Use Of Monensin To Regulate Glycosylation Of Recombinant Proteins

What is this patent about?

’760 is related to the field of recombinant protein production in mammalian cell cultures, specifically focusing on modulating the glycosylation patterns of therapeutic proteins. Glycosylation, the addition of sugar molecules to proteins, significantly impacts a protein's efficacy and safety. The background highlights the challenges in controlling glycosylation, particularly the high mannose glycoform content, which can affect a therapeutic antibody's clearance rate and immune response. Existing methods to manipulate glycosylation often involve altering media composition or culture conditions, which can negatively impact cell growth and protein production.

The underlying idea behind ’760 is to use monensin , an ionophore that disrupts intracellular pH gradients, to regulate the high mannose glycoform content of a recombinant protein produced in cell culture. The key inventive insight is that by carefully controlling the concentration and timing of monensin addition, the high mannose content can be increased without significantly compromising cell culture performance or protein yield. This approach provides a more targeted and controllable method for achieving a desired glycosylation profile.

The claims of ’760 focus on a method for regulating the high mannose glycoform content of a denosumab composition during a mammalian cell culture process. Specifically, the independent claim covers establishing a cell culture of genetically engineered Chinese Hamster Ovary (CHO) cells expressing denosumab in a serum-free medium within a bioreactor, maintaining these cells during a production phase, and introducing monensin into the CHO cell culture during this production phase at a concentration ranging from approximately 25 nM to 500 nM. The claim specifies that the addition of monensin leads to an increase in the high mannose denosumab glycoform content within the denosumab composition.

In practice, the method involves adding monensin to the cell culture medium at a specific concentration during the production phase. The concentration and timing of monensin addition are crucial for achieving the desired increase in high mannose content without negatively impacting cell growth or protein production. The patent describes various strategies for monensin addition, including single bolus doses, continuous feeding, and varying the concentration over time. The cell culture can be maintained using fed-batch or perfusion techniques to optimize cell density and protein yield.

’760 differentiates itself from prior approaches by offering a more direct and controllable method for modulating glycosylation. Unlike methods that rely on broad changes to media composition or culture conditions, monensin specifically targets the Golgi apparatus , where glycosylation occurs. By disrupting the pH gradient within the Golgi, monensin inhibits glycan processing enzymes, leading to an increase in high mannose glycoforms. This targeted approach allows for fine-tuning of the glycosylation profile without significantly affecting other aspects of cell culture performance or protein quality.