T

Chromatography

01 O3

Origin

Electrophoresis

Figure 7.4 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 by, for example, staining with ninhydrin. Two-dimensional separation of the peptides is far more likely to resolve each peptide completely from the others. In the case above, for example, chromatography (in the vertical dimension) alone would not have been sufficient to resolve peptides 1 and 3 fully. During biopharmaceutical production, each batch of the recombinant protein produced should yield identical peptide maps. Any mutation that 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 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 counterproductive, 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 staphy-lococci, 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 hGH, 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.

7.5.3 N-terminal sequencing

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 7.5). Although this technique was developed in the 1950s, advances in analytical methodologies now facilitate fast and automated determination of up to the first 100 amino acids from the N-terminus of most proteins, and usually requires a sample size of less than 1 ^mol to do so (Figure 7.6).

Analogous techniques facilitating sequencing from a polypeptide's C-terminus remain to be satisfactorily developed. The enzyme carboxypeptidase C sequentially removes amino acids from the C-terminus, but often only removes the first few such amino acids. Furthermore, the rate at which it hydrolyses bonds can vary, depending on what amino acids have contributed to bond formation. Chemical approaches based on principles similar to the Edman procedure have been attempted. However, poor yields of derivatized product and the occurrence of side reactions have prevented widespread acceptance of this method.

7.5.4 Analysis of secondary and tertiary structure

Analyses such as peptide mapping, N-terminal sequencing or amino acid analysis yield information relating to a polypeptide's primary structure, i.e. its amino acid sequence. Such tests yield no information relating to higher-order structures (i.e. secondary and tertiary structure of polypeptides, along with quaternary structure of multi-subunit proteins). Although a protein's three-dimensional conformation may be studied in great detail by X-ray crystallography or NMR spectroscopy, routine application of such techniques to biopharmaceutical manufacture is impractical, both from a technical and an economic standpoint. Limited analysis of protein secondary and tertiary structure can, however, be more easily undertaken using spectroscopic methods, particularly far-UV circular dichroism. More recently proton-NMR has also been applied to studying higher orders of protein structure.

Phenylisothiocyanate

Peptide

NH — C-NH - CH — C — N — CH — C — N-[etc

NH — C-NH - CH — C — N — CH — C — N-[etc

Phenylthiohydantoin- Shorter peptide amino acid derivative

Figure 7.5 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 mol l_1 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 acid identified by comparison to phenylthiohydantoin 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

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