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Year : 2018  |  Volume : 15  |  Issue : 2  |  Page : 107-117

Model of protein isoforms analysis by aqueous two-phase systems: Methodology importance in clinical biochemistry and biopharmaceutical production

Advanced Bioprocessing Centre, Institute for Environmental Health and Societies, Brunel University, London, United Kingdom

Date of Web Publication21-Jun-2018

Correspondence Address:
Rana M Hameed
Advanced Bioprocessing Centre, Institute for Environmental Health and Societies, Brunel University, London
United Kingdom
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/MJBL.MJBL_33_18

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Aqueous phase partitioning has a long history of applications to the analytical characterization of biomolecules. However, process applications have attracted the most interest in biotechnology where it has become widely recognized as a cost-effective technique. The application of aqueous two-phase systems (ATPSs) has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. This type of separation and purification system has also been successfully used for the separation of virus and virus-like particles. The advantage of this technique is that it may be used to monitor the aforementioned changes for purified proteins as well as for proteins in biological fluids, and that it is readily adaptable to automated high-throughput screening. However, the wide use of this technology has been diminished by the lack of a clear understanding of the factors and mechanisms that govern the behavior of proteins in these systems. It has prevented the development of analytical models that assist the rational design of these systems. This work has revised the development of ATPSs (preparation and sampling techniques), and also highlighted the knowledge gap in the ATPS.

Keywords: Application, aqueous two-phase system, biopharmaceutical production, clinical biochemistry, protein purification

How to cite this article:
Hameed RM. Model of protein isoforms analysis by aqueous two-phase systems: Methodology importance in clinical biochemistry and biopharmaceutical production. Med J Babylon 2018;15:107-17

How to cite this URL:
Hameed RM. Model of protein isoforms analysis by aqueous two-phase systems: Methodology importance in clinical biochemistry and biopharmaceutical production. Med J Babylon [serial online] 2018 [cited 2021 Jan 19];15:107-17. Available from: https://www.medjbabylon.org/text.asp?2018/15/2/107/234853

  Posttranslation Modifications of Proteins Top

Proteins are the most important biomolecules, playing an essential role in all biological processes. The genetic code, in the form of a sequence of nucleotides making up the chemical composition of DNA in any cell through a sequence of events termed transcription and translation, is finally expressed as the amino acid sequence of proteins. Proteins are involved in all essential cellular processes such as enzymes and structural components. The encoded information in DNA is first transferred to RNA in the form of messenger RNA (m-RNA) in a process known as transcription by complementary base pairing involving the enzyme RNA polymerase. Following transcription, the m-RNA is processed by ribosomes into the amino acid sequence comprising the protein backbone in a process known as translation. All proteins are made up of a sequence of amino acids linked together by peptide bonds. The unique sequence of the amino acid chain forms the primary structure of the protein. The unbranched polypeptide chains of amino acids are folded in different patterns to form the secondary structure linked by hydrogen bonds between C = O and H-N as for instance in α-helical and β-sheet structures.[1]

The protein has three-dimensional structures called tertiary structure that results from the folding of whole chains (secondary structure) into more compact structures driven by noncovalent interactions such as the burial of hydrophobic regions away from the aqueous environment (although membrane-bound proteins differ in this respect) but also involving specific interactions such as salt bridges, hydrogen bonds, and disulfide bonds. Quaternary structures may be formed when the protein is made up of multiple polypeptide chains (which could be homo- or heterodimer subunits) and/or with an inorganic compound (such as a heme group). The life cycle of the proteins is listed in [Figure 1]. Proteins have a diversity functions such as transport, storage, and catalysis and also have multiple functional groups such as thiols, amines, carboxyl, and many others.[1] Post-translation Modifications of the basic protein structure outlined above can occur during and after the “synthesis cycle” of the protein, i.e. during and after translation; the synthesis of protein on the ribosome from the mRNA template. These modifications result in changes in the conformation of the final protein and in its structure, function, and activity. Some proteins are modified shortly after translation, some are exposed to cleavage or addition of functional groups, and other modifications occur during protein maturation; furthermore, modifications may also arise during disease and during the manufacture of proteins, for instance, in the manufacture of therapeutic proteins.[1]
Figure 1: The synthesis cycle of the protein

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Posttranslational modifications (PTMs) of proteins are in general of two types involving either addition or cleavage of one or more groups at specific sites or links in the polypeptide chain. In addition, modifications may be reversible according to the nature such as in the catalytic activation or inactivation of the proteins, for example, phosphorylation, or it could be irreversible such as in targeting for lysosomal destruction. PTMs include N- and C-terminal modifications (acetylation, amidation, and methylation) and modification of individual side chains (phosphorylation, glycosylation, carbonylation, hydroxylation, and nitration).[1]

Briefly, the importance of the former PTMs as biomarkers in disease can be summarized: acetylation is one of the most common modifications of proteins and involves the introduction of an acetyl group by modification of the lysine residue. Acetylation of histone proteins has a significant effect on the regulation of the various transcription factors and cellular metabolism. Acetylation normally occurs as N-terminal modifications and it has been linked to many cardiovascular and neurological diseases, while C-terminal modifications generally occur through amidation. Both acetylation and amidation have an impact on the overall charge of the peptide and consequently on the solubility, stability, and biological activity of the proteins. Methylation of N-terminal amino groups is rare, but it has a remarkable influence in some diseases such as in the inflammatory and immune system response.[2]

In addition, modification by carbonylation and nitration has a tremendous impact on the formation of oxidative reactions and it is involved in many disorders, for instance, lung and cardiac diseases, cancer, and neurodegenerative diseases. Similarly, hydroxylation also has a significant relevance to the cellular physiology properties. Modification of proline by hydroxylation has an important effect on the activation of antioxidant defense that responds to decreases in available oxygen in the cellular environment.[2]

In bioprocessing, the PTMs have a vital role in biopharmaceutical manufacture, in manufacture of drug therapies, and additionally in drug response and disease diagnostics where the PTM could be used as a disease biomarker.[2] The common protein bioprocessing modifications include proteins folding and aggregation. Protein folding is most challenging in the production of therapeutic proteins. It results in accumulation of the protein or even folding to dimer, trimer, or high-molecular-weight (MW) forms. In general, protein folding may occur during the overloading of processes. However, the aggregation may result from reversible/irreversible reactions, hydrophobic interactions, and formation of covalent bonds between unpaired thiols. Another bioprocessing modification is the oxidation of some methionine residues which may be involved in loss of activity, for instance, oxidative damage in the case of α1-antitrypsin (which is used for the treatment of emphysema). In addition, the deamidation of asparagine residues is an isomerization modification which can result from long-term storage. This modification is involved in the formation of isoaspartate which has significant implications in reducing antibody reactivity and changing the activity of stem cell factors.[3]

The existence of PTMs has wide-ranging implications in many different sectors such as the development of cell based therapies, production of biotherapeutics, drug target proteins, and for the optimization of the quality and efficacy of bioprocesses.

Significant PTMs include phosphorylation and glycosylation which play a crucial role in the regulation of protein activity, stability, and function and may be strongly involved in biological processes and disease conditions.[2]

Phosphorylation is one of the most common reversible protein modifications which have an important effect on the solubility of proteins and in the regulation of many cellular processes including cell cycle, growth, and signal transduction pathways. The process of protein phosphorylation involves the donation of phosphate groups by Adenosine triphosphate (ATP) to specific amino acids having a hydroxylated side chain (serine, threonine, or tyrosine). The process is controlled by a protein kinase while dephosphorylation (removal of the phosphate group) is mediated by a phosphatase.

Several methods have been used to assess the protein phosphorylation; according to the previous studies, acrylamide-pendant Phos-tagTM ligand has been successfully used for the detection of phosphorylated proteins.[4]

Another method for the determination of phosphate content of the phosphorylated proteins is reagent-free ion chromatography. The method has been applied to the determination of phosphorylation of ovalbumin.[5]

A further chromogenic method named GelCode has been used to analyze gel-separated proteins that are phosphorylated at serine and/or threonine residues.[6]

Other studies have been carried out to develop a fluorescent-based phosphoprotein detection method for detecting phosphoproteins in polyacrylamide gels. This is based on immobilized metal ion affinity chromatography; it employs quercetin–aluminum (III)-appended complex as a fluoroprobe to selectively visualize phosphorylated proteins among total proteins.[7]

Glycosylation involves the modification of protein amino acid side chains by the addition of a carbohydrate moiety. The effect of the glycosylation of proteins is considered to be one of the most significant modifications which are important in protein folding, conformation, and stability.[1] Glycosylation is of increasing significance in bioprocessing as the type and extent of glycosylation depend on the organism in which the protein is expressed during heterologous expression and the fidelity of glycosylation may be highly significant in determining efficacy and potency.[8] On the other hand, glycation although not strictly glycosylation involves the linkage of a reducing sugar residue to the amino side chains of the N-terminus and residues such as lysine and histidine. This modification mostly happens during hyperglycemia in diabetes and results in various complications and the formation of less-functional biomolecules.[9]

Glycosylation is classified according the amino acid to which the carbohydrate is attached. N-linked glycosylation refers to the attachment of the carbohydrate moiety to the nitrogen atom of the imide group, usually the N- of an asparagine residue as shown in [Figure 2]. In bioprocessing, if the glycosylation pattern does not match the human native glycosylation, the therapeutic protein may be rejected by the immune system and may fail to reach the target tissue or result in an undesirable immune response.[8] N-linked glycosylation can be presented as widely different structures resulting in several kinds of linkages, for instance, the N-acetylneuraminic acid could be linked to either C3 or C6 of the galactose and produce a very different glycoprotein structure.
Figure 2: General types of N- glycan in mature glycoproteins[10]

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Other glycosylation classes are an O-linked glycosylation, which involves linkage to those amino acids containing a hydroxyl functional group that is linked to the additional carbohydrate, usually through the O- of the serine or threonine residue. Another form of glycosylation is C-linked glycosylation which refers to a different type of glycosylation since its represents the reaction of carbon–carbon bonds where the carbohydrate is usually linked to the C- of the indole ring of tryptophan.

Characterization of glycosylation is very complex due to differences in the level of glycosylation, for instance, the variety in the number and type of glycan, the type of the glycosylation class, and the site where the glycan is placed. In general, analysis of protein glycosylation could be studied by different methods as summarized in [Figure 3].
Figure 3: Summary of methods for analysis of protein glycosylation[11]

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Many methods are available to separate and detect protein structure relying on differences in their characteristic. These methods might maintain the native conformation of the protein or might not. However, there is no specific method which could be used to study the structure of all proteins.

The common methods employed in protein purification are concentrated salt solutions used in the salting-out of proteins; gel electrophoresis is widely applied in the separation of proteins based on differences in charge and size; adsorption chromatography is also a very important method to separate proteins base on their polarity, electrical charge, molecular size, or binding affinity; the separation could be performed using specific enzyme and antibody assays such as the enzyme-linked immunosorbent assay (ELISA) system; furthermore, gene sequences could be employed to determine the primary protein structure and the three-dimensional structures of proteins could be obtained by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and cryoelectron microscopy.[12]

It has been suggested that the difficult and complex task of characterizing PTMs and bioprocess variants could be replaced by a simple assay involving partition in aqueous two-phase systems (ATPSs). For some considerable time, there has been continued interest in the application of ATPS in downstream purification of biomolecules.[13] Partitioning in ATPS is generally deployed as a single-stage extraction process and has been represented as a good alternative to the traditional methods, involving solid-phase adsorption steps in the separation and purification of biomolecules.[14]

ATPSs have many distinctive features in comparison to traditional separations processes. The most important are the aqueous nature of both phases, few interactions with the substance to be separated, and a nondenaturing environment allowing these biological substances to maintain their structure and functions.[15]

Partitioning has frequently been employed as a large-scale separation, for analysis, and for the removal of contamination from chemical compounds, biological substances, and biopolymers such as peptides, proteins, and nucleic acid, with the advantage that it can rapidly effect an initial separation and purification procedure.[15]

Introduction to aqueous two-phase systems

ATPSs have been widely used as extraction and purification methods applied to the recovery of proteins and other biological macromolecules and particles.[16] The first report of ATPS was by the Dutch Microbiologist Beijerinck, who observed the formation of two liquid phases when solutions of gelatin and starch were mixed together.[17] Since the first discovery of these systems, the field has developed widely and many studies have been performed in ATPSs and multiphasic systems. Albertsson provided the first solid foundation for partitioning in ATPS and showed their potential for the separation of bio-macromolecules, cells, and organelles in a variety of ATPSs including polymer/polymer and polymer-salt systems.[16]

ATPSs are formed by aqueous mixtures of two or more polymers or of one polymer and a salt. Multiphase systems can form when mixtures of more than two polymers are mixed together above defined concentrations.[18]

When such components are mixed in water above certain concentrations, two immiscible phases are formed. Separation of the phases into top and bottom phases depends on differences in the density of the polymer solutions and their viscosity, and this largely determines the time required to achieve complete phase separation.[16]

Interactions between the substance to be analyzed and the components of the ATPS create a highly efficient system for solute partitioning.[16],[19]

The properties of the coexisting phases can be varied depending on the MW, concentration, and structure of the polymers. In addition, further chemical and physical factors may be varied through salt addition to control system charge and hydrophobicity. Differential partition of solutes having different phase preferences is based on their surface properties and the properties of the phase-forming components. Partitioning in ATPS has been the subject of several books and review papers.[16],[20],[21],[22],[23]

Walter also studied the factors which play an important role in affecting cell and particle partitioning. This early work showed the method to be well suited to the analytical and preparative separation of proteins.[24]

Phase system composition

Phase separation in solutions containing a mixture of polymers is common, and phase separation has been attributed to the high MW of the polymers combined with interactions occurring between segments of polymers; thus, in high-MW polymers, entropic contributions, which depend on the number of molecules, are small in comparison to enthalpic contributions which depend on the number of similar monomeric units. The separation system may also contain salts, which have a significant effect on the behavior of the system whose impact depends on both the type and the concentration of salt.[25]

Two types of ATPS are usually used:

  1. Polymer-polymer two-phase systems
  2. Polymer-salt two-phase systems.

Polymer-polymer two-phase systems normally consist of poly-ethylene-glycol (PEG) as one of the phase-forming polymers in ATPS because it is widely and cheaply available and easily forms a two-phase system with other neutral polymers as well as salts. The other common polymer is dextran which is a high-MW carbohydrate polymer consisting of alpha α-1,6 glycosidic linkages between glucose molecules, while branches begin from α-1,3 linkages. The selection of ATPS depends on the nature of the biomolecule and preparatively on the relative cost of the different systems selected for the separation.[25],[26] Salts can alter the physicochemical properties of the systems through hydrophobicity differences between the upper and lower phases and also by altering the distribution of ions between the phases, which consequently could affect the partitioning of analytes according to their molecular charge. Normally, salt is added to increase the selectivity of protein partitioning in the ATP methodology because it causes an uneven distribution in the system through the generation of a difference in electrical potential between the phases. The PEG/salt systems have mainly been used in large-scale enzyme extractions.[27],[28] PEG-salt systems have several advantages, including a greater difference in density between the phases and a lower viscosity compared with PEG-dextran systems as well as a considerably lower cost.

The essence of partitioning relies on the equilibrium distribution of added solutes into different phases. The most important factors which can affect the distribution are the physical and chemical properties of the system.[18]

Different ATPSs have been found to be suitable and provide high partitioning sensitivity for the purification and extraction of different target biological materials. [Table 1] lists some examples of ATPSs comprising both polymer/polymer and polymer/salt systems.
Table 1: Compositions of some of the early aqueous two-phase systems

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The composition of the phase system plays a crucial role in the partitioning process, because the wrong choice of phase composition could cause denaturation or aggregation of the partitioned substance. The goal of partitioning is to choose an appropriate phase system usually relatively close to the critical point to achieve the desired distribution and to be sensitive to the surface properties of the solute.[29]

Numerous ways to manipulate the partition coefficient have been found including alteration to the MW of one or both polymers and by the inclusion of various additives such as neutral salts.[16],[30]

Phase diagram

Phase separation of polymer/polymer and polymer-salt ATPS is characterized by the phase diagram in much the same way as conventional aqueous/organic systems used in conventional liquid-liquid extraction. The phase diagram defines the compositions in which the phase-forming species separate into discrete phases.

In other words, it is a map which indicates the phases present at a given temperature and composition. It is determined experimentally by recording and observing the cloud points over a range of compositions, the concentration of phase components in the top and bottom phases, and the ratio of phase volumes necessary to form a system with two phases that are in equilibrium.

The phase diagram consists of a binodal curve which is constructed by the determination of the compositions of top and bottom phases of a number of systems, in which the polymer concentrations are varied [Figure 4]. The binodal of the system is influenced by the MWs of the polymers; the lower the MW, the higher the concentrations required to form the two phases.[16]
Figure 4: Binodal curve: A and B are • Nodes which represent the final composition of the top and bottom phase, the points a1, a2, and a3 represent the total compositions of three systems lying on the same tie-line with different volume ratios. (Cp) The composition and volume of both phases are almost equal; Δx and Δy represent the difference in concentration of component X and Y between the two phases [19]

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The binodal information is required to calculate the weight percentage of each polymer in each phase and to define the models that predict partitioning of biomolecules. The binodal curve is determined by recording the weight of a series of systems prepared from stock solutions of known compositions, and the critical points of these systems are determined by the cloud point method. Finally, selected phase systems within the biphasic region are constructed by selecting defined mixture compositions which are made up at convenient scale, mixed, and allowed to settle. Upper and lower phase volumes are recorded, and the density of the phases was determined by pipetting numerous small samples of each phase and recording their weight. An empirical equation due to Mistry et al.[37] is often fitted to the binodal composition data by nonlinear least squares regression to yield the parameters of an empirical equation delineating the binodal curve.

The composition of the individual phases generated can be obtained using tie-lines. The simplest way to find a phase system composition suitable for partition of the substance of interest is by making a number of systems which differ in polymer concentration, salt additive, and pH.[16]

The tie-line length (TLL) is an important parameter allowing ordination of the partition coefficients of added solutes. The TLL reflects the solubility curve of the phase diagrams and is proportional to the composition of the two phases that exist in equilibrium with each other at this temperature. It also reflects the phase characteristics of a system and can be estimated using the equation 1:

TLL = √ΔX2+ ΔY2 (1)

Knowledge of the phase diagram enables specific systems to be made having selected values of TLL and volume ratio.[16]

Phase composition is related to the polymer density difference between the phases and a linear dependence has been found. The density of the phase composition has a crucial impact on the settling rate by the gravitational force on the phase droplets, the friction between the drops and phase will be high, that leads to the separation time being longer.[37] It is also the case that, whenever the TLL is increased, the difference between top and bottom composition becomes greater and the interfacial tension increases.[38]

The critical point is found on the binodal where the TLL = 0. Theoretically, at this point, phases have equal volume and composition and solutes are evenly distributed with a partition coefficient of one.[19]

The properties of the phase systems are affected by many factors such as the temperature and the type of polymers used and their MW. Very briefly, increasing the MW of one polymer will tend to make the binodal more asymmetrical. The higher the MW of the polymer, the lower the concentration required for phase separation. On the other hand, the effect of the temperature depends on the type of the polymer and differs for different phase diagrams.[16]

Polymer-polymer systems represent a phase separation characterized by an upper critical solution temperature, and polymer-salt systems are in general characterized by a lower critical solution temperature.

The distribution of an analyte between the phases can be expressed in terms of the partition coefficient K which is to be calculated as a ratio of the analyte concentration in the upper and lower phases.

K = (Analyte)Top/(Analyte)Bottom

The partition of an analyte between the two aqueous phases depends on its physicochemical properties as well as those of the two polymers.[16] The most important factors affecting protein partitioning in ATPS are analyte MW, charge, and surface properties, while for the phase-forming polymers, these are the polymer MW, phase composition, salt effects and affinity ligands attached to polymers, pH, and temperature.[16] To understand the mechanism of the aqueous two-phase behavior and the analyte partitioning, the fundamental theory of the system should be clarified. One force controlling the phase partition has been characterized as the free energy of transfer of the methylene group from the rich-salt lower phase to rich-PEG upper phase. This free energy represents just one contribution to the partition coefficient as the hydrophobicity of the analyte surface area.

Zaslavsky et al. suggested a method to estimate the relative hydrophobicity of the analyte in ATPS by the calculation of free energy change.[39] Based on the amino acids having different aliphatic side chains, the free energy was calculated using different partitioning systems.

The free energy of transfer depends on the nature of the phase diagram and the relative composition of the phases (equation 2).

ΔG = −RTlnK (C − C0) (2)

Where ΔG is the free energy change, R is gas constant; T is absolute temperature, C is concentration of system, and C0 is concentration of critical point.

The proposed general relationship between the partition coefficient and methylene groups was considered in the equation 3.

lnK = C + En (cH2) (3)

Where the K is the analyte partition coefficient, C is a constant related to the hydration properties of the phases, n is the number of carbons in chain, and E is the slope of linear plot of lnK versus n (cH2) and the constant represents the contribution of polar group present in the partitioned analyte.

By the same principle, many relationships can be characterized from the calculation of the free energy change from simple thermodynamic relations (equation 4).

ΔG0= −RTln = ΔH0 − TΔS0 (4)

Where lnK is a rate constant or an equilibrium constant, R is the universal gas constant, ΔH is the enthalpy, T is the temperature in Kelvin, and ΔS is the entropy. Such processes can often be broken down into simple additive contributions.

The TLL relationship with free energy may be varied from zero at the critical point to increasing values as TLL increases. In general, the free energy is a small value for polymer-polymer systems, but it is much larger for polymer-salt systems and can be varied by altering the composition of the phases. Thus, the chemical potential difference of the systems can be arranged to suit the surface potential of the added solute or particle. Since ΔG is a function characterizing the phase behavior and analyte partitioning, adjusting the phase composition by changing the phase components or increasing the TLL may result in achieving a desired separation.

Analytes, in general are varied in the hydrophobicity which results in differences in the analyte solvent interaction with the phase systems,[40] these interactions could be described based on different properties of the analyte, and often can be broken down into simple additive contributions (equation 5).

Some property = Cavity formation + polarity terms + hydrogen bonding terms + constant (5)

This equation represents a linear relationship to the free energy which is used widely in the several processes, for instance, partitioning in aqueous/organic systems and transport across biological membrane.

Analyte partitioning isotherm

The partition coefficient is defined as the ratio of the analyte concentration in the upper to its concentration in the lower phase. However, the partition coefficient may be influenced by a number of factors which will result in concentration-dependent behavior. Analytes may associate or dissociate in a concentration-dependent way or their solubility limits may be exceeded. This is analogous to the “overloaded condition” in analytical adsorption chromatography. In addition, single determinations of the partition coefficient may be subjected to a variable amount of error which may be minimized by multiple determinations of the distribution. However, this methodology does not reveal whether the partition isotherm is concentration dependent or subject to the effect of saturation or other nonidealities. Determination of the partition isotherm is a straightforward way of overcoming these problems or of revealing their existence. Ideally, in attempting to make analytical measurements, the isotherm should not be concentration dependent. The method of determining the isotherm has been recommended in the ATPS literature but is very rarely applied. In almost all cases, this method was adopted to determine the partition coefficient in the experimental parts of this work. The reliable method to measure the isotherm linearity is using a range of concentrations of the solute and partitioning these separately in systems having identical compositions. The partition coefficient is then determined by the slope of a linear regression of the concentration in the upper phase against the concentration in the lower phase.[23] Departures from linearity are then relatively easy to detect and indicate that there are problems in determining the partition coefficient which could lead to serious errors in any analytical application.

Advantages and limitations

ATPS have received considerable attention in the development of biotechnological processes. Conventional techniques of purification and separation are often perceived to be inefficient, expensive, and not fully scalable, and this has led to a search for alternative process steps.[41]

The application of ATPS that has attracted the most interest in biotechnology is its use in the isolation, extraction, and purification of proteins and other biological materials such as enzymes/proteins, nucleic acids, viruses, and cell organelles often from crude mixtures and homogenates. Suitably selected ATPSs provide mild conditions which do not change or denature biomolecular structures. Partition and separation of added solutes occur rapidly and the technique – as a form of liquid-liquid extraction – is potentially fully scalable.[19],[22]

However, there still remain some drawbacks to the widespread application of ATPSs.[42] Mayolo-Deloisa reported that there are two restrictions to limit the wide application of aqueous two-phase extraction. First, it is difficult to predict exactly the behavior of target proteins in the ATPS system. Second, monitoring the characteristics of proteins is a basic requirement for assessment of bioprocesses, and many assay systems may be adversely affected by the presence of high concentrations of polymers or salts.[43]

Factors determining by the partition coefficient

Any difference between the properties of the phases seems to convey the ability to discriminate between similar biomolecules. The analytical information which is provided by partitioning process is completely related to the interactions between the phases and the solute, and these interactions are known to be highly related to the solute structure, so the partition coefficient has emerged as a highly sensitive indicator of the characteristics of the solute.

In pathology, many of the proteins, hormones, enzymes, and peptides could be biological indicators or biomarkers of particular human diseases. This requires the analysis of protein compositions of samples from biological fluids or tissues by highly sensitive fractionation and analytical techniques. Many different approaches have been developed, but some disadvantages exist such as the neglect of protein interactions and conformational change. Biphasic partitioning for a mixture could reflect the differences between components dependent on their structural and/or functional characteristics. Such differences could be used as a marker to determine the physiological condition of a biological system. Zaslavsky gives an example of the application of the partitioning method to what appears to be a complex situation. The method involved the partitioning of plasma from patients with posttraumatic stress disorder in comparison to similar samples from a control group. The results indicated a difference in the overall distribution of total plasma proteins between patient samples and healthy controls.[44],[45],[46]

In another example, apotransferrin was used to prepare saturated solutions in the presence of a variety of ligands (Fe3+, Cu2+, Al3+, Bi3+, and Ca 2+) which were then analyzed in different ATPSs. Excess metal ions have been removed before partitioning using a centrifugal concentrator having a membrane characterized by a 3 KD MW cutoff. Protein concentrations were assayed by measuring the optical absorbance at 278 nm by UV/VIS spectrophotometry. After that, the partition coefficients for each solution were determined as the slope of the linear relationship for various dilutions of the transferrin samples, representing the concentration in the upper phase versus the concentration in the lower phase.

The results were presented as a range of partition coefficients for the binding of different ligands. The conclusion of this example was stated to be that the partition coefficient determined following binding of the different ligands reflected the conformational state resulting from the binding of different ligands which provided a unique signature for each species.[47],[48]

Another example of the application of ATPS partitioning as an analytical technique is provided by a method to determine the purity and homogeneity of recombinant human growth hormone (rhGH) again in a patent published by Zaslavsky.[49] rhGH was characterized by measuring its relative distribution between the phases of an ATPS (the partition coefficient), and this was shown to be highly correlated with changes in the biological potency and purity of the product.

Further examples, also due to Chait and Zaslavsky, include the determination of the partition ratio of prostate-specific antigen as a biomarker for prostate cancer and the ratio of glycated hemoglobin to total hemoglobin as an indicator of diabetes status.[50]

The K value may also represent an indicator for the purity of the solute similar to other physicochemical characteristics such as melting point of a pure compound.[51] Furthermore, partitioning may provide quantitative information about changes in solute structure which depends on the interaction between the solute and the coexisting phases.[16],[29],[52]

The use of multiple systems to characterize partition has also been proposed – on the assumption that there are meaningful differences in K between different ATPSs. This whilst more complicated than the use of a single K can statistically or graphically be used to produce a “signature” specific to a particular molecule (population of isoforms) which it is claimed can be used diagnostically in a technique termed solvent interaction analysis.[15]

On the other hand, the ratio of the amount of subpopulations of different molecules in a biphasic system can provide crucial information for medical diagnosis, quality control, pathology, toxicology, drug safety, and other applications.

Such biopolymers may differ by the number of certain isoforms such as phosphoforms and glycoforms. Many examples could be given of how the ratio of amounts of biomolecules or their subpopulations in a mixture could be considered clinically different from a reference sample. For example, modified forms of transferrin have been proposed as a marker of long-term alcohol abuse.[49]

In addition, Chait and Zaslavsky [53] reported that classic fractionation techniques to separate or characterize biomolecules have neglected two important aspects. First, generally, fractionation techniques cannot preserve protein-protein/protein-ligand interactions and are often unable to separate the mixture based on changes in conformation while ATPS may represent an advance in this field. The application of ATPS to quantify differences in the interactions of species using multiple biphasic systems has been claimed to enable identification of unique patterns of biomarkers for diagnostic applications.[53]

Partition in ATPS can reflect the structural and functional characteristics of biomolecules through interactions with the coexisting phases. Often, conformational changes in a biomolecule are associated with specific biological effects.

Such changes are often significant and may include changes to what other molecules may be bound by the biomolecule. Many biological processes are mediated by noncovalent interactions between a protein and another molecule, for instance, in the interaction between cellular receptors and their binding partners. In some cases, change in the binding partner can alter the function of the receptor, for example, the effect of different estrogenic compounds on the estrogen receptor where the binding of different ligands results in conformational change and changed activity, so determination of ligand binding can also be used to determine the function of receptor. One example of this is the effect of different estrogenic compounds on the estrogen receptor. In this case, different compounds resulted in different, distinct conformational changes and these different changes result in different activity and/or function of the estrogen receptor.[54]

Detecting protein structural modifications

Since 1958, when Albertsson made his first publication on the partitioning of a set of proteins having different MWs (1.3 × 104–9 × 106) in an ATPS, it has been followed by many studies attempting to understand the behavior of proteins in these systems.

Albertsson (1960) began the development of a protein partitioning theory and outlines the basis of partitioning which may be summarized as some points: using low MW of one polymer in the phase systems results in an increase in protein partitioning to the low-MW phase, dissociation of the protein may alter the partition of the protein. Partition is also strongly influenced by many factors such as the addition of salt where the partitioning changed dramatically with increasing concentration of salt from 1 to 5 M, polymer MW, the pH, and the net charge of the protein and its concentration. In the following years, ATPSs have become as an extremely attractive procedure to separate and purify biomolecules in large scale with maintaining the structure and have been a subject of several review papers.[55],[56]

It has been claimed that partitioning can distinguish between protein isoforms, so it can provide information about protein structural modifications. ATPSs can reflect the differences between components dependent on their structure and the corresponding electrostatic interaction with the biphasic systems.

Structures of protein are usually associated with their function. X-ray crystallography, NMR spectroscopy, and mass spectrometry are common methods used for determining structure. In addition, analytical techniques such as ELISA, while specific for particular species, can only give quantitative information and information on structure is minimal or absent because the signal in ELISA is the sum of signals arising from all isoforms. Partition, determined by specific assay such as ELISA, however, can give access to structural information since the partition coefficient contains information on the differential partition of the isoform population.[15]

Partition in ATPSs could be used as an index to provide information on the structural forms of proteins through the partition coefficient; this technique is based on differences in the interactions of proteins with the two phases mediated by differences in their properties. Differences in the nature of the two aqueous phases result in differences in distributions, enabling the detection of any changes in structure. Thus, the partition coefficient might represent a sensitive detection method for changes in structure and conformation.[57]

When combined with a suitable specific detection method, partitioning can distinguish between protein isoforms and thus provide information about structural modifications. This method consists of two obvious steps: (1) partitioning of the protein in a suitable biphasic systems and (2) examination of protein concentration in the aqueous phases by a protein-specific method.[15]

Thus, the technique can be used for classification and detection of changes to the structure of interest. This may represent a useful tool in various fields of biochemistry, molecular biology, cell biology, and especially biotechnology, and this represents the main goal in this work. It has been clearly demonstrated that in a molecularly simple system, the mixture composition of two different species can be determined from the measurement of the partition coefficient alone. We consider, for example, two species A and B having partition coefficients of 10 and 2, respectively. In a mixture comprising only A, the partition coefficient will be determined as 10, and in a mixture comprising only B, the partition coefficient will be determined as 2. Values intermediate between these two will be determined for all mixture compositions intermediate between these extremes. The resulting curve depends on the relative difference between the partition coefficients and the coefficient of determination of the assay.

Partitioning can distinguish between protein isoforms and combined with a specific detection assay seems capable of providing information about protein structural modifications since the partition coefficient represents the ratio of two different proteins such as the possibility of detecting the ratio of the carbohydrate deficient transferrin isoform from the total amount of the transferrin in a mixture. The separation of two sample populations depends on the difference in their K values and their relative abundance means the possibility of detecting the interactions of two isoforms. That will lead to the ability to quantify the amount of different isoforms in the mixture without initial separation.[58]

On the other hand, determination of the concentration of the analyte from their distribution in the phases represents an analytical signal for that particular compound since it is related to the interaction with the phase components. The interactions between two different macromolecules or macromolecule and a particle can be applied to study the quantitation.[59]

Highlighting the knowledge gap in the aqueous two-phase system

Since their first discovery and development, ATPS has been used as a very powerful technique for the purification and separation of many biomolecules. On the other hand, there is increased demand for new, more rapid, and more accurate bioanalytical techniques which can be exploited for studying the PTMs that may arise during the pathology of various disease conditions and to monitor product quality during biopharmaceutical production processes. Using ATPS as a biotechnological method to achieve this goal may represent an attractive approach to this problem; however, in ATPS, the behavior of the biomolecules is highly variable and many factors are involved in the partitioning process. This makes the prediction of the K values very complex and also studies of the partitioning of a modified protein using a protein model have rarely been undertaken. Finally, the effect of the protein modification process and the properties of the ATPS used in terms of examination of analytical problems could result in new and interesting applications.

  Conclusion Top

The practical application of protein partitioning in ATPS has been demonstrated in many cases, including a number of industrial applications, with excellent levels of purity and yield. The technique has also been successfully used for the separation of virus and virus-like particles and to monitor the PTMs of proteins as biomarkers of disease processes. In bioprocessing, the technique is readily adaptable to continuous processing and in analytical applications to automated high-throughput modes. However, the wider application of this technology is diminished by a lack of a clear understanding of the factors and mechanisms that govern the behavior of proteins in these systems which has slowed the development of analytical models to aid the rational design of these systems. Prediction of a model protein partition coefficients from structure has yet to be convincingly demonstrated; this is because protein crystal structures only partially represent the range of isoforms present in the native protein and some PTMs such as glycosylation are poorly represented in crystal structures and only by inference form the amino acid sequence or codon sequences of proteins in structure databases; therefore, this review highlighted the knowledge gap of the applications of ATPS.


First, I would like to acknowledge the Iraqi Ministry of Higher Education and Scientific Research and University of Karbala for funding and supporting this work. I also would like to thank my Brunel supervisors, Dr. Jonathan Huddleston, Dr. Sveltana Ignatova and Dr. Krishna Burugapalli. A very big thank for their huge help, support, and guidance. Furthermore, I am so grateful to Prof. Derek Fisher for introducing me to Brunel University London and the Advanced Bioprocessing Centre team.

Financial support and sponsorship

The study was funded by the Iraqi Ministry of Higher Education and Scientific Research and University of Karbala (grant No. 643 from 10/01/2013).

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1]


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