A recombinant CHO host expression cell line is used industrially to produce human Acid Alpha Glucosidase (GAA), the therapeutic enzyme used in the treatment of Pompe disease, a rare genetic disorder caused by glycogen accumulation in muscle cells. The production of GAA is challenging, in part due to protease degradation. To investigate these issues an independent cell line was first created. We then designed a two-step chromatographic purification process: first the clarified cell culture material is loaded onto an AIEX resin to capture the target protein. The eluate fraction is then dialysed into new buffer and loaded onto a Phenyl HIC resin exploiting the different hydropathicity between the two molecules. The AIEX step yields close to 70% of the target protein. Western Blot, GAA enzymatic diagnostic assay and MALDI tandem mass spectrometry have confirmed the presence of GAA in eluate fraction and its absence from other fractions.<br/>To investigate the protease content during this downstream process zymograms and protease inhibitor assays are utilised to map proteases present through this series of operations.<br/>A proteomic approach based on MS was taken to identification and revealed the presence of Cathepsin Z throughout the process. Cathepsin Z protease (also known as Cathepsin X) part of the lysosomal cysteine endopeptidase family, catalyses the release of C-terminal amino acid residues with broad specificity and is believed to be responsible of GAA degradation. Its separation was found to be challenging as Cathepsin Z and the therapeutic target enzyme behave similarly during chromatography due to similar isoelectric point values and hydrophobicity and hence tend to co-purify.<br/>Acknowledgements: Financial support is gracefully acknowledged from the EPSRC and BioMarin Pharmaceutical Inc.

Characterisation of proteases in anion exchange chromatography for the bioprocess of a therapeutic enzyme

251st National Meeting (2016)

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