Aqueous made by two water soluble polymers or a

Aqueous Two-Phase Extraction(ATPE) of proteins


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 “Affinity ligands attached to polymers”

student, Matricola: 0000812602

Dept of Chemical
Engineering, STEM-curriculum, University of Bologna, Italy.


is a process in which the extraction systems are made by two water soluble
polymers or a polymer and a salt above a certain concentration. This system then
separates into 2 Immiscible liquid phases so called the Extract (E phase) and
the Raffinate (R phase). One phase is rich in polymer 1 and the other phase is
rich in polymer 2 or a salt. The bio product to be extracted is distributed between
2 phases at equilibrium between the liquid immiscible phases and information
about the distribution of the solute (bio product) can be studied using
“Partition coefficient(k)”

Industrially, Poly
ethylene glycol(PEG) and Dextran. And salts of sulphate, phosphate, citrate etc
are most commonly used polymers and salts respectively for the ATPE process. Generally,
the concentration of polymer-polymer system is (say PEG-Dextran system) is
15%PEG and 10% Dextran and in case of Polymer-salt system the concentration is
about 15% PEG and 10% salt.

and need for ATPE: Bio-pharma process industries does not use
of organic solvent to extract of proteins/bio molecules as it is not a right
methodology. The reason is that some proteins are insoluble when there is an
organic and aqueous phase system. Also, some of the proteins easily get
denatured or degraded when contacted with organic solvents. It is a huge loss of
valuable products and the purpose of the extraction is lost. On the other hand,
Aqueous two-phase extraction is a non-denaturing and non-degrading technique
that can be applied to extract a wide range of biomolecules (For ex: proteins,
DNA, bio-nanoparticles, cells and cell organelles). This method has shown
fruitful results for the removal of undesirable contaminating by products namely
nucleic acids, polysaccharides and other suspended matted in the solution.
Researchers quoted that “Aqueous two-phase extraction (ATPE) at present is
considered as a feasible unit operation for extraction of monoclonal antibodies
or recombinant proteins”

In ATPE process the
concentration of polymer used is around 15%(W/W). Due to presence of high water content and low interfacial tension of the
systems, the system protects the proteins from getting denatured or degraded. On
a whole, the advantages of this technique are easy to scale-up, can be operated
as continuous operation and effective removal of undesired species etc.


affecting the biomolecule partitioning in ATPE:

In general, proteins
will partition to the top, less polar and more hydrophobic phase (PEG phase)
and the most soluble and particulate matter will partition and distribute to majority
to the lower phase. Separation of proteins from the solution can be done by
altering the partition coefficient by changing the average molecular weight of
the polymers, the type of ions in the system, the ionic strength of the salt
phase or by adding an additional salt of sodium and potassium. Some of the
important factors affecting the portioning are 1. Protein
molecular weight 2.Protein charge and surface properties 3.Polymers molecular
weight 4.Effect of salt and 5.Affinity
ligands attached dot the polymers (My field of interest)

Specific field of study

ATPE of antibodies using”Affinity
ligands attached to polymers”


Brief Discussion: To
be specific, I would like to discuss the paper one of the factor that
influences the biomolecule partitioning that is use of affinity ligands
attached to in ATPE process. Through the experimental studies, a comparison has
been done in between the conventional ATPE and ATPE process with use of
affinity ligands. It is found that the partition coefficient value has been one
and 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 affinity


Examples of bio-specific ligands: Ligands
from the Tiazine dyes, reactive dyes- Cibacron blue, Procian red, and Procion
yellow etc has been used for affinity separation processes for the extraction
of proteins by chemically modifying polymer. In majority of the cases, PEG is


Examples of processes (Research

Example 1: Use of PEG/dextran ATPE process, chemically modified PEG i.e.diglutaric
acid functionalized PEG to form ‘PEG–COOH’ has resulted great affinity to IgG and
maximum of IgGhas been recovered in the top phase (up to 89% till 93%).Compositions
being 20% (w/w) of PEG 150–COOH and 40% (w/w) PEG 3350–COOH.

Example 2: Extraction
of monoclonal antibodies by using PEG-Dextran system with a bio-specific ligand


Overview and Strategy of the process:
selectivity of extraction of proteins/antibodies or targeted biomolecule can be
maximized making use of concept of coupling. The coupling of a bio-specific
ligand to one of the polymers that can be easily modified can be used to hook
the targeted antibody and then obtain in one of the phases and then recover the
biomolecule by further processing.


In most of the
cases PEG is the polymer always chosen for this purpose. PEG is a polyether diol con be obtained in a range of different
molecular weights and is of low dispersity. PEG is a versatile molecule as it
can be used as the ligand-carrying polymer since it is easily chemically
modified to couple with a ligand using simple chemistry. Especially in
case of PEG and DEXTRAN polymers, the main objectives and focus lies on the active
hydroxyl groups present in the polymers


Depending upon the
type of the protein or antibody or genetic material to be extracted ligands are
coupled to the polymer accordingly. A large variety
of groups can be attached directly to the PEGs by means of alkylation
reactions, e.g. with alkyl halides, substitution reactions with acid chlorides,
anhydrides or cyanuric chloride, etc. Esterification reactions of PEGs by
reaction with anhydrides such as maleic, glutaric anhydrides etc. Modification
of PEG molecule with carboxylate groups (-COOH) is studied thoroughly due it
high recovery and selectivity. The processes is referred as covalent
coupling of affinity ligands to the selected polymer. Research
studies inferred that use of affinity ligands has shown promising results
especially increase in selectivity. A wide range of biomolecules have been effectively
undergone partition with several affinity ligands and at the end having maximum
extraction efficiency.


Once the phase separation takes place that
is after binding/hooking of bioproduct (antibody/proteins etc) to the
polymer-Ligand (say Top phase), it is important to recover the bioproduct in its
free form. We apply standard techniques such as gravity settling- decantation
method to recover TOP phase. Now to the TOP phase which contains polymer-ligand-bioproduct
molecule, the recovery of the bioproduct is done by addition of salt to the
system. Addition of salt results in 2 phase formation and partitioning of the
protein 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 in


To understand the situation in detail the
purpose of adding the salt is that-Salt competes with the bound ligand for the
protein’s binding/hooked site, but eventually salt dominates the interactions
between the ligand salt when compared with the interactions of
ligand-bioproduct which eventually results in releasing of the
bioproduct(protein/antibody) and shift into one of the phase (bottom phase).


Detail discussion using standard

describe the technique of Aqueous Two-Phase Extraction(ATPE) of antibodies
using “Affinity ligands attached to polymers”. I would take an example and discuss
in detail. The example I have considered for discussion is extraction of protein-IgG
using different polymer-polymer systems in which PEG is chemically modified
that addition of bio-specific ligands to the PEG. The effect of different
ligands in extracting antibodies compared in terms of extraction efficiency and


ATPE systems are prepared and one of the polymer-PEG is chemically modified in different

Case 1: PEG/Dextran ATPS system

Case 2: PEG-ligand/Phosphate system
modified polymer/salt ATPS

2.1. PEG
functionalized 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 in
pharma/drugs applications.

PEGs functionalized with amino groups PEG3350–diamine (PEG 3350–NH2)

It is
prepared by reaction of terminal hydroxyl groups of PEG 3350 using thionyl
chloride to obtain dichloride followed by reaction with potassium pthalamide and
hydrazine hydrate to form PEG 3350–NH2


The above system
are prepared and the proteins say pure proteins in certain concentration To
simulate the extraction of antibodies solutions just like in the real situation,
pure protein systems consisted of different IgG concentrations are used.The aim
of the problem is to extract the maximum amount of  IgG in case of different of PEG-ligand system.


The systems are
prepared and the ATPE is carried under standard conditions such as Temperature,
pH, time of operation and mixing. Phase separation that formation of 2 phases takes
place at equilibrium and the distribution of the protein/antibodies takes
places. After this step, the solution in respected phases are separated and the
concentration of protein “Quantification
of protein concentration” is done using different chromatographic
techniques such as size exclusion and affinity chromatography and the results
are analyzed.


Analysis of the results is based on the following parameters/aspects:

Partition coefficient(k): Defined as the
ratio of bio product concentration in the top phase to that of concentration of
the bioproduct in the bottom phase.

Yield of the bioproduct(Y) wrt (Top phase): Defined
as mass of the protein in the top phase to that of mass of protein added to the
ATPS system.

Selectivity(?): Selectivity is
as the ratio of the partition coefficient of IgG/targeted bio product to
partition of the contaminant proteins.


Important results and comments:

on the information I could gather through literature survey/Research articles I
could make the following understandings and inferences


Effect of salt concentration in ATPE
systems containing functionalized polymer Polymer-ligand/salt system

Effect of different ligands in binding
different proteins based on the charge and pH of solution

Comparison in terms of extraction
efficiency in conventional ATPE and ATPE process containing affinity ligands to
the chemical modified polymer


case of PEG/Dextran ATPS; At low
concentrations of the phosphate salt(10mM)
the partition coefficient k of the proteins has increased as pH increases. On
the contrary, at higher salt concentration and high pH (=9) due to fact that the protein becomes less positive and the
repulsions between protein and the phosphate ion increases (till 100mM) and there will more partitioning of IgG and
contaminant proteins towards the dextran rich phase. And the recovery is greater than 90%. But it is not
preferable to have a contaminant protein at the end of the process.

the case modified PEG/dextran ATPS, PEG-COOH Diglutaric acid ligands
display high affinity to IgG because of electrostatic interactions between the negatively
charged Diglutaric acid group and positively charged proteins. In fact, all IgG can be recovered in the top phase using
15- 20% (w/w) of PEG 150–COOH and 40% (w/w) of PEG 3350–COOH
are used. The maximum recovery that was reported was 93% using PEG 150-COOH 20%(w/w)

use of PEG 150 and PEG 33350 and their concentration can be changed accordingly
based on the extraction requirements. The number of modified PEG molecules will
have an impact in binding with proteins. Scaling up of process and process
optimization is very convenient in case of PEG with different molecular

case of PEG-NH2 polymer-ligand system, as PEG-NH2 increases and pH increases then
it implies that the charge of IgG becomes less positive and this increases interactions
between its negatively charged groups and the NH2 group of the functionalized
PEG. 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 high
concentration of PEG-NH2 is needed and this not a suggested practice as it
involves more cost and more functionalized PEG.

comparison between PEH-COOH and PEG-NH2, results are very fruitful in case of
PEG-COOH.1 order of magnitude

the PEG-COOH concentration(w/w) increases the selectivity of the IgG has
increased by 1 order of magnitude. In one of the research paper, the
selectivity was 1.4 at PEG-COOH (10% w/w) and IgG recovery about 89%(Top phase) and when concentration of PEG-COOH
is increased to 20%(w/w) selectivity
was 11.4 with a IgG recovery of 93%

addition of salts to a system
containing modified PEGs that is Tri methylamino-PEG, it is observed that the
addition of salts to the PEG/dextran ATPS has dampening effect of the affinity
ligand and protein interactions.

presence of salts at high concentration in systems containing functionalized
supports tends to reduce in the effect of the ligand by clouding the
electrostatic interaction between modified charged polymers and protein

I would like to comment that use of different
functionalized PEGs would maximize the recovery of human antibodies. But finding
the right functionalized PEG based on type of bioproduct to be recovered involves
use of advance organic chemistry, time, money and resources.



I would like to conclude by saying that, from research articles I
have 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 evident
that affinity ligands attached to polymer has been a successful technique in purification
of human antibodies, genetic material, cell organelles etc. And this technology
lays its way to efficient processes in bio-separations, downstream processing
especially in product purification of human