The document discusses aqueous two-phase extraction (ATP), a separation method used in downstream protein processing. ATP uses the partitioning of proteins between two immiscible aqueous phases formed by mixing polymers like PEG and dextran, or polymers and salts. Several factors affect partitioning, including biomolecule properties, polymer/salt concentrations, pH, and temperature. ATP has advantages over organic solvent extraction as it is milder and lowers interfacial stress on proteins. The document outlines applications of ATP in purifying pharmaceutical proteins, antibodies, enzymes, antibiotics, amino acids, and other compounds. It also summarizes a research paper on using ATP to purify lipase from rice bran.

1. Aqueous Two Phase
Extraction
Msc BT III 13005
13006
When water and water don’t mix

2. Introduction
• Aqueous two phase extraction (ATP) is one of the key separation
methods in downstream processing
• Its biocompatibility is one of the advantages in protein partitioning
with good selectivity at minimal protein loss
• ATP can be applied as a selective technique for a particular protein
separation by integrating with affinity ligands.
• ABS is an excellent method to employ for the extraction of proteins/
enzymes and other labile biomolecules from crude cell extracts or
other mixtures
• Aqueous two-phase systems are generally composed of a water
solution of two structurally distinct hydrophilic polymers or of one
polymer and certain salts

3. Principle:-
• In general, there are two major types of ATPS available, viz.,
polymer/polymer (e.g Polyethylene glycol/Dextran) and polymer/salt
(e.g Polyethylene glycol/phosphate) system.
• It is formed by mixing two different water-soluble polymers or one
water-soluble polymer and salt in water.
• When the limiting concentrations are exceeded, two immiscible
aqueous phases are formed.
• The limiting concentrations depend on the type of phase forming
components and on the pH, ionic strength and temperature of the
solution.

4. • PEG–dextran system
• The "upper phase" is formed by the more hydrophobic polyethylene
glycol (PEG), which is of lower density than the "lower phase,"
consisting of the more hydrophilic and denser dextran solution.
• Although PEG is inherently denser than water, it occupies the upper
layer. This is believed to be due to its solvent 'ordering' properties,
which excludes excess water, creating a low density water
environment.
• The degree of polymerisation of PEG also affects the
phase separation and the partitioning of molecules during extraction.

6. Factors Affecting
• Biomolecule concentration : Increase in protein conc and mol wt
decreases their separation in upper phase.
• Polymer concentration : Viscosity play important role here so for
extreme partition high content of polymer is to be chosen.
• Surface properties of biomolecule : surface net charge plays
important role in hydrophobicity or hydrophillicity of biomolecule.
for e.g. ; lysine and glutamic acid have relatively low hydrophobicity
and they partition favourably in the salt rich phase in the PEG/ salt
ATP systems.

7. • PH : In PEG/Salt system as pH changes from acidic to basic the protein
becomes less positive or more negative charge and negative charge
protein prefer upper phase.
• Temperature : At high temp it is easy to form two phase system with
small conc of PEG and salt . PEG/Dextran system will form two phase
system at low temp.
• Affinity ligand attach to polymers : PEG has many sites to which
other groups can attach . When hydrophobic group is attach the
hydrophobic protein and amino acid will be separated in the upper
phase.
• Salt : salt ion partition differently between the phases causing an
uneven distribution in the system that generates a difference in
electric potential between the phases.

8. Advantages
• They provide mild conditions that do not harm or denature
unstable/labile biomolecules
• The interfacial stress (at the interface between the two layers) is far
lower (400-fold less) than water-organic solvent systems used
for solvent extraction, causing less damage to the molecule to be
extracted
• The polymer layer stabilizes the extracted protein molecules,
favouring a higher concentration of the desired protein in one of the
layers, resulting in an effective extraction

9. • Specialised systems may be developed (by varying factors such as
temperature, degree of polymerisation, presence of certain ions etc. )
to favour the enrichment of a specific compound, or class of
compounds, into one of the two phases. They are sometimes used
simultaneously with ion-exchange resins for better extraction
• Separation of the phases and the partitioning of the compounds
occurs rapidly. This allows the extraction of the desired molecule
before endogenous proteases can degrade them.
• These systems are amenable to scale-ups, from laboratory-sized
setups to those that can handle the requirements of industrial
production. They may be employed in continuous protein-extraction
processes.

10. Application
THE PURIFICATION OF RECOMBINANT PHARMACEUTICAL PROTEINS:-
•Human insulin-like growth factor 1 (IGF-1) accu- mulates in medium and
cellular periplasmic space when expressed in E. coli with an endogenous
secretary signal sequence.
• Due to its heterogeneity in form and location, low yield of IGF-1 was
obtained using a typical recovery strategy.
•To enhance recovery yield, a new method, called in situ solubilization,
involves addition of chaotrope and reductant to alkaline fermentation broth
and provides re-covery of about 90% of all IGF-1 in an isolated super-natant.
Then, an aqueous two-phase extraction procedure was employed which
partitions soluble non-native IGF-1 and biomass solids into separate liquid
phases

11. THE PARTITIONING OF ANTIBODIES, ANTIGENS AND THEIR
COMPLEXES:
The Purification of Antibodies or Antigens:-
• As a technique for protein purification, an enteric coating polymer,
Eudragit S100, was employed as a ligand carrier .
• Eudragit was specifically partitioned to the top phase in the aqueous
two-phase system (PEG/Dextran).
• For application of this method to purification of recombinant protein
A, using human IgG coupled to Eudragit in an aqueous two-phase
system, 80% of protein A added was recovered with 81% purity

12. THE PURIFICATION OF PHARMACEUTICAL ENZYMES
•Glucose-6- phosphate dehydrogenase, the extraction of this enzyme in
a PEG-dextran aqueous two-phase system with the addition of
polymers carrying charged groups and affinity ligands.
• These polymers include DEAE-dextran, dextran sulfate etc. where the
ligands used were Cibacron Blue F3G-A and Procion Yellow HE-3G.
• The results showed that polyelectrolytes, forced into one of the phase
of the two-phase system by the choice of salts, could be used to affect
strongly the partitioning of the proteins.

13. THE PURIFICATION OF ANTIBIOTICS
•Most applications of aqueous two- phase are in the separation of
macromolecules.
•With the development of researches, it was found that some small
molecules have also the uneven distribution in the aqueous two- phase
systems, so aqueous two-phase systems found their application in the
purification of small molecules.
•The effects of average molecular weight of PEG, concentrations of PEG
and KH2PO4 and pH on the partition equilibrium of acetylspiramycin in
PEG/KH2PO4 aqueous two-phase systems. It was found that
acetylspiramycin partitioned unevenly in the aqueous two-phase
systems and could be purified by this technique.

14. THE PRODUCTION AND PARTITIONING OF LACTIC ACID
•During fermentation lactic acid inhibits growth of the organ-isms and
product formation.
•This can be avoided by extracting lactic acid from the broth. Further,
productivity can be improved by increasing the catalyst density in the
reactor.
• To meet all these requirements and to avoid detrimental effects of
conventional extraction media on bioconversion aqueous two-phase
systems are a possible alternative.

15. THE PARTITIONING OF AMINO ACIDS
AND OLIGOPEPTIDES:
• Amino acids are the constituents of proteins. The studies of the
partitioning of amino acids in aqueous two-phase system have great
significance not only for the purification of amino acids, but also to
the proteins
• The partition coefficients of several amino acids and peptides is
measured in poly(ethylene glycol)-magnesium sulfate (MgSO4)
aqueous two-phase systems.

16. THE PURIFICATION OF OTHER PHARMACEUTICALS
•The Purification of Pharmaceuticals from Plants
•Ecdysone and 20-hydroxyecdysone, hormones that regulate molting
cycles of anthropods, are of commercial interest as insecticides and
indicators of helminth and nematod parasitism and other medical
disorders in humans. Both molecules are steroids, but soluble in water
•The ecdysteroids were first partitioned in a two-phase system with a
UCON 50-HB-500-rich top phase and a dextran-rich bottom phase.
• In a two-phase system with an ethanol concentration of 20%,
recovery was 73.6% for ecdysone and 85.6% for 20-hydroxyecdysone.

17. The Partitioning of Porphyrin Compounds
•Porphyrin compounds are highly significant bio-logical molecules. They
play an important role in living processes such as photosynthesis and
transportation of oxygen.
•Hematoporphyrin derivatives could be used in the detection and
photodynamic therapy of tumors.
• The total content of uroporphyrin and coproporphyrin in a urine
sample is usually determined in clinical laboratories for diagnosis of
diseases.
•An aqueous two-phase system of cationic-anionic surfactant mixture is
used (ATP-CAS) for the extractions of porphyrin compounds.

18. Research Paper(Summary)
Aqueous two phase extraction – of Lipase from Rice Bran
•Due to various application of lipase in medical and industrial field, aqueous
two-phase extraction for the downstream processing of lipase has been
exploited.
• The influence of system parameters such as phase forming salts, molecular
weight of the phase forming polymer, system pH, tie line length, and phase
volume ratio on the partitioning behavior of lipase was evaluated.
•The aqueous two-phase system consisting of PEG 4000 and Magnesium
sulphate and it has shown better results with the purification of 1.648 fold.
As PEG 4000 with Magnesium sulphate has shown better results so further
work can be carried out using PEG 4000 as phase forming polymer.