Vpliv formulacijskih in procesnih parametrov na koloidno stabilnost liofiliziranih lipidnih nanodelcev

Gošek, Teja (author), Ahlin Grabnar, Pegi (mentor), Frieß, Wolfgang (co-mentor)

Kot zelo učinkovita v preprečevanju okužbe z boleznijo COVID-19 so se izkazala mRNA cepiva, pri katerih so molekule mRNA vključene v lipidne nanodelce (LND). Cilj magistrske naloge je bil razviti liofilizirano formulacijo LND s poudarkom na koloidni stabilnosti LND. V nalogi smo uporabili LND z lososovo DNA, ki smo jih pripravili sami, in LND iz cepiva Moderna. Velikost in porazdelitev velikosti delcev smo ovrednotili z metodo dinamičnega sipanja svetlobe. Za nekatere vzorce smo določili tudi učinkovitost vgradnje mRNA z metodo RiboGreen. Ovrednotili smo vplive različnih parametrov na koloidno stabilnost LND med zamrzovanjem: hitrost ohlajanja, polnilni volumen, pH pufra, ionsko moč in uporabo različnih potencialnih krioprotektantov. Analizirali smo številne pomožne snovi, da bi našli ustrezne lioprotektante. S primerjavo razlik v velikosti LND in polidisperznem indeksu (PDI) v različnih formulacijah po zamrzovanju in po celotnem procesu liofilizacije smo ovrednotili nestabilnosti, ki so posledica zamrzovanja in ostanek, do katerega pride med kasnejšimi fazami liofilizacije. Ugotovili smo, da hitrost ohlajanja ni vplivala na razlike v spremembi velikosti delcev in PDI. V primeru večjega polnilnega volumna so bile spremembe v velikosti LND po zamrzovanju manjše, vendar je bil PDI širši, kar bi lahko zakrilo spremembe v velikosti delcev. V natrijevem fosfatnem pufru in kalijevem fosfatnem pufru je prišlo do obsežne nestabilnosti LND. Nasprotno, smo v Tris pufru zaznali le manjšo porast velikosti delcev po zamrzovanju. Vpliv ionske moči smo določali v formulacijah z različnimi koncentracijami NaCl. V vzorcu s 300 mM NaCl je prišlo do obsežne agregacije, prav tako je do večjega poskoka v velikosti delcev prišlo v formulaciji s 150 mM NaCl. Pri manjši vsebnosti NaCl do teh sprememb ni prišlo. Kot krioprotektanti so obetavne rezultate pokazali kombinacija saharoze z obema, polimerom 188 (P188) in hidroksipropil-β-ciklodekstrinom (HP-β-CD) ter vzorec z 20% saharoze. Potencialni lioprotektanti so bili kombinacija saharoze tako s P188 kot s poloksamerom 407 (P407) ter kombinacija saharoze s polisorbatom 20 (PS20). Največjo učinkovitost vgradnje mRNA smo ovrednotili pri vzorcu s PVP 40 kDa, sledila sta vzorca s kombinacijo saharoze s P188 in P407.

• Language: Slovenian
• Keywords: lipidni nanodelci z mRNA, liofilizacija, zamrzovanje, koloidna stabilnost, krioprotektanti
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151146
• Publication date in RUL: 30.09.2023

Secondary language

• Language: English
• Title: Impact of process and formulation parameters on the colloidal stability of lyophilised lipid nanoparticles

Abstract

During COVID-19 pandemic, mRNA-LNP vaccines proved to be efficient in preventing COVID-19 infection. The aim of the master thesis was to develop a lyophilizable formulation of lipid nanoparticles (LNPs) with the main focus on colloidal stability of LNPs. Self-prepared LNPs with salmon DNA and LNPs from Moderna vaccine were used in the experiments. Particle size and size distribution were evaluated via DLS measurements. For some samples, also encapsulation efficiency of mRNA via RiboGreen assay was evaluated. Several experiments were performed to study effects of different parameters on the colloidal stability of LNPs during freeze-thawing. Following parameters were evaluated: cooling rate, filling volume, pH of the buffer, ionic strength and use of different potential cryoprotectants. Different excipients were analyzed to find appropriate lyoprotectants. Damage caused by freezing step compared to damage caused by the rest of lyophilization process was evaluated by comparing the differences in size and PDI of LNPs in different formulations after freeze-thawing and after whole lyophilization process. Comparing the change in size of LNPs and PDI after freeze-thawing, no visible change was detected if faster or slower cooling rate was used. When analyzing the effect of filling volume, smaller change in size of LNPs was obtained for samples with bigger filling volume. However, PDI was wider for these samples which could potentially mask the difference in size of LNPs after freezing. Regarding pH of formulation, sodium phosphate buffer and potassium phosphate buffer caused substantial LNPs instability. In contrast, only small differences in size of LNPs were detected in Tris buffer. Influence of the ionic strength was analysed using formulations with different concentrations of NaCl. A major size increase of LNPs was detected in a sample with 300 mM NaCl. Some aggregation occurred also in a sample with 150 mM NaCl. Samples with lower NaCl level did not result in pronounced changes in LNPs size. From the various cryoprotectants tested, promising results were obtained for formulations with combination of sucrose with either poloxamer 188 (P188) or hydroxypropyl-β-cyclodextrin (HP-β-CD) and for a sample with 20% sucrose. As potential lyoprotectants, combination of sucrose with both poloxamer 407 (P407) and P188 as well as combination of sucrose with polysorbate 20 (PS20) were determined. Highest encapsulation efficiency of mRNA was obtained in sample with PVP 40 kDa, followed by samples with combination of sucrose with P188 and P407.

• Keywords: mRNA lipid nanoparticles, lyophilization, freezing step, colloidal stability, cryoprotectants