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The development of a precipitation or crystallization step requires knowing the solubility of the target protein and its crystallization behavior in aqueous solutions at different pH, temperatures and in the presence of precipitating agents, especially salts. Within this work, a solubility model for proteins based on the second osmotic virial coefficient B 22 is developed. For this, a relation between protein solubility and B 22 was combined with the extended DLVO model. This solubility model was then used to model and also predict the protein solubility of lysozyme and monoclonal antibody for different salts, salt concentrations, and pH. The modeled as well predicted B 22 and protein solubility data of lysozyme in the presence of sodium chloride and sodium p-toluenesulfonate and of a monoclonal antibody in the presence of ammonium sulfate at different pH shows good agreement with experimental data. [ABSTRACT FROM AUTHOR]

A promising alternative to state of the art chromatographic separations of therapeutic proteins is the extraction of the target protein using an aqueous two-phase system ( ATPS). The use of an additional salt working as a displacement agent can influence the protein partitioning behavior in ATPS and thus enable a selective purification of the target protein. The selection of a suitable ATPS for protein extraction requires information concerning the protein-protein interactions (second osmotic virial coefficient B22) as well as the interactions between protein and solute (displacement agent and phase-forming components) (cross virial coefficient B23). In this work, the partitioning behavior and the precipitation affinity of immunoglobulin G (IgG) is considered within a polyethylene glycol ( PEG)-phosphate ATPS. The influence on IgG partitioning upon addition of NaCl and (NH4)2SO4 was investigated. In order to access the IgG precipitation affinity and the IgG partitioning behavior, the B22 and B23 values were determined for several combinations of solute [PEG, phosphate buffer, NaCl, and (NH4)2SO4] and IgG based on static light scattering measurements. A qualitative estimation of the IgG precipitation affinity and the suitability of a solute as potential displacement agent within the PEG-phosphate ATPS on the basis of the measured B22 and B23 values is presented. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3703-3709, 2015 [ABSTRACT FROM AUTHOR]

In this work the electrolyte perturbed chain statistical associating fluid theory (ePC-SAFT) is applied to model aqueous two-phase systems (ATPS) containing one of 6 different polymers and one of 8 different organic salts at temperatures between 278.15 K and 313.15 K. To accurately model the thermodynamic properties of organic-salt solutions, a novel modeling approach was applied, which accounts for the non-spherical shape of the anions. Applying this approach, 14 organic salt solutions have been modeled with an overall average relative deviation of 0.23% for solution densities and 1.51% for osmotic coefficients. The modeling of the polymers (PEG, PEGDME, PPG, and poly(ethylene glycol- co -propylene glycol)) has been carried out using a copolymer approach accounting for different molecular interactions of the polymer segments. Applying this approach, ATPS containing polymers and organic salts were modeled accurately. The overall absolute average deviation of the modeling with respect to the concentrations of the phase-forming components was 2.10 wt%. The influence of polymer molecular weight, polymer composition, kind of salt, pH, and temperature on the equilibrium composition and densities of the two phases was modeled correctly with ePC-SAFT. Moreover, it is shown that by applying ion-specific model parameters, ePC-SAFT is even capable of predicting ATPS containing salts which were not used for the parameter estimation. [ABSTRACT FROM AUTHOR]

Abstract: In this work the electrolyte perturbed chain statistical associating fluid theory (ePC-SAFT) was applied to model aqueous two-phase systems (ATPS) containing combinations of polyethylene glycol dimethyl ether (PEGDME), polypropylene glycol (PPG) and three poly(ethylene glycol-co-propylene glycol) copolymers in combination with 12 different inorganic salts for temperatures ranging from 278K to 333K. For the polymer modeling, a copolymer approach was applied splitting the polymer in different segment types accounting for their different molecular interactions. Using this approach allows for quantitative modeling of phase properties of aqueous homopolymer and copolymer solutions. ATPS consisting of water, salt and one out of the five polymers were successfully modeled by using only one binary interaction parameter between each polymer segment type and each ion. Applying these parameter set, the influence of (co)polymer composition, polymer molecular weight, and temperature on the phase composition and phase densities could be modeled accurately. [Copyright &y& Elsevier]

Abstract: In this work the ePC-SAFT equation of state is applied to model aqueous two-phase systems (ATPS) containing polyethylene glycol (PEG) and one of 16 different inorganic salts at temperatures between 277.15K and 333.15K. To ensure an accurate modeling of thermodynamic properties in PEG containing solutions, a novel modeling approach for PEG is applied considering different molecular interactions of PEG chain segments and PEG end-group segments. Applying this approach, the influence of PEG molecular weight, kind of salt, pH, as well as of temperature on the phase split as well as on the densities of the two phases can be modeled accurately. The overall absolute average deviation of the concentrations of the phase-forming components obtained by ePC-SAFT is 2.25wt%. Moreover, it could be shown that by applying ion-specific model parameters, ePC-SAFT is even capable of predicting ATPS which were not used for the parameter estimation. [Copyright &y& Elsevier]

The purification of therapeutic proteins is a challenging task with immediate need for optimization. Besides other techniques, aqueous 2-phase extraction (ATPE) of proteins has been shown to be a promising alternative to cost-intensive state-of-the-art chromatographic protein purification. Most likely, to enable a selective extraction, protein partitioning has to be influenced using a displacement agent to isolate the target protein from the impurities. In this work, a new displacement agent (lithium bromide [LiBr]) allowing for the selective separation of the target protein IgG from human serum albumin (represents the impurity) within a citrate–polyethylene glycol (PEG) ATPS is presented. In order to characterize the displacement suitability of LiBr on IgG, the mutual influence of LiBr and the phase formers on the aqueous 2-phase system (ATPS) and partitioning is investigated. Using osmotic virial coefficients ( B 22 and B 23 ) accessible by composition gradient multiangle light-scattering measurements, the precipitating effect of LiBr on both proteins and an estimation of both protein partition coefficients is estimated. The stabilizing effect of LiBr on both proteins was estimated based on B 22 and experimentally validated within the citrate–PEG ATPS. Our approach contributes to an efficient implementation of ATPE within the downstream processing development of therapeutic proteins. [ABSTRACT FROM AUTHOR]

The extraction of therapeutic proteins like monoclonal antibodies in aqueous two-phase systems (ATPS) is a suitable alternative to common cost intensive chromatographic purification steps within the downstream processing. Thereby the protein partitioning can be selectively changed using a displacement agent (additional salt) in order to allow for a successful purification of the target protein. Within this work a new shortcut strategy for the calculation of protein partition coefficients in polymer-salt ATPS is presented. The required protein-solute (phase-forming component, displacement agent) interactions are covered by the cross virial coefficient B 23 measured by composition gradient multi-angle light scattering (CG-MALS). Using this shortcut calculation allows for an efficient determination of the partition coefficients of the target protein immunoglobulin G ( IgG ) and the impurity human serum albumin ( HSA ) within PEG-citrate and PEG-phosphate ATPS independently on the protein concentration. We demonstrate that the selection of a suitable displacement agent allowing for a selective purification of IgG from HSA is accessible by B 23 . Based on the determination of the protein–protein interactions via CG-MALS covered by the second osmotic virial coefficient B 22 a further optimization of ATPS preventing protein precipitation is enabled. The results show that our approach contributes to an efficient downstream processing development. [ABSTRACT FROM AUTHOR]

The development of separation processes for polymers or proteins from aqueous solutions requires a high amount of experimental effort, including phase-equilibrium data such as solid-liquid, liquid-liquid or vapor-liquid equilibria. This effort can be reduced by means of thermodynamic models. This work presents a new method for parameter estimation and validation of thermodynamic models by means of static-light-scattering (SLS) measurements. In this work the Perturbed-Chain Statistical Associating Fluid Theory (PC-SAFT) was used to predict directly the SLS data (Rayleigh ratio) of macromolecular solutions. In a first step, SLS data were measured for binary water/polyethylene glycol (PEG molecular weight ranging from 2000 to 12000 g/mol) mixtures and for binary water/lysozyme mixtures. Applying pure-component PC-SAFT parameters from literature, the SLS data of these binary mixtures were successfully predicted with PC-SAFT. In a second step, one binary interaction parameter between lysozyme and PEG was adjusted to new experimental SLS data of buffered PEG/lysozyme/water solutions with PEG6000 concentration of 20 g/L. Finally, SLS data for buffered ternary PEG/lysozyme/water solutions with PEG of different molecular weights (2000–12000 g/mol) and different concentrations (1–50 g/L) were accurately predicted with PC-SAFT. Thus, (1) the proposed approach allows predicting SLS data, and (2) the method provides an access to the estimation of model parameters by means of experimental SLS data, which are accessible with much less effort than experimental phase-equilibrium data. [ABSTRACT FROM AUTHOR]

Reactive extraction has been shown as an applicable first step in the downstream processing for the recovery of dicarboxylic acids from aqueous solutions, leading to yields of X RE = 0.95 ± 0.05 for cis,cis -muconic acid. A next step towards a downstream processing concept is the examination of strategies to recover the dicarboxylic acid from the organic phase. A reasonable strategy has to lead to high yields and allow for a recycle of the organic phase for further reactive extraction steps. This work presents two recovery strategies for the cis,cis -muconic acid after reactive extraction. A pH-shift uses the strong pH dependency of the reactive extraction itself. A buffered aqueous phase as re-extraction phase leads to a yield of X REEX = 0.99 ± 0.080 at pH > 7. The second approach is the addition of water soluble amines as an additional reactive component. A complex of water soluble amines and the acid is re-extracted into an aqueous phase. Propylamine showed the best performance ( X REEX = 1 ± 0.069) of all water soluble amines investigated. An analysis of the distribution behavior of the water soluble amines showed that a recycle of the organic phase for further reactive extraction steps is feasible for both strategies. The results allow for a further development of an in situ downstream processing concept for biocatalytic produced dicarboxylic acids. [ABSTRACT FROM AUTHOR]