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such as deamidated species. An example of an important acidic peptide present in tryptic digests of humanised monoclonal antibodies is the T37 peptide located in the Fc constant region, which has been linked to the therapeutic efficacy of mAb-based drug products [25]. This peptide contains four acidic amino acid residues and only one basic amino acid (see Figure 6A), and hence is negatively charged over a wide pH range. When using a standard system and column with the acidic low ionic strength mobile phases which are optimal for LC/MS (water and acetonitrile containing 0.1% formic
Figure 5: Comparison of chromatograms for a standard versus a MaxPeak HPS column for the separ- ation of the MassPREP Oligonucleotide Standard, a mixture of five oligodeoxythymidines (dT) with 15 - 35 nucleotides. On each column, three consecutive injections were made of a mixture containing 10 pmol of each oligonucleotide. Acetonitrile gradient separations were carried out using ACQUITY UPLC Oligonucleotide BEH C18, 130 Å, 1.7 µm 2.1 x 50 mm Columns. The aqueous mobile phase was 25 mM hexylammonium acetate (pH 6.0), the column temperature was 60°C, and the flow rate was 0.4 mL/min. The peaks were detected by absorbance at 260 nm.
obtained using the standard system and column would not reflect the true amount of ATP in the sample. In addition, the precision would suffer because of the changing peak area with injection number. In contrast, when using both a system and column that incorporate MaxPeak HPS technology, consistent peak areas were observed from the first injection.
MaxPeak HPS technology has been found to be particularly valuable for separations of oligonucleotides [19], which are important for several high profile uses, including as vaccines [20], therapeutics [21], and diagnostics, e.g. as primers for the polymerase chain reaction (PCR) [22]. LC and LC/MS methods are important for quality control of these modalities [23]. An example of the effect of metal surfaces in a UPLC system and column on the separation of a mixture of oligonucleotides is shown in Figure 5. The sample contained a mixture of deoxythymidines (see Figure 2 for the chemical structure) with 15, 20, 25, 30 and 35 nucleotides, which were separated using either a standard UPLC column or a UPLC column that incorporated MaxPeak HPS technology. Three consecutive injections were made on both columns. With the standard column, low peak heights were observed in the first injection, and the heights gradually increased in subsequent injections. Thus, with a standard column, a number of conditioning injections are necessary before reproducible results may be obtained. However, even after extensive conditioning, accurate quantification may be challenging because the effects of conditioning are often transient [19]. In contrast, the column incorporating MaxPeak HPS technology gave reproducible
peak heights for all three injections. This demonstrates the reduced need for conditioning when using MaxPeak HPS technology.
Another important class of analytes that benefits from MaxPeak HPS technology is acidic peptides, that is, peptides that exhibit a net negative charge at neutral pH [24]. Such peptides may occur, for example, in the enzymatic treatment of proteins. The separation of proteolytic digests is a fundamental tool used to characterise therapeutic proteins, such as monoclonal antibodies (mAb). When coupled to mass spectrometry, these peptide maps provide information on the amino acid sequence, as well as the presence of related impurities
Figure 6. A) Amino acid sequence of tryptic peptide 37 from NIST mAb, with the four acidic residues shown in red and the basic residue in blue; B) Overlaid chromatograms showing the peak for this peptide using conventional (orange trace) versus MaxPeak HPS (blue trace) system and column hardware. Acetonitrile gradient separations were carried out employing mobile phases containing 0.1% (v/v) formic acid with ACQUITY UPLC CSH 130 Å C18 1.7 µm 2.1 x 100 mm columns. The column temperature was 60°C and the flow rate was 0.2 mL/min The peaks were detected using ESI+ MS (SIR mode). The minor peaks are from deamidated variants. The data were time-aligned to facilitate comparison.
Figure 7. Plot of tailing factors for tryptic peptide 37 from NIST mAb vs time using conventional (orange trace) versus MaxPeak HPS (blue trace) system and column hardware. The systems were washed with 30% phosphoric acid, then water, before carrying out the peptide separations. The test conditions are given in the caption for Figure 6.
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