Validation of a reversed-phase HPLC method for quantitative amino acid analysis.
A semi-automated method for amino acid derivatization and analysis has been validated for use in analysis of protein biopharmaceuticals. The method includes protein hydrolysis, o-phthalaldehyde derivatization, and reversed-phase high-performance liquid chromatography analysis in a general-purpose UV-visible high-performance liquid chromatography system. Amino-acid derivatization is performed automatically by the high-performance liquid chromatography autosampler right before injection. The required validation parameters, i.e., specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for bovine serum albumin and for a recombinant human Fab fragment. The method can be employed as an absolute quantification method for determination of extinction coefficients of recombinant proteins.
Bartolomeo
Maria Paola
MP
Milano Research Centre, Bracco Imaging SPA, Via Folli, 50, 20134 Milan, Italy.
Maisano
Federico
F
D000418:Albumins / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001331:Automation; D002417:Cattle; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D006868:Hydrolysis; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods*; D014466:Ultraviolet Rays; D009764:o-Phthalaldehyde / Q000737:chemistry
D000418:Albumins; D000596:Amino Acids; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D009764:o-Phthalaldehyde