Aqueous Two-Phase Extraction(ATPE) of proteins Using “Affinity ligands attached to polymers”ESWAR RAJA BABU PADARTHI, MSc 2nd yearstudent, Matricola: 0000812602Dept of ChemicalEngineering, STEM-curriculum, University of Bologna, Italy. Introduction:ATPEis a process in which the extraction systems are made by two water solublepolymers or a polymer and a salt above a certain concentration. This system thenseparates into 2 Immiscible liquid phases so called the Extract (E phase) andthe Raffinate (R phase). One phase is rich in polymer 1 and the other phase isrich in polymer 2 or a salt. The bio product to be extracted is distributed between2 phases at equilibrium between the liquid immiscible phases and informationabout the distribution of the solute (bio product) can be studied using”Partition coefficient(k)”Industrially, Polyethylene glycol(PEG) and Dextran.
And salts of sulphate, phosphate, citrate etcare most commonly used polymers and salts respectively for the ATPE process. Generally,the concentration of polymer-polymer system is (say PEG-Dextran system) is15%PEG and 10% Dextran and in case of Polymer-salt system the concentration isabout 15% PEG and 10% salt.Overviewand need for ATPE: Bio-pharma process industries does not useof organic solvent to extract of proteins/bio molecules as it is not a rightmethodology. The reason is that some proteins are insoluble when there is anorganic and aqueous phase system. Also, some of the proteins easily getdenatured or degraded when contacted with organic solvents. It is a huge loss ofvaluable products and the purpose of the extraction is lost. On the other hand,Aqueous two-phase extraction is a non-denaturing and non-degrading techniquethat can be applied to extract a wide range of biomolecules (For ex: proteins,DNA, bio-nanoparticles, cells and cell organelles).
This method has shownfruitful results for the removal of undesirable contaminating by products namelynucleic acids, polysaccharides and other suspended matted in the solution.Researchers quoted that “Aqueous two-phase extraction (ATPE) at present isconsidered as a feasible unit operation for extraction of monoclonal antibodiesor recombinant proteins”In ATPE process theconcentration of polymer used is around 15%(W/W). Due to presence of high water content and low interfacial tension of thesystems, the system protects the proteins from getting denatured or degraded. Ona whole, the advantages of this technique are easy to scale-up, can be operatedas continuous operation and effective removal of undesired species etc. Parametersaffecting the biomolecule partitioning in ATPE:In general, proteinswill partition to the top, less polar and more hydrophobic phase (PEG phase)and the most soluble and particulate matter will partition and distribute to majorityto the lower phase. Separation of proteins from the solution can be done byaltering the partition coefficient by changing the average molecular weight ofthe polymers, the type of ions in the system, the ionic strength of the saltphase or by adding an additional salt of sodium and potassium.
Some of theimportant factors affecting the portioning are 1. Proteinmolecular weight 2.Protein charge and surface properties 3.Polymers molecularweight 4.Effect of salt and 5.Affinityligands attached dot the polymers (My field of interest)Specific field of studyATPE of antibodies using”Affinityligands attached to polymers” Brief Discussion: Tobe specific, I would like to discuss the paper one of the factor thatinfluences the biomolecule partitioning that is use of affinity ligandsattached to in ATPE process.
Through the experimental studies, a comparison hasbeen done in between the conventional ATPE and ATPE process with use ofaffinity ligands. It is found that the partition coefficient value has been oneand two orders of magnitude greater than the conventional ATPE process.Implying a huge advantage in achieving maximum extraction efficiency up to 96%is (At specific polymer concentration by weight fraction) presence of affinityligands.
Examples of bio-specific ligands: Ligandsfrom the Tiazine dyes, reactive dyes- Cibacron blue, Procian red, and Procionyellow etc has been used for affinity separation processes for the extractionof proteins by chemically modifying polymer. In majority of the cases, PEG ismodified. Examples of processes (Researchstudies)Example 1: Use of PEG/dextran ATPE process, chemically modified PEG i.e.diglutaricacid functionalized PEG to form ‘PEG–COOH’ has resulted great affinity to IgG andmaximum of IgGhas been recovered in the top phase (up to 89% till 93%).Compositionsbeing 20% (w/w) of PEG 150–COOH and 40% (w/w) PEG 3350–COOH.Example 2: Extractionof monoclonal antibodies by using PEG-Dextran system with a bio-specific ligand’LYTAG-Z’.
Overview and Strategy of the process:Theselectivity of extraction of proteins/antibodies or targeted biomolecule can bemaximized making use of concept of coupling. The coupling of a bio-specificligand to one of the polymers that can be easily modified can be used to hookthe targeted antibody and then obtain in one of the phases and then recover thebiomolecule by further processing. In most of thecases PEG is the polymer always chosen for this purpose. PEG is a polyether diol con be obtained in a range of differentmolecular weights and is of low dispersity. PEG is a versatile molecule as itcan be used as the ligand-carrying polymer since it is easily chemicallymodified to couple with a ligand using simple chemistry. Especially incase of PEG and DEXTRAN polymers, the main objectives and focus lies on the activehydroxyl groups present in the polymers Depending upon thetype of the protein or antibody or genetic material to be extracted ligands arecoupled to the polymer accordingly.
A large varietyof groups can be attached directly to the PEGs by means of alkylationreactions, e.g. with alkyl halides, substitution reactions with acid chlorides,anhydrides or cyanuric chloride, etc. Esterification reactions of PEGs byreaction with anhydrides such as maleic, glutaric anhydrides etc. Modificationof PEG molecule with carboxylate groups (-COOH) is studied thoroughly due ithigh recovery and selectivity. The processes is referred as covalentcoupling of affinity ligands to the selected polymer. Researchstudies inferred that use of affinity ligands has shown promising resultsespecially increase in selectivity.
A wide range of biomolecules have been effectivelyundergone partition with several affinity ligands and at the end having maximumextraction efficiency. Once the phase separation takes place thatis after binding/hooking of bioproduct (antibody/proteins etc) to thepolymer-Ligand (say Top phase), it is important to recover the bioproduct in itsfree form. We apply standard techniques such as gravity settling- decantationmethod to recover TOP phase. Now to the TOP phase which contains polymer-ligand-bioproductmolecule, the recovery of the bioproduct is done by addition of salt to thesystem. Addition of salt results in 2 phase formation and partitioning of theprotein to one of the phase.
Therefore, we have 2 phases, in which one of the phase(mostly Bottom phase) being rich in the bio product and the other phase rich inPolymer-ligand. To understand the situation in detail thepurpose of adding the salt is that-Salt competes with the bound ligand for theprotein’s binding/hooked site, but eventually salt dominates the interactionsbetween the ligand salt when compared with the interactions ofligand-bioproduct which eventually results in releasing of thebioproduct(protein/antibody) and shift into one of the phase (bottom phase). Detail discussion using standardexampleTodescribe the technique of Aqueous Two-Phase Extraction(ATPE) of antibodiesusing “Affinity ligands attached to polymers”. I would take an example and discussin detail. The example I have considered for discussion is extraction of protein-IgGusing different polymer-polymer systems in which PEG is chemically modifiedthat addition of bio-specific ligands to the PEG.
The effect of differentligands in extracting antibodies compared in terms of extraction efficiency andselectivity. DifferentATPE systems are prepared and one of the polymer-PEG is chemically modified in differentways Case 1: PEG/Dextran ATPS systempolymer/polymerCase 2: PEG-ligand/Phosphate systemmodified polymer/salt ATPS2.1. PEGfunctionalized with diglutaric acid to obtain (PEG 150-COOH) and (PEG3350-COOH).The number 150 and 3350 etc. indicate the molecular weight of the PEG. The mol.
weight of PEG depends on type of application. For instance, PEG 3350 is used inpharma/drugs applications.2.
2.PEGs functionalized with amino groups PEG3350–diamine (PEG 3350–NH2) It isprepared by reaction of terminal hydroxyl groups of PEG 3350 using thionylchloride to obtain dichloride followed by reaction with potassium pthalamide andhydrazine hydrate to form PEG 3350–NH2 The above systemare prepared and the proteins say pure proteins in certain concentration Tosimulate the extraction of antibodies solutions just like in the real situation,pure protein systems consisted of different IgG concentrations are used.The aimof the problem is to extract the maximum amount of IgG in case of different of PEG-ligand system. The systems areprepared and the ATPE is carried under standard conditions such as Temperature,pH, time of operation and mixing.
Phase separation that formation of 2 phases takesplace at equilibrium and the distribution of the protein/antibodies takesplaces. After this step, the solution in respected phases are separated and theconcentration of protein “Quantificationof protein concentration” is done using different chromatographictechniques such as size exclusion and affinity chromatography and the resultsare analyzed. Analysis of the results is based on the following parameters/aspects:Partition coefficient(k): Defined as theratio of bio product concentration in the top phase to that of concentration ofthe bioproduct in the bottom phase.Yield of the bioproduct(Y) wrt (Top phase): Definedas mass of the protein in the top phase to that of mass of protein added to theATPS system.Selectivity(?): Selectivity isdefinedas the ratio of the partition coefficient of IgG/targeted bio product topartition of the contaminant proteins. Important results and comments:Basedon the information I could gather through literature survey/Research articles Icould make the following understandings and inferences Understandings· Effect of salt concentration in ATPEsystems containing functionalized polymer Polymer-ligand/salt system· Effect of different ligands in bindingdifferent proteins based on the charge and pH of solution· Comparison in terms of extractionefficiency in conventional ATPE and ATPE process containing affinity ligands tothe chemical modified polymer Inferences · Incase of PEG/Dextran ATPS; At lowconcentrations of the phosphate salt(10mM)the partition coefficient k of the proteins has increased as pH increases. Onthe contrary, at higher salt concentration and high pH (=9) due to fact that the protein becomes less positive and therepulsions between protein and the phosphate ion increases (till 100mM) and there will more partitioning of IgG andcontaminant proteins towards the dextran rich phase.
And the recovery is greater than 90%. But it is notpreferable to have a contaminant protein at the end of the process.· Inthe case modified PEG/dextran ATPS, PEG-COOH Diglutaric acid ligandsdisplay high affinity to IgG because of electrostatic interactions between the negativelycharged Diglutaric acid group and positively charged proteins. In fact, all IgG can be recovered in the top phase using15- 20% (w/w) of PEG 150–COOH and 40% (w/w) of PEG 3350–COOHare used. The maximum recovery that was reported was 93% using PEG 150-COOH 20%(w/w)· Alsouse of PEG 150 and PEG 33350 and their concentration can be changed accordinglybased on the extraction requirements. The number of modified PEG molecules willhave an impact in binding with proteins. Scaling up of process and processoptimization is very convenient in case of PEG with different molecularweights.· Incase of PEG-NH2 polymer-ligand system, as PEG-NH2 increases and pH increases thenit implies that the charge of IgG becomes less positive and this increases interactionsbetween its negatively charged groups and the NH2 group of the functionalizedPEG.
As a result, there is an increase in IgG recovery yield in the top phase. However,literature indicates that for total IgG recovery yield in the top phase highconcentration of PEG-NH2 is needed and this not a suggested practice as itinvolves more cost and more functionalized PEG.· Oncomparison between PEH-COOH and PEG-NH2, results are very fruitful in case ofPEG-COOH.1 order of magnitude· Asthe PEG-COOH concentration(w/w) increases the selectivity of the IgG hasincreased by 1 order of magnitude.
In one of the research paper, theselectivity was 1.4 at PEG-COOH (10% w/w) and IgG recovery about 89%(Top phase) and when concentration of PEG-COOHis increased to 20%(w/w) selectivitywas 11.4 with a IgG recovery of 93%· Inaddition of salts to a systemcontaining modified PEGs that is Tri methylamino-PEG, it is observed that theaddition of salts to the PEG/dextran ATPS has dampening effect of the affinityligand and protein interactions.· Thepresence of salts at high concentration in systems containing functionalizedsupports tends to reduce in the effect of the ligand by clouding theelectrostatic interaction between modified charged polymers and proteincharges.· I would like to comment that use of differentfunctionalized PEGs would maximize the recovery of human antibodies.
But findingthe right functionalized PEG based on type of bioproduct to be recovered involvesuse of advance organic chemistry, time, money and resources. ConclusionI would like to conclude by saying that, from research articles Ihave gone through I could observe that use of functionalized PEG in ATPE system,especially PEG-COOH has resulted in 93% recovery of IgG (Top phase) and selectivity is 50 to 60 times higher than non-functionalized systems. It is evidentthat affinity ligands attached to polymer has been a successful technique in purificationof human antibodies, genetic material, cell organelles etc. And this technologylays its way to efficient processes in bio-separations, downstream processingespecially in product purification of humanantibodies.