Download (direct link):
Peptide mapping entails exposure of the protein product to a reagent which promotes hydrolysis of peptide bonds at specific points along the protein backbone. This generates a series of peptide fragments. These fragments can be separated from each other by a variety of techniques, including one- or two-dimensional electrophoresis and, in particular, RP-HPLC. A standardized sample of the protein product, when subjected to this procedure, will yield a characteristic peptide fingerprint, or map, with which the peptide maps obtained with each batch of product can subsequently be compared. If the peptides generated are relatively short, a change in a single amino acid residue is likely to alter the peptide’s physicochemical properties sufficiently to alter its position within the peptide map (Figure 3.34). In this way single (or multiple) amino acid substitutions, deletions, insertions or modifications can usually be detected. This technique plays an important role in monitoring batch-to-batch consistency of the product, and also obviously can confirm the identity of the actual product.
The choice of reagent used to fragment the protein is critical to the success of this approach. If a reagent generates only a few very large peptides, a single amino acid alteration in one such peptide will be more difficult to detect than if it occurred in a much smaller peptide fragment. On the other hand, generation of a large number of very short peptides can be counter-productive, as it may prove difficult to resolve all the peptides from each other by subsequent chromatography. Generation of peptide fragments containing an average of 7-14 amino acids is most desirable.
The most commonly utilized chemical cleavage agent is cyanogen bromide (it cleaves the peptide bond on the carboxyl side of methionine residues). V8 protease, produced by certain staphylococci, along with trypsin, are two of the more commonly used proteolytic-based fragmentation agents.
Knowledge of the full amino acid sequence of the protein usually renders possible predetermination of the most suitable fragmentation agent for any protein. The amino acid sequence of human growth hormone, for example, harbours 20 potential trypsin cleavage sites. Under some circumstances it may be possible to use a combination of fragmentation agents to generate peptides of optimal length.
THE DRUG MANUFACTURING PROCESS 171
Figure 3.34. Generation of a peptide map. In this simple example, the protein to be analysed is treated with a fragmentation agent, e.g. trypsin (a). In this case, five fragments are generated. The digest is then applied to a sheet of chromatography paper (b) at the point marked ‘origin’. The peptides are then separated from each other in the first (vertical) dimension by paper chromatography. Subsequently, electrophoresis is undertaken (in the horizontal direction). The separated peptide fragments may be visualized, e.g. by staining with ninhydrin. 2-D separation of the peptides is far more likely to completely resolve each peptide from the others. In the case above, for example, chromatography (in the vertical dimension) alone would not have been sufficient to fully resolve peptides 1 and 3. During biopharmaceutical production, each batch of the recombinant protein produced should yield identical peptide maps. Any mutation which alters the protein’s primary structure (i.e. amino acid sequence) should result in at least one fragment adopting an altered position in the peptide map
N-terminal sequencing of the first 20-30 amino acid residues of the protein product has become a popular quality control test for finished biopharmaceutical products. The technique is useful as it:
• positively identifies the protein;
• confirms (or otherwise) the accuracy of the amino acid sequence of at least the N-terminus of the protein;
• readily identifies the presence of modified forms of the product in which one or more amino acids are missing from the N-terminus.
N-terminal sequencing is normally undertaken by Edman degradation (Figure 3.35). Although this technique was developed in the 1950s, advances in analytical methodologies now facilitate
Figure 3.35. The Edman degradation method, by which the sequence of a peptide/polypeptide may be elucidated. The peptide is incubated with phenylisothiocyanate, which reacts specifically with the N-terminal amino acid of the peptide. Addition of 6 M HCl results in liberation of a phenylthiohydantoin-amino acid derivative and a shorter peptide, as shown. The phenylthiohydantoin derivative can then be isolated and its constituent amino acids identified by comparison to phenylthiohydantion derivatives of standard amino acid solutions. The shorter peptide is then subjected to a second round of treatment, such that its new amino terminus may be identified. This procedure is repeated until the entire amino acid sequence of the peptide has been established
THE DRUG MANUFACTURING PROCESS 173