Determination of the amino acids in Cell Culture Media

The aim is that the validation of the analytical method should demonstrate that it is suitable for the determination of amino acids in cell culture media.

Sample Preparation and Analysis
Validation/Precision:

100 µL of the sample was mixed with 100 µL internal standard norleucine (10 µmol/mL) and 600 µL sample dilution buffer. 200 µL of precipitation solution were added and deposit in the refrigerator for 20 min for the protein precipitation. The sample was centrifuged for 5 min at 14000 rpm. Afterwards the supernatant was filtered with a membraSpin by centrifugation at 14000 rpm for five minutes. The particle free solution was used for the injection. For the analysis 100 µL of sample were diluted with 900 µL sample dilution buffer (dilution 1:100).

Accuracy/Recovery:

For the analysis 2 mL of sample and 2 mL of sample dilution buffer (including norleucin, 10  µmol/mL) were mixed and diluted with sample dilution buffer 1:50. The obtained stock solution was used for the dilution series of corresponding to 100 %, 80 %, 60 %, 40 %, 20 % and 10 %.

Table 1: Dilution series of stock solution for the validation of accuracy.

The samples were analyzed by the Amino Acid Analyzer ARACUS, manufactured and distributed by membraPure GmbH worldwide. ARACUS is using the classic routine analysis of amino acids by post-column derivatization with ninhydrin and the detection at 440 nm and 570 nm.

Validation/ Precision

The precision of the method was determined by repeatability (intra-day) (Table 2 & Table 3) and intermediate precision (inter-day) (Table 4 & Table 5). The intra-day precision was calculated as the relative standard deviation (RSD) of results from ten runs of the sample, during the same day, and the inter-day precision was studied by the sample preparation on ten different days. Ten sample solutions were prepared, and the standard deviation (SD) and RSD (coefficient of variation CV %) were calculated.

Table 2: Intra-day precision of amino acids in cell media concentration in nmol/g (n=10)

Table 3: Intra-day precision of amino acids in cell media concentration in mg/g (n=10)

Table 4: Inter-day precision of amino acids in cell media concentration in nmol/g (n=10)

Table 5: Inter-day precision of amino acids in cell media concentration in mg/g (n=10)

Accuracy (Recovery)

In this validation study, accuracy of the method has been investigated by calculating the recovery values obtained by analyzing the sample prepared with norleucine standard (100 µl/mL) corresponding to 100%, 80%, 60%, 40%, 20% and 10% (Table 6).

Table 6: Recovery values for dilution series for each amino acid, which were analysed in cell culture media.

Linearity

The linearity is important for the confirmation of the method’s sensitivity for the analysis of the amino acid concentration within a defined range.

Linearity is the ability to obtain test results which are directly proportional to the concentration of the amino acid. Linearity was determined by five injections of six different concentrations (injection volume 5 µl, 10 µl, 20 µl, 30 µl, 40 µl, 50 µl). The average peak areas were plotted against concentrations. Then linearity was evaluated using the calibration curve to calculate coefficient of correlation.Table 7). In general a value of correlation coefficient (R²) > 0.990 is considered as the evidence of an acceptable fit for the data to the regression line.

Table 7: Resulted linearity of plotting average peak area against different concentrations of amino acids.

Table 8: Limit of detection and limit of qualification for each amino acid, analysed in cell culture media.

Conclusion
The method was validated by using various validation parameters like accuracy, precision and linearity. All the validation parameters were found to be well within the acceptance criteria. It is shown, the method was precise, accurate, linear, reproducible and repeatable. These results show the method could find practical application for the analysis of the amino acids in cell culture media.

References

• Salazar, M. Keusgen, J. von Hagen, Amino Acids in the cultivation of mammalian cells, Amino Acids (2016) 48:1161-1171.