Overexpression Of N-Glycosylation Pathway Regulators To Modulate Glycosylation Of Recombinant Proteins

What is this patent about?

’186 is related to the field of recombinant protein production in mammalian cell cultures, specifically addressing the challenge of controlling glycosylation , a post-translational modification that significantly impacts the therapeutic efficacy of biopharmaceuticals like monoclonal antibodies. Glycosylation patterns, particularly the presence of high mannose glycoforms, can affect a protein's pharmacokinetics and effector functions, necessitating precise control during manufacturing.

The underlying idea behind ’186 is to engineer host cells to manipulate the N-glycosylation pathway directly. Instead of relying on environmental factors like media composition or temperature, the inventors propose genetically modifying cells to overexpress key enzymes and transporters involved in the glycosylation process. This approach aims to shift the glycosylation equilibrium, reducing undesirable high mannose glycoforms and promoting more complex, therapeutically beneficial glycan structures.

The claims of ’186 focus on a mammalian host cell engineered to regulate the high mannose glycoform content of a recombinant protein. This is achieved by transfecting the host cell to overexpress a protein involved in the N-glycosylation pathway, specifically Mgat1, Mgat2, Slc35a2 , or a combination thereof. The key outcome is a reduction in the high mannose glycoform content of the expressed recombinant protein to less than or equal to 10%.

In practice, the invention involves selecting a suitable mammalian host cell, such as a CHO cell, and introducing genetic material that causes the cell to overproduce one or more of the target proteins. This can be done before or after introducing the gene for the recombinant protein of interest. The modified cells are then cultured under standard conditions, and the resulting protein is harvested. The level of high mannose glycoforms is monitored to ensure it remains below the specified threshold.

The advantage of this approach is that it provides a more direct and predictable way to control glycosylation compared to traditional methods. By increasing the expression of enzymes like Mgat1 and Mgat2, the invention promotes the processing of high mannose glycans into more complex structures. Overexpressing Slc35a2, a UDP-Galactose transporter , ensures that the necessary building blocks are available for glycosylation. This genetic manipulation offers a stable and controllable means of tailoring the glycosylation profile of recombinant proteins, leading to improved therapeutic properties.