# US 11,441,138 B2 · Method for selection of high M6P recombinant proteins

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- Number: US 11,441,138 B2
- Document ID: US11441138B2
- Status: Granted
- Kind: B2
- Application number: 16/252,519
- Filed: 2019-01-18
- Published: 2019-12-19
- Granted: 2022-09-13
- Earliest claimed: 2016-03-30
- Assignees: Amicus Therapeutics, Inc.
- Applicants: Amicus Therapeutics, Inc.
- Inventors: Hung V. Do; Russell Gotschall
- Web page: https://patentgrep.com/patent/US11441138B2

## CPC

- C12N 9/2408: {acting on alpha -1,4-glucosidic bonds}
- A61P 3/08: for glucose homeostasis (pancreatic hormones A61P5/48)
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12Y 302/0102: Alpha-glucosidase (3.2.1.20)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES
- 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)

## Abstract

Methods for the production, capturing and purification of recombinant human lysosomal proteins are described. Such recombinant human lysosomal proteins can have high content of mannose-6-phosphate residues. Also described are pharmaceutical compositions comprising such recombinant human lysosomal proteins, as well as methods of treatment and uses of such recombinant human lysosomal proteins.

## Claims

### Claim 1

A method for producing purified recombinant human acid alpha-glucosidase (rhGAA) from a filtered medium, the method comprising
i) in a bioreactor, culturing host cells that secrete the rhGAA;
ii) removing medium from the bioreactor;
iii) filtering the medium to provide a filtrate;
iv) loading the filtrate onto an anion exchange chromatography (AEX) column;
v) eluting the rhGAA from the AEX column;
vi) applying the rhGAA eluted from the AEX chromatography column onto an immobilized metal affinity chromatography (IMAC) column; and
vii) eluting the rhGAA from the IMAC column, wherein at least 90% of the purified rhGAA binds to cation-independent mannose-6-phosphate receptor (CIMPR); wherein the purified rhGAA comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 2, wherein the purified rhGAA comprises a first potential N-glycosylation site, a second potential N-glycosylation site, a third potential N-glycosylation site, a fourth potential N-glycosylation site, a fifth potential N-glycosylation site, a sixth potential N-glycosylation site, and a seventh potential N-glycosylation site at amino acids corresponding to N84, N177, N334, N414, N596, N826, and N869 of SEQ ID NO:2, respectively, and wherein at least 50% of the molecules of the purified rhGAA comprise an N-glycan unit bearing bis-M6P at the first potential N-glycosylation site, at least 30% of the molecules of the purified rhGAA comprise an N-glycan unit bearing mono-M6P at the second potential N-glycosylation site, at least 30% of the molecules of the purified rhGAA comprise an N-glycan unit bearing bis-M6P at the fourth potential N-glycosylation site, at least 20% of the molecules of the purified rhGAA comprise an N-glycan unit bearing mono-M6P at the fourth potential N-glycosylation site, or a combination thereof.

### Claim 2 (depends on 1)

The method of claim 1, further comprising:
applying the rhGAA eluted from the IMAC column onto a third chromatography column; and
eluting the rhGAA from the third chromatography column.

### Claim 3 (depends on 2)

The method of claim 2, wherein the third chromatography column is selected from a cation exchange chromatography (CEX) column and a size exclusion chromatography (SEC) column.

### Claim 4 (depends on 2)

The method of claim 2, further comprising inactivating viruses in one or more of the rhGAA eluted from the AEX chromatography column, the rhGAA eluted from the IMAC column, and the rhGAA eluted from the third chromatography column.

### Claim 5 (depends on 2)

The method of claim 2, further comprising filtering the rhGAA eluted from the IMAC column or the rhGAA eluted from the third chromatography column.

### Claim 6 (depends on 1)

The method of claim 1, further comprising lyophilizing the purified rhGAA.

### Claim 7 (depends on 5)

The method of claim 5, further comprising lyophilizing the purified rhGAA.

### Claim 8 (depends on 1)

The method of claim 1, wherein the rhGAA is produced from Chinese hamster ovary (CHO) cells.

### Claim 9 (depends on 1)

The method of claim 1, wherein the purified rhGAA is suitable for enzyme replacement therapy for treating Pompe disease.