Modeling the residence time distribution of an end to end integrated biomanufacturing process

With the advancements in continuous manufacturing focused mainly on the development of individual unit operations, only a few end-to-end integrated continuous bioprocesses (ICB) have been reported. As the scope starts shifting also towards commercial applications, detailed process understanding is required for quality process design, process optimization and developing process control strategies. Please click Additional Files below to see the full abstract

Multiple Single-Unit Long-Term Tracking on Organotypic Hippocampal Slices Using High-Density Microelectrode Arrays

A novel system to cultivate and record from organotypic brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows for continuous recording of electrical activity of specific individual neurons at high spatial resolution while monitoring at the same time, neuronal network activity. For the first time, the electrical activity patterns of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied during several consecutive weeks at daily intervals. An unsupervised iterative spike-sorting algorithm, based on PCA and k-means clustering, was developed to assign the activities to the single units. Spike-triggered average extracellular waveforms of an action potential recorded across neighboring electrodes, termed ‘footprints’ of single-units were generated and tracked over weeks. The developed system offers the potential to study chronic impacts of drugs or genetic modifications on individual neurons in slice preparations over extended times

Mechanistic modeling of anion exchange chromatography on a case erythropoietin

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.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

Long-term, high-spatiotemporal resolution recording from cultured organotypic slices with high-density microelectrode arrays

A novel system to cultivate and record brain slices directly on high-density microelectrode arrays (HD-MEA) was developed. This system allows to continuously record electrical activity of selected individual neurons at high spatial resolution, while monitoring neuronal network activity at the same time. For the first time, properties of single neurons and the corresponding neuronal network in an organotypic hippocampal slice culture were studied over four consecutive weeks at daily intervals