Method for selection of high M6P recombinant proteins

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.