Recombinant FcRn and Variants Thereof for Purification of Fc-Containing Fusion Proteins
The invention is directed to methods of purifying Fc-containing molecules using a soluble neonatal Fc receptor (sFcRn). Native FcRn binds Fc-containing proteins at or below about pH 6.5 and releases them at or above about pH 7 and provides a much milder approach for capturing and purifying Fc-containing proteins, in particular, therapeutic Fc-containing proteins. Other embodiments of the invention provide modifications to alter the pH for binding and elution to the sFcRn, to modulate Fc-containing protein binding affinity, to affect sFcRn linkage to a support surface, or to improve the stability of sFcRn to conditions utilized in the methods of the invention.
1 . A method of purifying an Fc-containing protein from a sample, the method comprising:
(a) contacting a sample containing said Fc-containing protein with soluble neonatal Fc receptor (sFcRn) bound to a support surface under conditions that allow said Fc-containing protein to bind sFcRn;
(b) separating said Fc-containing protein from the sample;
(c) dissociating said Fc-containing protein from the sFcRn; and
(d) collecting said Fc-containing protein.
2 . The method of claim 1 , wherein said Fc-containing protein is an enzymatically-active protein:Fc fusion protein.
3 . The method of claim 1 , wherein said Fc-containing protein is a Factor VIII:Fc fusion protein.
4 . A method of increasing the biological activity of an Fc fusion protein in a sample, the method comprising:
(a) contacting a sample containing said fusion protein with soluble neonatal Fc receptor (sFcRn) bound to a support surface under conditions that allow said Fc fusion protein to bind sFcRn;
(b) separating said Fc fusion protein from the sample;
(c) dissociating said Fc fusion protein from the sFcRn; and
(d) collecting said Fc fusion protein.
5 . The method according to claim 4 , wherein said Fc fusion protein is selected from the group consisting of an enzymatically active protein:Fc fusion protein and a Factor VIII:Fc fusion protein.
6 . The method of claim 1 , wherein the percent purity, by weight, of said Fc-containing protein collected, compared to the sum weight of associated contaminants, is a percent purity selected from the group consisting of:
(a) at least about 20% pure;
(b) at least about 30% pure;
(c) at least about 40% pure;
(d) at least about 50% pure;
(e) at least about 60% pure;
(f) at least about 70% pure;
(g) at least about 80% pure;
(h) at least about 85% pure;
(i) at least about 90% pure;
(j) at least about 95% pure,
(k) at least about 98% pure; and
(l) at least about 99% pure.
7 . The method of claim 1 , wherein the percent recovery, by weight, of said Fc-containing protein collected, compared to the sum weight of Fc-containing protein in said sample, is a percent recovery selected from the group consisting of:
(a) at least about 20% recovery;
(b) at least about 30% recovery;
(c) at least about 40% recovery;
(d) at least about 50% recovery;
(e) at least about 60% recovery;
(f) at least about 70% recovery;
(g) at least about 80% recovery;
(h) at least about 85% recovery;
(i) at least about 90% recovery;
(j) at least about 95% recovery,
(k) at least about 98% recovery; and
(l) at least about 99% recovery.
8 . The method of claim 4 , wherein the fold increase in the biological activity of said Fc fusion protein collected, compared to the biological activity of Fc fusion protein in said sample, is a fold increase selected from the group consisting of:
(a) at least about 2-fold;
(b) at least about 5-fold;
(c) at least about 10-fold;
(d) at least about 20-fold;
(e) at least about 50-fold;
(f) at least about 100-fold;
(g) at least about 200-fold;
(h) at least about 500-fold;
(i) at least about 1000-fold;
(j) at least about 2000-fold;
(k) at least about 5000-fold; and
(l) at least about 10000-fold.
9 . The method of claim 1 , wherein one or both heavy chain (α-chain) or light chain (β2m) domains of said sFcRn are modified to increase or decrease the affinity of Fc-containing protein binding.
10 . The method of claim 9 , wherein said modification comprises a change selected from the group consisting of:
(a) one or more amino acid substitutions;
(b) one or more amino acid deletions;
(c) one or more amino acid insertions;
(d) a post-translational chemical modification; and
(e) a modification which increases the binding affinity of sFcRn to Fc-containing proteins.
11 . (canceled)
12 . (canceled)
13 . The method of claim 1 , wherein one or both heavy chain (α-chain) or light chain (β2m) domains of said FcRn are modified to modulate linkage to the support surface wherein said modification increases efficiency of Fc-containing protein purification.
14 . (canceled)
15 . The method according to claim 13 wherein the modification increases sFcRn linkage to the surface.
16 . (canceled)
17 . The method of claim 1 , wherein one or both heavy chain (α-chain) or light chain (β2m) domains of said FcRn are modified to increase the stability of sFcRn binding activity.
18 . The method of Claim 1 , wherein the sFcRn α-chain and β2m are chemically, photo, or thermally cross-linked.
19 . The method of claim 1 , wherein the sFcRn α-chain and β2m are covalently linked by an amino acid linker.
20 . The method according to claim 19 , wherein the amino acid linker comprises (GGGGS)n, wherein n is between 1 and 5.
21 . The method of claim 1 , wherein the sFcRn is a human sFcRn.
22 . The method of claim 1 , wherein the support surface comprises SEPHAROSE™, agarose, silica, collodion charcoal, sand, polystyrene, or methacrylate.
23 . The method of claim 1 , wherein the sFcRn is bound to a support surface through formation of a bond selected from the group consisting of:
(a) a covalent bond;
(b) an amide bond;
(c) an amine bond;
(d) a carbon bond;
(e) a disulfide bond;
(f) an ester bond;
(g) an ether bond;
(h) a thioether bond;
(i) a urea bond; and
(j) a thiourea bond.
24 . (canceled)
25 . The method of claim 1 , wherein the sFcRn is bound to a support surface through a chemically, photo, or thermally activated cross-link.
26 . The method of claim 1 , wherein said method comprises contacting the sample containing said Fc-containing protein with sFcRn at or below a pH selected from the group consisting of:
(a) about 7.0;
(b) about 6.9;
(c) about 6.8;
(d) about 6.7;
(e) about 6.6;
(f) about 6.5;
(g) about 6.4;
(h) about 6.3;
(i) about 6.2;
(j) about 6.1;
(k) about 6.0;
(l) about 5.9;
(m) about 5.8;
(n) about 5.7;
(o) about 5.6;
(p) about 5.5;
(q) about 5.0;
(r) about 4.5; and
(s) about 4.0.
27 . (canceled)
28 . The method of claim 1 , wherein said method comprises dissociating the Fc-containing protein from sFcRn at or above a pH selected from the group consisting of:
(a) about 7.0;
(b) about 7.1;
(c) about 7.2;
(d) about 7.3;
(e) about 7.4;
(f) about 7.5;
(g) about 7.6;
(h) about 7.7;
(i) about 7.8;
(j) about 7.9;
(k) about 8.0;
(l) about 8.1;
(m) about 8.2;
(n) about 8.3;
(o) about 8.4;
(p) about 8.5;
(q) about 9.0;
(r) about 9.5; and
(s) about 10.0.
29 . (canceled)
30 . The method of claim 1 , wherein said method comprises one or more washing steps prior to dissociating the Fc-containing protein from the sFcRn.