Interaction Between Biomolecules Studied by Phase Partition

Albertsson, Per‐Åke

doi:10.1002/9780470110492.ch1

Cited by 37 publications

(1 citation statement)

References 39 publications

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“…Some examples of recent preparative applications include: the continuous extraction of monoclonal antibodies using a multistage process [2], and the use of ATPS to improve the purification of Porcine Parvovirus Vaccine (PPV) in a PEGcitrate system [3]. Examples of analytical applications include the measurement of biomolecule interactions [4], the analysis of protein isoforms [5] and the pre-concentration of virus to improve assay detection limits [6].…”

Section: Introductionmentioning

confidence: 99%

Practical aspects of the automated preparation of aqueous two phase systems for the analysis of biological macromolecules

Hameed

Huddleston

Ignatova

2018

Journal of Chromatography B

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A robust strategy for the automated preparation of aqueous two-phase systems (ATPS) using a liquid handling sample processor was developed using gravimetric methods: to determine the accuracy of preparation. The major robotic control parameters requiring adjustment were; speed of aspiration and dispense; delay times following aspiration and dispense alongside measures to control cross-contamination during phase sampling. In general mixture compositions of both polymer/polymer and polymer/salt mixtures could be prepared with a target bias accuracy of less than 5%. However, we found that the bias accuracy with which systems of defined TLL and MR could be constructed was highly dependent on the tie line length of the ATPS and the geometrical form of the ATPS co-existence curve. For systems with a very low degree of curvature (PEG/salt systems here) increases in bias (accuracy) are appreciable at relatively long tie line lengths. Where the degree of curvature is more pronounced (PEG/dextran systems) closer approach to the critical point was possible without major effect on bias/accuracy. Application of the strategy to the measurement of the partitioning of phosphorylated and dephosphorylated forms of the model protein ovalbumin are reported. Differences in partition of phosphorylated (native) forms and dephosphorylated forms could be demonstrated. In a PEG/salt system this was manifest as a substantial decrease in solubility based on overall protein recovery derived from accurate knowledge of the system mass ratio. In a PEG/dextran system differences in partition coefficient could be demonstrated between phosphorylated and dephosphorylated forms.

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Section: Introductionmentioning

confidence: 99%

Practical aspects of the automated preparation of aqueous two phase systems for the analysis of biological macromolecules

Hameed

Huddleston

Ignatova

2018

Journal of Chromatography B

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Affinity partitioning: Development of mathematical model describing behavior of biomolecules in aqueous two‐phase systems

Cordes¹,

Floßdorf²,

Kula³

1987

Biotech & Bioengineering

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A mathematical model describing the affinity partitioning of macromolecules in aqueous two-phase systems has been derived. The model was used to calculate binding parameters that were compared against values deter mined by means of ultracentrifugation and fluorescence titration. The mathematical model and its modifications were found to describe satisfactorily the partition behavior of macromolecules with differing numbers of binding sites. It could be shown that in solutions containing PEG the binding behavior of FDH is changed fundamentally. The dissociation constants of FDH with PEG-blue in the presence and absence of PEG are different.

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Affinity partitioning of vancomycin

Godbole

Tsai

Clark

1991

Biotech & Bioengineering

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The use of the Flanagan and Barondes model(14) describing affinity partitioning as an aid in designing separation systems is discussed. Experimental studies are described for affinity partitioning of vancomycin, a glycopeptide antibiotic, in a water-methoxypolyethylene glycol-dextran system using methoxypolyethylene glycol-dextran system using methoxypolyethylene glycol bound D-alanyl-Dalanyl-D-alanine or D-alanyl-D-alanine as the reversible affinity ligand. Even for this ideal case of 1:1 binding interaction, the model only qualitatively predicts the affinity effect when all model parameters are measured independently. The discrepancy between measured and predicted values can be attributed to a difference in exposed surface of the free antibiotic and ligand compared to that in the bound state.The effect of experimentally varying model parameters is also described. It was determined that a polymers-ligand which partitions more strongly to the top phase would provide the most significant enhancement to this affinity partitioning system. Such an improvement can be made by increasing the molecular weight of the hydrophobicity of the polymer-ligand. A process for vancomycin recovery from fermentation broth using D--alanyl-D-alanine sepharose as affinity ligand is described.

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Interaction Between Biomolecules Studied by Phase Partition

Cited by 37 publications

References 39 publications

Practical aspects of the automated preparation of aqueous two phase systems for the analysis of biological macromolecules

Practical aspects of the automated preparation of aqueous two phase systems for the analysis of biological macromolecules

Affinity partitioning: Development of mathematical model describing behavior of biomolecules in aqueous two‐phase systems

Affinity partitioning of vancomycin

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