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 of producing a formulation comprising Apolipoprotein A-I (“ApoA-I”) protein complexed with lipids, comprising:
(a) culturing in a culture medium a mammalian host cell engineered to express an ApoA-I protein comprising:
(i) a signal sequence;
(ii) a propeptide sequence; and
(iii) an amino acid sequence comprising positions 25 to 267 of SEQ ID NO:1,
wherein the mammalian host cell comprises a nucleic acid encoding the ApoA-I protein operably linked to a constitutive promoter;
(b) recovering ApoA-I protein from the culture medium;
(c) purifying the recovered ApoA-I protein; and
(d) complexing the purified ApoA-I with sphingomyelin and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (“DPPG”) or a salt thereof to yield a composition comprising a population of lipoprotein complexes, wherein the sphingomyelin and the DPPG or the salt thereof are present in the composition in a 97:3 weight:weight ratio and the ApoA-I, the sphingomyelin and the DPPG or the salt thereof are present in the composition in a 1:2.7 ratio of weight ApoA-I:weight sphingomyelin and DPPG or salt thereof.
2. The method of claim 1 , wherein the mammalian host cell is a Chinese hamster ovary (“CHO”) cell.
3. The method of claim 2 , wherein the mammalian host cell is a CHO cell adapted for growth in serum-free medium.
4. The method of claim 1 , wherein yield of ApoA-I protein is at least 1 g/L in culture.
5. The method of claim 1 , wherein the mammalian host cell has an ApoA-I gene copy index of at least two.
6. The method of claim 5 , wherein the mammalian host cell has an ApoA-I gene copy index of 5-10-copies.
7. The method of claim 1 , wherein the promoter is a simian cytomegalovirus immediate early promoter.
8. The method of claim 1 , wherein the signal sequence comprises the amino acid sequence of SEQ ID NO:2.
9. The method of claim 1 , wherein the propeptide sequence comprises the amino acid sequence of SEQ ID NO:3.
10. The method of claim 1 , wherein the culture medium is serum-free.
11. The method of claim 1 , wherein the purifying comprises:
(A) contacting an ApoA-I containing solution with an anion exchange matrix under conditions such that the ApoA-I does not bind to the matrix;
(B) filtering the ApoA-I containing solution obtained in step (a) (A) through a membrane having a pore size sufficient to remove viruses or viral particles;
(C) passing the filtrate obtained in step (b) (B) through a first reverse phase chromatography column under conditions such that the ApoA-I binds to the matrix;
(D) eluting from the first reverse phase chromatography matrix a first ApoA-I containing reverse phase eluate using a gradient of increasing concentrations of an organic solvent;
(E) passing the first ApoA-I reverse phase eluate from step (d) (D) through a second reverse phase chromatography column under conditions such that the ApoA-I binds to the matrix; and
(F) eluting from the second reverse phase chromatography matrix a second ApoA-I containing reverse phase eluate using a gradient of increasing concentrations of an organic solvent.
12. The method of claim 11 , further comprising, before step (A), adjusting the pH of the ApoA-I containing solution to a pH range where ApoA-I does not bind to the anion exchange matrix in step (A).
13. The method of claim 11 , further comprising filtering the ApoA-I containing solution to remove cells and cell debris before step (A).
14. The method of claim 11 , further comprising diafiltering the second ApoA-I containing reverse phase eluate after step (F).
15. The method of claim 1 , wherein the formulation comprises no more than 10 picograms of mammalian host cell DNA per milligram of the ApoA-I protein.
16. The method of claim 1 , wherein the formulation contains no more than 200 ppm of non-aqueous solvent.
17. The method of claim 1 , wherein the complexing comprises thermal cycling the ApoA-I protein, the sphingomyelin, and the DPPG or salt thereof.
18. The method of claim 17 , wherein the thermal cycling is performed until lipoprotein complexes ranging in size from 4 nm to 15 nm, as measured by dynamic light scattering (DLS), are obtained.
19. The method of claim 17 , wherein the thermal cycling is performed until at least 90% of the ApoA-I protein is in lipoprotein complexes.