US2023099707A1PendingUtilityA1
Protein-based purification matrices and methods of using the same
Est. expiryFeb 19, 2040(~13.6 yrs left)· nominal 20-yr term from priority
C07K 19/00C07K 14/78G01N 33/566G01N 33/56983C07K 14/16B01D 15/3809C07K 2319/00C07K 1/22C12N 7/02C07K 14/15C07K 1/34C12N 7/00C07K 14/155C12N 2740/15051C12N 2740/15011G01N 33/53C07K 14/705C12N 2750/14151C12N 2710/10351
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Claims
Abstract
Provided herein are protein-based purification matrices and methods of use thereof to purify biologics and/or to remove contaminants from a composition. Methods of bringing two or more biologics in close proximity are also provided. The disclosed compositions and methods allow for faster, more efficient purification of a biologic compared to traditional affinity chromatography.
Claims
exact text as granted — not AI-modified1 . A method of purifying a biologic comprising contacting the biologic with a protein-based purification matrix;
wherein the biologic binds to the purification matrix to form a complex; wherein the size of the complex is increased by a first environmental factor; wherein the complex is separated from at least one contaminant on the basis of size; and wherein the biologic is separated from the purification matrix by a second environmental factor.
2 . The method of claim 1 , wherein the purification matrix comprises (i) a capture domain which binds to the biologic, and (ii) a polypeptide with phase behavior, wherein the capture domain is coupled to the polypeptide with phase behavior.
3 . The method of claim 2 , wherein the capture domain is coupled to the polypeptide with phase behavior via a linker.
4 . The method of claim 3 , wherein the linker is a peptide linker.
5 . The method of claim 4 , wherein the peptide linker comprises a protease cleavage site.
6 . The method of claim 3 , wherein the linker is a chemical linker.
7 . The method of claim 1 , wherein the purification matrix comprises a fusion protein comprising (i) a capture domain which binds to the biologic and (ii) a polypeptide with phase behavior.
8 . The method of any one of claims 2 - 7 , wherein the polypeptide with phase behavior is a resilin-like polypeptide.
9 . The method of any one of claims 2 - 7 , wherein the polypeptide with phase behavior is an elastin-like polypeptide.
10 . The method of any one of 2-7 or 9, wherein the polypeptide with phase behavior is a polymer containing a pentapeptide repeat having the sequence (Val-Pro-Gly-Xaa-Gly) n (SEQ ID NO: 10), or a randomized, scrambled analog thereof; wherein Xaa can be any amino acid except proline.
11 . The method of claim 10 , wherein n is an integer from 1 to 360, inclusive of endpoints.
12 . The method of any one of claim 2 - 7 or 9 , wherein the polypeptide with phase behavior comprises an amino acid sequence selected from:
a.
(SEQ ID NO: 1)
(GRGDSPY) n
b.
(SEQ ID NO: 2)
(GRGDSPH) n
c.
(SEQ ID NO: 3)
(GRGDSPV) n
d.
(SEQ ID NO: 4)
(GRGDSPYG) n
e.
(SEQ ID NO: 5)
(RPLGYDS) n
f.
(SEQ ID NO: 6)
(RPAGYDS) n
g.
(SEQ ID NO: 7)
(GRGDSYP) n
h.
(SEQ ID NO: 8)
(GRGDSPYQ) n
i.
(SEQ ID NO: 9)
(GRGNSPYG) n
j.
(SEQ ID NO: 11)
(GVGVP) n ;
k.
(SEQ ID NO: 12)
(GVGVPGLGVPGVGVPGLGVPGVGVP) m ;
l.
(SEQ ID NO: 13)
(GVGVPGVGVPGAGVPGVGVPGVGVP) m ;
m.
(SEQ ID NO: 14)
(GVGVPGWGVPGVGVPGWGVPGVGVP) m ;
n.
(SEQ ID NO: 15)
(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPGFGVPGVGVP) m ;
o.
(SEQ ID NO: 16)
(GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPGFGVPGVGVP) m ;
and
p.
(SEQ ID NO: 17)
(GAGVPGVGVPGAGVPGVGVPGAGVP) m ;
or a randomized, scrambled analog thereof;
wherein:
n is an integer in the range of 20-360, inclusive of endpoints; and
m is an integer in the range of 4-25, inclusive of endpoints.
13 . The method of any one of claim 2 - 7 or 9 , wherein the polypeptide with phase behavior comprises an amino acid sequence selected from:
(a)
(SEQ ID NO: 52)
(GVGVP) m ;
(b)
(SEQ ID NO: 57)
(ZZPXXXXGZ) m ;
(c)
(SEQ ID NO: 58)
(ZZPXGZ) m ;
(d)
(SEQ ID NO: 59)
(ZZPXXGZ) m ;
or
(e)
(SEQ ID NO: 60)
(ZZPXXXGZ) m
wherein m is an integer between 10 and 160, inclusive of endpoints, wherein X if present is any amino acid except proline or glycine, and wherein Z if present is any amino acid.
14 . The method of any one of claim 2 - 7 or 9 , wherein the polypeptide with phase behavior comprises an amino acid sequence selected from:
(a)
(SEQ ID NO: 53)
(GVGVPGVGVPGAGVPGVGVPGVGVP) m ;
or
(b)
(SEQ ID NO: 55)
(GVGVPGVGVPGLGVPGVGVPGVGVP) m ;
wherein m is an integer between 2 and 32, inclusive of endpoints.
15 . The method of any one of claim 2 - 7 or 9 , wherein the polypeptide with phase behavior comprises an amino acid sequence selected from:
(a) (GVGVPGVGVPGAGVPGVGVPGVGVP) m (SEQ ID NO: 53), wherein m is 8 or 16;
(b) (GVGVPGAGVP) m (SEQ ID NO: 54), wherein m is an integer between 5 and 80, inclusive of endpoints; or
(c) (GXGVP) m (SEQ ID NO: 56), wherein m is an integer between 10 and 160, inclusive of endpoints, and wherein X for each repeat is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine.
16 . The method of any one of claims 2 - 15 , wherein the capture domain comprises the sequence of any one of SEQ ID NOS: 24-49, 62-72, 77-148, 167, 170, and 171, or a sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative thereto.
17 . The method of any one of claims 2 - 15 , wherein the binding of the biologic to the purification matrix is reversible.
18 . The method of any one of claims 2 - 15 , wherein the binding of the biologic to the purification matrix is non-covalent.
19 . The method of any one of claims 2 - 15 , wherein the binding of the biologic to the purification matrix is covalent.
20 . The method of any one of claims 1 - 19 , wherein the biologic is a lipid, a lipopolysaccharide, a cell, a protein, a nucleic acid, a carbohydrate, or a viral particle.
21 . The method of claim 20 , wherein the nucleic acid is a DNA or an RNA.
22 . The method of claim 17 , wherein the viral particle is an adenovirus particle, an adeno-associated virus (AAV) particle, a lentivirus particle, a retrovirus particle, a poxvirus particle, a measles virus particle, or a herpesvirus particle.
23 . The method of any one of claims 1 - 28 , wherein the biologic has a diameter between 1 nm and 100 μm, inclusive of the endpoints.
24 . The method of any one of claims 1 - 23 , wherein the method is completed in about 0.5 to about 24 hours.
25 . The method of any one of claims 1 - 24 , wherein the separation of the complex from the at least one contaminant can be observed visually with an unaided eye.
26 . The method of any one of claims 1 - 25 , wherein the increase in the size of the complex is at least a 2-fold increase.
27 . The method of any one of claims 1 - 26 , wherein the first environmental factor comprises one or more of:
a. a change in one or more of temperature, pH, salt concentration, concentration of the purification matrix, concentration of the biologic, or pressure; b. the addition of one or more surfactants, cofactor, vitamin, molecular crowding agents, reducing agents, oxidizing agents, enzymes, or denaturing agents; or c. the application of electromagnetic or acoustic waves.
28 . The method of any one of claims 1 - 26 , wherein the second environmental factor comprises one or more of:
a. a change in one or more of temperature, pH, salt concentration, concentration of the purification matrix, concentration of the biologic, or pressure; b. the addition of one or more surfactants, molecular crowding agents, reducing agents, enzymes, denaturing agents, cofactor, vitamin, or oxidizing agents; or c. the application of electromagnetic or acoustic waves.
29 . The method of any one of claims 1 - 28 , wherein the separation on the basis of size is performed using tangential flow filtration, membrane chromatography, analytical ultracentrifugation, high performance liquid chromatography, membrane chromatography, normal flow filtration, acoustic wave separation, centrifugation, counterflow centrifugation, and fast protein liquid chromatography.
30 . The method of any one of claims 1 - 29 , wherein the at least one contaminant is selected from a solvent, an endotoxin, a protein, a peptide, a nucleic acid, and a carbohydrate.
31 . The method of any one of claims 1 - 30 , wherein the purification yield of the biologic is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
32 . The method of any one of claims 1 - 31 , wherein the biologic is purified to at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% purity.
33 . The method of any one of claims 1 - 32 , wherein the purification matrix has an amino acid sequence of any one of SEQ ID NOS: 173 and 176-178, or a sequence having at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative thereto.
34 . A method of increasing yield of a biologic during production thereof, the method comprising culturing biologic-producing cells in the presence of a purification matrix.
35 . A method of stabilizing a biologic during production thereof, the method comprising culturing biologic-producing cells in the presence of a purification matrix.
36 . A method of stabilizing a biologic during purification thereof, the method comprising contacting the biologic with a purification matrix during purification thereof.
37 . A method of stabilizing a biologic during storage thereof, the method comprising storing the biologic in the presence of a purification matrix.
38 . A method of increasing the shelf-life of a biologic, the method comprising storing the biologic in the presence of a purification matrix.
39 . The method of any one of claims 34 - 38 , wherein the biologic reversibly binds to the purification matrix to form a complex.
40 . The method of any one of claims 34 - 38 , wherein the purification matrix is present at a concentration of at least about 10 μM.
41 . A method of bringing a first biologic into proximity with a second biologic, the method comprising contacting the first biologic with a first protein-based purification matrix and contacting the second biologic with a second protein-based purification matrix;
wherein the first biologic binds to the first purification matrix to form a first complex; wherein the second biologic binds to the second purification matrix to form a second complex; and
wherein an environmental factor brings the first complex and the second complex into proximity with one another.
42 . A method of separating a first biologic from a second biologic, the method comprising contacting the first biologic with a first protein-based purification matrix and contacting the second biologic with a second protein-based purification matrix; wherein the first biologic binds to the first purification matrix to form a first complex; wherein the second biologic binds to the second purification matrix to form a second complex; and
separating the first biologic from the second biologic by applying an environmental factor.
43 . A method of purifying a biologic, the method comprising contacting the biologic with a protein-based purification matrix;
wherein the biologic binds to the matrix to form a complex; wherein the size of the complex is increased; wherein the complex is separated from at least one contaminant on the basis of size; and wherein the biologic is separated from the matrix by an environmental factor.
44 . A method of removing a contaminant from a composition comprising a biologic, the method comprising contacting the contaminant with a protein-based purification matrix;
wherein the contaminant binds to the matrix to form a complex; wherein the size of the complex is increased by a first environmental factor; wherein the complex is separated from the biologic on the basis of size; and wherein the contaminant is separated from the matrix by a second environmental factor.Cited by (0)
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