Amino Acid Analysis, Past, Present and Future

Introduction

Following on from the discovery of the structure of DNA by Watson and Crick and the subsequent completion of the human genome sequencing project, the attempt to understand the relationship between genes and their protein products in different tissues, development, and disease has renewed scientific interest in protein chemistry.

The studies of genetics and protein sciences that once diverged into separate disciplines have now converged together heralding the birth of proteomics, as a new science. Protein-based pharmaceuticals are entering the drug development pipeline, making compositional analysis and protein quantitation by amino acid analysis necessary to ensure accurate characterization of recombinant proteins, amino acids, and peptides in development, bulk, and final products.

Amino Acid Analysis

Amino acid analysis is the gold standard technique used to determine the amino acid composition or content of proteins, peptides, and other pharmaceutical preparations. It represents one of the most commonly used techniques for quantitating proteins and peptides that does not depend on the specific structural features of the polypeptide. The method is completely independent of shape, charge, or protein function because the protein is completely degraded.

Amino acid analysis can be performed on aqueous solutions containing free amino acids or on proteins and peptides following hydrolysis to quantitate the total amount of each amino acid in the sample or to determine the ratio of the amino acids in the sample. The technique allows for less common amino acids to be quantified and for very small quantities of amino acids to be measured.

Biochrom Amino Acid Analysers

Biochrom Ltd, based on the prestigious Science Park in Cambridge, UK has been manufacturing and supporting amino acid analysis instruments on a global basis for well over 30 years. The Biochrom 30 Amino Acid Analyzer is a dedicated system that uses ion exchange chromatography with postcolumn ninhydrin-based detection to achieve high accuracy quantitative amino acid analysis. Separation of the amino acids on an ion-exchange column is accomplished through a combination of changes in pH, cation strength, and temperature.

When the amino acids react with ninhydrin, the reactant has a characteristic purple or yellow colour. Amino acids, except imino acids, give a purple colour and show the maximum absorption at 570 nm. The imino acids such as proline give a yellow colour and show the maximum absorption at 440 nm. The postcolumn reaction between ninhydrin and the amino acid eluted from the column is monitored at 440 and 570 nm, and the chromatogram obtained is used for the determination of the amino acid composition. The concentration of the amino acid is proportional to the absorbance of the resting solution.

History of Amino Acid Analysis

The study of amino acids has a long history. It was in the early 1900’s that the 1902 Nobel Laureate, Emil Fischer showed how amino acids are bound to each other forming polypeptides, the building blocks of proteins. Shortly after this, research into proteins and amino acids showed that the proteins of humans and animals contain 20 amino acids, 10 of which cannot be synthesised from the human body, but must be supplied as part of the diet. Following this research, the most important next step was to identify the amino acids present and determine their quantitative levels.

The first breakthrough in the separation of amino acids by chromatography came from the work of Martin and Synge, who first described partition chromatography, for which they won the Chemistry Nobel Prize in 1952. Synge’s subsequent work used columns packed with starch, which he used to establish the sequence of amino acids in peptides leading to the structural elucidation of gramicidin S, a cyclic decapeptide.

Stein and Moore pioneered new methods of chromatography for analysing amino acids and small peptides obtained by the hydrolysis of proteins. In 1958 they helped develop the first automated amino acid analyser, a machine that greatly facilitated the analysis of the amino acid sequences of proteins. In 1959 Moore and Stein used the new machine to make the first determination of the complete chemical structure of the enzyme, ribonuclease.

From what took months in 1946, weeks in 1950, once automated, analysis took a single day. Analysis time was reduced due to improvements in the ion-exchange process; improved sulphonated resins with narrower particle sizes has meant higher flow rates without loss of resolution.

Stein and Moore, in order to commercialise their new automated amino acid analyser partnered with the Spinco division of Beckman and built with them, the first Beckman Amino Acid Analyser in 1958. In subsequent years, improvements in resin and the use of a single column allowed analysis time to be reduced further to 4 hours for a simple hydrolysate.

Now nearly 50 years later, although the underlying principles of amino acid analysis have remained the same, analysis times for full profiles are less than 2 hours and can be as little as 10 minutes for certain amino acids families. Improvements over the years have focused on increasing the speed of data output, increasing sensitivity and minimizing operator time.

Current amino acid analysers are bench top units with “load and go” operation. The operator can load several samples at a time, a programme controls the sample injections, buffer changes and column regeneration and the PC receives outputs from the colorimeter, identifies the peaks and calculates the concentration of the amino acids. No additional derivitisation procedures are required. The ergonomic design of the amino acid analyser enables the user at eye level to check the chemical levels in the bottles and to perform routine maintenance with ease.

A wide variety of sample types can be analysed using a range of available programmes and columns for the pharmaceutical, clinical and food and feedstuffs application areas. The instruments are sold and supported worldwide, with engineers and scientist dedicated to amino acid analysis providing service and application support. The Biochrom 30 Amino Acid Analyser is IVD approve for the diagnosis of PKU, FDA approved and IS0 13485 certified.

Current applications

Today amino acid analysers are used in a wide range of application areas. As protein-based pharmaceuticals are moving into the drug development pipeline, more emphasis is being placed on their use in this environment.

Applications include:

Quantify peptides and proteins
Confirm synthetic peptide structures
Confirm recombinant protein structures
Identify proteins based on amino acid composition
Design fragmentation strategies
Detect odd amino acids

Amino acid analysis allows researchers to determine the amino acid composition and the quantification of the sample for both synthesized proteins and peptides aswell as samples prior to sequencing. The knowledge of the unordered amino acid composition of a protein and peptide is an important first step in primary structure determination – this information can be used to help the discovery of errors in the sequencing process or to distinguish between ambiguous results.

Amino acid analysis can be used to assess coupling of peptides to carrier proteins. The quantitation of which is aided by incorporating a non-naturally occurring amino acid such as nor-leucine or beta-alanine to the peptide. The technique represents an essential tool in the quality control of peptide synthesis and for the identification of an unusual amino acid or a modification.

Exact knowledge of protein quantities is required for further protein chemistry applications. Amino acid analysis is a suitable tool for precise determination of protein quantities but also provides detailed information regarding the relative amino acid composition and free amino acids.

Relative amino acid composition gives a characteristic profile or protein, which is often sufficient for identification of a protein. Often used as decision support for choice of proteases for protein fragmentation.

QA/QC Laboratory

Amino acid analysers in pharmaceutical QA/QC laboratories require installation and operational qualification (IQOQ) testing, which is a service offered by Biochrom on the sale of the Biochrom 30 Amino Acid Analyzer. Chromatographic data aquisition systems must also be fully validated, including performance qualification (PQ) as well as IQ and OQ. If results are used for pharmaceutical formal release applications, those tests also require full validation. The validation procedures are more time consuming but do provide the necessary requirements for analytical testing in a modern QA/QC pharmaceutical lab.

Future Developments

Protein-based drugs are continually making their way into the marketplace, so compositional analysis and protein quantitation by amino acid analysis will be a requirement for some time. A dedicated amino acid analyser is considered the gold standard for this type of work because of sensitivity and reproducibility.

More information on the Biochrom 30 Amino Acid Analysers can be found on www.biochrom.co.uk