Reduction of high mannose glycoforms from monoclonal antibodies by affinity chromatography using a recombinant prokaryotic lectin

Glycan profiles of monoclonal antibodies are critical quality attributes for their use as therapeutic agents. However, the control of glycosylation can prove difficult during large-scale manufacture. An on-going problem is the increase in high-mannose glycans (HM) that can occur unexpectedly under cell culture conditions that disrupt the glycosylation pathway. We have developed a method for removal of high-mannose glycans from a heterogeneous mixture of mAb glycoforms purified from a bioprocess. This involves the use of a prokaryotic lectin (RPL-Man2) bound to agarose beads to selectively bind reversibly to mAbs carrying HM-glycans. The unbound flow-through from this step contains the remainder of the mAb preparation with a reduced HM-glycan content. In this paper we demonstrate the application of this method using two distinct antibodies derived from Chinese hamster ovary (CHO) cells purposely grown under conditions that enhanced the content of HM-glycans. The presence of an elevated level of mannose in the culture of the producer cells as well as supplementation with a specific mannosidase inhibitor resulted in purified mAbs containing significantly higher levels of HM-glycans than would be expected normally under standard conditions. We showed that HM-glycans in the purified mAbs were reduced significantly by the application of lectin chromatography. Analysis by both UPLC using fluorescence detection as well as by mass spectrometry showed that the removal of the HM-glycoforms was selective and did not affect the profile of the remaining glycoforms. This method has potential for use in large-scale biomanufacturing for the reduction or elimination of HM-glycoforms of mAbs.