Affinity partitioning of proteins tagged with choline-binding modules in aqueous two-phase systems

Maestro, Beatríz; Velasco, Isabel; Castillejo, Isabel; Arévalo-Rodrı́guez, Miguel; Cebolla, Ángel; Sanz, Jesús M.

doi:10.1016/j.chroma.2008.08.106

Cited by 31 publications

(18 citation statements)

References 37 publications

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“…One advantage of the recombinant approach is that relatively small MW affinity tags of a few amino acids can be constructed which provide for enhanced partition, as well as affinity, ion‐exchange or hydrophobic interaction chromatography performance [43]. A fourth affinity possibility is chemically modifying the target molecule to adjust its partition behavior [44, 45]. All of the above affinity approaches have advantages and disadvantages in regard to the need to produce generic (based on charge or hydrophobic interactions for example) or specific affinity ligands, and isolate affinity ligands from targets downstream.…”

Section: Strengths – Advantages Of Atpss For Downstream Processingmentioning

confidence: 99%

“…There are many other emergent ATPS‐based purification strategies that, while still not being ready for industrial application, show great potential for future industrial implementation. Examples include: (i) the combination of a magnetic separation and ATPS, allowing a dual‐step purification after exposing the collected top phase to a magnetic field [99–101]; (ii) the continuous extraction of fermentation products in extractive fermentation [102–106]; (iii) use of recombinant peptide fusions to enhance ATPS partition [38, 45, 107, 108]; and (iv) simultaneous extraction and purification of biomolecules from solid matrices [109].…”

Section: Opportunities – Atps Partitioning In Relation To Other Modmentioning

confidence: 99%

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Partitioning in aqueous two‐phase systems: Analysis of strengths, weaknesses, opportunities and threats

Soares

Azevedo

Alstine

et al. 2015

Biotechnology Journal

131

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For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.

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Section: Strengths – Advantages Of Atpss For Downstream Processingmentioning

confidence: 99%

Section: Opportunities – Atps Partitioning In Relation To Other Modmentioning

confidence: 99%

Partitioning in aqueous two‐phase systems: Analysis of strengths, weaknesses, opportunities and threats

Soares

Azevedo

Alstine

et al. 2015

Biotechnology Journal

131

View full text Add to dashboard Cite

show abstract

“…ATPSs composed of (polymer + salt + water) ternary systems have been widely used for the partitioning and purification of proteins, human antibodies, enzymes, and other biological macromolecules [1][2][3][4][5][6][7]. Also, Greve and Kula [8] have used the alcohol-saltwater ATPSs to extract the salt from (polymer + salt + water) protein extraction systems to minimize the environmental pollution as well as to economize on the use of chemicals.…”

Section: Introductionmentioning

confidence: 99%

Thermodynamic investigation of the ATPSs composed of some (aliphatic alcohol+sodium carbonate+water) ternary systems

Nemati‐Kande¹,

Shekaari²

2013

The Journal of Chemical Thermodynamics

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“…This feature has been explored in different works for the purification of LYTAG‐tagged proteins using PEG two‐phase systems. Indeed, Maestro and collaborators showed that LYTAG binding to PEG molecules can be used to accumulate the tagged protein in the PEG‐rich phase, whereas addition of choline reverses this interaction and directs the protein to the PEG‐poor phase . The affinity of LYTAG for choline had been explored previously as an affinity tag for the purification of tagged‐proteins by adsorption to amine‐containing chromatographic resins (e.g.…”

Section: Introductionmentioning

confidence: 99%

LYTAG‐driven purification strategies for monoclonal antibodies using quaternary amine ligands as affinity matrices

Campos-Pinto

Capela

Silva-Santos

et al. 2017

J of Chemical Tech & Biotech

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BACKGROUND: Monoclonal antibodies are becoming a leading class of biopharmaceuticals but to increase their accessibility by the general population, new production processes must be developed in particular for the downstream processing. RESULTS: In this work, an alternative and innovative affinity chromatographic method using quaternary amine matrices is proposed. Separation is driven by the dual affinity ligand LYTAG-Z, composed of a choline binding polypeptide (LYTAG) and the synthetic antibody binding Z domain. A two-elution method was developed for the purification of mAbs and the performance of different anion exchangers containing quaternary amines that act as choline analogues -CIMmultus Q, Q Sepharose and gPore Q -were tested and compared, with both CIMmultus Q and Q Sepharose allowing a recovery of more than 94% of mAbs from a CHO cell supernatant with a purity greater than 95%.An integrated platform combining an initial affinity extraction step for the clarification and capture of mAbs and a subsequent chromatographic separation using Q-matrices for the polishing of mAbs is also proposed. LYTAG-Z triggers the extraction of 94.7 ± 1.7% mAbs to the PEG-rich phase, as opposed to 26.9 ± 0.6% in the absence of the ligand, using 7% PEG 3350 and 6% dextran 500 k. Further purification using Q Sepharose allowed a mAb recovery of 95.3 ± 1.4% with a purity level of 91.4 ± 13.0%. CONCLUSION: An integrated platform based on two purification steps -affinity extraction and affinity chromatography -results in an overall process yield of 90%, allowing the processing of mAbs directly from a non-clarified CHO cell culture. ATPS formulationPEG/dextran aqueous two-phase systems consisting of 7% (w/w) PEG 3350 Da and 6% (w/w) dextran 500 kDa were prepared by weighing the appropriate amounts of each component, namely PEG from stock solutions of 50% (w/w), dextran from a stock solution of 25% (w/w), biological sample and water, to a final weight of 10 g.The biological sample (pure human serum antibodies or CHO cell culture supernatant) was loaded at 35% (w/w). The LYTAG-Z ligand was loaded to the systems at double the mass concentration of the antibody (to a concentration of 100 mg L -1 of antibody, the concentration of LYTAG-Z was 200 mg L -1 ), to assure a molar ratio /jctb CHO genomic DNA quantification CHO genomic DNA (gDNA) content was determined by Quant-iT™ PicoGreen ® dsDNA reagent. Genomic DNA standards were prepared by serial dilution in elution buffer of genomic DNA extracted wileyonlinelibrary.com/jctb © 2017 Society of Chemical Industry J Chem Technol Biotechnol 2018; 93: [1966][1967][1968][1969][1970][1971][1972][1973][1974] The chromatographic profiles obtained with both convective flow devices are present in Fig. 3(A), and are very similar to the one obtained with Q Sepharose (in Fig. 2(A)), with (i) a large peak in the FT corresponding to most impurity proteins, (ii) small elution peaks obtained during low pH elution, and (iii) a larger peak

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Affinity partitioning of proteins tagged with choline-binding modules in aqueous two-phase systems

Cited by 31 publications

References 37 publications

Partitioning in aqueous two‐phase systems: Analysis of strengths, weaknesses, opportunities and threats

Partitioning in aqueous two‐phase systems: Analysis of strengths, weaknesses, opportunities and threats

Thermodynamic investigation of the ATPSs composed of some (aliphatic alcohol+sodium carbonate+water) ternary systems

LYTAG‐driven purification strategies for monoclonal antibodies using quaternary amine ligands as affinity matrices

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