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. A population of lipoprotein complexes, each comprising
(a) a lipid fraction consisting of a sphingomyelin and, optionally, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](DPPG); and
(b) an apolipoprotein fraction consisting essentially of an Apolipoprotein A-I (“ApoA-I”);
wherein
(i) at least 95% by weight of the apolipoprotein in the population is in complexed form;
(ii) at least 95% by weight of the lipid in the population is in complexed form;
(iii) at least 95% of the population is in a single peak in a gel permeation chromatogram;
(iv) the population has an apolipoprotein fraction-to-phospholipid fraction ratio of 1:2.7 by weight; and
(v) the weight ratio of the sphingomyelin to DPPG, when present, is 97:3.
2. The population of claim 1 , further characterized by at least one of the following characteristics:
(a) at least 95% of the lipoprotein complexes are in the form of particles of 4 nm to 15 nm in size as measured by dynamic light scattering (“DLS”);
(b) at least 95% by weight of ApoA-I in said population is in mature form;
(c) no more than 5% by weight of ApoA-I in said population is in immature form; and
(d) no more than 10% by weight of ApoA-I in the population is in truncated form.
3. The population of claim 1 , further characterized by at least one of the following characteristics:
(a) no more than 5% of each of methionine 112 and methionine 148 in said ApoA-I in said population is oxidized;
(b) no more than 4% of the amino acids of the ApoA-I in said population is deamidated;
(c) the population contains no more than 1 EU of endotoxin per milligram of ApoA-I;
(d) the population contains no more than 25 picograms host cell DNA per milligram of ApoA-I; and
(e) the population contains no more than 100 nanograms host cell protein per milligram of ApoA-I.
4. The population of claim 1 wherein said ApoA-I is a human ApoA-I protein.
5. The population of claim 1 wherein said ApoA-I is a recombinant ApoA-I.
6. The population of claim 1 , wherein said lipid fraction consists of 97 weight % sphingomyelin and 3 weight % DPPG.
7. The population of claim 6 in which the sphingomyelin is egg-sphingomyelin.
8. The population of claim 6 , wherein the lipid has a peroxide value of less than 5 meq 0/kg.
9. The population of claim 1 , wherein the amino acid sequence of said ApoA-I consists of amino acids 25 to 267 of SEQ ID NO: 1.
10. The population of claim 9 , wherein said lipid fraction consists of 97 weight % sphingomyelin and 3 weight % DPPG.
11. The population of claim 10 in which the sphingomyelin is egg-sphingomyelin.
12. The population of claim 1 , wherein said lipid fraction consists of sphingomyelin.
13. The population of claim 9 , wherein said lipid fraction consists of sphingomyelin.
14. A pharmaceutical composition comprising the population of claim 1 and one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
15. The pharmaceutical composition of claim 14 , comprising lipoprotein complexes having 2 ApoA-I molecules per lipoprotein complex.
16. The pharmaceutical composition of claim 15 , comprising a lipoprotein complexes having 3 or 4 ApoA-I molecules or ApoA-I equivalents per lipoprotein complex.
17. A pharmaceutical composition which consists essentially of the population of claim 1 and one or more pharmaceutically acceptable carriers, diluents and/or excipients.
18. A unit dosage form comprising a therapeutically effective amount of a pharmaceutical composition of claim 17 .
19. A pharmaceutical composition comprising the population of claim 9 and one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
20. A pharmaceutical composition comprising the population of claim 10 and one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
21. A pharmaceutical composition comprising the population of claim 11 and one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
22. A pharmaceutical composition comprising the population of claim 12 and one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
23. A pharmaceutical composition comprising the population of claim 13 and one or more pharmaceutically acceptable carriers, diluents, and/or excipients.
24. A method for treating a dyslipidemic disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a population of lipoprotein complexes according to claim 1 .
25. A method for treating a dyslipidemic disorder, comprising:
(a) administering to a subject an initial dose of 1 mg/kg to 12 mg/kg of a population of lipoprotein complexes according to claim 1 ;
(b) determining whether the subject's triglyceride, VLDL-cholesterol and/or VLDL-triglyceride is elevated to more than two fold 4, 8, 12, 24, 48, 72, 168, 336 or 504 hours after said administration; and
(c) if the subject's triglyceride, VLDL-cholesterol and/or VLDL-triglyceride is elevated to more than two fold of the pre-dosing levels, repeating said administration but at a lower dose, and if the subject's triglyceride, VLDL-cholesterol and/or VLDL-triglyceride is not elevated to more than two fold of the pre-dosing levels, then repeating said administration at an equivalent or greater dose.