Non-chromatographic preparative purification of enhanced green fluorescent protein

Hekmat, Dariusch; Maslak, Dominik; Roman, Matthias Freiherr von; Breitschwerdt, Peter; Ströhle, Christoph; Vogt, Alexander; Berensmeier, Sonja; Weuster‐Botz, Dirk

doi:10.1016/j.jbiotec.2014.11.027

Cited by 9 publications

(2 citation statements)

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“…Current DSP of insulin production employ three times crystallization in the train, producing purest insulin with a purity higher than 98% [6]. In recent years, crystallization as a purification method for mAbs [27,28], digestion product of full-length antibody [29,30], and other recombinant proteins [31][32][33][34] have been reported.…”

Section: Continuous Crystallization To Enhance Crystal Size Distribut...mentioning

confidence: 99%

Improving the reproducibility of size distribution of protein crystals produced in continuous slug flow crystallizer operated at short residence time

Hadinoto

2021

Chemical Engineering Science

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Section: Continuous Crystallization To Enhance Crystal Size Distribut...mentioning

confidence: 99%

Improving the reproducibility of size distribution of protein crystals produced in continuous slug flow crystallizer operated at short residence time

Hadinoto

2021

Chemical Engineering Science

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Section: Continuous Crystallization To Enhance Crystal Size Distribut...mentioning

confidence: 99%

Advanced strategies to enhance the performances of crystallization and precipitation of biopharmaceuticals

Pu¹

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Recent years have seen significant growth in the production titer of upstream of biopharmaceuticals, a level that could lead to diminishing returns upon increasing due to the downstream processing (DSP) bottleneck. The capture and polishing steps, representing the most laborious and expensive aspects of the DSP, are currently mainly dominated by pack-bed chromatography. However, due to the high cost of adsorbent and the high operation expenditure of chromatography-based theology, anything but chromatography (ABC) methodologies started to gain interest in the field as alternative method for purifying biopharmaceuticals. Precipitation and crystallization are classic unit operations that can provide promising alternatives in DSP. The aim of this dissertation is to develop advanced crystallization and precipitation strategies to improve process efficiency and product quality of biopharmaceuticals using bioactive Van glycopeptide and protein LYZ as the model biopharmaceuticals In the first study, we investigated the phase behavior of salting-out roomtemperature crystallization of glycopeptide antibiotics to determine crystallization conditions (i.e., pH, salt and peptide's concentrations, incubation time) to avoid needle-shaped crystal formation. Batch crystallizations of the prominent crystal habits identified from the phase behavior study (i.e., needle and non-needle) were subsequently performed. The batch crystallization's products were evaluated in their production yield and capacity, purity, size distribution, thermal stability, interfacial water content, dissolution profile, and antibiotic activity. The results showed that octahedral crystals were the predominant habit produced across the range of pH, salt, and peptide concentrations investigated. Needle crystal formation could be avoided by precise controls of pH and salt concentration. Octahedral crystals were produced at a significantly higher yield than needle crystals. The octahedral and needle crystals exhibited many similar properties but with distinct thermal stability and dissolution profiles.The second study compared the antisolvent (with organic solvent) and salting-out methods in their precipitation efficiency and product qualities. The phase behavior study showed that heavy precipitates composed of nanoparticles were the

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Differential Precipitation and Solubilization of Proteins

Ryan¹

2016

Methods in Molecular Biology

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Summary/AbstractDifferential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physico-chemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g. His-Tag, Size Exclusion etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during over-expression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilised so as to reform functional, correctly folded proteins. Here, a typical protein extraction, precipitation and selective resolubilisation procedure is outlined, based on a recombinantly expressed protein.

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Non-chromatographic preparative purification of enhanced green fluorescent protein

Cited by 9 publications

References 32 publications

Improving the reproducibility of size distribution of protein crystals produced in continuous slug flow crystallizer operated at short residence time

Improving the reproducibility of size distribution of protein crystals produced in continuous slug flow crystallizer operated at short residence time

Advanced strategies to enhance the performances of crystallization and precipitation of biopharmaceuticals

Differential Precipitation and Solubilization of Proteins

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