Aqueous two-phase systems for protein separation: Phase separation and applications

Asenjo, Juan A.; Andrews, Bárbara A.

doi:10.1016/j.chroma.2012.03.049

Cited by 274 publications

(151 citation statements)

References 22 publications

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“…This liquid-liquid extraction (LLE) technology has been successfully reported in recent times as a primary stage unit operation in the downstream processing of several biological products including therapeutic proteins, such as antibodies [4,10,11,[15][16][17][18][19][20][21][22][23][24]. Its technical feasibility at large scale has been, however, demonstrated for the downstream processing of just few biological products [25][26][27]. This may be attributed, on one hand, to the limited knowledge of the mechanism of solute partitioning in ATPS, and in another hand, to the disadvantages of batch operations as well as to the difficulties associated with the implementation of ATPE processes in a continuous mode of operation [25].…”

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confidence: 99%

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

Rosa

Azevedo

Sommerfeld

et al. 2013

Biotechnology Journal

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An aqueous two‐phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back‐extraction, and washing) was set up and validated in a pump mixer‐settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155‐fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22‐fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.

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mentioning

confidence: 99%

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

Rosa

Azevedo

Sommerfeld

et al. 2013

Biotechnology Journal

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“…The addition of PEG to accelerate flocculent settling can also result in product precipitation, flocculent interaction, or product phase separation into the flocculent. 23,33 This mechanism of product loss can be circumvented by dispensing the flocculant and additive aid, or the conditioning of the cell broth prior to flocculation. Adjusting the cell broth pH or conductivity resulted in a significant decline in PDADMAC flocculation performance for all conditions tested (Fig.…”

Section: Discussionmentioning

confidence: 99%

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

McNerney

Thomas

Senczuk

et al. 2015

mAbs

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(2015) PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies, mAbs, 7:2, 413-427, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids ( 15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.

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“…98 ATPS has increased in popularity owing to its associated mild purification capabilities, and in addition, associated denaturation and loss of biological activity are rare. 100 Furthermore, the polymers used can promote protein stability, and the ability to separate the protein of interest in one step is highly attractive. 100 The two phases have different hydrophobic properties and hence, cellular debris, particulate matter, and contaminants such as BSA and HCPs tend to move to the lower, more polar phase, while the protein of interest moves to the upper, less polar layer, which is more hydrophobic.…”

Section: Aqueous Two-phase Partitioningmentioning

confidence: 99%

“…98 ATPS involves the formation of two immiscible liquid phases, which can be achieved when polymers such as PEG are mixed with dextran or other polymers or salts (eg, phosphate, sulfate, or citrate) under particular conditions. [98][99][100] The partitioning property of a protein depends on its surface properties (charge, hydrophilicity, and hydrophobicity) and on the physicochemical properties of the two liquid phases. 98 ATPS has increased in popularity owing to its associated mild purification capabilities, and in addition, associated denaturation and loss of biological activity are rare.…”

Section: Aqueous Two-phase Partitioningmentioning

confidence: 99%

Technology advancements in antibody purification

Murphy¹,

Devine²,

O’Kennedy³

2016

ANTI

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Abstract:The separation of antibodies from complex mixtures can be achieved using chromatographic or non-chromatographic techniques. The purification of antibodies using chromatography involves the separation of antibodies or antibody-derived molecules present in complex mixtures by passing them through a solid phase (eg, silica resin or beads, monolithic columns, or cellulose membranes) and allowing the antibodies to bind or pass through depending on whether "bind-and-elute" or "flow-through" chromatographic methods are employed. Chromatographic methodologies can incorporate different separation techniques, such as affinity-tag binding, ion-exchange, size-exclusion chromatography, or immunoaffinity chromatography. However, in a concerted effort to become less reliant on chromatography-based separations owing to the high cost of large-scale production, non-chromatographic-based techniques such as precipitation, flocculation, crystallization, filtration, and aqueous two-phase partitioning are now becoming more popular. This review details current chromatographic and non-chromatographic methodo logies used for the purification of antibodies and expands on technological advancements and practical uses that have recently been reported.

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Aqueous two-phase systems for protein separation: Phase separation and applications

Cited by 274 publications

References 22 publications

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

Continuous purification of antibodies from cell culture supernatant with aqueous two‐phase systems: From concept to process

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

Technology advancements in antibody purification

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