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Amino Acid Level Analysis by Reversed Phase HPLC

It is a regulatory requirement that the amino acid composition is determined. In general, consolidated and automated methods are widely employed but all methods consist of four main steps:

1. Hydrolysis   The amino acids are liberated by hydrolysis in a highly acidic environment.  This converts asparagine (Asn) to aspartate (Asp) and glutamine (Gln) to glutamate (Glu) and completely destroys tryptophan (Trp).  Non-proteinogenic amino acids such as norvaline and/or norleucine are added as internal standards.
2. Labelling   The seventeen remaining proteinogenic amino acids are then labelled (derivatized) with a suitable UV/fluorotag – o-phthaldehyde (OPA) for all primary amino acids and Fluorenylmethyloxycarbonyl (FMOC) for the secondary amino acid of proline
3. Separation   Chromatographic separation is then performed on a C18 stationary phase under basic mobile phase conditions using a wide polarity gradient
4. Detection/Quantification   The separated amino acids are detected on either a UV or a more sensitive fluorescence detector
A typically encountered chromatogram is shown in Figure1

Figure 1: Typical reversed phase chromatogram of a mAb amino acid composition

 

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