Solubilization of proteins in aqueous two-phase extraction through combinations of phase-formers and displacement agents

Kress, Christian; Sadowski, Gabriele; Brandenbusch, Christoph

doi:10.1016/j.ejpb.2016.11.016

Cited by 8 publications

(6 citation statements)

References 25 publications

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“…S2. The miscibility gap widened with addition of NaCl, which was also found in literature, , . This fact goes along with an increase of the PEG concentration in the PEG phase and the citrate concentration in the citrate phase while the tie line slope remains constant .…”

Section: Results Und Discussionsupporting

confidence: 83%

“…The miscibility gap widened with addition of NaCl, which was also found in literature, , . This fact goes along with an increase of the PEG concentration in the PEG phase and the citrate concentration in the citrate phase while the tie line slope remains constant . During a back‐extraction, the recycled PEG phase contains NaCl to which citrate phase without NaCl is added leading to an increase of the PEG phase volume and simultaneously a decrease of the citrate phase volume.…”

Section: Results Und Discussionsupporting

confidence: 76%

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Multistage Processing of Tunable Aqueous Polymer Phase Impregnated Resins (TAPPIR®)

et al. 2018

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A promising concept of aqueous two-phase extraction (ATPE) is the Tunable Aqueous Polymer Phase Impregnated Resins (TAPPIR ) technology. Thereby, one phase of an aqueous two-phase system (ATPS) is immobilized inside porous solids, which are then dispersed in the second aqueous phase. Beside extraction performance, the reusability of the impregnated solids for several cycles decides upon the competitiveness of TAPPIR . Multistage extraction experiments using a polyethylene glycol 4000/citrate ATPS to partition lysozyme were investigated for TAPPIR and ATPE. A stable impregnation was demonstrated for multiple cycles. Moreover, it was found that multistage extraction processes should not be designed on basis of the first extraction as the yield changes in the following stages.

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Section: Results Und Discussionsupporting

confidence: 83%

Section: Results Und Discussionsupporting

confidence: 76%

Multistage Processing of Tunable Aqueous Polymer Phase Impregnated Resins (TAPPIR®)

et al. 2018

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“…This behaviour has been ascribed to the ambivalent character of PEG400 as precipitant and stabilizer 35,47 . 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 .…”

Section: Resultsmentioning

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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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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“…Within ATPE, the partitioning of the target molecules is dictated by the selection of appropriate phase formers that create the respective ATPS . In order to tune the partitioning, a displacement agent, mostly a neutral salt, is often added to selectively displace the target biomolecule into the preferred phase while it is desired that the contaminants remain in the other phase (depending on their physio-chemical properties). ,,− Several groups investigated the influence of sodium chloride (NaCl) on salt-polyethylene glycol (PEG) ATPS and showed that a selective partitioning of the target biomolecule Immunoglobulin G (IgG) into the PEG-rich phase was possible, obtaining a yield of higher than 98.5%. , Besides NaCl, Kress et al (2016) investigated the influence of lithium bromide (LiBr) as a new displacement agent added to trisodium 2-hydroxypropane-1,2,3-tricarboxylate (trisodium citrate, (Na 3 Cit))–PEG2000 ATPS . LiBr allows for selective separation of IgG from the contamination human serum albumin (identified as a representative impurity) within Na 3 Cit–PEG2000 ATPS with an IgG yield of 74.6% …”

Section: Introductionmentioning

confidence: 99%

“…6,11,17−19 ATPS and showed that a selective partitioning of the target biomolecule Immunoglobulin G (IgG) into the PEG-rich phase was possible, obtaining a yield of higher than 98.5%. 18,20 Besides NaCl, Kress et al (2016) investigated the influence of lithium bromide (LiBr) as a new displacement agent added to trisodium 2-hydroxypropane-1,2,3-tricarboxylate (trisodium citrate, (Na 3 Cit))−PEG2000 ATPS. 19 LiBr allows for selective separation of IgG from the contamination human serum albumin (identified as a representative impurity) within Na 3 Cit−PEG2000 ATPS with an IgG yield of 74.6%.…”

Section: Introductionmentioning

confidence: 99%

Influence of Sodium Chloride and Lithium Bromide on the Phase Behavior of a Citrate–Polyethylene Glycol 2000 Aqueous Two-Phase System

2020

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Several studies have shown that aqueous two-phase extraction using an aqueous two-phase system (ATPS) is a promising purification technology for biomolecules (e.g., monoclonal antibodies). To beneficially influence the partitioning of the target biomolecule, a displacement agent can be added to the ATPS. Within this work, the influence of the displacement agents, sodium chloride (NaCl) and lithium bromide (LiBr), on the phase behavior of two ATPSs containing triammonium citrate and polyethylene glycol 2000 (PEG2000) and tripotassium citrate and PEG2000 was determined and compared to previous measurements for trisodium citrate−PEG2000 ATPS. Therefore, the tieline length (TLL), tie-line slope (TLS), and the size of the biphasic region of the ATPS were investigated at 298.15 K, 101.3 kPa, and pH 7. The results show that the addition of NaCl to the ATPS increases the TLL up to 47.4%, while the TLS stays constant. In contrast, the addition of LiBr causes a decrease in the TLS up to 34.5%, while the TLL remains constant. These results are in agreement with previous measurements performed for trisodium citrate−PEG2000 ATPS. Prospectively, the investigations enable a mechanistic understanding of the displacement agent effect on the phase behavior and transfer of this information to other organic salt−polymer ATPSs to improve their extraction performance.

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Solubilization of proteins in aqueous two-phase extraction through combinations of phase-formers and displacement agents

Cited by 8 publications

References 25 publications

Multistage Processing of Tunable Aqueous Polymer Phase Impregnated Resins (TAPPIR®)

Multistage Processing of Tunable Aqueous Polymer Phase Impregnated Resins (TAPPIR®)

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

Influence of Sodium Chloride and Lithium Bromide on the Phase Behavior of a Citrate–Polyethylene Glycol 2000 Aqueous Two-Phase System

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