Downstream processing of an alkaline phosphatase

• US 10,570,380 B2
• Filed: 01/26/2015
• Issued: 02/25/2020
• Est. Priority Date: 01/24/2014
• Status: Active Grant

• Current Assignee: AM-Pharma B.V.
• Original Assignee: AM-Pharma B.V.
• Inventors: Jonk, Luigi Johannes Cornelius, Connor, Stephen Edward, Van Den Berg, Erik Jan, Van Elsas, Andrea, Shukla, Abhinav Alok, Horne, Heather Bethea, Cook, Susan, Kelly, Timothy Martin, Dowling, Victoria Anne, Ramaroson, Mialy Fanjamalala
• Primary Examiner(s): Singh, Satyendra K
• Application Number:
  US15/113,699
• Publication Number:
  US 20170009217A1
• Time in Patent Office: 1,856 Days
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 47/10 Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
• CPC Class Codes: A61K 47/12 Carboxylic acids; Salts or anhydrides thereof
• CPC Class Codes: A61K 47/22 Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
• CPC Class Codes: A61K 47/26 Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 39/00 General protective or antinoxious agents
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 301/03001 Alkaline phosphatase (3.1.3.1)

Abstract

The invention relates to the field of downstream processing (DSP) of an alkaline phosphatase (AP). More specifically, it relates to a method for reducing host cell protein content in a composition comprising AP. The invention further relates to a composition comprising an AP and a reduced content of a host cell protein.

Claims

1. A method for isolating an alkaline phosphatase (AP) comprising:

(a) providing a solid phase comprising a ligand of formula (I);

2. A method according to claim 1, wherein the wash buffer comprises less than 1 mM NaCl.

3. A method according to claim 1, wherein the elution buffer comprises less than 1 mM NaCl.

4. A method according to claim 1, wherein the washing buffer comprises about 40 mM Arg.

5. A method according to claim 1, the method further comprising:

(i) providing a second solid phase comprising a second ligand of formula (II);

6. A method according to claim 5, wherein said second washing buffer comprises between 1-20% glycerol.

7. A method according to claim 5, wherein (i)-(ii) precedes (a)-(d).

8. A method according to claim 1, further comprising at least one further purification step selected from the group consisting of anion exchange chromatography, ultrafiltration/diafiltration, viral filtration, and hydrophobic interaction chromatography, and any combination thereof.

14. A method according to claim 1, wherein the HCP is a cathepsin-like protein and/or wherein said alkaline phosphatase is obtained from a cell-based expression system.

9. A method for producing a composition comprising an isolated alkaline phosphatase (AP) comprising an amino acid sequence that is 100% identical to SEQ ID NO:

1, the method comprising

dissolving or diluting said AP in a buffer, which results in a composition having a pH of between 6.5 and 7.5 and comprising between 200-300 mM sorbitol, and/or between 10-40% glycerol, and/or between 5-40 mM histidine, and/or between 10-40 mM citrate, or any combination thereof;

wherein there is no visible particle formation in said composition, using a stability test at 2-8°

C. for 2 months.

10. A method according to claim 9, wherein the composition comprises less than 100 ppm host cell protein (HCP).

16. A method according to claim 10, wherein the composition comprises AP obtained by a method comprising:

(a) providing a solid phase comprising a ligand of formula (I);

11. A composition comprising an isolated alkaline phosphatase obtained from a cell-based expression system, wherein the alkaline phosphatase comprises an amino acid sequence that is 100% identical to SEQ ID NO:

1, wherein the composition comprises less than 100 ppm of a host cell protein (HCP) and wherein the composition does not show visible particle formation during stability testing at 2-8°

C. for 2 months.

12. A composition according to claim 11, wherein the composition has a pH of between 6.5-7.5, and comprises between 10-40 mM citrate or between 5-40 mM histidine.

13. A composition according to claim 12, wherein the composition comprises between 200-300 mM sorbitol and/or between 10-40% glycerol.

15. A method for treating a disease or condition that can be improved by the administration of alkaline phosphatase, the method comprising administering to an individual in need thereof a composition according to claim 11.

17. A composition according to claim 11, wherein the HCP is a cathepsin-like protein.

18. The composition according to claim 11, wherein the alkaline phosphatase is recombinant.

19. A composition according to claim 12, wherein the pH is about 7.