Martin Samonig Applications Scientist Thermo Fisher Scientific
Alexander Schwahn European Support Expert Thermo Fisher Scientific
Ken Cook Ph.D. EU Bio-Separations Expert Thermo Fisher Scientific
Mike Oliver Product Manager Thermo Fisher Scientific
Remco Swart Ph.D. Director Product Manager Thermo Fisher Scientific
Rowan Moore Vertical Marketing Manager Thermo Fisher Scientific
Kelly Broster Pharma & Biopharma Vertical Marketing Manager Thermo Fisher Scientific

This is the third article in a series of four, describing the characterization of protein-based bio-therapeutics by bottom-up analysis at peptide level (peptide mapping analysis). This article summarizes the requirements for chromatographic separations.

Reproducibility and associated confidence in results is of paramount importance in ensuring accurate peptide mapping.  Improvements in reproducible sample preparation, as shown with the Thermo Scientific™ SMART Digest™ kit, need to be complemented across the entire workflow. The chromatographic separation stage has a significant impact on the quality and of results that can be obtained, as well as throughput. Both instrument and column considerations are critical to success in this area.

Peptide mapping has typically employed “long” gradients to separate highly complex peptide-mapping samples, with run times often taking hours to complete. However, the duration of separation is often at odds with the needs of high-throughput biopharmaceutical environments. Fast peptide-mapping achieved during the cell-line development/clone selection phase can expedite the transfer of candidate compounds into the drug process development phase. The advantages of an increase in speed must not come at the expense of the quality of the analytical results. By ensuring this, the method could also be transferred into the QC environment.

In order to achieve these requirements, the column and UHPLC system employed must provide high levels of reproducibility and robustness coupled with the ability to deliver fast and efficient separations.

Column Considerations for Peptide Mapping

Protein digest samples are often very complex and consequently gradient conditions and columns with high peak capacity are required to achieve adequate separation. To achieve high peak capacity, small particle sizes and long columns are typically employed along with long run times and shallow gradients. These long run-times are undesirable due to throughput considerations, as well as from a sensitivity perspective due to in-column peak dilution.

Sensitivity is of particular importance for the identification of low-level peptides carrying potential modifications and also when peptide mapping is performed with ultra-violet (UV) detection alone. The risk is that low-level components will not be detected. Therefore, high peak capacity is required while balancing a total run time which is reasonably short.

The use of elevated temperatures has a major impact on the peak capacity of peptides separated by reversed phase (RP) chromatography. Temperature increases from 40 °C up to 80 °C can lead to >20% increase in peak capacity for a 30-minute gradient. Peptides have increased diffusion rates at higher temperatures, ultimately producing narrower peaks, and thus delivering higher peak capacity peptide maps. The use of mobile phase pre-heating can also reduce peak broadening and retention time variation through the prevention of thermal differences inside the column.

The choice of stationary phase also has an impact on the quality of data achieved for peptide mapping. Peptide separations normally employ RP columns with silica-based C18 stationary phases with near-zero silanophilic activities which result in superior separation with minimal peak broadening. The use of formic acid as a mobile-phase additive also provides additional advantages for MS detection. Compared to TFA, formic acid causes lower ion suppression and consequently results in higher sensitivity for MS-based methods. In contrast, UV-only methods benefit from the use of TFA as an ion-pairing reagent, often resulting in improved peak separations compared to formic acid.

UHPLC System Considerations for Peptide Mapping

Proteins and peptides by their nature may have an affinity to interact with metal surfaces and might adsorb, therefore, the UHPLC system used should be “biocompatible”. Reproducibility and robustness are critical for peptide-mapping experiments and the UHPLC systems used must be capable of delivering highly stable, precise flow-rates and gradients in order to deliver highly reproducible retention times (RTs) (Figure 1). 

Highly reproducible peptide maps and retention times also enable confidence in direct comparisons of samples for monitoring of conditional differences (Figure 2). The ability to compare reduced and non-reduced peptide samples provides information on disulfide bond locations within the molecule, resulting in a greater understanding of the protein structure.

Figure 1. Retention time repeatability of a peptide separation

Chromatographic reproducibility is also of particular importance where UV detection is used for standalone detection, and assignments are made solely based on analyte elution time. Confidence in the reproducibility of the protein digestion and separation allows the facile transfer of methods from LC-MS platforms to LC-UV (Figure 3).

There are a number of other considerations which can have a dramatic impact on the results obtained, and the applicability of the system to throughput requirements. The choice of the autosampler can affect reproducibility; this can be mitigated by using systems which offer sample loop pre-compression to minimize pressure changes. Does the column oven offer a wide temperature range and high stability? Does the sample manager offer sample capacities which meet throughput requirements?

Figure 2. Comparison of reduced and non-reduced forms of rituximab

The choice of pump also needs to be considered based on the application requirement. For high-throughput environments requiring fast peptide-map run times, a binary high-pressure pumping system can offer lower gradient delay volumes and therefore faster re-equilibration and re-injection times. The ability to increase flow rates by high-pressure capabilities allows faster run times. By varying the pressure and flow rate, gradients can be reduced from >30 minutes to just 5 minutes. A 1,000-bar pressure system would be capable of achieving the 13-minute gradient, and a 1,500-bar system would have the pressure capabilities to achieve the 5-minute separation shown in Figure 4.

Figure 3. Overlaid chromatograms of the total ion current (TIC) and the UV trace at 214 nm of a rituximab sample digested with a Thermo Scientific™ SMART Digest™ kit

For all five gradient times tested, from 30 minutes down to 5 minutes, very good separation is achieved while maintaining sequence coverage of 100% for both the light and heavy chain of rituximab.

For separations where throughput is not as important and system flexibility is needed, a quaternary pumping UHPLC system is also appropriate. Both options need to deliver low dispersion, robust and reliable high flow rates, and generate low baseline noise.

In the next article in the series, we will investigate the role of mass spectrometry in the peptide-mapping workflow.

Figure 4. Reduction in chromatographic run-time for rituximab while maintaining 100% sequence coverage

Additional Resources
1. Technical Note: High Resolution Peptide Mapping for Biopharmaceutical Analysis
2. Application Note: Providing the Highest Retention Time and Peak Area Reproducibility for Maximal Confidence in Peptide Mapping Experiments. Application Note: LC-UV-MS Peptide Mapping Development for Easy Transfer to LC-UV QA/QC.
3. ‘Ultra’ Biotherapeutic Characterisation. Innovations in Pharmaceutical Technology, April 2016, pages 32-37

Martin Samonig is application scientist, LC-MS; Alexander Schwahn is European support expert for biopharma industry; Ken Cook is EU bio-separations expert; Mike Oliver is product manager, sample preparation and Accucore LC products; Remco Swart is director, product manager, HPLC & LC-MS solutions; Rowan Moore is pharma & biopharma vertical marketing manager; Kelly Broster is pharma & biopharma vertical marketing manager at Thermo Fisher Scientific.

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