Method for selection of high M6P recombinant proteins
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.
1 . A method for producing purified recombinant human acid alpha-glucosidase (rhGAA), wherein the purified rhGAA comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 2, 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 a first chromatography column to capture the rhGAA, wherein the first chromatography column is an anion exchange chromatography (AEX) column, wherein the filtrate is not loaded onto a chromatography column prior to the AEX column; and
v) eluting the rhGAA from the AEX column,
wherein the purified rhGAA comprises at least 3.0% of the total N-glycans of the rhGAA are in the form of a bis-mannose-6-phosphate (bis-M6P) N-glycan and wherein at least 90% of the purified rhGAA binds to cation-independent mannose-6-phosphate receptor (CIMPR).
2 . The method of claim 1 , wherein at least 90% of the purified rhGAA comprises an N-glycan carrying mono-M6P or bis-M6P.
3 . The method of claim 1 , wherein the purified rhGAA comprises seven potential N-glycosylation sites.
4 . The method of claim 2 , wherein the rhGAA has seven potential N-glycosylation sites that are located at amino acids corresponding to N84, N177, N334, N414, N596, N826, and N869 of SEQ ID NO:2, respectively.
5 . The method of claim 3 , wherein at least 50% of the purified rhGAA comprise an N-glycan unit bearing bis-M6P at the first potential N-glycosylation site, at least 30% of the purified rhGAA comprise an N-glycan unit bearing mono-M6P at the second potential N-glycosylation site, at least 30% of the purified rhGAA comprise an N-glycan unit bearing bis-M6P at the fourth potential N-glycosylation site, or at least 20% of the purified rhGAA comprise an N-glycan unit bearing mono-M6P at the fourth potential N-glycosylation site.
6 . The method of claim 1 , further comprising:
applying the rhGAA eluted from the AEX column onto a second chromatography column; and
eluting the rhGAA from the second chromatography column.
7 . The method of claim 6 , wherein the second chromatography column is an immobilized metal affinity chromatography (IMAC) column, a cation exchange chromatography (CEX) column, or a size exclusion chromatography (SEC) column.
8 . The method of claim 1 , further comprising inactivating viruses in the rhGAA eluted from the AEX column.
9 . The method of claim 6 , further comprising inactivating viruses in the rhGAA eluted from the second chromatography column.
10 . The method of claim 1 , further comprising filtering the purified rhGAA to provide a filtered product.
11 . The method of claim 10 , further comprising filling a vial with the filtered product.
12 . The method of claim 1 , further comprising lyophilizing the purified rhGAA.
13 . The method of claim 1 , wherein the host cells comprise Chinese hamster ovary (CHO) cells.
14 . The method of claim 1 , wherein the purified rhGAA comprises 3.0-6.0 moles of M6P per mole of rhGAA.
15 . The method of claim 1 , wherein the purified rhGAA comprises 3.0-8.0 moles of sialic acid residues per mole of rhGAA.
16 . The method of claim 1 , wherein the purified rhGAA comprises 3.0-6.0 moles of M6P and 3.0-8.0 moles of sialic acid residues per mole of rhGAA.
17 . The method of claim 1 , wherein the purified rhGAA comprises on average at least 1 mol bis-M6P per mol of protein.
18 . The method of claim 1 , wherein the purified rhGAA comprises on average 1.3 mol bis-M6P per mol of protein.
19 . The method of claim 1 , wherein the purified rhGAA comprises at least 17% of the total N-glycans of the rhGAA are in the form of a bis-M6P N-glycan.
20 . The method of claim 1 , wherein the purified rhGAA comprises 4.0 moles of sialic acid residues per mole of rhGAA.