Patent SG-11201903890Q-A (SG)
Title: Adeno-associated virus purification methods
Inventor: FIEDLER CHRISTIAN
IPC: C12N7/00
Filed: 2017-11-03
Abstract: WO 18/ 128688 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date 12 July 2018 (12.07.2018) WIPO I PCT onion °nolo Oil OH 1101 En oimIE (10) International Publication Number WO 2018/128688 Al (51) International Patent Classification: C12N 7/00 (2006.01) (21) International Application Number: PCT/US2017/059967 (22) International Filing Date: 03 November 2017 (03.11.2017) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 62/417,775 04 November 2016 (04.11.2016) US (71) Applicants: BAXALTA INCORPORATED [US/US]; 1200 Lakeside Drive, Bannockburn, IL 60015 (US). BAX- ALTA, GmbH [CH/CH]; Zaehlerweg 4, 6300 Zug (CH). (72) Inventors: FIEDLER, Christian; Raaberbahngasse 18/36, 1100 Vienna (AT). GRILLBERGER, Leopold; Fin- sterergasse la, 1220 Vienna (AT). HASSLACH- ER, Meinhard; Vorgartenstrasse 221/1/7, 1020 Vienna (AT). KRAUS, Barbara; Fasangasse 21, 2120 Obern- dorf (AT). MITTERGRADNEGGER, Dominik; Fan- ny-Mintz Gasse 3/142, 1020 Vienna (AT). REUBERG- ER, Stefan; Seefeldergasse 6/13, 1220 Vienna (AT). SCHAFHAUSER, Horst; Erlaaerstrasse 33, 1230 Vienna (AT). BENDIK, Marian; Gabcikova 4, 84105 Bratislava (SK). (74) Agent: SULLIVAN, Nicole R.; Troutman Sanders LLP, 875 Third Avenue, New York, NY 10022 (US). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (54) Title: ADENO-ASSOCIATED VIRUS PURIFICATION METHODS FIGURE 1 Triple transfection of HEK293 cells (Example 1) Harvesting and clarifying supernatant (SN) of HEK293 culture (Example 2) OPTIONAL TEFL ultra and diafiltration (Example 3) Buffer exchange and concentration adjustment Negative AEX chromatography (Example 4) Purification step: Removal of typically more than 60% of the unwanted impu es (e.g. host cell proteins) TFF2: ultra and diafiltration (Example 5) Buffer exchange and concentration adjustment Sucrose Gradient Ultracentrifugation (Examples 6, 7, 9, & 14) Removal of particles containing truncated and/or incomplete Vector DNA Solvent/Detergent treatment (Example 8) Inactivation of lipid enveloped viruses AEX chromatography polishing step (e.g. on Fractogel TMAE) (Example 8) Removal of particles with incomplete protein composition and oligomerized structures Nanofiltration (Example 10 & 11) Removal of all viruses greater than the pore size of the nanofilters (57) : Provided herein are methods of producing an adeno-associated virus (AAV) product, methods of purifying adeno-as- sociated virus, and methods of purifying full AAV capsids from a concentrated AAV fraction comprising empty AAV capsids and full AAV capsids. [Continued on next page] WO 2018/128688 Al MIDEDIM11110111111111111111111111111111111111111111111111111110111110111111111111 EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: with international search report (Art. 21(3)) before the expiration of the time limit for amending the claims and to be republished in the event of receipt of amendments (Rule 48.2(h))