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(12) Patent Application Publication (10) Pub. No.: US 2010/0197888 A1 Adib Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2010/0197888 A1 Adib Et Al US 2010O197888A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0197888 A1 Adib et al. (43) Pub. Date: Aug. 5, 2010 (54) METHOD FOR MANUFACTURING LINEAR Related U.S. Application Data POLYETHYLENIMINE (PED FOR TRANSFECTION PURPOSE AND LINEAR PE (60) Provisional application No. 60/952,993, filed on Jul. OBTANED WITH SUCH METHOD 31, 2007. Publication Classification (75) Inventors: Abdennaji Adib, Illkirch (FR): (51) Int. Cl. Fabrice Stock, Lingolsheim (FR): C08G 73/06 (2006.01) Patrick Erbacher, Illkirch (52) U.S. Cl. ........................................................ 528/423 Graffenstaden (FR) (57) ABSTRACT Correspondence Address: The invention concerns a method of synthesising and prepar BANNER & WITCOFF, LTD. ing linear polyethylenimine (PEI) for use as a transfection 1100 13th STREET, N.W., SUITE 1200 vector, and the product obtained with such a method. It com prises drying a monomer 2-ethyl-2-oxazoline and polymeris WASHINGTON, DC 20005-4051 (US) ing said monomer for obtaining poly (2-ethyl-2-oxazoline) (PEOX) by: using acetonitrile as solvent, adding a dried ini (73) Assignee: Polyplus Transfection, Ilkirch tiator of the reaction of polymerisation, and mixing them Graffenstaden (FR) altogether, purifying said obtained PEOX by evaporation, while performing at least three times successive washing/ (21) Appl. No.: 12/671,312 precipitation steps with methanol and diethyl ether and cor responding filtrations, in order to obtain (i), by performing (22) PCT Fled: Jul. 31, 2008 1H-NMR tests, correct identification of said PEOX polymer, confirmation of absence of monomer to a level <1.0% and (86) PCT NO.: PCT/B2O08/OO2339 confirmation of absence of solvent to a level <5.0% and (ii), by performing Gel Permeation Chromatography, a mean of S371 (c)(1), molecular weight (Mw)>23,000 Da and polydispersity (Mw/ (2), (4) Date: Apr. 8, 2010 Mn) of said PEOX-1.5, hydrolysing said PEOX. Patent Application Publication Aug. 5, 2010 Sheet 1 of 6 US 2010/O197888A1 S & O sc WO C - CO N CN On - WO N P N Patent Application Publication Aug. 5, 2010 Sheet 2 of 6 US 2010/O197888A1 MONOMER 21 O 37 24- 3 39 PEI 23 O 26. 27 PEOX 3 28 3 2 33 3 6 FIG.2 Patent Application Publication Aug. 5, 2010 Sheet 3 of 6 US 2010/0197888A1 500 4.50 4.00 350 300 250 200 S00 450 4.00 350 300 2SO 200 Patent Application Publication Aug. 5, 2010 Sheet 4 of 6 US 2010/0197888A1 50 52 53 49 S1 Patent Application Publication Aug. 5, 2010 Sheet 5 of 6 US 2010/0197888A1 FIG.7 Patent Application Publication Aug. 5, 2010 Sheet 6 of 6 US 2010/0197888A1 64. 65 66 67 7 6 5 4 3 2 1 ppm US 2010/O 197888 A1 Aug. 5, 2010 METHOD FOR MANUFACTURING LINEAR use as a transfection vector comprising the steps of from a POLYETHYLENIMINE (PED FOR determined quantity of monomer 2-ethyl-2-oxazoline at a TRANSFECTION PURPOSE AND LINEAR PE purity Superior to 99%, thoroughly drying said quantity of OBTANED WITH SUCH METHOD monomer, and polymerising said quantity of monomer for obtaining poly(2-ethyl-2-oxazoline) (PEOX) by: 0017 after thorough drying of a predetermined quantity of 0001. The present invention concerns the manufacture and acetonitrile, using said acetonitrile as solvent in said quantity quality control of linear Polyethylenimine (PEI) for transfec of dried monomer, while adding a predetermined quantity of tion applications. thoroughly dried initiator of the reaction of polymerisation, 0002 The invention also relates to a product obtained with and mixing them altogether, Such manufacturing method, and more specifically for appli 0018 purifying said obtained PEOX by evaporation to cation in vivo including but not limitated to nuclear acid based therapy. remove said solvent, while performing at least three times 0003. This application is a non provisional application Successive washing/precipitation steps with methanol and concerning and claiming priority of earlier U.S. provisional diethyl ether and corresponding filtrations, application 60/952,993. said operations of drying, polymerising, and purifying being 0004 Polyethylenimine (PEI) is an organic macromol arranged to obtain (i), by performing H NMR tests, correct ecule with a high cationic-charge-density potential. Every identification of said PEOX polymer, confirmation of absence third atom of PEI is an amino nitrogen that can be protonated. of monomer to a level <1.0% and confirmation of absence of PEI can ensnare DNA, and, owing to the close location of solvent to a level <5.0% and (ii), by performing Gel Perme many linkeramino groups, PEI retains a Substantial buffering ation Chromatography, a mean of molecular weight (Mw) capacity at virtually any pH. >23,000 Da and polydispersity (Mw/Mn) of said PEOX-1.5, 0005 PEI alone is a highly efficient vector for delivering (0019 hydrolysing said PEOX with hydrochloric acid for DNA plasmids both in vitro and in vivo. obtaining said PEI sufficiently efficiently to have, by per 0006 PEI compacts DNA into positively charged particles forming H-NMR tests, an amount of residual side chains or capable of interacting with anionic proteoglycans at the cell propionic acid <5% and to identify the PEI as a single peak. Surface and facilitating entry of the particles by endocytosis. 0020. By thoroughly drying a specific quantity of mono Positively charged particles attach to anionic cell-Surface pro mer, acetonitrile or the initiator, one should understand teoglycans at the cell surface and are subsequently spontane obtaining, just before use, a reduction of the humidity below ously endocytosed (Boussifet. al., 1995). PEI also possesses 10 ppm of water, which can be obtained by drying on calcium the unique property of acting as a “proton sponge' and this hydride over 48 h and then by distillation and collecting the buffers the endosomal pH and protects DNA from degrada monomer above the temperature of 129°C. tion, once it has entered the cell. Sustained proton influx also 0021. The present invention also proposes advantageous induces endosomal osmotic Swelling and rupture which pro embodiments including, but not limited to, one and/or a plu vides an escape mechanism for DNA particles to the cyto rality of the following features: plasm (Boussif, et. al., 1995: Behr, 1997). 0022 the mean of molecular weight (Mw) of the PEOX 0007. In summary, PEI-based delivery systems mimic is such as 40,000 DaKMw<54,000 Da; some of the key properties of viruses, such as DNA conden 0023 the monomer/initiator ratio is about 500. sation/protection and endosome escape. By about one should understand +5%; 0008. Several manufacturing methods exist for PEI. 0024 the monomer/initiator ratio is 480; 0009. This is indeed due to the fact that such polymer has been used in a plurality of fields of the Industry since many (0.025 the monomer is at a Purity Superior to 99.95%; years. 0026 the initiator is mixed with the acetonitrile before 0010. However, when used for transfection purpose, the addition to the monomer; efficiency of such PEI is often not good, i.e. less than 10% of 0027 the polymerisation is performed during more the manufactured products succeed in transfer into a cell. than 20 hours at a temperature superior to 85°C.; 0011. In particular there is low efficiency for high molecu 0028 the temperature of polymerisation is superior or lar weight of PEI, i.e. >10,000 Da. equal to 105° C.: 0012. Furthermore, and as soon as such product is to be 0029 after the first filtration, the residue is washed used in the medical field and more particularly for genetic freely with a solvent such as MeOH, and after addition therapy with high standard of manufacturing such as the GMP of diethyl ether, the poly (2-ethyl-2-oxazoline) is natu standard, exceptional efficiency and quality are requested. rally separated as oil from solution, the overall solvent is 0013 The present invention aims to solve this problem decanted and said washing and separation is repeated at and allows great efficiencies 55%) with high molecular least four times before drying in vacuo; weight and a low polydispersity. 0030 the hydrolysing step comprises removing from 0014 For this purpose, it is an object of the present inven the reaction mixture the discharged propionic acid tion to provide an improved method of synthesising and pre obtained by azeotropic distillation regularly and during paring a linear polyethylenimine (PEI) which renders pos at least one day, while monitoring the process of reaction sible an efficiency and reliability of the transfection better by 'H-NMR spectroscopy; than the ones already known, with higher quality. 0.031 the residue obtained at the end of the process of 0015. In one preferred embodiment, the quality is the stan reaction is diluted in water and evaporated at least three dard one for GMP (Good Manufacturing Product). times to remove traces of propionic acid, then the resi 0016. More precisely, the invention proposes a method of due is dissolved again in water and filtered before lyo synthesising and preparing linear polyethylenimine (PEI) for philisation; US 2010/O 197888 A1 Aug. 5, 2010 0032 the filtration is provided through a sterile mem highly controlled polymerization, provides polymers having brane with a dimension of mesh between 0.20 um and determined Mn near the theoretical Mn and with a low poly 0.25 um, particularly a sterile cellular acetate mem dispersity index. brane. 0047 Classical yields of polymerization were in the range 0033. However the filtration as such is not sterile, which is of 55 to 95% when the molecular weights of PEOX were therefore not involving additional cost, and this contrarily to expected from 1,000 to 10,000 Da (Hoogenboom et al., the general opinion of the man skilled in the art, for whom 2003).
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