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

This is the second 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 advantages of new approaches to protein digestion which can address many of the challenges faced today.

Peptide mapping is a critical workflow in bio-therapeutic protein characterization and is essential for elucidating the primary amino acid structure of proteins. Within a bioproduction, this is necessary for manufacturing process monitoring and quality control (QC) as it enables product comparability testing. This allows identification of any product-related impurities, such as deamidation and/or oxidation following any formulation, manufacturing process, or storage change.

In order to generate a peptide map, the protein is digested into its constituent peptides typically via an in-solution based enzyme reaction. These approaches are often subject to a number of issues that are not aligned to the needs of the modern biopharmaceutical laboratories, which are tasked with providing high-quality analytical results, often in high-throughput, regulated environments. These issues include: 

1. Complex procedures–prior to digestion the protein must be denatured. This is done in order to unravel and open the protein structure to allow for optimal enzyme interaction. This requires multiple steps, including disulfide bond reduction, and free sulphydryl alkylation. 

2. Reproducibility of results–due to the complexity of the process and the chemicals involved (potential for introduction of chemically induced posttranslational modifications [PTMs]), there is the potential for poor reproducibility which has a significant effect on the confidence in the analytical results produced.

3. Speed of analysis–depending on the size and complexity of the protein being analyzed, the digestion process can take up to 24 hours to complete. This, combined with the complexity of the process, does not lend itself to automation and high-throughput processing. 

Figure 1. Heat-stable, immobilized enzyme design.

In order to meet these challenges, a number of sample preparation options have shown promise; in particular the Thermo Scientific™ SMART Digest™ Kit, which is a heat stable, immobilized resin-based enzyme design (Figure 1).

The SMART Digest kit enables a simplified process, where the heat stable enzyme allows the protein to be unfolded at elevated temperatures without the need for chaotropes such as urea. This, combined with pre-mixed buffers, helps reduce the process to a few simple steps (Figure 2).

Figure 2. Simple procedure using the SMART Digest kit.

Due to the simplified procedure and reduced number of chemicals, the new approach allows for a highly reproducible digestion process regardless of operator or experience. Therefore, eliminating the need for complex denaturation, reduction, and alkylation steps. Figure 3 shows UV chromatogram overlay from three separate digestions from the same mAb, conducted by three individual operators. The peptide maps generated perfectly overlap with an average RSD for the peak area of less than 5%.

In comparison with traditional in-solution digestion methods, similar levels for all modifications are detected, and no significant trends of increased or decreased PTMs in any of the conditions are observed with the SMART Digest kit. It is noteworthy that for many modification sites, e.g., deamidation of asparagine residues, the amount detected in the resin-based digest sample was actually lower compared to in-solution digest samples (Figure 4, 5 & 6). 

Figure 3. UV chromatogram overlay from three separate SMART digestions from the same mAb, conducted by three individual operators. The 15 marked peptides in each sample were used for inter-user/inter-day RSD value calculations.


Figure 4. Relative abundance of 85 identified modifications, including oxidation, double oxidation, glycation, glycosylation, NH3 loss, isomerization, lysine truncation, methylation, dimethylation, and carbamylation. 


Figure 5. Relative abundance of 12 identified oxidations (A) and 5 deamidations (B) in different runs with various digestion methods. 


Figure 6. Relative amount of total deamidation and oxidation modifications measured for the six different digest conditions (Normalization to results produced by the SMART Digest kit, 15 min). 

Due to the excess of resin-based, heat-stable enzyme in the SMART Digest kit, the speed of digestion can be greatly accelerated, providing procedures which can be performed in just a few minutes or hours, compared to overnight. This not only speeds up workflows, but significantly reduces method development time. The time of digestion is easily optimized by taking time-points for analysis until no intact protein or very large peptides remain. In the example below, it can be seen that for carbonic anhydrase full digestion is achieved in 5 minutes (Figure 7).

This new approach to protein digestion facilitated by the novel heat stable, immobilized enzyme design allows for a significant improvement in speed, simplicity, and reproducibility of results. However, other factors across the workflow also have a significant influence on the quality of results obtained. We shall investigate these in the subsequent articles.

Figure 7. Time course experiment for optimized digestion time for Carbonic Anhydrase.

Additional Resources
1. SMART Digest and SMART Digest ImmunoAffinity (IA) Technical Guide
2. SMART Digest Kit User Manual
3. Application Note: SMART Digest compared to classic in-solution digestion of rituximab for in-depth peptide mapping characterization
4. SMART Digest Kit Digestion Time Calculator
5. Webinar: Take the SMART Route to Protein Digestion – How to increase reproducibility whilst reducing preparation time

Martin Samonig is an application scientist LC-MS, Alexander Schwahn is the European support expert for biopharma industry, Dr. 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, and Rowan Moore is pharma & biopharma vertical marketing manager at Thermo Fisher Scientific.

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