High-Resolution Protein Separation with < 0.2% Difference in Molecular Weight: Presto Column Distinguishes Between Bovine and Human Serum Albumins

Human and bovine serum albumin differ by only two amino acids. This paper highlights the high-resolution capabilities of the Presto column to separate these proteins.

Successful separation of large, closely related proteins poses a significant challenge, especially with the low efficiency of current methods, such as size-exclusion chromatography (SEC). For example, human serum albumin (HSA) and bovine serum albumin (BSA) are both reasonably large proteins, with high molecular weight (MW) of 65 kDa, differing by only two amino acids. Such a small difference (< 0.2%) is well beyond the resolution capabilities of SEC.

Addressing this challenge, we introduced our unique Presto column which is packed with non-porous, 2 µm, C18 modified silica particles. This high resolution material is offered here as an alternative solution for difficult separations that occur at high molecular weights. A note on performance characteristics and method optimization is also discussed as a comparison between a fast separation of albumin (short column, steep gradient), and a slower separation (longer column, shallow gradient).

Experimental Conditions

Separations were run using Presto FF-C18 columns (50 × 4.6 mm or 250 × 4.6 mm); mobile phases: (A) water + 0.1% trifluoroacetic acid, (B) acetonitrile + 0.1% trifluoroacetic acid. Gradients were 0 to 10 min, 0–80% B for 50 × 4.6 and 250 × 4.6 mm column; and 30–40% B for 250 × 4.6 mm column.

Results and Discussion

Chromatograms and retention times (RT) and full widths at peak half max (FWHM) of bovine and human serum albumins separated using 50 mm column with 0–80% B gradient, 250 mm column with 0–80% gradient, and 250 column with 30–40% B gradient are shown in Figures 1a–c and Figures 1d–f.

Figure 1: Bovine serum albumin: a) 50 mm length column, 0–80% B, b) 250 mm length column, 0–80% B, c) 250 mm column length, 30–40% B. Human serum albumin d) 50 mm length column, 0–80% B, e) 250 mm length column, 0–80% B, f) 250 mm column length, 30–40% B.

Qualitatively, both BSA and HSA present excellent peak shape for a short (50 mm) column under 0–80% B gradient, Figures 1a and 1d. The peak characteristics (RT and FWHM) are very similar for both BSA and HSA. The longer (250 mm) column under the same conditions predictably produces extended retention time and slightly improves separation (Figures 1b and 1e).

A striking difference is observed when a longer (250 mm) column is used along with a shallower (30–40% B) gradient (Figures 1c and f). Both BSA and HSA peaks exhibit new and distinct features, for example, BSA appear to consist of two overlapping peaks, while HSA has a single major peak that both fronts and tails. The retention time differences are even more pronounced, providing close to baseline resolution. Clearly, these conditions produce best separation (highest resolution) among the three sets tested.

Conclusion

Our unique non-porous, 2 µm particle C18 Presto FF-C18 produced very interesting separation results as a function of column length and gradient employed. Based on these, the column can be used in a wide variety of applications. Examples span from fast medium resolution runs to slower high-resolution runs. In particular, the optimized last set allows unprecedented separation of two large molecules (ca. 65 kDa) that vary by only two amino acids. This is equivalent to less than 0.2% difference in mass, a result better by at least an order of magnitude compared to SEC.

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