IQVIA recently released a comprehensive overview of the US pharmaceutical market. The report highlighted that despite recent approvals of biosimilars, they are used less than one-third of the time when available. Additionally, at most recent conferences, there have been several dedicated sessions discussing the challenges developing, manufacturing and commercializing biosimilars. Dahodwala and Sharfstein have briefly explained many of these challenges, and the FDA’s Biosimilar Action Plan along with research/manufacturing cooperatives like the Advanced Mammalian Biomanufacturing Innovation Center (AMBIC) and the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) have launched various initiatives to address these concerns.
From a regulatory standpoint, the glycosylation profile of a proposed biosimilar is a critical quality attribute (CQA) that needs to match its originator biologic since it affects a drug’s half-life, efficacy, and safety. Glycosylation can be altered by a variety of methods (e.g., cell line and clone selection, genetic engineering, culture variables). However, due to intellectual property restrictions, it would be convenient to have a toolbox of variables to implement when in early stage process development. In the paper titled “Impact of Cell Culture Media Additives on IgG Glycosylation Produced in Chinese Hamster Ovary Cells,” the authors first provide a useful list of small molecules that have been shown to modulate glycosylation, their proposed mechanisms of action, and references. Moreover, they carried out their own analysis of these compounds using two relevant cell lines (CHO K1 and CHO DG44) on two different process development culture platforms (Spin tubes and a Sartorius ambr® 15 system). Their goal was to provide this information “to serve as a starting point to help biotechnology scientists define a good supplementation strategy to target a specific biosimilar quality profile without sacrificing titer.”
The table below provides a brief summary of the many culture additives tested in the study. Glycosylation profiles at different timepoints were screened by traditional approaches (CGE-LIF and UPLC-MS) and broken down into total fucosylation, total mannosylation, total galactosylation, total sialylation and total GlcNAc (N-acetylglucosamine).
Type of Glycosylation
Culture additives tested (Some at Multiple concentrations)
|Mannosylation||Deoxymannojirimycin, deoxynomannojirimycin, high osmality, kifunensine, mannose, mannostatin A, monensin, raffinose, sucrose, tagatose|
|Fucosylation||2F-PerAcFuc, DMSO, mycophenolic acid, Reactive Red 120|
|Galactosylation||Ammonium chloride, cytidine + fucose + uridine, galactose + manganese chloride (Mn), galactose + Mn + uridine (UMG), Mn|
|Sialylation||UMG, UMG + AC4ManNAc, UMG + CuCl2, UMG + DANA, UMG + dexamethasone, UMG + fetuin, UMG + glycerol, UMG + hydrocortisone, UMG + ManNAC, UMG + ManNaz, UMG + ManNAc + DANA|
Out of 19 different culture conditions, supplementation with kifunensine exhibited the highest increase in total mannosylation which included a significant increase in the Man9 species. Tagatose, deoxymannojirimycin, deoxynojirimycin, and mannose were also shown to yield double digit percentage increases in total mannosylation with preferences in some Man species over others. In 11 different culture conditions, the fucosyltransferase inhibitor 2‐F‐peracetyl fucose (2F‐PerAcFuc) reduced fucosylation significantly more than Reactive Red 120 or mycophenolic acid. 2F‐PerAcFuc acted in a dose‐dependent manner and was more than 10x as effective than the other culture additives. The addition of ammonium chloride decreased total galactosylation, with the monogalactosylated species being affected the most. On the other hand, a custom media blend containing uridine, Mn and galactose increased galactosylation by more than 10%. With the increase in galactosylation on the antibodies, there was also a concurrent increase in sialylation, which was expected because higher galactosylation is critical for increases in sialic acid binding. In combination with the three-component uridine, Mn and galactose mixture, dexamethasone supplementation increased both the total amount of sialylation and disialytlation the most in 23 culture conditions. Glycerol addition in the feed of the culture supplemented with the three-component uridine, Mn and galactose mixture decreased the sialic acid content the most when normalized to the control conditions.
Ultimately, the authors provided a comprehensive analysis of potential cell culture media additives that may affect the glycosylation profile of a monoclonal antibody. Using these small molecules, biosimilar manufacturers may be able to navigate around intellectual property restrictions to shape a glycosylation profile to match the targeted originator biologic.