Overexpression of N-glycosylation pathway regulators to modulate glycosylation of recombinant proteins

What is this patent about?

’627 is related to the field of recombinant protein production and mammalian cell culture engineering. Specifically, it addresses the technical challenge of controlling post-translational modifications, such as glycosylation, which significantly impact the efficacy, safety, and metabolic clearance of therapeutic antibodies produced in systems like Chinese hamster ovary (CHO) cells.

The underlying idea behind ’627 is that the accumulation of undesirable high mannose glycoforms can be suppressed by genetically enhancing the cell’s internal processing machinery rather than relying solely on external media adjustments. By overexpressing specific enzymes and transporters in the N-glycosylation pathway, the inventor discovered that the cell can more efficiently convert simple mannose structures into complex glycans, thereby ensuring a more consistent and human-like protein product.

The claims of ’627 focus on a method for regulating high mannose glycoform content to levels at or below 10% by transfecting host cells to overexpress Mgat1, Mgat2, or the UDP-Galactose transporter Slc35a2. The independent claims specifically cover these genetic modifications within the context of two distinct industrial scales: one utilizing alternating tangential flow (ATF) for perfusion cultures and another utilizing fed-batch culture systems, followed by the harvesting and purification of the resulting protein.

In practice, the invention works by increasing the abundance of N-acetyl-glucosaminyltransferase-1 and 2, which act as gatekeepers in the Golgi apparatus. These enzymes facilitate the transition from immature high-mannose species to complex, branched sugar chains. The addition of the Slc35a2 transporter further supports this by ensuring an adequate supply of nucleotide sugar substrates is available within the Golgi lumen to fuel these enzymatic reactions.

This approach differs from prior methods that typically relied on trial-and-error adjustments to media composition, pH, or temperature to manage glycosylation. Unlike those environmental tweaks, which often inadvertently reduce overall antibody productivity or cell viability, the genetic engineering approach described here provides a targeted mechanism to lower high mannose content without compromising the growth performance or final yield of the bioreactor.