Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 190

Knowledge of the full amino acid sequence of the protein usually renders possible pre-
determination 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 fi rst 20-30 amino acid residues of the protein product has become
a popular quality control test for fi nished biopharmaceutical products. The technique is useful, as
it:

• positively identifi es the protein;

• confi rms (or otherwise) the accuracy of the amino acid sequence of at least the N-terminus of

the protein;

• readily identifi es the presence of modifi ed 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 fi rst 100 amino acids from the N-terminus of most
proteins, and usually requires a sample size of less than 1 µmol 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 fi rst 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 informa-
tion 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, how-
ever, 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.