Methods For Increasing Mannose Content Of Recombinant Proteins

What is this patent about?

’085 is related to the field of recombinant protein production in mammalian cell cultures, specifically addressing the challenge of controlling the glycosylation patterns of therapeutic proteins. Glycosylation, the addition of sugar molecules to proteins, significantly impacts a protein's efficacy and safety. Achieving a consistent and desired glycosylation profile, particularly the level of high mannose glycoforms, is crucial for therapeutic protein development.

The underlying idea behind ’085 is that the mannose-to-total-hexose ratio in the cell culture medium can be manipulated to predictably upregulate the high mannose glycoform content of a recombinant protein. By carefully controlling the relative amounts of mannose and other hexose sugars (like glucose) in the medium, the cell's glycosylation machinery can be influenced to produce proteins with a higher proportion of high mannose structures.

The claims of ’085 focus on a method for controlling the Mannose-5 glycoform content of denosumab molecules, measured as a percentage, and increased compared to a control value. The method involves establishing a CHO cell culture in a bioreactor with cells expressing denosumab, initially in a medium without mannose. The cells are grown until a viable cell density of at least 3x10^6 cells/mL is reached, then switched to a second medium containing both mannose and glucose, with a mannose-to-total-hexose ratio between 0.1 and 0.4 . Finally, the denosumab molecules are recovered.

In practice, the method involves a two-stage culture process. Initially, the CHO cells are grown in a standard medium to achieve a high cell density. Once the desired cell density is reached, the culture is switched to a specialized medium with a controlled mannose-to-total-hexose ratio. This shift in the nutrient environment alters the cell's metabolic pathways, specifically influencing the glycosylation enzymes responsible for adding and processing sugar molecules on the recombinant protein. The denosumab molecules are then harvested and purified.

Prior approaches to controlling glycosylation often involved trial-and-error adjustments to various culture parameters like temperature, pH, or osmolality. ’085 differentiates itself by providing a more direct and predictable method based on the precise control of sugar ratios in the culture medium. This approach allows for fine-tuning the high mannose glycoform content of the recombinant protein without significantly impacting cell growth or productivity, offering a more robust and scalable solution for therapeutic protein manufacturing.