Generation of site‐distinct N‐glycan variants for in vitro bioactivity testing

Abstract

Glycosylation, a critical product quality attribute, may affect the efficacy and safety of therapeutic proteins in vivo. Chinese hamster ovary fed‐batch cell culture batches yielded consistent glycoprofiles of a Fc‐fusion antibody comprizing three different N‐glycosylation sites. By adding media supplements at specific concentrations in cell culture and applying enzymatic glycoengineering, a diverse N‐glycan variant population was generated, including high mannose, afucosylated, fucosylated, agalactosylated, galactosylated, asialylated, and sialylated forms. Site‐specific glycosylation profiles were elucidated by glycopeptide mapping and the effect of the glycosylation variants on the FcγRIIIa receptor binding affinity and the biological activity (cell‐based and surface plasmon resonance) was assessed. The two fusion body glycosylation sites were characterized by a high degree of sialic acid, more complex N‐glycan structures, a higher degree of antennarity, and a site‐specific behavior in the presence of a media supplement. On the other hand, the media supplements affected the Fc‐site glycosylation heterogeneity similarly to the various studies described in the literature with classical monoclonal antibodies. Enzymatic glycoengineering solely managed to generate high levels of galactosylation at the fusion body sites. Variants with low core fucosylation, and to a lower extent, high mannose glycans exhibited increased FcγRIIIa receptor binding affinity. All N‐glycan variants exhibited weak effects on the biological activity of the fusion body. Both media supplementation and enzymatic glycoengineering are suitable to generate sufficient diversity to assess the effect of glycostructures on the biological activity.

Tractable Method for Rapid Quality Assessment of Therapeutic Antibodies in Harvested Cell Culture Fluid based on FcγRIIIa-Immobilized Magnetic Microspheres

“…The extraction mechanism could be interpreted as the Fe 3 O 4 @PDA@PAMAM-FcγRIIIa composites are directly added into the complex HCCF sample serving as MSPE adsorbents. IgG species with high FcγRIIIa-binding affinity could be selectively captured by the adsorbents under neutral conditions, whereas other low FcγRIIIa-binding species are barely captured, based on the fact that different IgG variants show significant differences in the FcγRIIIa-binding affinity, generally 2–10 2 fold of dissociation constant ( K D ) values. , By magnetic separation, the expected high FcγRIIIa affinity IgG species are isolated together with Fe 3 O 4 @PDA@PAMAM-FcγRIIIa composites, while low FcγRIIIa affinity IgG species, host cell-related substances, and other culture medium-related substances are simultaneously removed. The unexpected adsorbates, formed by inevitable nonspecific interactions, are eliminated through the subsequent washing step with neutral buffers.…”

Immobilized enzyme cascade for targeted glycosylation

“…We also demonstrated the ability of our in-house immobilized GalT to modify the galactosylation profile of three full-length IgGs. Driving galactosylation of IgGs in vitro has been demonstrated on multiple occasions, highlighting the importance of such an endeavor 39,[53][54][55] . In contrast to previous work, our strategy enabled enzyme reusability for four cycles, demonstrating activity over a time span of more than 80 h. This result shows the potential for implementation of SUGAR-TARGET in large-scale industrial processes, including for the continuous modification of glycoproteins.…”

Innovative Metrics for Reporting and Comparing the Glycan Structural Profile in Biotherapeutics

“…Antibody fusion molecule and recombinant FSH products were processed according to the procedures previously reported in [101,102], respectively.…”