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Method Abstract 371

371 / AMINO ACIDS ANALYSIS OF CELL CULTURE MEDIA

Pickering Laboratories specializes in the manufacturing of cation-exchange columns and eluants for Amino Acid Analysis. Post-column derivatization with Ninhydrin offers unmatched selectivity and reproducibility of the analysis for most challenging matrixes.

Pinnacle PCX post-column derivatization system allows analysts to combine eluant gradient capabilities of modern HPLC instruments with column temperature gradients. We introduce new accelerated Amino Acids Analysis method for physiological samples that utilizes temperature and eluant gradients.

Cell cultures are widely used to produce biopharmaceuticals and other biologically active compounds. The composition of the cell culture media affects the yield and structure of the desired products and must be carefully optimized. Cell culture media is typically composed of mixtures of amino acids, vitamins, carbohydrates, inorganic salts as well as different peptides, proteins and other compounds. As the cells grow, they consume nutrients and release target biopharmaceuticals as well as waste products.

Amino Acids serve as the building blocks of proteins, as well as intermediates in many metabolic pathways. Amino Acids are typically added to cell culture media to provide nutritional requirements for the cells. Monitoring and adjusting Amino Acid composition is an essential part of optimizing the manufacturing process to ensure high quality and optimum yield of the final product.

Amino Acids Analysis using cation-exchange chromatography with post-column Ninhydrin derivatization allows for easy determination of Amino Acid concentrations in many complex matrices, including cell culture media. The post-column method is very sensitive, reproducible and rugged. It has been and continues to be a method of choice for laboratories running biological samples, protein, peptides and foods analysis. Most chemical compounds present in the media do not interfere with analysis, so the majority of samples only need diluting with citric buffer and filtering before analysis. If serum is added to the media, then the proteins need to be precipitated using either Seraprep™ solution or ultrafiltration.

Pickering Laboratories, Inc. offers the complete solution for Amino Acids Analysis, including post-column derivatization instruments, columns, eluants, reagents and standards. The Pinnacle PCX derivatization system has a programmable column oven to allow for shorter run times and easy method optimization.
 

METHOD

Analytical Conditions

Column: High-efficiency Lithium cation-exchange column,
       4.6 x 75 mm, Catalog Number 0354675T
Flow Rate: 0.55 mL/min
Mobile Phase: See method in Table 1

Post-column Conditions

Post-column System: Pinnacle PCX
Reactor Volume: 0.5 mL
Reactor Temperature: 130 °C
Flow Rate: 0.3 mL/min
Injection Volume: 10-50 uL
Detection: UV/VIS 570 nm for primary amino acids,
       440 nm for secondary amino acids
 

HPLC PROGRAM

TIME

1700-1125 %

Li365 %

Li375 %

RG003%

0

100

0

0

10

100

0

0

19

40

60 

0

0

32

0

100 

0

0

43

0

100 

0

0

43.1 0 0 100 0
57 0 0 100 0
57.1 0 0 70 30
72 0 0 70 30
72.1 100 0 0 0
84.0 100 0 0 0

COLUMN OVEN PROGRAM

TIME

TEMP ºC

0

34

6

34

17

65

25

70

70

70

71

34

MA 371A
MA 371B