Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli

Lindner, Robert; Moosmann, Anna; Dietrich, Alexander; Böttinger, Heiner; Kontermann, Roland E.; Siemann‐Herzberg, Martin

doi:10.1016/j.jbiotec.2014.10.003

Cited by 14 publications

(11 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This was in contrast to what was observed by Lindner et al. for anti‐epidermal growth factor receptor scFv with p I of 7.4, which likely has a different spatial distribution of electrostatic potential and spatial hydrophobicity. As a result of poor recovery from Capto MMC™, MEP HyperCel™ was chosen as a candidate for capture.…”

Section: Resultscontrasting

confidence: 99%

“…No scFv protein was found in the FT and recovery from the resin was only possible at both high conductivity and high pH such as during CIP procedure with 0.5 M sodium hydroxide. This was in contrast to what was observed by Lindner et al [20] for anti-epidermal growth factor receptor scFv with pI of 7.4, which likely has a different spatial distribution of electrostatic potential and spatial hydrophobicity. As a result of poor recovery from Capto MMC TM , MEP HyperCel TM was chosen as a candidate for capture.…”

Section: Capture Of Scfv From Hek293 Feedstockcontrasting

confidence: 99%

“…Furthermore, Lindner et al. developed a four‐step chromatographic purification process for an anti‐epidermal growth factor receptor scFv produced periplasmatically in Escherichia coli . In the Lindner et al.…”

Section: Introductionmentioning

confidence: 99%

“…Gagnon et al [19] highlighted the challenges with purification of mAb-fragments by conventional methods and concluded that MM chromatography is a valuable alternative when conventional methods are insufficient [19]. Furthermore, Lindner et al [20] developed a four-step chromatographic purification process for an anti-epidermal growth factor receptor scFv produced periplasmatically in Escherichia coli. In the Lindner et al study, capture was performed by cation hydrophobic MM chromatography using Capto MMC TM , whereafter intermediate purification and polishing were performed by conventional anion-and cation-exchange chromatography, respectively.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single‐chain variable fragment

Sakhnini

Pedersen

León

et al. 2019

Engineering in Life Sciences

View full text Add to dashboard Cite

Single‐chain variable fragments (scFv) are widely used in several fields. However, they can be challenging to purify unless using expensive Protein L‐based affinity adsorbents or affinity tags. In this work, a purification process for a scFv using mixed‐mode (MM) chromatography was developed by design of experiments (DoE) and proteomics for host cell protein (HCP) quantification. Capture of scFv from human embryonic kidney 293 (HEK293) cell feedstocks was performed by hydrophobic charge induction chromatography (MEP HyperCel™), whereafter polishing was performed by anion hydrophobic MM chromatography (Capto Adhere™). The DoE designs of the polishing step included both binding and flow‐through modes, the latter being the standard mode for HCP removal. Chromatography with Capto Adhere™ in binding‐mode with elution by linear salt gradient at pH 7.5 resulted in optimal yield, purity and HCP reduction factor of 98.9 > 98.5%, and 14, respectively. Totally, 258 different HCPs were removed, corresponding to 84% of identified HCPs. The optimized conditions enabled binding of the scFv to Capto Adhere™ below its theoretical pI, while the majority of HCPs were in the flow‐through. Surface property maps indicated the presence of hydrophobic patches in close proximity to negatively charged patches that could potentially play a role in this unique selectivity.

show abstract

Section: Resultscontrasting

confidence: 99%

Section: Capture Of Scfv From Hek293 Feedstockcontrasting

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Optimizing selectivity of anion hydrophobic multimodal chromatography for purification of a single‐chain variable fragment

Sakhnini

Pedersen

León

et al. 2019

Engineering in Life Sciences

View full text Add to dashboard Cite

show abstract

“…A combination of these methods allowed the extraction of 70% of scFv from E. coli biomass [240]. Some physical, chemical, and biochemical methods are known, mainly from the isolation of the periplasmic fraction in a small scale.…”

Section: Downstream Processingmentioning

confidence: 99%

Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

View full text Add to dashboard Cite

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

show abstract