Modeliranje časovne in temperaturne odvisnosti različno nabitih proteinskih variant

KRALJ, NIKA (author), Ravnik, Miha (mentor), Kuzman, Drago (co-mentor)

Magistrsko delo obravnava časovno spreminjanje relativnih deležev t.i. variant monoklonskih protiteles z različnimi naboji, posebej pod vplivom različnih temperatur, kot pomembno pri uporabi v bioloških zdravilih. Variante - efektivno enake proteine, vendar z različnimi naboji - delimo na kisle in bazične, ki se od glavnih razlikujejo v vrednosti izoelektrične točke oziroma neto naboju. Spremembe med variantami lahko vplivajo na kvaliteto in učinkovitost zdravila, zato je razumevanje njihovega časovnega spreminjanja nujno za učinkovitejši razvoj in proizvodnjo zdravila. Visoke temperature pospešijo degradacijske procese in omogočajo v krajšem času spoznati potek časovnega spreminjanja variant. Za dobljene meritve sem iz tipičnih stabilnostnih študij uvedla efektivne modele, ki opisujejo prehajanje med variantami in vključujejo tako reverzibilne kot ireverzibilne prehode med variantami. Izkazalo se je, da dane meritve najbolje opiše model, ki vključuje reverzibilno prehajanje iz glavnih variant v kisle in bazične variante, za validacijo in uvedbo ostalih pa je ključna omejenost merskih podatkov. Uporabnost modela se izkaže pri časovni ekstrapolaciji podatkov, kjer dobimo primernejše napovedi spreminjanja variant, kot pri uporabi linearne ekstrapolacije. Ker je količina podatkov, vzetih iz tipičnih stabilnostnih študij, omejena, nam kompleksni modeli določijo le grobo oceno parametrov. Za natančnejšo oceno bi potrebovali večje število podatkov ali alternativni preprostejši model. Uspešna, vendar ne povsem natančna je tudi uporaba modela, ki predpostavlja le ireverzibilno prehajanje iz glavnih v kisle in bazične variante. Pri danih formulacijah so bile kisle variante temperaturno pogojene in so se njihove vrednosti hitreje povečevale skladno s povišanjem temperature. Spreminjanje bazičnih variant ni bilo temperaturno odvisno. Prisotnost temperaturne odvisnosti omogoča uporabo Arrheniusove zveze, ki opisuje temperaturno odvisnost konstante hitrosti reakcije. Na podlagi modelskih rezultatov ireverzibilnih prehajanj med variantami, je bilo moč določiti Arrheniusovo zvezo, ki dobro opiše časovno spreminjanje temperaturno pogojenih variant. V splošnem magistrsko delo temelji na uporabi matematično fizikalnih pristopov za opis kompleksnih proteinskih procesov in prispeva k svetovnemu izzivu razvoja novih bioloških zdravil.

• Language: Slovenian
• Keywords: proteini, dinamične enačbe, monoklonska protitelesa, stabilnost, časovno spreminjanje variant, temperaturna odvisnost, Arrheniusova zveza
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FMF - Faculty of Mathematics and Physics
• Year: 2020
• PID: 20.500.12556/RUL-119858
• COBISS.SI-ID: 28209411
• Publication date in RUL: 12.09.2020

Secondary language

• Language: English
• Title: Modelling of temporal and temperature variability of protein charged variants

Abstract

This Master thesis explores time dependence of monoclonal antibody charge variants specially as affected by different temperatures, and in relevance for biopharmaceutical drug development. Variants -- effectively, same protein, but with a different net charge -- emerge as acidic and basic, and differ from the main ones in the value of isoelectric point or net charge. Conversion of variants may influence quality and effectiveness of therapeutics, therefore the understanding of their transformation through time is crucial for a more efficient therapeutic development and production. High temperatures accelerate the degradation processes and allow for understanding the time variation of variants. I introduced effective models that describe the conversion of variants for the measurements obtained from typical stability studies and also include both reversible and irreversible conversions between variants. The best model to describe the given data turned out to be the one involving a reversible conversion from the main variants to the acidic and basic ones, whereas other models would require more accessible data. The model is well capable of time extrapolation of data, which gives us more appropriate results comparing to the ones obtained by linear extrapolation. Because the amount of data taken from typical (experimental) stability studies is limited, complex models give us only a rough estimate of the parameters. For a more accurate estimate, more data or an alternative, simpler model would be required. Model assuming only an irreversible conversion from the main to the acidic and basic variants is also successful, to a good degree. In the given formulations, the acid variants are temperature conditioned and their values increase more rapidly with increasing temperature, whereas the basic variants are not temperature dependent. Temperature dependence allows for the use of the Arrhenius equation, which relates the temperature dependence with the reaction rate constant. Based on the model results of irreversible conversion between variants, it was possible to determine the Arrhenius equation coefficients, which well describes the time dependence of temperature conditioned variants. More generally, this work is based on the use of physical and mathematical approaches for modelling of complex protein processes and is a contribution towards the world challenge of developing novel biological drugs.

• Keywords: proteins, rate equations, monoclonal antibodies, stability, time dependence of variants, temperature dependence, Arrhenius equation