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Comparative Study
. 2010 May-Jun;2(3):320-34.
doi: 10.4161/mabs.2.3.11802. Epub 2010 May 17.

Differences in N-glycan structures found on recombinant IgA1 and IgA2 produced in murine myeloma and CHO cell lines

Esther M Yoo  1 , Li J Yu, Letitia A Wims, David Goldberg, Sherie L Morrison
Affiliations
Comparative Study

Differences in N-glycan structures found on recombinant IgA1 and IgA2 produced in murine myeloma and CHO cell lines

Esther M Yoo et al. MAbs. 2010 May-Jun.

Abstract

The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. The choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. These glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHO cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galalpha(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHO cells contained glycans that were more similar to those found on human IgA. Expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHO cells was significantly lower than that previously reported for serum IgA1. These data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential.

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Figures

Figure 1
Figure 1
Glycosylation of human IgA. IgA1 has N-linked carbohydrate addition sites in CH2 (Asn263) and in CH3 (Asn459) and o-linked carbohydrate in its hinge region. IgA2 lacks linked carbohydrates, but has additional N-linked sites in CH1 (Asn166) and in CH2 (Asn337). the IgA2m2 and the IgA2n allotypes have a fifth N-linked site in CH1 (Asn211). ▪, N-linked glycan; O, O-linked glycan.
Figure 2
Figure 2
Negative ion MALDI-TOF analysis of acidic N-glycans released from rIgAs produced in murine myeloma cell line Sp2/0. the likely glycan structures are shown as a schematic and the mass of the glycans is expressed as m/z. (A) IgA1, (B) IgA2m1, (C) IgA2m2, (D) IgA2n.
Figure 2
Figure 2
Negative ion MALDI-TOF analysis of acidic N-glycans released from rIgAs produced in murine myeloma cell line Sp2/0. the likely glycan structures are shown as a schematic and the mass of the glycans is expressed as m/z. (A) IgA1, (B) IgA2m1, (C) IgA2m2, (D) IgA2n.
Figure 3
Figure 3
Positive ion MALDI-TOF analysis of neutral N-glycans released from rIgAs produced in murine myeloma cell line Sp2/0. the likely glycan structures are shown as a schematic and the mass of the glycans is expressed as m/z. (A) IgA1, (B) IgA2m1, (C) IgA2m2, (D) IgA2n.
Figure 3
Figure 3
Positive ion MALDI-TOF analysis of neutral N-glycans released from rIgAs produced in murine myeloma cell line Sp2/0. the likely glycan structures are shown as a schematic and the mass of the glycans is expressed as m/z. (A) IgA1, (B) IgA2m1, (C) IgA2m2, (D) IgA2n.
Figure 4
Figure 4
Negative ion MALDI-TOF analysis of acidic N-glycans released from rIgAs produced in CHO cells. The likely glycan structures are shown as a schematic and the mass of the glycans is expressed as m/z. (A) IgA1, (B) IgA2m2.
Figure 5
Figure 5
positive ion MALDI-TOF analysis of neutral N-glycans released from rIgAs produced in CHO cells. the likely glycan structures are shown as a schematic and the mass of the glycans is expressed as m/z. (A) IgA1, (B) IgA2m2.
Figure 6
Figure 6
Positive ion MALDI-TOF analysis of neutral N-glycans released from rIgAs produced in mutant CHO cell lines defective in glycan processing. The likely glycan structures are shown as a schematic and the mass of the glycans is expressed as m/z. (A) IgA1 produced in Lec2, (B) IgA2m2 produced in Lec2, (C) IgA1 produced in Lec8, (D) IgA2m2 produced in Lec8.
Figure 6
Figure 6
Positive ion MALDI-TOF analysis of neutral N-glycans released from rIgAs produced in mutant CHO cell lines defective in glycan processing. The likely glycan structures are shown as a schematic and the mass of the glycans is expressed as m/z. (A) IgA1 produced in Lec2, (B) IgA2m2 produced in Lec2, (C) IgA1 produced in Lec8, (D) IgA2m2 produced in Lec8.
Figure 7
Figure 7
WAX chromatography of N-linked glycans from IgA1, IgA2m1 and IgA2n glycans produced in murine myelomas. The peaks were assigned by comparison with glycan standards isolated from bovine fetuin (data not shown). N, neutral glycans; 2AB, free 2-AB label; Mono, mono-sialylated glycans; Di, di-sialylated glycans.
Figure 8
Figure 8
WAX chromatography of N-linked glycans from IgA1, and IgA2m2 produced in CHO cells. The peaks were assigned by comparison with glycan standards isolated from bovine fetuin (data not shown). N, neutral glycans; 2AB, free 2-AB label; Mono, mono-sialylated glycans; Di, di-sialylated glycans.
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