Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

• Published in: Amino acids Vol. 44; no. 4; pp. 1225 - 1231
• Main Authors: Biermann, Michael, Bardl, Bettina, Vollstädt, Sebastian, Linnemann, Julia, Knüpfer, Uwe, Seidel, Guido, Horn, Uwe
• Format: Journal Article
• Language: English
• Published: Vienna Springer Vienna 01.04.2013; Springer Nature B.V
• Subjects: Analytical Chemistry; Antibodies - genetics; Antibodies - metabolism; Biochemical Engineering; Biochemistry; Biomedical and Life Sciences; Chromatography, High Pressure Liquid - methods; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Fermentation; Industrial Microbiology; Life Sciences; Neurobiology; Norleucine - analysis; Norleucine - metabolism; Original; Original Article; Proteomics; Valine - analogs & derivatives; Valine - analysis; Valine - metabolism; Norvaline; Norleucine; Bioprocess design; Recombinant antibody; Ultra-high performance liquid chromatography; Biopharmaceutical fermentation
• DOI: 10.1007/s00726-013-1459-3
• ISSN: 0939-4451

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho -phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub -2 μm particle C 18 reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C 18 reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.