In this study, we used an ACQUITY UPLC H-Class Bio System coupled to a SYNAPT G2-Si for mAb subunit characterization. This application demonstrates the benefit of using the BioResolve RP mAb Polyphenyl Column which was purposefully designed and contains solid core, silica-based particles surrounded by a porous layer (i.e., SCP) that reduces intra particle diffusion distances of the chromatographed proteins to decrease peak band broadening compared to what is frequently seen using fully-porous particles. 450Å pores are contained within the coated particle and the total particle size is 2.7 µm. These attributes contribute to the packed column’s ability to deliver outstanding intact mAb and mAb subunit component resolution, recovery, and low injection-to-injection carryover.3-4
Chromatographic examples are shown for the Waters® mAb Subunit Standard (P/N: 186008927), a new material that is manufactured from reduced, IdeS-digested NIST mAb and available for use as a benchmarking, proficiency testing, or system suitability tool. The respective theoretical masses for the Fc/2, LC, and Fd’ fragments in this standard are 23790.81 Da, 23127.51 Da, and 25705.89 Da. It was found that each of these three subunits could be baseline resolved using a 10 minute gradient (Figure 1), and a 2.1 x 50 mm column (BioResolve RP) or 1.7 x 50 mm column (leading C4). As shown in Figure 1, the BioResolve RP mAb Polyphenyl Column was capable of yielding favorable chromatographic separations using two different MS-friendly mobile phases: one based on 0.1% formic acid and another on 0.02% TFA. The enhanced resolution provided by the BioResolve RP mAb Polyphenyl Column, resulted in improved peak widths and baseline resolved impurity-peaks compared to the leading C4 column used in the analysis.
To achieve these exemplary results, care was given to optimize the mass load of the analysis at a level of 0.5 µg. Separate experimentation has shown that the recommended on-column loading for the employed 2.1 mm I.D. column is between 0.05 and 2.50 µg and that it is important to consider this variable to reach optimal performance for detecting low-level species without saturating the MS detector. With these conditions, extracted ion chromatograms for each subunit yielded narrow, baseline-resolved peaks; the peak widths at half height reported in Figure 1 corroborate this observation. These sharp chromatographic peaks yielded high quality deconvoluted mass spectra, such as the one corresponding to the Fc/2 peak (Figure 2). Applying this analysis to the entire chromatographic profile led to the generation of a detailed view of the sample along with a list of masses and relative abundance information for the most abundant protein modifications of the Waters mAb Subunit Standard (Table 1).
The chromatographic resolution of these example assays also revealed several additional, low abundance peaks, which have been labeled as 1 through 6 in Figure 1 for the BioResolve RP mAb Polyphenyl Column. It is from the resolution afforded by the BioResolve RP mAb Polyphenyl Column that a baseline separation could be achieved to support the identification of MS-based ‘product-related’ impurities. For instance, peaks 2 and 5 can be attributed to a fragment of the Fd’ subunit (PQ[1-88]D) that is present in the standard and represented by an average mass of 9832.2 Da.5
