Patent SG-11201809903W-A (SG)
Title: Method for purification and activation of botulinum neurotoxin
Inventor: DONG MIN
IPC: C12N9/52
Filed: 2017-05-16
Abstract: INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property 11111111111111111111111111111111111111111111111111111111111111111111111111111111 Organization International Bureau (10) International Publication Number (43) International Publication Date ....... WO 2017/201105 Al 23 November 2017 (23.11.2017) WIPO I PCT (51) International Patent Classification: PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, C07K 14/33 (2006.01) C12N 9/52 (2006.01) SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, CO7K 1/22 (2006.01) TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (21) International Application Number: (84) Designated States (unless otherwise indicated, for every PCT/US2017/032985 kind of regional protection available): ARIPO (BW, GH, (22) International Filing Date: GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, 16 May 2017 (16.05.2017) UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (25) Filing Language: English EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (26) Publication Language: English 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, (30) Priority Data: KM, ML, MR, NE, SN, TD, TG). 62/336,958 16 May 2016 (16.05.2016) US Declarations under Rule 4.17: (71) Applicant: PRESIDENT AND FELLOWS OF HAR- — as to applicant's entitlement to apply for and be granted a VARD COLLEGE [US/US]; 17 Quincy Street, Cam- patent (Rule 4.1700) bridge, Massachusetts 02138 (US). — as to the applicant's entitlement to claim the priority of the (72) Inventors: DONG, Min; 12 Croft Lane, Weatogue, Con- earlier application (Rule 4.17(iii)) necticut 06089 (US). BARKHO, Sulyman; 111 Heath Published: Street, Apt. 1, Jamaica Plain, Massachusetts 02130 (US). _ with international search report (Art. 21(3)) TAO, Liang; 32 Queensberry Street, Boston, Massachu- — sequence listing part of description (Rule 5.2(a)) setts 02215 (US). — with = Agent: JARRELL, Brenda Herschbach et al.; Choate, = (74) Hall & Stewart LLP, Two International Place, Boston, Massachusetts 02110 (US). _ Designated States (unless otherwise indicated, for every (81) = 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, 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, = = Title: METHOD FOR PURIFICATION AND ACTIVATION OF BOTULINUM NEUROTOXIN (54) = = = Neutral-alkaline pH pH 6 = — BoNT NTNHA NTNHA BoNT complex = \ If) ..v FIG. 6A 0 1-1 C 1-1 (57) : Disclosed herein are methods for the isolation and purification of a botulinum neurotoxin (BoNT) protein, or a polypep- el tide comprising a receptor binding domain of BoNT, from a solution. The method comprises contacting the solution containing the Cr: protein or polypeptide to a matrix which has attached thereto a non-toxic non-hemagglutinin (NTNHA) under conditions appropriate for Il binding, washing the matrix to thereby remove unbound materials, and eluting the protein or polypeptide with a solution that dissociates © the bound protein from the NTNHA. Conditions appropriate for binding are a pH of less than 7.5 (e.g, 6). Conditions appropriate for ei dissociation are a pH greater than or equal to 7.5 (e.g., 8). Compositions specific to the methods are also disclosed.