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Ugotavljanje konformacijskih sprememb monoklonskih protiteles in razvoj modela za napovedovanje njihove stabilnosti : magistrska naloga
Markoja, Uroš (Author), Kristl, Julijana (Mentor) More about this mentor... This link opens in a new window, Winter, Gerhard (Co-mentor), Svilenov, Hristo (Co-mentor)

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Abstract
Monoklonska protitelesa (mAb) uporabljena kot biološka zdravila, so skupina bioloških zdravil, katerih pomembnost v zadnjih treh desetletjih strmo narašča. Večina zdravil z mAb na trgu se nahaja v obliki tekočih formulacij v katerih pa mAb razpadajo po poteh, ki jih je težko predvideti. Za določitev konformacijske stabilnosti proteinov v raztopinah predstavlja kemična denaturacija alternativen pristop termičnemu stresu. Namen magistrske naloge je bil proučiti konformacijske spremembe modelnega IgG1 monoklonskega protitelesa v prisotnosti kemičnih denaturantov z uporabo različnih eksprerimentalnih in teoretičnih pristopov. Poleg tega smo želeli izboljšati v literaturi že znano metodo testiranja stabilnosti z Ab tako, da bi lahko hitreje in enostavneje zaznali razlike v njihovi strukturi. Proučevali smo stabilnost modelnega mAb pod vplivom najpogostoje uporabljenih denaturantov (gvanidinijevega hidroklorida (GuHCl) in sečnine) in treh alternativnih denaturantov (gvanidinijevega tiocianata, N-metil sečnine in N-etil sečnine), in sicer tako eksperimentalno kot matematično. Merili smo spremembo intenzitete intrinzične fluorescence kot posledico razvijanja mAb pri pH 4,5 do 8,5 v 24 urah in 15 dneh pri 24 različnih koncentracijah denaturanta. Iz denaturacijskih krivulj (odvisnost signala intrinzične fluorescence od koncentracije denaturanta in časa) smo izračunali ΔG in polovične čase t1/2 razvitja z uporabo eksperimentalnega in matematičnega modeliranja. Za evaluacijo napovednih metod stabilnosti smo izvedli pospešene stabilnostne teste pri 40°C v 12-ih tednih. Izmed preučevanih denaturantov povzroča le GuHCl zadovoljivo razvijanje mAb in je lahko uporabljen kot denaturant na celotnem spektru testiranih puferskih raztopin. Pri merjenju kinetike razvitja v prvih 24 urah smo dokazali, da polovični časi t1/2 korelirajo s spremembami proste energije ΔG iz denaturacijskih krivulj s Pearsonovim korelacijskim koeficientom 0,78. Za razvrstitev pufernih raztopin z mAb po stabilnosti, osnovano na t1/2, potrebujemo trikrat manj proteina v primerjavi z uveljavljenim termičnim pristopom, kjer je izračunan ΔG in daje vrednosti z manjšo standardno deviacijo. Razviti pristop z določitvijo t1/2 bi lahko nadomestil standardne izračune ΔG. Pri spremljanju kemične denaturacije v 15-ih dneh smo opazili spremembe signala intrinzične fluorescence v tranzicijski regiji denaturacijske krivulje. Meritve z dinamičnim sipanjem svetlobe so pokazale, da je to najverjeneje posledica aggregacije. S prileganjem signala intrinzične fluorescence s pseudo prvim redom kinetike aggregacije smo pridobili aggregacijske čase tagg. Opazovali smo tudi majhne premike signala intrinzične fluorescence v pred-tranzicijski regiji pri ~1M koncentraciji GuHCl po inkubaciji do 15 dni. Ti premiki so zelo dobro korelirali (Pearsonovov korelacijski koeficient 0,89) s kinetičnimi konstantami razpada monomera pri pospešenih stabilnostnih študijah pri 40 °C v 12 tednih. Merjenje majhnih premikov intrinzične fluorescence mAb v pred-tranzicijski regiji bi tako lahko bilo novo orodje za napoved izzida pospešenih stabilnostnih testov.

Language:English
Keywords:monoclonal antibody therapeutic proteins protein stability chemical denaturation fluorescence spectroscopy kinetic simulation
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:FFA - Faculty of Pharmacy
Year:2017
Publisher:[U. Markoja]
Number of pages:XIV, 85 f.
UDC:615.32.011(043.3)
COBISS.SI-ID:4412785 This link opens in a new window
Views:110
Downloads:47
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Secondary language

Language:Slovenian
Title:Tracking of conformational changes of monoclonal antibodies of prediction models for their stability
Abstract:
Monoclonal antibodies (mAbs) used as biological drugs are a class of biopharmaceuticals that has rapidly expanded over the past three decades. The majority of therapeutic products on the market that are based on mAbs are liquid formulations, in which the mAbs can have complex degradation pathways that are difficult to predict. Chemical denaturation is an alternative approach to thermal stress for determination of conformational stability (denaturation) of proteins (such as mAbs) in solution. The purpose of this project was to investigate the conformational changes of a model IgG1 mAb in the presence of chemical denaturants using both experimental and theoretical approaches. Moreover, we wanted to improve upon the stability-testing approaches in the literature for mAbs through the determination of faster and easier approach(es) to indicate differences in mAb stabilities. We studied the stability of the model mAb under the influence of two widely used denaturants (guanidine hydrochloride [GuHCl], urea) and three alternative denaturants (guanidine thiocyanate, N-methyl-urea, N-ethyl-urea), both experimentally and mathematically. We measured the changes in the intrinsic fluorescence signals due to the unfolding of the mAb under different denaturant concentrations at pH 4.5 to pH 8.5 over 24 h and 15 days. From the denaturation curves (intrinsic fluorescence versus denaturant concentration and time), we calculated the denaturant-concentration-independent changes in the Gibb’s free energy (ΔG) and the half times (t1/2) of unfolding, again, experimentally and through mathematical modelling. To evaluate the predictions of these stability-indicating methods, we also performed accelerated stability studies at 40 °C over 12 weeks. GuHCl was the only investigated denaturants that provided complete data and can be used as a denaturant in buffer solutions of this mAb. By tracking the kinetics of the unfolding over the first 24 h, we show that t1/2 correlates with ΔG from chemical denaturation curves, with a Pearson’s correlation coefficient of 0.78. The formulation stability ranking based on t1/2 required a third of the protein used in the approach where ΔG was calculated, and provided data with smaller standard deviations. This approach using the calculation of t1/2 can therefore substitute for the standard ΔG calculations. When the chemical denaturation was tracked over 15 days, shifts were seen for the intrinsic fluorescence signals in the transitional regions of the denaturation curves. Dynamic light scattering measurements showed that these are most likely due to the formation of aggregates. By fitting the intrinsic fluorescence signal changes to a pseudo first-order kinetic model of aggregation, the aggregational times (tagg) were obtained. Small shifts in the intrinsic fluorescence signals were also seen in the pre-transitional region at ~1 M GuHCl following incubations of up to 15 days. These shifts correlated well (Pearson’s correlation coefficient, 0.89) with the mAb monomer degradation rates in accelerated stability studies at 40 °C over 12 weeks. Measurements of the intrinsic fluorescence (F350/330) shifts therefore provide a novel stability-indicating tool to predict the outcomes of accelerated stability studies.

Keywords:monoklonska protitelesa kemična denaturacija stabilnostne študije stabilnost mAb

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