Amino Acid Analysis

Determination and control of critical nutrients in cell culture is important to maximize protein yield and provide a consistent manufacturing process. The Waters UPLC®Amino Acid Analysis (AAA) Solution is a turnkey solution that provides accurate, high throughput amino acid analysis.

The UPLC Amino Acid Analysis Solution meets the specific demands of analyzing amino acids for protein characterization, cell culture monitoring, and nutritional analysis of foods and feeds. This end-to-end system is designed to deliver the accuracy and reliability you need to be successful.

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UPLC Amino Acid Analysis Solution

  • AccQ•Tag™ Ultra derivatization and separation chemistries including quality-control tested columns, reagents, and eluents
  • ACQUITY UPLC® (binary), ACQUITY UPLC H-Class (quaternary), or ACQUITY UPLC H-Class Bio (quaternary) system with a Tunable UV Detector, with enhanced chromatographic resolution and maximum-sensitivity detection
  • Empower™ Software pre-configured projects, methods and report formats
  • Connections INSIGHT® ISDP instrument diagnostics to ensure continuous, consistent, and reliable operation in your lab
  • Installation, Operation, and Performance Qualification (IQ/OQ/PQ) protocols that ensure every element of the system is performing, for reproducible results day-to-day, instrument-to-instrument, lab-to-lab, around the world.

The UPLC Amino Acid Analysis Solution provides you with control over the major factors that commonly cause variability in amino acid analysis. From optimized instrumentation and quality controlled chemistries to standardized software projects, methods, report templates, and qualification tools, you will rely on a method that is both rugged and reliable – a comprehensive and detailed approach that ensures successful analysis of every sample.

The UPLC AAA System brings together industry-leading technologies—instrumentation, chemistries, software, qualification tools and services – that are specifically optimized for exceptional, application-focused performance. Designed by scientists for scientists, these solutions remove the guesswork in choosing the best solution for your application’s needs.

Key Technologies

Importance of Amino Acids

Analysis of amino acids is required in several areas of research, and it is a fundamental tool in product analysis. The application imposes different requirements on the analytical method because the amino acids play different roles.

  • Amino acids are the basic constituents of proteins. Qualitative and quantitative analysis of the amino acid composition of hydrolyzed samples of pure proteins or peptides is used to identify the material and to directly measure its concentration
  • Amino acids are also intermediates in metabolic pathways, often not directly involving proteins. The amino acids are measured as elements of physiological and nutritional studies. This has proven particularly important in monitoring the growth of cells in cultures, as used in the production of biopharmaceuticals
  • Similar considerations lead to the analysis of foods and feeds to ensure that nutritional requirements are met

With the UPLC® Amino Acid Analysis Solution, you benefit from better resolution and sensitivity, all achieved in a shorter analysis time than with conventional methodologies:

  • Method robustness minimizes variability
  • Better resolution translates into accurate results
  • UPLC delivers greater throughput than traditional HPLC
  • Protein characterization accelerates for comparison of amino acid composition deduced from the gene sequence for a product or its natural counterpart
  • Monitoring cell culture media for critical nutrients allows you to optimize expression efficiencies and to make timely product harvesting decisions
  • Avoid costly reformulations primary nutritional components

Optimized Peak Identification

  • AccQ•Tag™ Ultra Chemistries used in the UPLC Amino Acid Analysis Solution are a comprehensive and fully-tested set of reagents, columns, and eluents optimized for use with the ACQUITY UPLC, ACQUITY UPLC H-Class, and ACQUITY UPLC H-Class Bio Systems. With superior resolution of all amino acids, you will confidently achieve peak identification and quantitation, which in turn assures the accuracy of your results.
  • This chemistry is based on Waters’ widely-used and understood AccQ•Tag derivatizaton method. Primary and secondary amino acids react with a single reagent, AccQ•Fluor™, in a high-throughput batch process resulting in exceptionally stable derivatives. High resolution separations are achieved using pre-qualified AccQ•Tag Ultra UPLC columns and mobile phases. Derivatized amino acids are quantified to sub-picomole levels with single wavelength UV detection.

Performance Qualification

Qualification services available for the UPLC Amino Acid Analysis Solution meet the most stringent regulatory requirements. Installation, Operation, and Performance Qualification (IQ/OQ/PQ) protocols ensure that every element of the system is performing according to its intended use. An additional PQ, specific for amino acid analysis, is also available to verify performance and ensure accurate results.

Resolution

Analysis of a Hydrolysate Standard at 10 pmol/mL
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The UPLC Amino Acid Analysis Solution provides high resolution to ensure method robustness with good quantitation. The short cycle time is obtained without compromising accurate results.

Efficiency

Efficient, Reliable Foods and Feed Sample Analysis: Soybean Meal Hydrolysate

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Two samples of hydrolyzed soybean meal were analyzed. The upper trace shows the untreated sample while the lower trace shows the results of performic acid oxidation. Note the appearance of cysteic acid and methionine sulfone in the oxidized sample.

Key Applications

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.

Flexible Methodology

Analysis of Hydrolysate and Cell Culture Standards

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The upper trace shows the analysis of a hydrolysate standard at 10 pmol/mL, and the lower trace a standard mixture of the larger number of amino acids found in cell culture media, also at 10 pmol/mL. The adaptation of the method requires only a change in dilution of the Eluent A Concentrate supplied as part of the total system solution.

Cell Culture Analysis

Direct Cell Culture Sample Analysis

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Aliquots of cell culture media were taken directly from a bioreactor producing a biopharmaceutical protein. Samples were taken over a period of six days and no special sample preparation was required before the amino acid analysis shown here. The amino acid concentration trends can be plotted with growth as well as the response to feeding.