Kvantitativno določanje vsebnosti prostih aminokislin v bioprocesni brozgi z avtomatsko derivatizacijo na visokotlačnem tekočinskem kromatografu

Urh, Maja (author), Jamnik, Polona (mentor), Vidmar, Tilen (comentor)

Analitska metoda, ki bi omogočala določanje prostih aminokislin med bioprocesom v bioprocesni brozgi, bi pripomogla k hitrejši optimizaciji in prilagajanju njene sestave. S tem bi znatno vplivali na izboljšanje bioprocesa in kakovosti končnega produkta. V ta namen smo v okviru magistrske naloge testirali metodo, kjer se aminokisline najprej derivatizirajo z reagentoma orto‐ftaldialdehidom (OPA) in 9-fluorenilmetil kloroformatom (FMOC). Sledi ločba derivatov z reverzno fazno kromatografijo na ultra visokotlačnem tekočinskem kromatografskem (UHPLC) sistemu. Metodo smo optimizirali s ciljem doseči čim višjo stopnjo avtomatizacije, zato vsi koraki (redčenje, derivatizacija, ločba in detekcija) potekajo direktno na UHPLC sistemu. Ustreznost metode smo preverili z validacijskimi parametri. Z rezultati smo pokazali, da smo uspešno optimizirali in dosegli zastavljeno avtomatizacijo metode, prav tako smo potrdili doseženost validacijskih parametrov z uporabo standardnih raztopin. Pri analizah bioprocesnih vzorcev smo potrdili primernost metode za določanje primarnih aminokislin in oteženo kvantifikacijo sekundarnih aminokislin. Vzrok so nespecifični kromatografski vrhovi in variabilnost kromatograma aminokislin, derivatiziranih s FMOC reagentom. Za najbolj problematične nespecifične vrhove smo ugotovili in potrdili, da so posledica samega OPA reagenta in reakcij OPA reagenta z neznanimi komponentami v bioprocesni brozgi. Kljub temu ostaja težava pri kromatogramu sekundarnih aminokislin, saj kromatografski vrhovi niso optimalno ločeni, njihova detekcija pa je variabilna. Metoda potrebuje nadaljnjo optimizacijo, da bo dosegla vse kriterije sprejemljivosti določanja celotnega željenega spektra aminokislin (primarne in sekundarne aminokisline) z željeno avtomatizacijo.

• Language: Slovenian
• Keywords: aminokisline, kromatografska metoda, razvoj, OPA, FMOC, avtomatizacija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [M. Urh]
• Year: 2021
• PID: 20.500.12556/RUL-126532
• UDC: 606:62:577.112.34:602.4:543.544(043.2)
• COBISS.SI-ID: 61759491
• Publication date in RUL: 27.04.2021

URH, Maja, 2021, Kvantitativno določanje vsebnosti prostih aminokislin v bioprocesni brozgi z avtomatsko derivatizacijo na visokotlačnem tekočinskem kromatografu [online]. Master’s thesis. Ljubljana : M. Urh. [Accessed 18 May 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=126532

Secondary language

• Language: English
• Title: Quantitative determination of free amino acids in bioprocessed broth with automatic derivatization by high pressure liquid chromatography system

Abstract

An analytical method that allows determination of free amino acids in bioprocessed broth during the bioprocess would help to optimize and adjust its composition on-line. This could lead a significant impact on improvement of the bioprocess parameters and the quality of the final product. For this purpose, we tested the method, where amino acids are derivatized with reagents ortho-phthalaldehyde (OPA) and (9-Fluorenylmethyl) chloroformate (FMOC), followed by separation of the derivatives by reverse phase chromatography on an ultra high pressure liquid chromatography (UHPLC) system. The method was optimized in the way to achieve the highest possible level of automation, therefore all steps (dilution, derivatization, separation and detection) are performed directly in the autosampler compartment of the UHPLC system. The suitability of the method was evaluated according to the pre-defined validation parameters. Our results are showing that we successfully optimized and achieved targeted level of automation of the method, and we fulfilled the validation parameters via standard solutions. Analysis of bioprocess samples confirmed the suitability of OPA and FMOC method for the determination of primary amino acids. However quantification of secondary amino acids is more challenging due to non-specific chromatographic peaks and chromatogram variability of amino acids derivatized with FMOC reagent. The cause of the most problematic nonspecific peaks was identified and confirmed to be due to the OPA reagent itself and the reactions of the OPA reagent with unknown components in the bioprocess media. Nevertheless, the main issue remains determination of secondary amino acids, whereas the chromatographic peaks are not optimally separated and their detection is variable. The method needs further optimization to achieve all the acceptance criteria for determining the full spectrum of amino acids (primary and secondary amino acids) with the desired level of automation.

• Keywords: amino acids, chromatographic method, development, OPA, FMOC, automation