Mehanistično modeliranje anionske izmenjevalne kromatografije na primeru eritropoetina

SENČAR, JURE (author), Maček Lebar, Alenka (mentor), Belič, Aleš (comentor)

Eritropoetin (EPO) je glikoproteinski hormon, ki vpliva predvsem na proizvodnjo rdečih krvničk v kostnem mozgu. Najpogostejše bolezensko stanje, ki ga zdravimo z EPO-m, je anemija – pomanjkanje rdečih krvničk v krvnem obtoku. Molekulo EPO-a sestavlja proteinska veriga na katero so na štirih mestih vezani sladkorji (glikani). To jo uvršča med glikoproteine. Različne podvrste EPO-a imajo različno in vivo biološko aktivnost, zato je reguliranje deleža podvrst v končni zdravilni učinkovini ključnega pomena. Delež podvrst v proizvodnji regluiramo z izmenjevalno ionsko kromatorgrafijo (IEC). Pri obravnavanem koraku izoforme, ki imajo enak skupni naboj, obravnavamo kot posamezne podvrste. Imenujmo jih nabojne izoforme. V magistrski nalogi je izveden mehanistični matematični model, ki opisuje elucijo nabojnih izoform v odvisnosti od elucijskega gradienta. Mehanistični model je postavljen s programskim paketom CADET. Za opis vezave je uporabljen standardni izmenjevalni model, za opis transporta pa splošni hitrostni model. Izmed difuznih parametrov je s prilagajanjem oblike elucijskega vrha določen filmski difuzijski koeficient za vsako nabojno izoformo ter aksialni disperzijski koeficient za kromatografski nosilec. Vezavni parametri so določeni na podlagi Yamamoto metode. Postavljeni model zelo dobro opisuje retencijske čase vrhov, slabše pa trend širine vrhov. Izkaže se, da je ločba neodvisna od oblike gradienta, kar nakazuje na večje število glikoform znotraj posamezne nabojne izoforme.

• Language: Slovenian
• Keywords: CADET, ionska izmenjava, standardni izmenjevalni model, SDM, splošni hitrostni model, GRM, tekočinska kromatografija visoke ločljivosti, HPLC, biološko podobno zdravilo, nabojne izoforme
• Work type: Master's thesis/paper
• Organization: FE - Faculty of Electrical Engineering
• Year: 2016
• PID: 20.500.12556/RUL-80815
• Publication date in RUL: 03.03.2016

Secondary language

• Language: English
• Title: Mechanistic modeling of anion exchange chromatography on a case erythropoietin

Abstract

Erythropoietin (EPO) is glycoprotein hormone influencing the red blood cell production in bone marrow. It is most widely used for treatment of anemia – a lack of red blood cells in bloodstream. EPO molecule is composed of protein backbone with four glycosylation sites. Isoforms with different glycosylation patterns exist and show different in vivo bioactivity. Thus it is crucial to regulate such isoform ratios in drug substance. An ion exchange chromatographic step (IEC) is employed to set the specified net charge isoform ratio. Net charge isoforms in the context are defined as isoforms with the same net charge. In this work the chromatographic step is investigated by mechanistic modeling approach. The mechanistic model simulates the elution of net charge isoforms at different elution gradient slopes. The mechanistic model combines the standard displacement model for binding and general rate model for transport phenomena. The model is implemented within the CADET software framework. The binding parameters are obtained by Yamamoto method. Among transport parameters, the axial dispersion and film diffusion are taken into account. Their value is obtained by fitting the simulated peak shapes to the experimental ones. The model successfully describes the retention times of net charge isoforms. However it underperforms in describing the peak broadening across different gradient slopes. Further investigation reveals that the separation is essentially independent of gradient length. This phenomena implies many subspecies with different binding properties within the single net charge isoform.

• Keywords: CADET software, ion exchange, standard displacement model, SDM, general rate model, GRM, high pressure liquid chromatography, HPLC, biosimilar drug, erythropoietin, charge isoforms