Protein-based purification matrices and methods of using the same
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.
1 . A method of purifying a viral particle comprising:
(i) contacting the viral particle with a fusion protein comprising (a) at least one low-density lipoprotein receptor (LDLR) or an extracellular domain thereof; and (b) at least one polypeptide with phase behavior,
wherein the fusion protein binds to the viral particle and forms a complex;
(ii) contacting the complex with a first environmental factor;
(iii) separating the complex from at least one contaminant; and
(iv) separating the viral particle from the fusion protein by contacting the complex with a second environmental factor.
2 . The method of claim 1 , wherein the LDLR or an extracellular domain thereof comprises the sequence of any one of SEQ ID NO: 73-76 or 168-169, or a sequence with at least 90% identity thereto.
3 . The method of claim 1 , wherein the LDLR or an extracellular domain thereof comprises the sequence of any one of SEQ ID NO: 73-76 or 168-169, or a sequence with at least 95% identity thereto.
4 . The method of claim 1 , wherein the LDLR or an extracellular domain thereof comprises the sequence of any one of SEQ ID NO: 73-76 or 168-169, or a sequence with at least 97% identity thereto.
5 . The method of claim 1 , wherein the LDLR or an extracellular domain thereof comprises the sequence of any one of SEQ ID NO: 73-76 or 168-169, or a sequence with at least 99% identity thereto.
6 . The method of claim 1 , wherein the LDLR or an extracellular domain thereof comprises the amino acid sequence of any one of:
(a) amino acids 2-860 of SEQ ID NO: 73;
(b) amino acids 2-768 of SEQ ID NO: 74;
(c) amino acids 2-40 of SEQ ID NO: 75;
(d) amino acids 2-38 of SEQ ID NO: 76;
(e) SEQ ID NO: 168; or
(f) SEQ ID NO: 169.
7 . The method of claim 1 , wherein the LDLR or an extracellular domain thereof comprises the amino acid sequence of SEQ ID NO: 169.
8 . The method of claim 7 , wherein:
the polypeptide with phase behavior comprises the amino acid sequence of (GVGVP)m, wherein m is 80.
9 . The method of claim 1 , wherein the polypeptide with phase behavior is an elastin-like polypeptide (ELP).
10 . The method of claim 9 , wherein the ELP is a polymer containing a pentapeptide repeat having the sequence (Val-Pro-Gly-Xaa-Gly)n (SEQ ID NO: 10);
wherein Xaa can be any amino acid except proline; and
wherein n is an integer from 1 to 360, inclusive of endpoints.
11 . The method of claim 1 , wherein the polypeptide with phase behavior comprises an amino acid sequence selected from:
(a) (GRGDSPY)n (SEQ ID NO: 1);
(b) (GRGDSPH)n (SEQ ID NO: 2);
(c) (GRGDSPV)n (SEQ ID NO: 3);
(d) (GRGDSPYG)n (SEQ ID NO: 4);
(e) (RPLGYDS)n (SEQ ID NO: 5);
(f) (RPAGYDS)n (SEQ ID NO: 6);
(g) (GRGDSYP)n (SEQ ID NO: 7);
(h) (GRGDSPYQ)n (SEQ ID NO: 8);
(i) (GRGNSPYG)n (SEQ ID NO: 9);
(j) (GVGVP)n (SEQ ID NO: 11);
(k) (GVGVPGLGVPGVGVPGLGVPGVGVP)m (SEQ ID NO: 12);
(l) (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 13);
(m) (GVGVPGWGVPGVGVPGWGVPGVGVP)m (SEQ ID NO: 14);
(n) (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGEGVPG FGVPGVGVP)m (SEQ ID NO: 15);
(o) (GVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGKGVPG FGVPGVGVP)m (SEQ ID NO: 16); and
(p) (GAGVPGVGVPGAGVPGVGVPGAGVP)m (SEQ ID NO: 17);
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.
12 . The method of claim 1 , wherein the polypeptide with phase behavior comprises an amino acid sequence selected from:
(a) (GVGVP)m (SEQ ID NO: 52);
(b) (ZZPXXXXGZ)m (SEQ ID NO: 57);
(c) (ZZPXGZ)m (SEQ ID NO: 58);
(d) (ZZPXXGZ)m (SEQ ID NO: 59); or
(e) (ZZPXXXGZ)m (SEQ ID NO: 60);
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.
13 . The method of claim 1 , wherein the polypeptide with phase behavior comprises an amino acid sequence selected from:
(a) (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 53); and
(b) (GVGVPGVGVPGLGVPGVGVPGVGVP)m (SEQ ID NO: 55);
wherein m is an integer between 2 and 32, inclusive of endpoints.
14 . The method of claim 1 , wherein the polypeptide with phase behavior comprises an amino acid sequence selected from any one of:
(a) (GVGVPGVGVPGAGVPGVGVPGVGVP)m (SEQ ID NO: 193), 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; and
(c) (GXGVP)m (SEQ ID NO: 56), wherein m is an integer between 10 and 160, inclusive of endpoints, wherein each X is independently selected from the group consisting of glycine, alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, lysine, arginine, aspartic acid, glutamic acid, and serine.
15 . The method of claim 1 , wherein the polypeptide with phase behavior comprises the amino acid sequence of (GVGVP)m, wherein m is 80.
16 . The method of claim 1 , wherein contacting the viral particle with the fusion protein comprises adding the fusion protein to tissue culture media of cells producing the viral particle.
17 . The method of claim 1 , wherein the first environmental factor is selected from the group consisting of: a change in temperature, a change in pH, a change in salt concentration, a change in the concentration of the fusion protein, a change in the concentration of the viral particle, a change in pressure, the addition of a surfactant, the addition of a cofactor, the addition of a vitamin, the addition of a molecular crowding agent, the addition of a reducing agent, the addition of an oxidizing agent, the addition of an enzyme, the addition of a denaturing agent, the application of electromagnetic waves, the application of acoustic waves, or a combination thereof.
18 . The method of claim 1 , wherein the second environmental factor is selected from the group consisting of: a change in temperature, a change in pH, a change in salt concentration, a change in the concentration of the fusion protein, a change in the concentration of the viral particle, a change in pressure, the addition of a surfactant, the addition of a cofactor, the addition of a vitamin, the addition of a molecular crowding agent, the addition of a reducing agent, the addition of an oxidizing agent, the addition of an enzyme, the addition of a denaturing agent, the application of electromagnetic waves, the application of acoustic waves, or a combination thereof.
19 . The method of claim 1 , comprising separating the complex from at least one contaminant by a method selected from the group consisting of: 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.
20 . The method of claim 1 , wherein the first environmental factor is the addition of salt.
21 . The method of claim 1 , wherein a purification yield of the viral particle is at least 70%.