Composition comprising a mixture of CD95-Fc isoforms
The present invention relates to a composition comprising a mixture of fusion protein isoforms, each fusion protein comprising an extracellular CD95 domain or a functional fragment thereof or an Fc domain or functional fragment thereof, formulations providing such composition in a stable form as well as a method for producing such a composition.
1. A composition comprising a mixture of fusion protein isoforms, said fusion protein comprising (i) at least an extracellular cluster of differentiation 95 (CD95) domain or a functional fragment thereof and (ii) at least an Fc domain or a functional fragment thereof, distributing within a pI range of 4.0-8.5, wherein the isoforms are different forms of the same fusion protein differing by a single amino acid substitution and/or by post-translational modification, and the mixture inhibits apoptosis.
2. The composition according to claim 1 , wherein the CD95 is a human CD95 and the Fc is a human Fc.
3. The composition according to claim 1 , wherein (i) is located at the N-terminus of the fusion protein.
4. A composition comprising a mixture of fusion protein isoforms, wherein the fusion protein is APG101 (the amino acid sequence of 26-400 of SEQ ID NO: 1), a polypeptide having at least 95% identity to APG101 and/or a functional fragment of APG101.
5. The composition according to claim 1 , wherein the pI range is 4.5-7.8.
6. The composition according to claim 5 , wherein the pI range is 5.0-7.5.
7. The composition according to claim 1 , wherein 0.0-5.0mol % of said isoforms are high molecular weight forms of dimers and/or aggregates.
8. The composition according to claim 1 , wherein the isoforms comprise sialic acids.
9. The composition according to claim 1 , further comprising N-terminally shortened isoforms that are shortened in the N-terminal signal sequence.
10. The composition according to claim 9 , wherein the N-terminally shortened isoforms are N-terminally truncated by 16, 20, or 25 amino acids.
11. The composition according to claim 1 , wherein the Fc domain or functional fragment thereof is N-linked glycosylated.
12. The composition according to claim 1 , wherein 80-99 mol % of said isoforms are N-terminally blocked and/or 1-20 mol % of said isoforms have a free N-terminus.
13. The composition according to claim 1 , comprising N-terminally blocked isoforms.
14. The composition according to claim 13 , wherein the N-terminally blocked isoforms are blocked by pyro-Glu modification.
15. A pharmaceutical formulation comprising the composition of claim 1 , and a pharmaceutical acceptable carrier.
16. The composition according to claim 1 , wherein the post-translational modification is addition of sialic acids, Fc-based glycosylation, and/or pyro-Glu-modification.