A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system

Becker, W.; Scherer, Anne; Faust, Christine; Bauer, David Kornblüh; Scholtes, Simone; Rao, Ercole; Hofmann, Joachim; Schauder, Rolf; Langer, Thomas

doi:10.1016/j.pep.2018.08.003

Cited by 15 publications

(4 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Importantly, observed high neutralization potencies were not caused by multimerization of CODV-Ig variants, as evidenced by size-exclusion chromatography (SEC) of one-step protein A affinity-purified samples ( Supplementary Figure 6 ). In addition, improvements in functional activities and production titers seen at the level of micro-scale expression supernatants were confirmed at protein level for selected variants purified from large-scale expression cultures as previously described 27 (data not shown).…”

Section: Resultssupporting

confidence: 63%

An end-to-end automated platform process for high-throughput engineering of next-generation multi-specific antibody therapeutics

Furtmann

Schneider

Spindler

et al. 2021

mAbs

View full text Add to dashboard Cite

Next-generation multi-specific antibody therapeutics (MSATs) are engineered to combine several functional activities into one molecule to provide higher efficacy compared to conventional, mono-specific antibody therapeutics. However, highly engineered MSATs frequently display poor yields and less favorable drug-like properties (DLPs), which can adversely affect their development. Systematic screening of a large panel of MSAT variants in very high throughput (HT) is thus critical to identify potent molecule candidates with good yield and DLPs early in the discovery process. Here we report on the establishment of a novel, format-agnostic platform process for the fast generation and multiparametric screening of tens of thousands of MSAT variants. To this end, we have introduced full automation across the entire value chain for MSAT engineering. Specifically, we have automated the in-silico design of very large MSAT panels such that it reflects precisely the wet-lab processes for MSAT DNA library generation. This includes mass saturation mutagenesis or bulk modular cloning technologies while, concomitantly, enabling library deconvolution approaches using HT Sanger DNA sequencing. These DNA workflows are tightly linked to fully automated downstream processes for compartmentalized mammalian cell transfection expression, and screening of multiple parameters. All sub-processes are seamlessly integrated with tailored workflow supporting bioinformatics. As described here, we used this platform to perform multifactor optimization of a next-generation bispecific, cross-over dual variable domain-Ig (CODV-Ig). Screening of more than 25,000 individual protein variants in mono- and bispecific format led to the identification of CODV-Ig variants with over 1,000-fold increased potency and significantly optimized production titers, demonstrating the power and versatility of the platform.

show abstract

Section: Resultssupporting

confidence: 63%

An end-to-end automated platform process for high-throughput engineering of next-generation multi-specific antibody therapeutics

Furtmann

Schneider

Spindler

et al. 2021

mAbs

View full text Add to dashboard Cite

show abstract

“…Antibodies were purified using protein A MabSelect for adalimumab, CaptureSelect IgM (Thermo Fisher Scientific) for all IgM molecules (with or without J‐chain), or CaptureSelect IgA (Thermo Fisher Scientific) for IgA molecules. After the capture step, the proteins were desalted and immediately loaded on a size exclusion chromatography column essentially as described before [20]. For expression of the pIgR (UniProt: P01833) and FcαμR (UniProt: Q8WWV6), DNA coding for amino acids 20–564 (pIgR) and 17–450 (FcαμR) were cloned with a C‐terminal His8‐tag into an expression vector and expressed as described above.…”

Section: Methodsmentioning

confidence: 99%

Functional studies with IgM and IgA immunoglobulins: binding to pIgR, FcαμR, FcμR, and CDC activities

Beyer,

Sommerfeld,

Grandien

et al. 2024

APMIS

Self Cite

View full text Add to dashboard Cite

IgMs are the first antibodies produced by the immune system upon encounter of a possible pathogen and are one of five antibody subclasses in humans. For IgG, the most intensively studied antibody class, the N‐linked glycosylation site located in the Fc‐domain is directly involved in high affinity binding to the respective receptors and initiation of corresponding immune response. IgM molecules have five N‐glycosylation sites and one N‐glycosylation site in the J‐chain, which can be incorporated in IgM or IgA molecules. There is only limited knowledge available concerning the function of these N‐glycosylations in IgMs. To address this question, we produced IgM molecules lacking a particular N‐glycosylation site and tested these variants as well as IgA molecules for binding to the known receptors: the polymeric immunoglobulin receptor (pIgR), the dual receptor for IgA and IgM, FcαμR, and the specific receptor for IgM, FcμR. The single glycosylation sites did not show an impact on expression and multimerization, except for variant N402Q, which could not be expressed. In SPR measurements, no major impact on the binding to the receptors by particular glycosylation sites could be detected. In cellular assays, deglycosylated variants showed some alterations in induction of CDC activity. Most strikingly, we observed also binding of IgA to the FcμR in the same affinity range as IgM, suggesting that this might have a physiological role. To further substantiate the binding of IgA to FcμR we used IgA from different origins and were able to confirm binding of IgA preparations to the FcμR.

show abstract

“…15 ), SG scientists modified these systems (sometimes using existing laboratory equipment like peristaltic pumps) to improve their functionality [3] , [4] , [5] , [6] . This type of work is continued to this day by scientists, as well as ÄKTA engineers that have developed newer versions [7] , [8] , [9] . While multiple FPLC systems could be run in parallel, this is an expensive alternative to manually processing several protein purifications by gravity column.…”

Section: Hardware In Contextmentioning

confidence: 99%

REVOLVER: A low-cost automated protein purifier based on parallel preparative gravity column workflows

et al. 2022

View full text Add to dashboard Cite

A fully automated three-step protein purification procedure for up to five samples using the NGC chromatography system

Cited by 15 publications

References 16 publications

An end-to-end automated platform process for high-throughput engineering of next-generation multi-specific antibody therapeutics

An end-to-end automated platform process for high-throughput engineering of next-generation multi-specific antibody therapeutics

Functional studies with IgM and IgA immunoglobulins: binding to pIgR, FcαμR, FcμR, and CDC activities

REVOLVER: A low-cost automated protein purifier based on parallel preparative gravity column workflows

Contact Info

Product

Resources

About