Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 3, 2000, pp. 191

Biotecnología Aplicada, Volume 17, July-September 2000, p. 191

The Glycosylation of Immunoglobulin (IgG) in Bioreactors

Michael Butler, Jeremy Kunkel, David Jan, Norman Huzel, James Jamieson

Departments of Microbiology and Chemistry, University of Manitoba, Winnipeg, MB, Canada R3T 2N2

From a selection of papers from Biotecnología Habana`99 Congress.
November 28-December 3, 1999.

Code Number: BA00055

Introduction

Most secreted proteins from mammalian cells are glycoproteins having a significant carbohydrate moiety. The glycan structures are important for many properties including solubility, stability and in vivo bioactivity. The consistency of these structures is essential for any glycoprotein designated for therapeutic use. Immunoglobulin (IgG) produced from B-lymphocytes or hybridomas contains approximately 2-3% of carbohydrate by weight. A conserved N-linked glycosylation is present at Asn-297 on the Fc portion of the heavy chain constant region (CH2). The glycans attached to each chain are located in the interstitial space between the two CH2 domains which are constrained by two (in humans) or three (in mice) disulfide bridges between the heavy chains in the hinge region.

Glycosylation is also described in the Fab region of 30% of human serum IgG. The consensus structure of the N-glycan is a complex biantennary form with a core fucose. The Fab glycan has a low level of sialylation whereas the Fc domain glycan has minimal sialylation. The terminal galactosylation of the nonsialylated antennae is variable. The three distinct structures of biantennary glycans are G0, G1 and G2 having zero, one or two terminal galactose residues.

Under certain pathological conditions (notably rheumatoid arthritis) the degree of galactosylation of human IgG is significantly decreased [1].

Methods

A murine hybridoma (CC9C10) that secretes an IgG1 with an affinity for bovine insulin was grown in a serum-free medium. The IgG was shown to be glycosylated only on the heavy chain, corresponding to the conserved site (Asn-297). Continuous cultures were established in a 2L bioreactor (LH 210) with a controlled dilution rate, pH, dissolved oxygen (DO), stirring rate and temperature [2].

Results

Cell culture parameters may affect the pattern of immunoglobulin glycosylation. One example is the effect of DO on the glycans of IgG isolated from cell culture of a murine hybridoma during steady state continuous cultures in which DO was controlled as a constant culture parameter. The secreted MAb was isolated by immunoaffinity chromatography and subjected to either enzymatic deglycosylation by PNGase or chemical deglycosylation by hydrazinolysis. The isolated samples of N-glycans were analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE) and high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). These analyses showed that the predominant glycan structure was biantennary, asialo, core fucosylated and with variable galactosylation [3]. The N-linked glycosylation occurred on the heavy chain. There was no evidence for light chain glycosylation or for O-linked glycosylation. A shift towards increased galactosylation was observed at higher DO. At a DO at, and above, 50% air saturation, the galactosylation pattern of the glycans isolated from the secreted antibody resembled that found in normal human serum. However, by decreasing the DO to 10% air saturation there was a significant decrease in the level of galactosylation which then resembles more closely the pattern found in serum from rheumatoid arthritis.

Conclusion

The concentration of dissolved oxygen is one of many parameters that may affect the glycosylation of a secreted protein in a mammalian cell culture system. The normal level of immunoglobulin galactosylation decreases significantly below 50% DO of air saturation in a bioreactor.

References

Copyright Elfos Scientiae 2000

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