Osmotic Virial Coefficients as Access to the Protein Partitioning in Aqueous Two-Phase Systems

Kress, Christian; Brandenbusch, Christoph

doi:10.1002/jps.24602

Cited by 12 publications

(5 citation statements)

References 21 publications

(49 reference statements)

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“…Kress et al 48 observed the decrease in B 22 of an immunoglobulin G (IgG) when increasing PEG2000, sodium citrate and NaCl solute concentration, if used separately. However, the mutual effect of PEG and salts resulted in a shift of B 22 from negative to almost neutral values when increasing the NaCl amount if used together 32 . This behaviour has been explained by a solubilizing effect of IgG in PEG2000 induced by NaCl until a maximum salt concentration.…”

Section: Resultsmentioning

confidence: 98%

“…Protein partitioning measurements. Anti-CD20 repartition between aqueous and PEG phases has been estimated by the method described by Kress et al 32 . A solution (5 mL) containing Na 2 SO 4 1.1 M (13.54 wt.%) and PEG400 10.46% V/V (10.22 wt.%) in HEPES 0.1 M at pH 7.4, was added with 200 μL Anti-CD20 solution 10.3 mg mL −1 in the same buffer.…”

Section: Methodsmentioning

confidence: 99%

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On the Aggregation and Nucleation Mechanism of the Monoclonal Antibody Anti-CD20 Near Liquid-Liquid Phase Separation (LLPS)

Pantuso

Mastropietro

Briuglia

et al. 2020

Sci Rep

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The crystallization of Anti-CD20, a full-length monoclonal antibody, has been studied in the PEG400/ na 2 So 4 /Water system near Liquid-Liquid Phase Separation (LLPS) conditions by both sitting-drop vapour diffusion and batch methods. In order to understand the Anti-CD20 crystallization propensity in the solvent system of different compositions, we investigated some measurable parameters, normally used to assess protein conformational and colloidal stability in solution, with the aim to understand the aggregation mechanism of this complex biomacromolecule. We propose that under crystallization conditions a minor population of specifically aggregated protein molecules are present. While this minor species hardly contributes to the measured average solution behaviour, it induces and promotes crystal formation. The existence of this minor species is the result of the LLPS occurring concomitantly under crystallization conditions. A thorough understanding of protein aggregation behaviour in solution is crucial for the controlled manufacture and formulation of therapeutics and for the development of crystallization-based purification strategies for biological drugs 1-4. Unfortunately, while the state diagram of protein-solvent systems can provide indications about the phase behaviour of biomacromolecules and their stability in solution, the intimate mechanism underlying protein nucleation and crystal growth have not been fully understood yet and, in many cases, it remains elusive. Protein aggregation is directly related to protein-protein interactions and may be strongly related to protein crystallization behaviour 5. Since different types of intermolecular forces are involved in protein interactions, such as electrostatic, van der Waals and hydrophobic 6 , environmental factors like pH, ionic strength and the presence of additives, can drastically alter the intermolecular interaction profile of proteins in solution 7,8. Colloidal and conformational stabilities are the two main factors that govern the existence of a protein aggregate in a solution 9,10. Colloidal stability is a delicate balance between repulsive and attractive forces between proteins, while conformational stability is related to the free energy difference between two molecular states, folded and partially or totally unfolded. Several investigations have found that the melting temperature (T m) and the osmotic second virial coefficient (B 22) can respectively account for the protein conformational 11 and colloidal 12 stability. Both T m and B 22 can be experimentally determined by using correspondingly Dynamic Light Scattering (DLS) and Static Light Scattering (SLS) methods 13. Aggregation induced by thermal unfolding can be monitored by measuring the average hydrodynamic diameter value as a function of temperature, while B 22 describes the first deviation from ideal behaviour of dilute colloidal solutions 14. A positive or negative value of B 22 accounts for a net repulsive or attractive interaction balance, respectively, and could therefore be used t...

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Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

On the Aggregation and Nucleation Mechanism of the Monoclonal Antibody Anti-CD20 Near Liquid-Liquid Phase Separation (LLPS)

Pantuso

Mastropietro

Briuglia

et al. 2020

Sci Rep

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“…In literature, several modeling approaches for the phase behavior of ATPS are suggested as the application of osmotic virial coefficient. , The application of virial coefficients in the model approach has the advantage that proteins can be included in the model. Furthermore, Gibbs Excess Energy models , or equations of state (EOS) , can be applied to model the phase behavior of ATPS.…”

Section: Introductionmentioning

confidence: 99%

Polymeric Aqueous Two Phase System with an Immobilized Phase by Cross-Linking

Fischlschweiger

Zeiner

2022

Ind. Eng. Chem. Res.

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This work studies the modeling of polymeric aqueous two-phase systems (ATPS) with an immobilized phase. As the model system, the ATPS composed of polyethylene glycol (PEG) and Dextran was analyzed, whereas Dextran was immobilized. The model approach is based on the Koningsveldt–Kleintjens model forming the backbone of the swelling model. To model the ATPS with the immobilized phase, a novel incompressible elastic term is introduced considering the elastic limit of the dextran chains. At first, the ATPS with free PEG and dextran chains was modeled with the Koningsveldt-Kleintjens model and a good accordance to the experimental data was found. Afterward, the Koningsveldt-Kleintjens model was combined with the developed elastic term. A sensitivity analysis of the model was conducted to identify the important model parameters. To verify the model, experiments were performed to analyze the swelling of the immobilized dextran and the sorption of the amino acid l-serine in the immobilized phase. Thereby, the swelling and the sorption of l-serine in the immobilized phase could be modeled in good accordance to the experimental data.

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“…With a wide range of ILs able to form ABS and the complex nature of proteins, it is problematic to generalize and predict the behavior of proteins using these systems, and thus, it is necessary to establish well‐known models that will help the understanding of those mechanisms. Currently, there are mathematical and computational models that can help to predict some aspects of the formation and partitioning process of ABS, such as regression analysis, molecular dynamics (MD) simulations, conductor like screening model for real solvents (COSMO‐RS) and perturbed chain‐statistical associating fluid theory (PC‐SAFT) . As example, the partitioning behavior of two amino acids (tyrosine and leucine) using IL‐based ABS was successfully predicted using regression analysis with information of ABS characteristics and quantitative structure activity relationship (QSAR) of three other amino acids (tryptophan, phenylalanine and valine) .…”

Section: Introductionmentioning

confidence: 99%

“…As example, the partitioning behavior of two amino acids (tyrosine and leucine) using IL‐based ABS was successfully predicted using regression analysis with information of ABS characteristics and quantitative structure activity relationship (QSAR) of three other amino acids (tryptophan, phenylalanine and valine) . The use of osmotic virial coefficients as access to the protein (immunoglobulin G, IgG) partitioning in PEG‐phosphate ABS (without IL as phase forming agent) was also demonstrated, and although this approach has not yet been applied to systems composed of ILs, it seems to be a feasible option to give further insights about the mechanisms of protein partitioning in systems with ILs. MD simulations are also applied in the study of IL‐based ABS formation, providing further insights to the mutual solubilities of ILs, salts and polymers, and then the main interactions governing the phase demixing at molecular level .…”

Section: Introductionmentioning

confidence: 99%

Liquid–liquid extraction of biopharmaceuticals from fermented broth: trends and future prospects

Santos

Santos-Ebinuma

Pessoa

et al. 2017

J of Chemical Tech & Biotech

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Biopharmaceuticals are one of the most important groups of biotechnological products, representing one-quarter of all pharmaceutical sales and providing suitable and efficient medical care for many previously untreatable diseases. However, their production and purification usually require complex processes, resulting in extremely expensive final products. Thus, the development of newer, simpler and cheaper methods for biosynthesis and purification of these biocompounds, as well as proper integration between these stages, are crucial to reduce their commercial costs and to increase the scale of biopharmaceuticals' medical use. One solution for this concern relies on the proper integration between upstream and downstream processing applying liquid-liquid extraction systems as an intermediate stage. Recently, several works reported that a proper choice of liquid-liquid systems such as aqueous biphasic systems (ABS) can be a suitable platform to simplify, reduce the number of the downstream stages, improve the recovery and purification yields and, consequently, decrease biopharmaceuticals' manufacturing costs. This review will explain the general concept of biopharmaceuticals, their main production and purification methods, particularly, exploring the use of ABS as effective and integrative platforms for their recovery and purification, and providing further insights into the future trends and prospects in the field.

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Osmotic Virial Coefficients as Access to the Protein Partitioning in Aqueous Two-Phase Systems

Cited by 12 publications

References 21 publications

On the Aggregation and Nucleation Mechanism of the Monoclonal Antibody Anti-CD20 Near Liquid-Liquid Phase Separation (LLPS)

On the Aggregation and Nucleation Mechanism of the Monoclonal Antibody Anti-CD20 Near Liquid-Liquid Phase Separation (LLPS)

Polymeric Aqueous Two Phase System with an Immobilized Phase by Cross-Linking

Liquid–liquid extraction of biopharmaceuticals from fermented broth: trends and future prospects

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