# US 11,998,587 B2 · Lipoprotein complexes and manufacturing and uses thereof

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- Number: US 11,998,587 B2
- Document ID: US11998587B2
- Status: Granted
- Kind: B2
- Application number: 18/365,328
- Filed: 2023-08-04
- Published: 2023-11-23
- Granted: 2024-06-04
- Earliest claimed: 2011-02-07
- Assignees: Abionyx Pharma SA
- Applicants: Abionyx Pharma SA
- Inventors: Jean-Louis Dasseux; Rose Ackermann; Daniela Carmen Oniciu
- Web page: https://patentgrep.com/patent/US11998587B2

## CPC

- A61K 38/1709: {from mammals}
- A61K 38/17: from animals; from humans {(enzyme inhibitors A61K38/005)}
- A61K 38/18: Growth factors; Growth regulators
- A61K 45/00: Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K 47/50: the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/06: Antihyperlipidemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- C07K 1/04: on carriers {(C07K1/003, C07K1/006 take precedence)}
- C07K 1/042: {characterised by the nature of the carrier}
- C07K 14/775: Apolipopeptides
- Y10T 428/2982: Particulate matter [e.g., sphere, flake, etc.]
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the mammalian host cell is a Chinese hamster ovary (“CHO”) cell.

### Claim 3 (depends on 2)

The method of claim 2, wherein the mammalian host cell is a CHO cell adapted for growth in serum-free medium.

### Claim 4 (depends on 1)

The method of claim 1, wherein yield of ApoA-I protein is at least 1 g/L in culture.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mammalian host cell has an ApoA-I gene copy index of at least two.

### Claim 6 (depends on 5)

The method of claim 5, wherein the mammalian host cell has an ApoA-I gene copy index of 5-10-copies.

### Claim 7 (depends on 1)

The method of claim 1, wherein the promoter is a simian cytomegalovirus immediate early promoter.

### Claim 8 (depends on 1)

The method of claim 1, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO:2.

### Claim 9 (depends on 1)

The method of claim 1, wherein the propeptide sequence comprises the amino acid sequence of SEQ ID NO:3.

### Claim 10 (depends on 1)

The method of claim 1, wherein the culture medium is serum-free.

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 11)

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).

### Claim 13 (depends on 11)

The method of claim 11, further comprising filtering the ApoA-I containing solution to remove cells and cell debris before step (A).

### Claim 14 (depends on 11)

The method of claim 11, further comprising diafiltering the second ApoA-I containing reverse phase eluate after step (F).

### Claim 15 (depends on 1)

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.

### Claim 16 (depends on 1)

The method of claim 1, wherein the formulation contains no more than 200 ppm of non-aqueous solvent.

### Claim 17 (depends on 1)

The method of claim 1, wherein the complexing comprises thermal cycling the ApoA-I protein, the sphingomyelin, and the DPPG or salt thereof.

### Claim 18 (depends on 17)

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.

### Claim 19 (depends on 17)

The method of claim 17, wherein the thermal cycling is performed until at least 90% of the ApoA-I protein is in lipoprotein complexes.