OBTAINING A TRADITIONAL IMMUNOMODULATOR IN THE FORM OF GUM JELLIES: FROM LABORATORY TO PILOT SCALE

Abstract

Fermentation process scale-up, from plate to 3.6 L InforsHT bioreactors, nanoparticulation optimization via microfluidization (from 40 mL, LM-20, to 1000 mL, M110EH), upscale process to achieved composition and casting of (over 1000 pcs/lot) jellies. The immunomodulatory effect was evaluated by in vitro tests on HEK293 cells transfected with Toll-like receptor 2 or 4 genes (TLR2, TLR 4), stability studies under normal and accelerated conditions were also performed. Scale-up processes from laboratory level to pilot level are vital to ensure effective knowledge and/or technology transfer. [...]in addition to the scaling process, the maintenance of immunomodulatory properties and shelf stability of the product and intermediates was verified.

Objectives. This study presents the bacterial fermentation and formulation methods upscaling process for an inactivated and lysed polybacterial immunomodulatory product under the form of gummy jellies.

Materials and Methods. Bacterial mass for 13 heat-inactivated Gram-positive and Gramnegative strains (DO 6 McF) was obtained. The bacterial suspensions were lysed with sodium deoxycholate, 48 hours, after which a concentrated polybacterial mixture was obtained, nanoparticulated by microfluidization, sterilized by filtration and used as raw material. Fermentation process scale-up, from plate to 3.6 L InforsHT bioreactors, nanoparticulation optimization via microfluidization (from 40 mL, LM-20, to 1000 mL, M110EH), upscale process to achieved composition and casting of (over 1000 pcs/lot) jellies. The immunomodulatory effect was evaluated by in vitro tests on HEK293 cells transfected with Toll-like receptor 2 or 4 genes (TLR2, TLR 4), stability studies under normal and accelerated conditions were also performed.

Results and Discussion. Scale-up processes from laboratory level to pilot level are vital to ensure effective knowledge and/or technology transfer. Several difficulties were encountered and resolved during scaling. Moreover, in addition to the scaling process, the maintenance of immunomodulatory properties and shelf stability of the product and intermediates was verified.

Conclusions. Within a multiannual project it was possible to obtain gummy jellies of a classic immunomodulator. The formulation, as well as the fermentation process, were scaled up to pilot to support adequate knowledge transfer. This allowed the in detail analyze of the lysing agent and active substance (vitamin C) behavior, the immunomodulatory effects and the stability. The information obtained is very valuable considering on the one hand the excessive use of antibiotics, antibiotic resistance and the need to stimulate the innate human system against diseases, and on the other hand the debate opened by the European Medicines Agency regarding the use of bacterial lysates as medicine without a characterization supported.

Acknowledgements. The study was carried out within the PSCD - IMUNO II - MApN project, funded by Ministry of Defense, 2020-2024

Obiective. In acest studiu este prezentat procesul de scalare a metodelor de fermentatie bacterianá si formulare a unui produs imunomodulator polibacterian inactivat si lizat sub forma de jeleuri gumate.

Materiale si metode. Masa bacteriana pentru 13 tulpini Gram-pozitive si Gram-negative inactivatá termic si dozatá (DO 6 McF) a fost obtinutá. Suspensiile bacteriene au fost lizate cu deoxicolat de sodiu, 48 ore, ulterior fiind obtinut un amestec polibacterian concentrat, nanoparticulat prin microfluidizare, sterilizat prin filtrare si folosit ca materie prima. A fost realizatd scalarea procesului de fermentatie, de la cresterea pe pláci la bioreactoare InforsHT cu о capacitate de 3,6 L, optimizarea procesului de nanoparticulare prin microfluidizare (de la 40 mL, LM-20, la 1000 mL, M110EH), scalarea procesului de formulare de obtinere a compozitiei de jeleuri si optimizarea procesul de turnare jeleuri (peste 1000 buc/sarja). Efectul imunomodulator a fost evaluat prin teste in vitro pe celule HEK293 transfectate cu gene Toll-like receptor 2 sau 4 (TLR2, TLR 4), studiu de stabilitate a fost in conditii normale si accelerate.

Rezultate si discutii. Procesele de scalare de la nivel de laborator la nivel de pilot sunt vitale pentru a asigura un transfer de cunostinte si/sau tehnologic eficient. Mai multe dificultati au fost intámpinate si rezolvate pe parcursul scalarii. Mai mult, pe lángá procesul de scalare, s-a verificat mentinerea proprietátilor imunomodulatoare si a stabilitätii la raft sub diferite conditii a produsului si intermediarilor.

Concluzii. In cadrul proiectului IMUNO II, multianual, a fost posibilá obtinerea de jeleuri gumate a unui imunomodulator clasic. Formularea, dar si procesul de fermentatie, au fost scalate pana la pilot pentru a sustine un transfer de cunostinte adecvat. Aceasta a permis analiza in detaliu comportamentul agentului de liza si a substantei active (vitamina С), efectele imunomodulatoare si stabilitatea. Informatiile obtinute sunt foarte valoroase considerand pe de o parte, folosirea in exces a antibioticelor, rezistenta la antibiotice si nevoia de a stimula sistemul imun innáscut, si de cealalta parte, dezbaterea deschisá de Agentia Europeaná a Medicamentelor privind folosirea lizatelor bacteriene ca medicament fara o caracterizare sustinuta.

Sursa de finantare : Studiul a fost desfásurat in cadrul proiectului PSCD - IMUNO II - MApN, 2020-2024.

Sidebar

Cantacuzino National Military Medical Institute for Research and Development, Bucharest, Romania