AAA Analysis for Biopharmaceutical Applications
Accurate, precise, and robust amino acid analysis is required as fundamental information in several kinds of investigational projects as well as in routine QC and process monitoring. In biochemical structural analysis, the amino acid composition of hydrolyzed proteins is used to confirm sequence and to determine extinction coefficients. In contrast to measurements of protein bound amino acids, free amino acids are important indicators of metabolic status and genetic origin. Such analyses are required for diverse applications such as optimizing cell culture conditions in the production of biopharmaceuticals as well as process monitoring in industrial scale fermentations.
The demand of these applications led to the invention of amino acid analyzers and the continuous improvement of the technology over several decades. A turnkey, total application solution was developed. The technique based on a well-characterized derivatization protocol followed by a high resolution chromatographic separation. The amino acids are derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) under largely aqueous conditions. The derivatives are separated using UltraPerformance Liquid Chromatography (UPLC). The enhanced resolution of UPLC ensures a robust chromatographic method that reliably gives accurate and precise measurements.
This technique has been widely adopted by biopharmaceutical laboratories since its introduction in 2007. This UPLC Amino Acid Analysis System solution is now available for Waters ACQUITY UPLC (binary) and ACQUITY UPLC H-Class and H-Class Bio (quaternary) configurations and detection protocols, with documented linearity, accuracy, and precision. The continuous enhancement of this application solution ensures the most convenient and efficient generation of reliable amino acid analyses.
AAA for Proteins, Cell Culture Media, and Feeds
The analysis of amino acids is essential in a variety of investigational projects as well as in routine QC and process monitoring. Successful analysis requires consideration of sample handling, separation and detection of the amino acids. The UPLC Amino Acid Analysis System is intended to provide accurate and precise analysis over a wide range of sample types. The amino acids are derivatized by reaction with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and separated using reversed phase UPLC. The suitability of this method has been validated across several applications.
The amino acid composition of acid hydrolyzed pure proteins was compared to expected values from the known sequence. Amino acids as metabolic indicators were measured in growing cell culture media and various foods and beverages. The nutritional content of feeds was also determined after hydrolysis. Optimization of handling for each sample type provided more reliable results. The extreme sensitivity of the derivatization and separation techniques simplified some aspects of sample preparation while facilitating optimization of others. (See Posters under Literature tab)
Particularly for feed samples, the use of microwave hydrolysis techniques provided a practical addition to the overall analytical process. Feed samples were subjected to acid hydrolysis both with and without performic acid oxidation as well alkaline hydrolysis for a full profile of amino acid and, therefore, protein content. Sample handling throughput was much higher with this approach and was compatible with the analytical throughput of the system.
These experiments demonstrate the suitability of sample handling procedures and the amino acid analysis system
