WO2022009136A4

WO2022009136A4 - Method of encapsulation of an active protein using electrodeposition techniques, |an antibacterial composition containing the active protein and polymer and its|use for the production of veterinary medicinal products

Info

Publication number: WO2022009136A4
Application number: PCT/IB2021/056130
Authority: WO
Inventors: Lukasz BZDZION; Jakub GRZESIAK; Karol Wrzeszcz; Adam Junka
Original assignee: Bioceltix Spolka Akcyjna
Priority date: 2020-07-10
Filing date: 2021-07-08
Publication date: 2022-06-16
Also published as: WO2022009136A2; WO2022009136A3; PL434626A1

Abstract

The subject of invention is a method of encapsulating an active protein using electrodeposition techniques, characterised in that it comprises the following steps: (a) establishing a primary mesenchymal cell culture containing 2,000-5,000 source tissue cells and a serum-supplemented culture medium; (b) maintaining the cell culture established in step (a) for 280-340 hours until the culture surface is fully covered by the cultured cells; (c) obtaining a culture fluid from the above of the cultured cells; (d) purifying the culture fluid obtained in step (c) from cell debris and suspended cells by centrifuging said fluid with a force of 300 to 1200 x g; (e) transferring the upper liquid phase from above the sediment to a new vessel; (f) gently mixing the purified liquid phase obtained in step (e) with an aqueous solution of polyvinyl alcohol; (g) adding ethyl alcohol to the mixture obtained in step (f) while stirring continuously; (h) the material obtained in step (g) is deposited on the collector surface by means of electro spinning or electrospraying. Another subject of invention is an antibacterial composition containing an active protein and a polymer, characterised in that it contains ethyl alcohol, wherein the active protein is a fibrous, fully water-soluble material containing proteins released by mesenchymal cells, including cathelicidin at an amount from 270 to 1230 pg/100mg of dry weight of the composition, and the polymer is an aqueous solution of polyvinyl alcohol. Another subject of invention is an use of the antibacterial composition according to invention for the preparation of veterinary medicinal products.

Claims

AMENDED CLAIMS received by the International Bureau on April 14, 2022 Method of encapsulating an active protein using electrodeposition techniques, characterised in that the active protein is a fibrous, fully water-soluble material containing proteins released by mesenchymal cells from a primary culture, comprising cathelicidins at an amount from 990 to 1230 pg/100 mg of dry weight of the composition, said method_comprises the following steps:

(a) establishing a primary mesenchymal cell culture obtained directly from frozen tissue isolate or from direct inoculation of the tissue isolate, and not subjected to further culture passages and containing 2,000-5,000 source tissue cel Island a serum-supplemented culture medium;

(b) maintaining the cell culture established in step (a) for 280-340 hours until the culture surface is fully covered by the cultured cells;

(c) obtaining a culture fluid from the above of the cultured cells;

(d) purifying the culture fluid obtained in step (c) from cell debris and suspended cells by centrifuging said fluid with a force of 300 to 1200 x g;

(e) transferring the upper liquid phase from above the sediment to a new vessel;

(e’) purification of liquid phase from proteins greater than 50kDa by filtering;

(f) gently mixing the purified liquid phase obtained in step (e) with an aqueous solution of polyvinyl alcohol;

(g) adding ethyl alcohol to the mixture obtained in step (f) while stirring continuously;

(h) the material obtained in step (g) is deposited on the collector surface by means of electrospinning or electro spraying. Method according to claim 1, characterised in that establishing the culture in step (a) is performed using a culture medium selected from the group consisting of DMEM, DMEM-Ham’s F-12, IMDM. The method according to any preceding claim from 1 to 2, characterised in that the mesenchymal cells used in step a) are mesenchymal stromal cells derived from adipose tissue, bone marrow or Wharton’s jelly.

AMENDED SHEET (ARTICLE 19) Method according to claim 3, characterised in that the mesenchymal cells are mesenchymal cells of species selected from the group consisting of dogs, cats, horses and sheep. Antibacterial composition obtained by method according to any of preceding claims from 1 to 4, containing an active protein and a polymer, characterised in that it contains 5% of ethyl alcohol, 47.5% of the active protein and 47.5% of polymer, wherein the active protein is a fibrous, fully water-soluble material containing proteins released by mesenchymal cells from a primary culture, obtained directly from frozen tissue isolate or from direct inoculation of the tissue isolate, and not subjected to further culture passages, comprising cathelicidin at an amount from 990 to 1230 pg of cathelicidin/ 100 mg of dry weight of the composition, and the polymer is an aqueous solution of polyvinyl alcohol; wherein the composition is produced by electrodeposition of the active protein admixed with the polymer and ethyl alcohol. Antibacterial composition according to claim 5, characterised in that the polymer is a 30% aqueous solution (300mg/ml) of polyvinyl alcohol. Antibacterial according to any preceding claim from 5 to 6, characterised in that the mesenchymal cells are mesenchymal stromal cells derived from adipose tissue, bone marrow or Wharton’s jelly. Antibacterial composition according to claim 7, characterised in that the mesenchymal cells are mesenchymal cells of species selected from the group consisting of dogs, cats, horses and sheep. Use of the antibacterial composition according to any preceding claim from 5 to 8 for the preparation of veterinary medicinal products.

AMENDED SHEET (ARTICLE 19)