Transient pentameric IgM fulfill biological function-Effect of expression host and transfection on IgM properties.

Julia Hennicke,Nicole Thielens,Wai Li Ling,Linda Schwaigerlehner,Isabelle Bally,Renate Kunert,Jean-Baptiste Reiser,Clemens Grünwald-Gruber

doi:10.1371/journal.pone.0229992

Abstract

Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.

Highlights

Immunoglobulins of subclass M (IgM) are considered as the main actors in the protection against humoral microbial infection and in the mediation of cell debris clearance by activating and controlling the complement classical pathway and inflammation
To assess if the transient expression is a suitable alternative to stable expression, production of IgM was investigated in both systems
Comparison of stable expression in CHO DG44 and transient expression in HEK293E showed that similar maximum viable cell concentrations result in comparable amounts of the respective IgM antibody variant (Fig 1A), transient expression often shows reduced product concentration compared to stable production

Summary

Introduction

Immunoglobulins of subclass M (IgM) are considered as the main actors in the protection against humoral microbial infection and in the mediation of cell debris clearance by activating and controlling the complement classical pathway and inflammation. This crucial role theoretically makes them relevant for therapeutic applications. Clinical trials of metastatic melanoma patients with a human anti-GD2 IgM (MORAb-028) were already started, the study was terminated due to a lack of product availability (ClinicalTrials.gov Identifier: NCT01123304, update: July 2019). Production of IgMs is a cost-intensive and tedious process no matter if isolated from serum or recombinantly expressed in stable cell lines [1,2,3].

Methods

Results

Discussion

Conclusion