Methods For Increasing Mannose Content Of Recombinant Proteins

What is this patent about?

’595 is related to the field of recombinant protein production, specifically addressing the challenge of controlling glycosylation patterns in therapeutic antibodies produced in mammalian cell cultures. A key quality attribute of these antibodies is the level of high mannose (HM) glycoforms, which can affect their pharmacokinetic properties. Traditional methods for manipulating HM content, such as altering media composition or temperature, often have unpredictable effects on cell culture behavior and antibody productivity.

The underlying idea behind ’595 is to modulate the mannose content of recombinant proteins by carefully controlling the availability of glucose in the cell culture medium while providing an alternative carbon source. The key inventive insight is that by limiting glucose and supplementing with either galactose or sucrose, the high mannose glycoform content, particularly Mannose 5 (Man5), can be increased without significantly compromising cell growth, viability, or protein production.

The claims of ’595 focus on a method for modulating mannose 5 on an immunoglobulin molecule during a mammalian cell culture process. This involves establishing a cell culture in a bioreactor, limiting the amount of glucose such that the spent medium has a concentration of about 0 to 3 g/L, and supplementing the cell culture with a feeding medium comprising galactose, such that the concentration of galactose in the resulting spent medium is above 2.5 g/L. The claims specifically mention IgG2 , denosumab, and panitumumab as target immunoglobulins.

In practice, the method involves first establishing a cell culture in a bioreactor using standard techniques. The glucose concentration is then carefully managed, often by switching to a perfusion medium with a lower glucose concentration. The spent medium is monitored to ensure that glucose levels remain within the desired range. Simultaneously, galactose or sucrose is added to the culture medium to provide an alternative energy source for the cells. This approach allows for fine-tuning of the glycosylation process, leading to a higher proportion of high mannose glycoforms in the final product.

The differentiation from prior approaches lies in the specific combination of glucose limitation and alternative carbon source supplementation. While previous methods relied on broad adjustments to culture conditions, ’595 provides a more targeted approach to manipulating glycosylation. By precisely controlling glucose levels and providing galactose or sucrose, the method achieves a balance between promoting high mannose glycoform production and maintaining desirable cell culture parameters, resulting in a more efficient and predictable process for producing therapeutic antibodies with desired quality attributes.