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 method for preparing lipoprotein complexes, comprising:
(a) cooling a starting suspension comprising a lipid component and a protein component from a temperature in a first temperature range to a temperature in a second temperature range, wherein
(i) the lipid component consists essentially of particles of lipids formed by homogenization;
(ii) the protein component consists essentially of Apolipoprotein A-I (ApoA-I) that has not been subject to homogenization and, optionally, a lipid in an amount that is 10% or less by weight of the total amount of lipid in the starting suspension;
(b) heating the cooled suspension of (a) from a temperature in said second temperature range to a temperature in said first temperature range;
(c) cooling said heated suspension of (b) from a temperature in said first temperature range to a temperature in said second temperature range; and
(d) repeating steps (b) and (c) until at least 90% of the protein component is incorporated into lipoprotein complexes, thereby forming lipoprotein complexes.
2. The method of claim 1 , wherein step (d) comprises repeating steps (b) and (c) at least three times.
3. The method of claim 1 , wherein step (d) comprises repeating steps (b) and (c) until at least 95% of the protein component is incorporated into lipoprotein complexes.
4. The method of claim 1 , wherein step (d) comprises repeating steps (b) and (c) until lipoprotein complexes of 4 nm to 15 nm in diameter, as measured by gel filtration chromatography, are obtained.
5. The method of claim 1 , wherein said second temperature range includes temperatures no less than 10 degrees below and no more than 5 degrees above the transition temperature of said lipid component.
6. The method of claim 1 , wherein said lipid component comprises neutral lipids.
7. The method of claim 6 , wherein the neutral lipids consist essentially of sphingomyelin.
8. The method of claim 6 , wherein the starting suspension comprises negatively charged lipids.
9. The method of claim 8 , wherein the negatively charged lipids consist essentially of 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (“DPPG”).
10. The method of claim 9 , wherein the neutral lipids consist essentially of sphingomyelin.
11. The method of claim 8 , wherein the negatively charged lipids are pre-complexed with the ApoA-I, and wherein the method further comprises the step of forming the protein component by a process comprising combining the ApoA-I and the negatively charged lipids.
12. The method of claim 1 , wherein the lipid:protein molar ratio in said starting suspension is from about 2:1 to about 200:1.
13. The method of claim 1 , further comprising the step of forming said starting suspension by a process comprising combining the lipid component and the protein component.
14. The method of claim 13 , wherein the lipid component and the protein component are each preheated at a temperature in said first temperature range.
15. The method of claim 13 , which further comprises, prior to the step of forming the starting suspension, a step of forming the lipid particles using high pressure homogenization.
16. The method of claim 1 , in which the lipid component consists essentially of lipid particles, said lipid particles being: at least 45 nm in diameter, as measured by gel permeation chromatography; and up to 500 nm in diameter, as measured by gel permeation chromatography.
17. The method of claim 1 , wherein the starting suspension contains negatively charged lipid, neutral lipid and lipid-binding peptides in a molar ratio ranging from 2-6 (negatively charged lipid) : 90- 120 (neutral lipid) : 1 (lipid-binding peptide, lipid binding protein or mixtures thereof).
18. The method of claim 1 , which further comprises the step of lyophilizing the resulting lipoprotein complexes.
19. The method of claim 1 , wherein the lipid component consists essentially of particles of lipids formed by high pressure homogenization.
20. The method of claim 1 , wherein the starting suspension does not contain detergent.
21. A method for making a pharmaceutical composition, comprising:
(a) preparing a population of lipoprotein complexes according to the method of claim 1 ; and
(b) combining said population of lipoprotein complexes with one or more pharmaceutically acceptable excipients.
22. The method of claim 1 , which does not comprise a size fractionation step after step (d).
23. A method for making a pharmaceutical composition, comprising:
(a) preparing a population of lipoprotein complexes according to the method of claim 22 ; and
(b) combining said population of lipoprotein complexes with one or more pharmaceutically acceptable excipients.
24. The method of claim 19 , wherein the high pressure homogenization comprises microfluidization.
25. The method of claim 21 , wherein the one or more pharmaceutically acceptable excipients comprises one or more isotonicity agents.
26. The method of claim 25 , wherein the one or more isotonicity agents comprise sucrose and/or mannitol.
27. The method of claim 23 , wherein the one or more pharmaceutically acceptable excipients comprises one or more isotonicity agents.
28. The method of claim 27 , wherein the one or more isotonicity agents comprise sucrose and/or mannitol.
29. The method of claim 1 , wherein the ApoA-I(:lipid weight ratio in said starting suspension ranges from 1:2.6 to 1.3.