Amino acid Analysis (AAA) is a procedure used to determine the mole percent and/or quantity of each individual amino acid in a protein or peptide. There are four steps in Amino Acid Analysis (AAA)

• Hydrolysis
• Derivatization
• Separation of derivatized amino acids
• Data interpretation and calculations

Hydrolysis: Blue Stream offers several hydrolysis techniques for the preservation / determination of specific amino acids. Several amino acids such as tryptophan and tyrosine, and to a lesser extent threonine and serine, are destroyed under common 6N acid hydrolysis conditions. Blue Stream offers alternatives to the 6N hydrolysis condition to preserve these more labile residues. To determine cysteine, the test article must be reduced and carboxymethylated. Blue Stream will provide this sample preparation step upon request.

Derivatization: Blue Stream conducts the derivitization of the residues for Amino Acid Analysis via utilization of a pre-column PITC derivatization. This pre-column derivitization relies on the coupling reaction of phenylisothiocyanate (PITC) with both primary and secondary amino acids to form phenylthiocarbamyl (PTC) derivatives. Other derivatization procedures utilizing fluorogenic agents, such as OPA or fluorescamine, can also be performed upon request.

Chromatographic Separation and Data Analysis: The separation of the amino acids is performed using Reverse Phase HPLC w/ appropriate data collection and analysis software. The data calculation and interpretation may be conducted using a variety of systems. Additional information for Blue Stream's Amino Acid Analysis (AAA) options is available upon request.

Amino Acid Analysis (AAA) may be used for:

• Characterization information for a CMC program
• Lot Release testing for determination of specific "specialized" amino acids where they are a crucial indicator of protein function: gammacarboxyglutamic acid
• In conjunction with A280, quantitative Amino Acid Analysis (AAA) can be used for the experimental determination of an extinction coefficient
• Quantification of a protein or total protein in a mixture
• The determination of free amino acids in cell culture media