This Small Business Innovation Research (SBIR) Phase I project seeks to create a cost-effective, fungal strain appropriate for manufacturing glycosylated therapeutic proteins for use in humans. Fungi are promising alternatives to mammalian cells as manufacturing hosts because they are rapid, cheap systems that can perform eukaryotic post-translational modifications. Unfortunately, fungi synthesize glycoproteins with non-human glyco-structures, which often compromises the pharmacokinetic behavior of that protein in humans. This can be solved by engineering the N-glycosylation biosynthetic pathway to make human glycan structures. "Glycan engineering" has previously been applied to fungal species not well suited for commercial production. This proposal utilizes the filamentous fungus Myceliophthora thermophila (C1) that is well validated for ultra-high expression level (50-100g/L) at industrial scale (50,000-150,000L), and seeks to engineer a strain/s of C1 with the human G0 N-glycan (especially important for human therapeutic antibodies). C1's glycoprofile is close to that of humans; therefore, the strategy proposed requires minimal engineering. Specifically, a two-step engineering strategy that involves the genomic knockout of one endogenous gene and the knock-in of three novel N-glycan enzymatic functions should yield the desired G0 structure. Because so little genomic engineering is anticipated, the robust fermentation and expression characteristics of C1 are expected to remain intact.
The broad impact/commercial potential of this project is in its effect on lowering the cost of manufacturing biopharmaceutical drugs. Approximately one-fourth of the new drugs entering the market are biopharmaceuticals, with annual global sales projected to surpass $157B by 2015. As the market for therapeutic proteins and antibodies expands so will bottlenecks in production. There are commercial pressures (e.g., patent expiry) and government pressures (e.g., regulation to mitigate rising healthcare costs) to develop faster, better, and cheaper methods for the manufacture of biological drugs. Despite years of work, the current systems represent incremental but not step-fold improvements over those used earlier. This proposal introduces a potentially game changing filamentous fungus host expression system, Myceliophthora thermophila that has been well vetted for industrial enzyme manufacturing, and has the potential to eclipse other production systems thereby making major improvements in time-to-clinic, and cost of goods. Further, this proposal will extend our knowledge of the filamentous fungi by confirming the extent of genomic conservation among the N-glycan biosynthetic pathways, and also help to elucidate the subtle differences of M. thermophila that might make it the most tenable host for the production of human biotherapeutics.
Increasing demand for recombinant proteins and especially those called ‘glycoproteins’ (proteins with post-translationally added sugar structures) has focused research on protein expression hosts for producing these proteins at cost-effective levels. Yeasts and fungi are promising candidates because they provide rapid and cheap systems that can perform eukaryotic post-translational modifications. Unfortunately, yeasts and fungi modify their glycoproteins with non-human, glyco-structures, which is often detrimental to the pharmacokinetic behavior of that protein, or the optimal functioning of the drug. This problem can be solved by engineering the host’s N-glycosylation biosynthetic pathway to produce human glycan structures. Previous work has utilized hosts not particularly well suited for commercial production. The work done under this grant utilizes the filamentous fungus Myceliophthora thermophila (C1) that is well validated for ultra-high expression level (50-100g/L) at industrial scale (50,000-150,000L), and seeks to engineer a strain(s) of C1 with the human G0 N-glycan (especially important for human therapeutic antibodies). C1’s glycoprofile is surprisingly close to human; therefore the strategy requires minimal engineering. Specifically, a risk-mitigated, two-step engineering strategy that involves the genomic knockout of one endogenous gene and the knock-in of three novel N-glycan enzymatic functions was devised to yield the desired G0 structure. Because so little genomic engineering is anticipated, the robust fermentation and expression characteristics of C1 are expected to remain intact. This interim report documents the successful completion of the first phases of the project. All genes encoding the enzymatic functions necessary for this engineering project have been obtained. New selection markers, which are critical tools when introducing multiple genes into the genome, have been identified and confirmed. All cloning has been completed. First transformations have been successfully completed. The phenotype and behavior of the resulting clones are consistent with successful re-engineering of the first steps of our strategy. Further, and as anticipated, there is no apparent decrease in viability of the newly engineered C1 strains. The remaining transformations are underway and we anticipate completion within the remaining time frame.
