Production and Characterization of Engineered mAb 26.4 Variants

Vectors encoding recombinant mAb 26.4 isoforms were produced as previously described (13 (link), 14 (link)). Briefly, cDNA fragments encoding the 26.4 variable H and L chain regions were codon optimized for mammalian expression and subcloned in-frame with the cDNA sequence of the human IgG1 constant H and L chain regions in pcDNA3.1 expression vectors. In addition to the mAb 26.4.WT, the effector silent variant 26.4.AAAG (L234A, L235A, N297A, P329G) and 26.4.H435A with reduced FcRn binding (15 (link), 16 (link)) were produced. mAb 26.4.WT was also produced containing altered N-glycans, low fucose (LF; 26.4.WT.LF), and high galactose (HG; 26.4.WT.HG). The mAb variants were produced using FreeStyle 293-F cells (Thermo Fisher Scientific, Waltham, MA). Variants with modified N297-linked N-glycans were produced by supplying a fucose decoy, 2-deoxy-2-fluoro-L-fucose, posttransfection to decrease fucosylation, or by adding 5 mM D-galactose (Sigma-Aldrich, Burlington, MA) and expression vector encoding β−1,4-galactosyltransferase 1 to the transfection mix to increase galactosylation. The mAb variants were purified using a Protein A HiTrap HP affinity column (GE Healthcare, Chicago, IL), and their integrity was verified by SDS-PAGE. Fc glycosylation, including galactosylation and fucosylation, was assessed by mass spectrometry.

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Mørtberg T.V., Zhi H., Vidarsson G., Foss S., Lissenberg-Thunnissen S., Wuhrer M., Michaelsen T.E., Skogen B., Stuge T.B., Andersen J.T., Newman P.J, & Ahlen M.T. (2022). Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in Mice: Biochemical and Cell Biological Characterization of Isoforms of a Human Monoclonal Antibody. ImmunoHorizons, 6(1), 90-103.

Publication 2022

FreeStyle 293-F cells D-galactose Protein A HiTrap HP affinity column

2 deoxy 2 fluoro l fucose A protein Cdna Cells Codon Frame Fucose Galactose Galactosyltransferase Glycans Glycosylation Human Igg1 Isoforms Mammalian Mass spectrometry Sds page Transfection Vector

Corresponding Organization :

Other organizations : University Hospital of North Norway, UiT The Arctic University of Norway, Versiti Blood Center of Wisconsin, Sanquin, University of Amsterdam, Oslo University Hospital, University of Oslo, Netherlands Metabolomics Centre, Leiden University Medical Center, Norwegian Institute of Public Health

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Variable analysis

independent variables

MAb 26.4.WT
MAb 26.4.AAAG (L234A, L235A, N297A, P329G)
MAb 26.4.H435A with reduced FcRn binding
MAb 26.4.WT containing altered N-glycans, low fucose (LF; 26.4.WT.LF)
MAb 26.4.WT containing altered N-glycans, high galactose (HG; 26.4.WT.HG)

dependent variables

Integrity of mAb variants verified by SDS-PAGE
Fc glycosylation, including galactosylation and fucosylation, assessed by mass spectrometry

control variables

Human IgG1 constant H and L chain regions in pcDNA3.1 expression vectors
FreeStyle 293-F cells (Thermo Fisher Scientific, Waltham, MA) used for mAb variant production
Protein A HiTrap HP affinity column (GE Healthcare, Chicago, IL) used for mAb variant purification

controls

Positive control: mAb 26.4.WT
Negative controls not explicitly mentioned

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