	US 12,233,119 B2
	Composition for oral or nasal delivery of tetanus, diphtheria, and pertussis vaccine alone or in combination using neurotoxin associated proteins
Bal Ram Singh, Rehoboth, MA (US)
Assigned to Prime Bio, Inc., North Dartmouth, MA (US)
Filed by Prime Bio, Inc., North Dartmouth, MA (US)
Filed on Sep. 14, 2023, as Appl. No. 18/467,682.
Application 18/467,682 is a division of application No. 15/064,651, filed on Mar. 9, 2016, granted, now 11,771,752.
Prior Publication US 2024/0131137 A1, Apr. 25, 2024 Prior Publication US 2024/0226262 A9, Jul. 11, 2024
Int. Cl. A61K 39/02 (2006.01); A61K 39/00 (2006.01); A61K 39/08 (2006.01); A61K 39/395 (2006.01); A61P 31/04 (2006.01); C07K 14/19 (2006.01); C07K 14/235 (2006.01); C07K 14/33 (2006.01); C07K 14/34 (2006.01); C12N 15/62 (2006.01); C12N 15/66 (2006.01); C12N 15/70 (2006.01)

CPC A61K 39/099 (2013.01) [A61K 39/0018 (2013.01); A61K 39/08 (2013.01); A61K 39/39525 (2013.01); A61P 31/04 (2018.01); C07K 14/19 (2013.01); C07K 14/235 (2013.01); C07K 14/33 (2013.01); C07K 14/34 (2013.01); C12N 15/62 (2013.01); C12N 15/66 (2013.01); C12N 15/70 (2013.01); A61K 2039/542 (2013.01); A61K 2039/6031 (2013.01); A61K 2039/622 (2013.01)]

6 Claims

1. A process for the preparation of a second-generation tetanus toxoid vaccine, comprising the steps of:

a) inducing an E. coli culture at OD₆₀₀=0.5 by adding 0.2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG);

b) growing the culture at 14-16° Celsius (C) for 14 to 20 hours;

c) suspending the culture in 25 mM phosphate buffer containing 200 mM sodium chloride;

d) adding 1% of (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) to the phosphate buffer and resuspending the culture in said phosphate buffer;

e) sonicating the culture for a period of 3 minutes (at 5 sec on/off pulse) at 4° C. on cold beads;

f) centrifuging the culture for 45 to 90 minutes;

g) collecting and purifying the supernatant using Nickel-Nitrilotriacetic acid (Ni-NTA) affinity column with an eluant; and

h) combining the eluant into a pool with contaminated bands and concentrating using centrifugal filters to isolate one or more detoxified domains of a tetanus neurotoxin (DrTeNT) heavy chains proteins.