Methods For Increasing Mannose Content Of Recombinant Proteins

What is this patent about?

’972 is related to the field of recombinant protein production in mammalian cell cultures, specifically addressing the challenge of controlling the high mannose glycoform content of therapeutic proteins like antibodies. Achieving a desired range of high mannose glycoforms is crucial because it affects the pharmacokinetic properties of these proteins. Existing methods for manipulating glycoform content, such as altering media composition or temperature, are often unpredictable and can negatively impact cell culture behavior and productivity.

The underlying idea behind ’972 is that the mannose to total hexose ratio in the cell culture media can be manipulated to predictably upregulate the high mannose glycoform content of a recombinant protein. By carefully controlling this ratio, the invention provides a method to influence the glycosylation process within the cell, leading to a higher proportion of high mannose structures on the produced protein. This approach offers a more targeted and controllable way to achieve the desired glycoform profile.

The claims of ’972 focus on a cell culture media composition and methods for using it. Claim 1 covers a cell culture media containing mannose, where the mannose to total hexose ratio is between 0 and 1.0. Claims 3 and 4 cover methods for increasing or upregulating the high mannose glycoform content of a recombinant protein by establishing a mammalian cell culture in a bioreactor with media initially lacking mannose, then maintaining or perfusing the culture with media containing mannose within the specified ratio. Claim 12 specifies the use of an ultrafilter or microfilter for alternating tangential flow.

In practice, the invention involves first establishing a mammalian cell culture, such as a CHO cell culture, in a bioreactor using a standard cell culture media that does not contain mannose. As the cells enter the production phase, the culture is then maintained or perfused with a media where the mannose to total hexose ratio is carefully controlled to be greater than 0 but less than 1.0. This can be achieved by supplementing the media with mannose while adjusting the concentration of other hexoses, such as glucose, to maintain the desired ratio. The perfusion process ensures a continuous supply of nutrients and removal of waste products, supporting high cell densities and protein production.

The differentiation from prior approaches lies in the predictable and scalable nature of the high mannose glycoform modulation. Unlike previous methods that relied on trial and error, this invention provides a direct correlation between the mannose to total hexose ratio and the resulting high mannose glycoform content. This allows for precise control over the glycosylation process without significantly impacting cell growth or antibody production. Furthermore, the method has been shown to be applicable across different cell lines and scalable to larger bioreactor volumes, making it a robust and valuable tool for therapeutic protein development.