvanceBio Peptide Mapping column generating 57 peptide peaks.
The new 2.7 um AdvanceBio Peptide Mapping column has been introduced to fulfill a critical segment in biotherapeutic characterizations, for generating both rapid and highly efficient peptide maps at low liquid chromatography (LC) system pressures. Using superficially porous chromatographic media, the AdvanceBio Peptide Mapping columns achieve substantial improvements in peptide mapping during very fast run times, while still maintaining high efficiency peak performance.

Rapid mAb Tryptic Digest Peptide Mapping by HPLC–UV




To highlight this performance, the AdvanceBio Peptide Mapping column was used for the rapid LC–MS analysis of a monoclonal antibody (mAb) tryptic digest. Traditionally, shallow gradients of 2 h or longer are employed for mapping mAb peptide maps to achieve the desired baseline performance. The longer run times ensure that optimum resolution is achieved and that critical PTM peak information is not sacrificed during the shorter run. However, the AdvanceBio Peptide Mapping column has the flexibility to increase analysis speed without sacrificing separation performance. The UV–LC AdvanceBio column separations in Figure 1 demonstrate this flexibility. The top chromatogram provides an example of a more common peptide map generated during 75 min on a 2.1 150 mm column at a standard flow rate (relative to column internal diameter) of 0.2 mL/min. The separation provides excellent baseline resolution with 59 mAb tryptic digest peaks resolved over the entire gradient profile. In the bottom chromatogram comparison, a 2.1 100 mm AdvanceBio column was used with a very steep gradient at a high flow rate of 0.6 mL/min but at a fraction of the runtime in under 14 min. In this separation, the mAb tryptic peak count has remained relatively unchanged (57 peaks) while baseline resolution, peak shapes, sensitivity, and selectivity have not been compromised. In addition, the column back pressure remained well under 500 bar making this rapid and highly efficient separation achievable on traditional high perfomance liquid chromatography (HPLC) instrumentation.


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