FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality

Abstract

During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc.

Interaction of cell culture process parameters for modulating mAb afucosylation

“…genetic engineering of recombinant cell lines (Louie et al, 2016;Yamane-Ohnuki et al, 2004); (b) addition of enzyme inhibitors (Allen et al, 2016;Okeley et al, 2015); (c) modification of the levels of cofactors and substrates involved in glycosylation, including supplementation with alternative sugars (Hossler et al, 2014;Hossler et al, 2017); and (d) fine-tuning of cell culture process parameters (Konno et al, 2012). Of the many cell culture process parameters known to impact glycosylation previously (Hossler, Khattak, & Li, 2009), osmolality was shown to affect afucosylation (Konno et al, 2012).…”

“…This change in afucosylation can have a considerable impact on the potency of a mAb: a~1% increase in afucosylation can increase ADCC activity by~14-28%(Louie et al, 2016;Thomann et al, 2016). This change in afucosylation can have a considerable impact on the potency of a mAb: a~1% increase in afucosylation can increase ADCC activity by~14-28%(Louie et al, 2016;Thomann et al, 2016).…”

Cell Culture Processes for Biopharmaceutical Manufacturing

“…For example, knocking out Fut8 or FX genes can achieve fully afucosylated glycoforms 19 . FX knockout cell line can be supplementated with fucose to express antibodies with desired ratio of fucosylated to afucosylated glycans 20 . Host cells may also be engineered to reduce some host cell proteins susceptible to be retained as impurity during downstream processing which may have an adverse impact on the product during processing or storage 21 .…”

Afucosylated Biosimilars need Afucosylation Specific Assays

“…The work of a number of groups over the years has been directed at the development of afucosylation specific assays [6][7][8][9]. Using controlled mixtures involving homogeneous afucosylated and fucosylated antibodies to control overall afucosylation content, Chung et al [6] first demonstrated the existence of a linear dose-response curve between activity and afucosylation content.…”

“…Many of the technical difficulties associated with acquiring the reagents needed to implement afucosylation specific assays at low afucosylation content appear to have been solved by the elegant work of Louie and coworkers. Louie et al [9] used a FX KO CHO cell line and controlled fucose feeding to produce antibodies with different levels of afucosylation. This method allowed them to use a single cell line thus enabling the expression of different levels of afucosylated antibodies without significantly altering the overall glycoform distribution.…”