Prevention Of Disulfide Bond Reduction During Recombinant Production Of Polypeptides

What is this patent about?

’294 is related to the field of recombinant protein production, specifically addressing the problem of disulfide bond reduction in polypeptides, such as antibodies, during the harvesting and purification process from recombinant host cell cultures. Maintaining the integrity of disulfide bonds is crucial for the proper folding and biological activity of these proteins, particularly multi-chain antibodies with inter-chain disulfide linkages. The background highlights the importance of mammalian cell culture, especially CHO cells, for producing therapeutic proteins, but acknowledges the challenges in preserving protein conformation during large-scale manufacturing.

The underlying idea behind ’294 is that the reduction of disulfide bonds during recombinant protein production is primarily caused by an active thioredoxin (Trx) system present in the harvested cell culture fluid (HCCF). This system, comprising thioredoxin, thioredoxin reductase, and NADPH, facilitates the reduction of disulfide bonds. The inventive insight is to inhibit this Trx system, or upstream components that supply it with necessary cofactors, to prevent unwanted disulfide bond reduction and maintain the structural integrity of the target polypeptide.

The claims of ’294 focus on a method for producing an antibody by expressing it in a Chinese Hamster Ovary (CHO) recombinant host cell culture, and following a production phase of the cell culture, sparging the pre-harvest cell culture fluid of the recombinant host cell with air to inhibit reduction of a disulfide bond in the antibody during processing, wherein the antibody is a therapeutic monoclonal antibody, and wherein the air sparging is continued until the amount of dissolved oxygen (dO2) in the pre-harvest cell culture fluid is at least 10%.

In practice, the invention involves supplementing the pre-harvest or harvested cell culture fluid with a thioredoxin inhibitor or employing measures that indirectly inhibit thioredoxin activity. Direct inhibitors target thioredoxin or thioredoxin reductase, while indirect methods include air sparging to oxidize reducing agents or lowering the pH to slow down thiol-disulfide exchange. The air sparging is continued until the amount of dissolved oxygen (dO2) in the pre-harvest cell culture fluid is at least 10%.

This approach differs from prior art by directly addressing the root cause of disulfide bond reduction, the active thioredoxin system, rather than merely attempting to re-oxidize reduced bonds later in the process. By inhibiting the Trx system or its upstream components, the invention provides a proactive method for preserving the structural integrity and biological activity of recombinant polypeptides, particularly antibodies, during large-scale manufacturing. This leads to a more efficient and reliable production process, yielding higher quality therapeutic proteins.