Vpliv izbranih pomožnih snovi na zniževanje viskoznosti raztopin monoklonskih protiteles

Kolman, Nina (avtor), Ahlin Grabnar, Pegi (mentor), Bjelošević Žiberna, Maja (komentor)

V zadnjih letih je zdravljenje z monoklonskimi protitelesi v obliki subkutanih injekcij močno naraslo zaradi številnih prednosti pred ostalimi načini parenteralne aplikacije. Volumen raztopine je pri subkutani aplikaciji omejen na največ 2 ml, kar vodi do potrebe po visokih koncentracijah monoklonskih protiteles (nad 100 mg/ml). Visoka koncentracija monoklonskih protiteles (mAb) lahko vodi do visoke viskoznosti in slabše stabilnosti bioloških makromolekul, kar poleg optimizacije proizvodnega procesa, npr. filtriranja, mešanja, narekuje tudi visoke sile iztisa raztopine v podkožje, kar lahko predstavlja bolečino za bolnika. V magistrski nalogi smo želeli ugotoviti, katere pomožne snovi učinkovito znižajo viskoznost raztopine izbranega mAb s koncentracijo 150 mg/ml. Preskusili smo raztopine 20 različnih spojin s koncentracijo 25 mM, med katerimi so bili aminokisline, mimetiki aminokislin ter di- in tripeptidi. Koncentracijo monoklonskih protiteles smo določali spektrofotometrično in viskoznost z viskozimetrom na čipu. Ugotovili smo, da so vse testirane spojine v določenem obsegu znižale viskoznost raztopin z monoklonskimi protitelesi, vendar je le 14 spojin znižalo viskoznost pod sprejemljivo vrednost za subkutano aplikacijo (20 mPas). Najučinkoviteje so viskoznost znižale spojine, ki so delovale tako na elektrostatske kot tudi na hidrofobne interakcije. Od prisotnih funkcionalnih skupin je imela na znižanje viskoznosti raztopine mAb največji vpliv karboksilna, najmanjši pa hidroksilna skupina. Pomemben je tudi položaj funkcionalnih skupin na aromatskem obroču, in sicer so najobetavnejše spojine vsebovale funkcionalno skupino na para mestu obroča. Pri di- in tripeptidih smo ugotovili, da ima največji vpliv spojina, ki vsebuje dva pirolidinska obroča, najmanjšega pa spojine, ki so vsebovale glicinski ostanek. V vseh raztopinah smo izmerili tudi porazdelitev velikosti molekul mAb in ugotovili, da med učinkovitostjo spojin za znižanje viskoznosti raztopin mAb in stabilnostjo mAb ni korelacije. Za dodatno zagotovitev stabilnosti monoklonskih protiteles bi bilo v raztopine smiselno dodati stabilizatorje, kot je saharoza. Ugotovili smo, da viskoznost raztopine narašča eksponentno s koncentracijo saharoze, kar je treba upoštevati pri razvoju formulacij z monoklonskimi protitelesi. Raztopini saharoze smo dodali tudi arginin in histidin ter ugotovili, da nimata vpliva na njeno viskoznost.

• Jezik: Slovenski jezik
• Ključne besede: monoklonska protitelesa, subkutana aplikacija, zniževanje viskoznosti, aminokisline, prolin
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-152240
• Datum objave v RUL: 14.11.2023

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The influence of selected excipients on the viscosity reduction of monoclonal antibodies’ solutions

Izvleček

In recent years, monoclonal antibody treatment in the form of subcutaneous injections has grown significantly due to its many advantages over other parenteral routes of administration. The volume of the solution is limited to a maximum of 2 ml in subcutaneous administration, which leads to the need for high concentrations of monoclonal antibodies (above 100 mg/ml). High concentrations of monoclonal antibodies (mAbs) can lead to high viscosity and poorer stability of the biological macromolecules, which, in addition to the optimisation of the production process, e.g., filtering, mixing, also requires forceful injection of the solution into the subcutaneous tissue, which can be painful for the patient. In the master's thesis we wanted to find out which excipients effectively lower the viscosity of a solution of a selected mAb with a concentration of 150 mg/ml. We tested solutions of 20 different compounds at a concentration of 25 mM, including amino acids, amino acid mimetics, and di- and tripeptides. The concentration of monoclonal antibodies was determined spectrophotometrically, and the viscosity was determined using an on-chip viscometer. We found that all tested compounds lowered the viscosity of the monoclonal antibody solutions to some extent, but only 14 compounds lowered the viscosity below an acceptable value for subcutaneous administration (20 mPas). The most effective viscosity lowering was achieved by compounds acting on both electrostatic and hydrophobic interactions. Of the functional groups present, the carboxyl group had the greatest effect on lowering the viscosity of the mAb solution, while the hydroxyl group had the least effect. The position of the functional groups on the aromatic ring is also important, with the most promising compounds containing a functional group at the para position of the ring. For di- and tripeptides, we found that compounds containing two pyrrolidine rings had the greatest influence, while compounds containing a glycine residue had the least. We also measured the size distribution of the mAb particle for all solutions and found that there was no correlation between the efficacy of the compounds in lowering the viscosity of the mAb solutions and the stability of the mAb. To further ensure the stability of monoclonal antibodies, it would be useful to add stabilisers such as sucrose to the solutions. We have found that the viscosity of the solution increases exponentially with sucrose concentration, which should be taken into account when developing monoclonal antibody formulations. We also added arginine and histidine to the sucrose solution and found that they had no effect on the viscosity of the sucrose solution.

• Ključne besede: monoclonal antibody, subcutaneous application, viscosity reduction, amino acids, proline