CHO cell line specific prediction and control of recombinant monoclonal antibody N‐glycosylation

Abstract

Here we demonstrate that it is possible to predict and control N-glycan processing of a secreted recombinant monoclonal antibody during manufacturing process development using a combination of statistical modelling and comparative measurement of cell surface glycans using fluorescent lectins. Using design of experiments--response surface modelling (DoE-RSM) methodology to adjust the relative media concentrations of known metabolic effectors of galactosylation (manganese, galactose, and uridine) we have shown that β1,4-galactosylation of the same recombinant IgG4 monoclonal antibody produced by different CHO cell lines can be precisely controlled in a cell line specific manner. For two cell lines, monoclonal antibody galactosylation could be increased by over 100% compared to control, non-supplemented cultures without a reduction in product titre and with minimal effect on cell growth. Analysis of galactosylation effector interactions by DoE-RSM indicated that Mn²⁺ alone was necessary but not sufficient to improve galactosylation, and that synergistic combinations of Gal and Urd were necessary to maximize galactosylation, whilst minimizing the deleterious effect of Urd on cell growth. To facilitate rapid cell culture process development we also tested the hypothesis that substrate-level control of cellular galactosylation would similarly affect both cell surface and secreted monoclonal antibody glycans, enabling facile indirect prediction of product glycan processing. To support this hypothesis, comparative quantitation of CHO cell surface β1,4-galactosylation by flow cytometry using fluorescent derivatives of RCA and ConA lectins revealed that substrate-controlled variation in monoclonal antibody galactosylation and cell surface galactosylation were significantly correlated. Taken together, these data show that precision control of a complex, dynamic cellular process essential for the definition of protein product molecular heterogeneity and bioactivity is possible. Moreover, real-time, or near real-time control can be enabled by facile, rapid measurement of cell surface biomarkers of cellular biosynthetic capability.

Tailoring recombinant protein quality by rational media design

“…process information (such as viable cell density, productivity) to increase the mechanistic knowledge by integrating further important characteristics besides the media composition in the framework of glycosylation modulation 122,[280][281][282] . At later stages of process development, such additional (dynamic) characteristics are likely to play a key role in building predictive process models.…”

“…A decrease of Man5 and hence higher abundance of galactosylated species were observed at increasing Mn 2+ concentration: 0.25-1.0 µM 63 . A recent DoE experiment with post-seeding supplementation of 0-40 µM Mn 2+ , 0-20 µM uridine (Urd) and 0-100 µM galactose (Gal) led to the conclusion that Mn 2+ was necessary but not sufficient to improve galactosylation, and that synergistic combinations of Urd and Gal maximized galactosylation 122 . The resulting osmolality increase at high galactose concentrations tested may however be toxic to the cells and hence harm the culture.…”

“…Manganese (Mn 2+ ) favors sialylation according to a publication describing a correlation between the enhancement of sialylated G1 N-glycans and the supplementation of Mn 2+ (0-40 µM) in the presence of Urd and Gal during the culture 122 . While improved sialylation of IFN-γ was the result of medium supplementation with the specific sialic acid precursor N-acetylmannosamine (ManNAc) at 20 mM in a CHO cell line (cotransfected with genes for dihydrofolate reductase) 134 , it remained unchanged for human tissue inhibitor of metalloproteinases 1 (TIMP-1) at the same ManNAc concentration in GS-NS0 and GS-CHO cells 135 .…”

“…As a nonspecific component, galactose also influences other glycosylation steps including galactosylation 122 . This finding was further confirmed in GS-CHO cell line expressing the human tumor necrosis factor receptor linked to the Fc portion of human IgG1.…”

Cell Culture Processes for Biopharmaceutical Manufacturing

“…89). Improvement in either one of the two aspects alone may or may not be sufficient 90 since supplementation of all, i.e. uridine, galactose and Mn 2+ which targets both the availability of NSD substrate and enzymatic activity, usually achieves better galactosylation 91 .…”