Use Of Monensin To Regulate Glycosylation Of Recombinant Proteins
Patent No. US11130980 (titled "Use Of Monensin To Regulate Glycosylation Of Recombinant Proteins") was filed by Amgen Inc on Oct 30, 2014.
What is this patent about?
’980 is related to the field of recombinant protein production, specifically addressing the challenge of controlling glycosylation patterns in therapeutic proteins produced in mammalian cell cultures. Glycosylation, the addition of sugar molecules to proteins, significantly impacts a protein's efficacy and safety. Achieving a desired glycosylation profile, particularly the level of high mannose glycans, is crucial for optimizing therapeutic properties of monoclonal antibodies (mAbs). Current methods for manipulating glycosylation are often cell line and molecule-specific, and can negatively impact cell culture performance.
The underlying idea behind ’980 is to use monensin , an ionophore that disrupts intracellular pH gradients, to modulate the glycosylation pathway within cells producing recombinant proteins. By carefully controlling the exposure of cells to monensin, the inventors discovered that the high mannose glycoform content of secreted proteins, particularly mAbs, can be increased. This is achieved by interfering with the normal processing of glycans in the Golgi apparatus, leading to an accumulation of high mannose structures on the protein.
The claims of ’980 focus on a method for increasing the high mannose glycoform content of a secreted recombinant protein containing an immunoglobulin Fc region during a mammalian cell culture process. The method involves establishing a mammalian cell culture expressing the protein in a serum-free medium within a bioreactor, maintaining the cells during a production phase, and then contacting the cell culture with an effective amount of monensin to increase the high mannose glycoform content of the secreted recombinant protein.
In practice, the method involves carefully controlling the concentration and duration of monensin exposure. The inventors found that a single bolus dose or continuous feeding of monensin at nanomolar concentrations can significantly increase high mannose content without compromising cell growth, viability, or protein yield. The timing of monensin addition is also important, with addition during the production phase proving most effective. The process can be implemented in various cell culture systems, including fed-batch and perfusion cultures.
This approach differs from prior methods that rely on manipulating media composition, osmolality, or temperature, which often have inconsistent effects and can negatively impact cell culture performance. By directly targeting the glycosylation machinery with monensin, the invention provides a more universal and controllable method for modulating high mannose glycoform content. The inventors demonstrated that this method can be applied to various cell lines and antibody products, offering a valuable tool for optimizing the therapeutic properties of recombinant proteins.
How does this patent fit in bigger picture?
Technical landscape at the time
In the early 2010s when ’980 was filed, recombinant proteins, such as monoclonal antibodies, were typically produced in mammalian cell cultures, often CHO cells. At a time when glycosylation was known to affect the efficacy of therapeutic antibodies, methods for controlling glycosylation patterns, such as high mannose content, were actively sought. When systems commonly relied on media composition and culture conditions to modulate glycosylation, identifying a universal mechanism that could increase high mannose glycoforms without compromising cell culture performance was non-trivial.
Novelty and Inventive Step
The examiner allowed the claims because the applicant provided data showing that a specific concentration of monensin, when added to the cell culture medium, increases the high mannose glycoform content of a secreted recombinant protein. Prior art references disclosed that monensin can inhibit protein production, including proteins with high mannose content, and did not teach or recognize that a specific concentration of monensin could increase the high mannose glycoform content of a secreted recombinant protein. The examiner concluded that a person skilled in the art would not have been motivated to select the specific concentration of monensin claimed to increase the high mannose glycoform content of a secreted recombinant protein.
Claims
This patent contains 26 claims, with claim 1 being the only independent claim. Independent claim 1 is directed to a method of increasing the high mannose glycoform content of a secreted recombinant protein comprising an immunoglobulin Fc region during a mammalian cell culture process by contacting the cell culture with monensin. The dependent claims generally specify details and variations of the method described in independent claim 1, including monensin concentration, timing of monensin addition, and cell culture maintenance.
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Description
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US11130980
- Application Number
- US15033559
- Filing Date
- Oct 30, 2014
- Status
- Granted
- Expiry Date
- Apr 5, 2035
- External Links
- Slate, USPTO, Google Patents