IgM characterization directly performed in crude culture supernatants by a new simple electrophoretic method

Vorauer-Uhl, Karola; Wallner, Jakob; Lhota, Gabriele; Katinger, Hermann; Kunert, Renate

doi:10.1016/j.jim.2010.05.003

Cited by 18 publications

(29 citation statements)

References 28 publications

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“…3). This phenomenon was previously reported by Vorauer-Uhl et al (2010). Following the hypothesis, that maybe the IgM-012 protein has some folding problems, which would explain the lower specific productivity, as well as the difference in abundance of pentamers in supernatants in IgM-012 producing cell lines, signs for any kind of ER stress (elevated levels of GRP78, PDI, XBP-1 and calnexin proteins and their transcript levels) were analyzed.…”

Section: Discussionmentioning

confidence: 61%

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

et al. 2014

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Despite the fact, that monoclonal antibodies are the fastest growing group of biopharmaceuticals in development, this is not true for the IgM class, which remains as enigmatic as ever. While more examples of usefulness of IgMs for medical applications are emerging, their recombinant production is still not common. In our study, stable monoclonal IgM producing CHO DG44 and HEK 293 cell lines, expressing two model IgM molecules (IgM-617 and IgM-012) were established. Recombinant cell lines were compared in regard of specific productivity, specific growth rate, maximal achieved antibody titer, gene copy numbers and transcription levels of transgene. IgM-617 cell lines were identified as high while IgM-012 clones were low producers. Although differences in gene copy numbers as well as in transcription levels were observed, they did not seem to be a limitation. Levels of relevant endoplasmic reticulumstress related proteins were analyzed and no indications of unfolded protein response were detected. This could indicate that the difference in the intrinsic protein stability of our model proteins (as was previously observed on purified samples) might cause lower yields of IgM-012. Transcriptomics and/or proteomics follow up studies might be necessary for identification of potential bottlenecks in IgM producing cell lines.

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Section: Discussionmentioning

confidence: 61%

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

et al. 2014

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“…The challenges in recombinant IgM production are an adequate productivity, homogenous polymerization, purity, structural integrity and biological function. Even though pentamers and hexamers are the predominant forms of IgM in human serum, aggregated or incomplete polymers were described for recombinantly produced IgMs resulting in reduced product quality [2,[4][5][6]. Another important quality attribute is the glycosylation since it may affect IgM secretion, cytolytic activity, immunogenicity or pharmacokinetics [7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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“…How-ever the detection range of such gels is narrow and lower molecular weight fractions are not visible. Recently, Vorauer-Uhl proposed an optimized method to analyze and quantify the IgM oligomerization status using gradient gels combined with Sypro ® Ruby staining and densitometric evaluation [34]. This optimized method is capable of analyzing multimers, isoforms and fragments simultaneously in molecular range of 25 -1200 kDa.…”

Section: Product Quality and Biological Functionmentioning

confidence: 99%

Recombinant IgM expression in mammalian cells: A target protein challenging biotechnological production

Mäder¹,

Chromikova²,

Kunert³

2013

ABB

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For many years, the potential of immunoglobulin M (IgM) antibodies was not fully understood because of characteristics different to the well-known immunoglobulin G type like low target affinity, cross reactivity and complex protein structure. In the meanwhile IgMs have been positively evaluated for their use as therapeutic agent in the mucosal environment but also in serum to eradicate upcoming tumor cells and invading antigens. Therefore IgM class of antibodies will play a significant role in clinical applications but also diagnosis in the future. To evaluate the full potential of this kind of antibody molecules large amounts of high quality product will be needed. In this review the focus is set on the biotechnological aspect of producing IgM class antibodies recombinantly in mammalian cells. Current achievements in expression and purification of this molecule are highlighted and compared.

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IgM characterization directly performed in crude culture supernatants by a new simple electrophoretic method

Cited by 18 publications

References 28 publications

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

Evaluating the bottlenecks of recombinant IgM production in mammalian cells

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

Recombinant IgM expression in mammalian cells: A target protein challenging biotechnological production

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