High-Throughput Amino Acid Analysis using Tecan Automated Preparation

The objective of this application note is to demonstrate the equivalency of manual preparations of AccQ●Tag-labelled amino acids to those prepared using a Tecan Freedom EVO 100/4 automation platform with an Amino Acid Cell Culture Standard Kit.

Benefits

• Sample preparation robustness and equivalency via a Tecan automation platform
• Elimination of risks associated with human error and contamination
• Method standardization and transfer between multiple sites

Amino acids are the primary building blocks of proteins, and the sequence in which they present is unique to each protein or peptide. Amino acids are essential for the growth and repair of cells. Determining amino acid composition is of significant relevance in fields dealing with bio-pharmaceutical preparations and protein hydrolysates. In cell culture media, the concentration of amino acids depends on the cells’ metabolic and transport requirements. Monitoring and optimizing the amino acid components of bioreactor media is essential for ensuring the best growing conditions for the cells.¹ This makes amino acid analysis a requirement in the pharmaceutical industry. Waters Amino Acid Cell Culture Standard Kit, which contains nine additional amino acids critical in cell cultures, complements a 17-Amino Acid Hydrolysate Standard. The addition of the cell culture amino acids is important as they include key indicator amino acids that are essential for cell growth (Table 1).

Derivatization of amino acids is a key step in amino acid analysis. Waters UPLC Amino Acids Solution consists of a pre-column AccQ●Tag Ultra Derivatization Kit, as well as an optimal system configuration with certified column and eluents to enhance reproducibility. Automation is an ever-growing field in sample preparation. Automation platforms reduce variability compared to manual preparations and increase laboratory efficiency.² Waters automation compatible AccQ●Tag Ultra Derivatization Kit provides the increased volumes of reagents required for automated preparation.

In this application note, we will demonstrate the equivalency of manual preparations of AccQ●Tag-labelled amino acids to those prepared using a Tecan Freedom EVO 100/4 automation platform with an Amino Acid Cell Culture Standard Kit.

Design Factors

A.      Optional Script Features

The scripts for the Tecan Freedom EVO 100/4 were created with barcode export which provides users the functionality of building sample lists in their chromatography data system from the exported data, minimizing mistakes commonly made when manually inputting data. The functionality of the Tecan script is enhanced by their ability to perform dilution of standards with a reference range of 500–0.5 µM (Cystine 250–0.25 µM). Additionally, cell culture samples can be diluted on deck using the available worklist import function. Selection of sample number and well starting position provides the user the advantage of running with the AccQ●Tag Ultra Derivatization Automation Kit for 32, 64, or 96 samples. There is also the flexibility to include the Norvaline Internal Standard (p/n: 186009301) as an optional feature when preparing samples.

B.      AccQ●Tag Ultra Derivatization Automation Kit (p/n: 186009232)

The AccQ-Tag Ultra Derivatization Automation Kit scales up the reagent volumes necessary for use with the automation systems due to their increased dead volume requirements. The volumes of reagents provided allow for the preparation of 96 samples and in a 3 x 32 sample format.

C.      Labware

The manual preparation of amino acid samples with the AccQ●Tag Derivatization Kit is performed using Waters Total Recovery Vials. In order to make this automation compatible, these glass vials were replaced with a 96-well collection plate (p/n: 186002481). Extensive feasibility testing was conducted to support the labware change, and no impact to product performance was detected.

D.       Experimental Design

A minimum of three lots of eluents, columns, and AccQ●Tag Ultra Derivatization Automation Kits were evaluated during testing in order to demonstrate the assay’s robustness. Details of this can be found in the section on robustness. Different analysts (n = 4) were also evaluated throughout the study to demonstrate the independence of the automated workflow from the user.

The automation preparation method using the Tecan Freedom EVO 100/4 was assessed and compared to a pre-existing manual preparation method for robustness and equivalency. Performance characteristics were monitored across three preparations and three-replicate injections of each preparation, at three concentration levels (10 µM, 200 µM, and 400 µM) to determine the accuracy and precision (retention time, analyte peak area, and concentration) and linearity of the results. The internal standard-Norvaline was used in all experiments. The use of a Norvaline internal standard best compensates for the variability generated in sample hydrolysis and amino acid analysis.

Figure 3 shows a representative chromatogram of the cell culture standard at 10 pmols on column.

Precision

In liquid chromatography, retention time is the primary method of identifying chromatographic peaks. As demonstrated in Table 2, the %CV for retention time was ≤0.3%, showing good equivalence across both methods. Consistent analyte area across injections and preparations is an excellent indicator of the repeatability of both the sample preparation and analysis methods. The data in Table 2 demonstrates the excellent repeatability of the automated AccQ●Tag preparation method and indicates a maximum %CV across all analytes of ≤2.8% for the manual preparation and ≤1.8% for the Tecan preparation.

Accuracy

Accuracy was assessed at concentrations of 10, 200, and 400 µM using three preparations at each concentration, and three-replicate injections of each preparation. For the 10 µM panel, the %Recovery for each amino acid prepared using the Tecan automation method was within ±20% of the target concentration.* For the panels at 200 and 400 µM, the %Recovery for each amino acid prepared using the Tecan automation method was within ±15% of the target concentration.* This recovery data, as well as the precision data in Table 3, demonstrates the suitability of the Tecan automated preparation platform as a considerable, time-saving alternative to manually derivatizing amino acids.

The recovery of the cell culture standard when compared to NIST SRM 2389a was also analyzed as a means of independent assessment. A 200 µM panel was prepared from the cell culture standard using the Tecan automated preparation method and recovery calculated using a single NIST standard (prepared at 250 µM concentration [a 1 in 10 dilution of SRM]), by the existing manual method but analyzed in the same UPLC analytical run. The Tecan automated preparation %Recovery ranged from 94% for lysine to 110% for aspartic acid. This demonstrates the accuracy of the Waters Amino Acid Cell Culture Standard when compared to SRM.

*Serine was an outlier for the 10 µM panel and was not within the ±20% specifications set for the 10 µM panel. The 3 µM difference was likely due to a small amount of contamination during the preparation. Aspartic acid was an outlier in the 200 µM panel and did not meet the ±15% specifications, but was within 20% of the target value. All other analytes met specification.

Linearity

Linearity was assessed using a cell culture standard prepared at seven concentration levels for each amino acid across a range of 0.5–500 µM (cystine 0.25–250 µM). All analytical runs were assessed for linearity and all met the criteria of r² > 0.995 with no point deviation from the expected concentration by >15% for calibrators 2–7 (2.5–500 µM) and >20% for calibrator one (0.5 µM). The data was consistent between manual and automated preparation methods and no trends were observed.

The performance characteristic of precision, accuracy, and linearity were used to determine the equivalence of the Tecan automation versus manual preparations. The results indicate good overall agreement between the two sample preparation methods for the UPLC Amino Acid Analysis Solution, however there are significant advantages to automation that must also be considered when performing a comparative analysis:

• Preparation time for the Tecan automated instrument was significantly shorter than the manual counterpart, highlighting the efficiency of automated sample preparation versus manual preparation. The complete sample preparation time for a 96-sample run, including a standard dilution step, is <1 hour for the Tecan automation platform.
• The automation method developed did not require manual intervention during the run, thus allowing the analyst time to perform other laboratory tasks.
• Automation reduces the risk of human error and contamination.
• Automation removes analyst-to-analyst variation, allowing laboratories and companies to standardize analysis methods and facilitate method transfer between multiple sites.

The automated analysis of amino acids using an AccQ●Tag Ultra Derivatization Automation Kit is a promising alternative to the current manual preparation method for laboratories that suffer from time and resource constraints. The results generated show good agreement in terms of precision, accuracy, and linearity.

1. Salazar, A.; Keusgen, M.; von Hagen, J. Amino Acids in the Cultivation of Mammalian Cells. Amino Acids, 2016, 48 (5), 1161–1171.
2. Saitoh, S.; Yoshimori, T. Fully Automated Laboratory Robotic System for Automating Sample Preparation and Analysis to Reduce Cost and Time in Drug Development Process. Journal of the Association for Laboratory Automation, 2008, 13 (5), 265–274.