Divide and Conquer Methods for Protein Characterization

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Improved techniques in peptide mapping aid in quality control of protein therapeutics.

In the biopharmaceutical arena, a growing number of companies are working on protein therapeutics. More and more traditional biotechnology companies are working on second- or third-generation protein therapeutics (primarily antibodies) while many pharmaceutical companies are developing generic versions of first-generation drugs. For many blockbuster protein therapeutics such as erythropoietin (EPO), G-CSF, interleukin-2 (IL-2), and growth hormone, patent protection has expired or will do so in the next five years. While the economic incentive for developing generic alternatives for these blockbusters is clear, many interested companies lack expertise in biopharmaceutical manufacturing and quality control testing procedures for such protein therapeutics.

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Figure 1: Tryptic digest peptide maps of bovine and canine cytochrome c run on a Jupiter Proteo 250×4.6 mm column. Sample preparation conditions were not optimized: reagent grade trypsin and a standard heating block were used and the reaction was not quenched upon completion. Note how the varying degrees of digestion make it difficult to identify any contrasts in the peptide map due to sequence difference between the two proteins. (Source: Phenomenex Inc.)

Peptide mapping (also known as enzymatic protein digestion) is one test where many groups struggle in developing a robust and reproducible analytical method. While the technique can be powerful in detecting and identifying minute changes in a protein therapeutic, poor sample preparation and faulty chromatographic methodology can minimize the utility of this mapping. This article discusses some simple insights that can vastly improve the results of peptide mapping techniques. A thorough understanding of the sample preparation process is critical to the success of these methods. Some often-overlooked chromatographic principles also are discussed.

Finding quality proteases
The goal of proteolytic analysis is to compare the peptides generated during an enzymatic digestion of a protein sample to a known standard. Because enzymatic digestion is a dynamic process, any good peptide map procedure should be performed side-by-side with a reference standard protein. To guarantee results, it’s important to use a freshly-prepared, high-quality proteolytic enzyme. This reagent should be HPLC- or sequencing-grade, and trypsin is the most commonly-used proteolytic enzyme. In order to guarantee similar specific proteolytic activity for every digest, the enzyme should not be reused. Lower-quality, proteolytic enzymes may contain other protease activities that can lead to irreproducible results between digests. This is particularly true of low-quality trypsin, which contains chymotrypsin and other proteases. After the addition of proteolytic enzyme, proper mixing must be performed for the enzyme to react with all protein present to ensure homogeneous digestion of the target. Fixed digestion time and the use of proper temperature control, achieved with a circulating water bath, ensure similar degrees of

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Figure 2: Tryptic digest peptide maps of bovine and canine cytochrome c run on a Jupiter Proteo 250x 4.6 mm column. In these peptide maps, high-quality trypsin and water bath heater were used for digestion. Samples were also quenched with acid prior to injection on the HPLC. Note that early eluting peptides closely match in retention time and intensity. The only difference between the maps are in the late eluting peaks were there are amino acid difference between the two species. (Source: Phenomenex Inc.)

digestion for every analysis. Finally, proper quenching of a proteolytic reaction is crucial to ensure that all samples have equal digestion time. A common mistake among first-time users is to assume that digestion stops upon removal of the samples from a water bath incubator. In fact, digestion continues, albeit at a lower reaction rate, unless the reaction is quenched. Because many enzymes have a limited pH activity range, addition of acid after digestion will prevent proteolytic reactions from progressing further.

The effects that proper sample preparation technique can have on the utility of peptide mapping are illustrated in Figures 1 and 2. In Figure 1, two different cytochrome c samples are digested using low-quality trypsin with poor sample preparation technique. Samples were not quenched after digestion and were run sequentially on HPLC. In the resulting chromatograms, it is difficult to conclude whether or not the results are actually due to differences between the species of cytochrome c.

Chromatographic methods
In Figure 2, proper sample preparation technique using high-quality trypsin and controlled digestion conditions were used. Samples were quenched with acid before injection on HPLC. Specific contrasts due to amino acid differences between the two proteins are easily seen by peptide mapping. This one example shows how proper sample preparation techniques are crucial to the collection of useful structural information from a peptide map.

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Figure 3: Peptide map of human erythropoietin run on a Jupiter 300 3µm C18 150 x 2 mm HPLC column. Note broad peaks corresponding to the major glycopeptides of EPO versus the non-glycosylated peaks that are very sharp. Peptide mapping by reversed phase HPLC can potentially visualize differences in glycoforms in a glycoprotein. (Source: Phenomenex Inc.)

In addition to thorough sample preparation techniques, a good understanding of proper HPLC operations is important for obtaining useful peptide maps. A well-maintained HPLC is mandatory, as is the use of a column heater to obtain reproducible retention times run-to-run. Also important is the use of a high-quality HPLC column specifically designed for protein and peptide separations. Another suggestion is to obtain columns from three different production batches for proper method validation. Often-overlooked are the run conditions themselves. Ideally, the gradient method used should start with a low percentage of organic buffer (no less than 3% organic to avoid equilibration issues) and end slightly above where the last peak elutes. Another recommended step is to include a wash with high organic in the gradient (90% organic is usually acceptable) before re-equilibration. Finally, performing a blank gradient before any peptide maps ensures that the column is properly equilibrated. Improper equilibration run-to-run can lead to retention time shifts for certain peptides, which can make identifying differences difficult.

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Figure 4: Peptide map of human interleukin-2 run on a Jupiter 4µm Proteo 150 x 2.0 mm column. The lower chromatogram is of the non-reduced tryptic digest. The upper chromatogram is of the same digest reduced with DTT. Note the peak corresponding to the one disulfide of IL-2 at 29 minutes in the lower trace. In the reduced peptide map (upper trace), the disulfide peptide corresponds to two peaks (27 and 29.5 minutes) that are released upon reduction. (Source: Phenomenex Inc.)

In addition to ensuring the best mechanics of peptide mapping, actual understanding of the protein being analyzed is useful in designing an optimal method. Standard enzymatic digestion using trypsin will work well for many biogeneric proteins (EPO, IL-2, and others) and some examples are shown in Figure 3 and 4. A tryptic digest of EPO is shown in Figure 3. Note the broad cluster of peaks at 15 and 21 minutes retention time that correspond to the major glycopeptides of erythropoietin. While such a digest may be adequate for QC testing purposes, a digest using an enzyme other than trypsin, such as Glu-C, might be appropriate to obtain more structural information on the different glycopeptides. In the case of IL-2 (Figure 4, lower trace), the map from the tryptic digest has a wide distribution of peptides with different retention times. Reduction using dithiothreitol after digest (Figure 4, upper trace) can elucidate the disulfide containing peptide (the peptide at 29 minutes in the non-reduced map shifts to two peaks at 27 and 29.5 minutes upon reduction). As is shown in both of these examples, peptide mapping can be useful in identifying critical post-translational modifications of protein therapeutics. The integration of a mass spectrometer can make the mapping even more useful as sequence and mass information can provide positive identification for all the observed peptides in an enzymatic digest.

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Figure 5: Peptide map of lysozyme run on a Jupiter 3µm C18 150 x 2 mm column. The lower chromatogram is the digested protein using standard conditions; the upper chromatogram shows a sample first reduced and alkylated before digestion. Note that the non-reduced lysozyme is resistant to proteolytic digestion. Upon reduction of all the cysteines, the protein easily digests. Such techniques are often required for proteins with complex disulfide structures. (Source: Phenomenex Inc.)

Most proteins are amenable to direct digestion as shown above. However, there are some cases where tight folding and complex disulfide structures can make the analytes resistant to enzymatic digestion. Such protease resistance is often seen with growth factors and other proteins found in enzymatically-rich environments. To analyze such proteins requires additional steps to generate a useful peptide map. Cysteines must be reduced and alkylated in a denaturing environment so the protein unfolds and can be digested by trypsin or another proteolytic enzyme. Typically, researchers will use high concentrations of a chaotrope (urea or guanidine) to unfold a protein while breaking disulfide bonds with a reducing reagent (dithiothreitol or ?-mercaptoethanol). Free cysteines are often then modified with an alkylation reagent to avoid reforming of disulfide bonds. The sample is then diluted or desalted to reduce the chaotrope concentration to a level where proteolytic enzymes are active, then digested and analyzed. An example of the utility of performing this procedure is shown in Figure 5; lysozyme is protease-resistant using normal digestion conditions (Figure 5, lower trace) upon reduction and alkylation (Figure 5, upper trace) a useful peptide map is generated.

Summary
Peptide mapping is a useful method for detecting minute changes in a protein structure and is a required analytical quality control method for any protein therapeutic drug. As shown in this article, attention to sample preparation and analysis techniques is crucial to realizing the utility of this powerful method. Knowledge of the protein being analyzed is also important to design the best analysis technique. Alternative enzymes or even unfolding protocols may be necessary to maximize the structural information that a peptide map method provides.

About the Authors
Michael McGinley is the Biochromatography product manager at Phenomenex with 20 years of experience in the life science and pharmaceutical industries. He is a well-published expert in the fields of protein biochemistry and chromatography.

Vita Knudson is a research scientist at Phenomenex involved in new product and application development for the biochromatography business unit. Her decade of protein purification experience is currently focused on better solutions for characterizing proteins.