Methods For Increasing Mannose Content Of Recombinant Proteins

What is this patent about?

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

The underlying idea behind ’568 is to modulate the HM glycoform content, particularly Mannose 5 (Man5), by carefully controlling the carbon source available to the cells. The core insight is that by limiting glucose in the cell culture medium and supplementing with an alternative carbon source like galactose or sucrose, the cells can be directed to produce proteins with a higher proportion of Man5, while still maintaining acceptable cell growth and protein production levels.

The claims of ’568 focus on a method for modulating mannose 5 on a recombinant protein during a mammalian cell culture process. This involves establishing a cell culture in a bioreactor, limiting the amount of glucose so that the spent medium has a concentration of 0 to 3 g/L, and supplementing the culture with a feeding medium containing either galactose (resulting in a spent medium concentration of 4 to 8 g/L) or sucrose (resulting in a spent medium concentration of 16-24 g/L).

In practice, the method involves first establishing a cell culture under standard conditions with sufficient glucose to promote cell growth and protein production. Once the culture reaches a desired density or production phase, the glucose supply is reduced, and galactose or sucrose is added to the medium. The concentration of glucose in the spent medium is monitored to ensure that it remains within the target range of 0 to 3 g/L. This forces the cells to utilize the alternative carbon source, which in turn affects the glycosylation machinery and increases the proportion of Man5 on the produced protein.

This approach differs from prior methods that rely on broad changes to the cell culture environment. By specifically targeting the carbon source, ’568 provides a more precise and controllable way to influence glycosylation. The use of galactose allows the cells to continue producing the desired protein, while sucrose appears to exert an osmolality-related effect on cell metabolism and glycosylation. This allows for fine-tuning of the Man5 content without significantly compromising cell viability or protein titer, offering a valuable tool for biopharmaceutical manufacturing.