Microcalorimetric Study of the Adsorption of PEGylated Lysozyme and PEG on a Mildly Hydrophobic Resin: Influence of Ammonium Sulfate

Engineering in Life Sciences

2014

Self Cite

Interaction of macromolecules in aqueous salt‐containing solution with a hydrophobic adsorbent is studied by adsorption equilibrium measurements and by independent isothermal titration calorimetry. The macromolecules are native as well as mono‐, di‐, and tri‐PEGylated lysozyme and four pure PEGs. The hydrophobic adsorbent is Toyopearl PPG‐600M. The salt is sodium chloride. The sodium chloride concentration in the aqueous 25 mM sodium phosphate buffer is varied from 2000 to 4500 mM at pH 7.0 and 25°C. PEGylation of the lysozyme is carried using 5 and 10 kDa PEG chains. The molar enthalpy of adsorption Δhp ads is calculated from the adsorption equilibrium and the calorimetric data. The results show that the adsorption of the PEGylated lysozyme is caused by both the interaction of the lysozyme and the interaction of the PEG chains with the adsorbent, respectively, but the interaction of the lysozyme is stronger than that of PEG. The comparison of the results of the present study on the influence of sodium chloride with a corresponding study on the influence of ammonium sulfate shows that the adsorption mechanism changes upon the variation of the salt. The knowledge of the adsorption mechanisms supports the systematic development of chromatographic purification steps.

Section: Resultssupporting

confidence: 93%

Section: Discussionsupporting

confidence: 51%

Section: Resultsmentioning

confidence: 62%

“…The release of water from the hydration shells of the hydrophobic resin and the PEGylated lysozymes is endothermic. This water release also results in an increase in entropy .…”

Section: Resultsmentioning

confidence: 99%

“…From the molar enthalpy of adsorption

Δ h_{p}^{ads}

, the molar enthalpy of adsorption at infinite dilution

Δ h_{p}^{ads \infty}

can be obtained from an extrapolation of the data according to . The results, shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

Influence of sodium chloride on hydrophobic adsorption of PEGylated lysozyme

Werner

Blaschke

Engineering in Life Sciences

2014

Self Cite

Influence of mixed electrolytes on the adsorption of lysozyme, PEG, and PEGylated lysozyme on a hydrophobic interaction chromatography resin

Hackemann

Werner

2017

Biotechnology Progress

Self Cite

In a recent work (Werner A and Hasse H in J Chromatogr A 2013;1315:135) the influence of mixed electrolytes on the adsorption of the macromolecules lysozyme, PEG and di-PEGylated lysozyme on a hydrophobic resin has been studied, but only at one overall ionic strength (3000 mM). The present work, therefore, extends these studies to other ionic strengths (2400 and 2700 mM), and explores the application of a model to predict the entire data set. The adsorbent is Toyopearl PPG-600M. The solvent is a 25 mM aqueous sodium phosphate buffer at pH 7.0. The studied salts are sodium chloride, sodium sulfate, ammonium chloride and ammonium sulfate. Pure salts as well as binary and ternary mixtures of these salts with varying ratios of the amounts of the salts are studied at 25 °C. The loading of the adsorbent increases with increasing salt concentration for all macromolecules. Synergetic effects of the mixed electrolytes are observed. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1104-1115, 2017.

Mathematical modeling of adsorption isotherms in mixed salt systems in hydrophobic interaction chromatography

Hackemann

2018

Biotechnology Progress

Self Cite

Hydrophobic interaction chromatography (HIC) is often used for purifying proteins. The prediction of the adsorption equilibria in such systems is a difficult task as they depend on the pH value and the influence of the salts, which are added for controlling the solvent properties. This is especially tedious when salt mixtures are used, which can have positive or negative cooperative effects on the adsorption. A mathematical model to describe these complex effects of the salts was recently introduced by our group. In that model, the influence of the salts is described by a Taylor series expansion in the individual ion molarities. However, in our previous work, only the loading of the adsorbent at a given constant protein concentration in the liquid phase c = 0.01 mM was considered and correlated as a function of the salt composition at constant ionic strength. In the present study, that model is extended to other protein concentrations and it is shown that the Taylor series expansion works well for a wide range of c . The model parameters are found to depend on c , but only in a simple way. The dependence of the model parameters on c was correlated using a new three parameter equation. From that correlation, the loading q can be determined for any value of c . Hence, the entire adsorption isotherm is known for any salt composition. The approach is illustrated using experimental data from previous studies of our group on the adsorption of bovine serum albumin, lysozyme, polyethylene glycol, and di-PEGylated lysozyme on the mildly hydrophobic resin Toyopearl PPG-600 M. The pH values are between 4.0 and 7.0. Binary and ternary mixtures of ammonium chloride, sodium chloride, ammonium sulfate, and sodium sulfate as well as the single salts are used at overall ionic strengths between 1,500 and 4,200 mM. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018.