Lipoprotein complexes and manufacturing and uses thereof
The present disclosure relates to lipoprotein complexes and lipoprotein populations and their use in the treatment and/or prevention of dyslipidemic diseases, disorders, and/or conditions. The disclosure further relates to recombinant expression of apolipoproteins, purification of apolipoproteins, and production of lipoprotein complexes using thermal cycling-based methods.
1. An Apolipoprotein A-I (“ApoA-I”) formulation comprising: (a) an ApoA-I protein comprising the amino acid sequence of amino acids 25 to 267 of SEQ ID NO: 1, (b) sphingomyelin, and (c) 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (“DPPG”) or a salt thereof, wherein:
(a) the ApoA-I, the sphingomyelin, and the DPPG or the salt thereof are in the form of lipoprotein complexes;
(b) the sphingomyelin and the DPPG or the salt thereof are present in a 97:3 weight:weight ratio; and
(c) the ApoA-I, the sphingomyelin and the DPPG or the salt thereof are present in a 1:2.7 ratio of weight ApoA-I:weight sphingomyelin and DPPG or salt thereof.
2. The formulation of claim 1 , wherein the ApoA-I is recombinant.
3. The formulation of claim 2 , wherein the ApoA-I is produced by a mammalian host cell engineered to express an ApoA-I protein.
4. The formulation of claim 3 , wherein the ApoA-I is produced by a Chinese hamster ovary (CHO) cell.
5. The formulation of claim 4 , wherein the CHO cell is a CHO-S cell.
6. The formulation of claim 3 , which comprises no more than 10 picograms of host cell DNA per milligram of the ApoA-I protein.
7. The formulation of claim 3 , which comprises no more than 100 nanograms of host cell protein per milligram of the ApoA-I protein.
8. The formulation of claim 1 , which comprises lipoprotein complexes 4 nm to 15 nm in size as measured by dynamic light scattering (“DLS”).
9. The formulation of claim 1 , wherein at least 95% of the ApoA-I in the formulation is in mature form.
10. The formulation of claim 1 , wherein no more than 3% of methionine and tryptophan residues in the ApoA-I in the formulation are oxidized.
11. The formulation of claim 1 , wherein the formulation contains no more than 0.1 EU of endotoxin per milligram of the ApoA-I.
12. The formulation of claim 1 , wherein no more than 15% of the amino acids in the ApoA-I are deamidated.
13. The formulation of claim 1 , wherein the formulation contains no more than 200 ppm of non-aqueous solvent.
14. The formulation of claim 13 , wherein the formulation contains no more than 50 ppm of acetonitrile.
15. The formulation of claim 1 , wherein the sphingomyelin is egg sphingomyelin.
16. The formulation of claim 1 , wherein the sphingomyelin is palmitoylsphingomyelin.
17. The formulation of claim 1 , further comprising 4% (w/w) sucrose and 2% (w/w) mannitol.