The document discusses the production and purification of monoclonal antibodies (mAbs) using aqueous two-phase systems (ATPS), highlighting their effectiveness despite high production costs. It details the use of a dual ligand, lytag-z, for mAbs purification from complex media, demonstrating significant improvements in partitioning and purity of antibodies. The results indicate that employing ATPS can enhance yield and efficiency in mAbs recovery while offering a simplified purification process.

1. Presented by:
Nagendra P
16PBT204
M.Tech Pharmaceutical Biotechnology
INSTITUTE OF CHEMICAL TECHNOLOGY

2. MonoclonalAntibodies
Monoclonal antibodies (mAbs) are established medicines that are being safely
produced by big pharma companies in quantities up to several tons per year.
Despite their effectiveness and safety as therapeutic agents, mAbs are amongst the
most expensive drugs on the market
Given their low potency and increasing market potential, large amounts of pure
mAbs are thus required.
This increasing product demand has been challenging biotechnologists to increase
production capacity, improve purification technologies and reduce manufacturing
costs.

3. Up to now, protein A affinity chromatography has been considered the most
effective technology in the downstream processing of mAbs.
It accounts for more than 50% of the entire downstream processing costs.
In fact, the current DSP technologies are unable to cope not only with the
high titers, but also with the large cell densities, which are challenging
traditional solid–liquid technologies, resulting in poor yields.

4. What isAqueous Two Phase Extraction?
 To extract biologically active polymers such as proteins, enzymes and nucleic acids
it is necessary to have a two-phase system, which will not denature the
biopolymers.
 The separation of two or more components from a liquid mixture into two
immiscible liquid phases, based on their unequal solubility is known as aqueous
two phase extraction.
 Aqueous two phase partitioning of biomolecules is a well established process which
was first introduced by Albertson.
 It is a type of liquid – liquid extraction method which makes use of two aqueous
phases.
 In aqueous bi-phase extraction, the basic principle involves the differential
partitioning of desired proteins in to two immiscible phases extracted from an
aqueous feed containing a complex mixture of proteins. Both the phases contain
distinct polymers or polymer and salt and are relatively rich in water (80-85%
content) and do not denature proteins and other biopolymers.

5. It has many advantages like
simple and benign technique (presence of more than 80% water in both phases),
rapid separation with little denaturation (volatile organic components are not used),
rapid mass transfer (low interfacial tension),
selective separation (affinity partition) and
easy scale up.
Therefore, ATPS has been applied in several fields of biotechnology such as recovery of proteins,
enzymes, biopharmaceuticals and extractive fermentation.
Two types of aqueous two-phase systems are commonly used:
1. Polymer-polymer two-phase system (e.g Polyethylene glycol/Dextran)
2. Polymer-salt two-phase system (e.g Polyethylene glycol/phosphate)
A polymer-polymer two phase system can for instance be obtained by mixing dextran and PEG at
a certain composition. By adding specific amounts of these polymers to an aqueous feed phase
(which contains the solute), two aqueous phases, one rich in PEG and the other rich in dextran is
obtained.

7. Phase Diagram
The miscibility range and the formation of aqueous two-phase systems between two
phase forming polymer-polymer or polymer-solute combinations depends on their
concentration ratios and it is discussed with the help of phase diagrams.
A mixture represented by point B with a composition of 85% water+5% PEG+10%
dextran, is below the binodal curve and is not stable as a homogeneous phase.
It will separate into two phases, one phase with 90% water+10% PEG (represented by
point C in the phase diagram) and a second phase with 80% water+ 19% dextran + 1%
PEG (represented by point D in the phase diagram).

9. Present discussion
Aqueous two-phase systems (ATPS) allow for the simultaneous purification,
concentration, and clarification of bioproducts by direct extraction of the target
product from the cell culture media in a system composed by an aqueous solution
of two polymers or a polymer and a salt.
This paper demonstrated the use of an affinity dual ligand, the LYTAG-Z, which
is composed of a choline binding polypeptide tag (C-LytA) fused to the synthetic
antibody binding Z-domain, for mAbs purification from complex media using
PEG and EOPO containing ATPSs.

10. C-LytA
 C-LytA is the C-terminal region of the LytA amidase enzyme
isolated from Streptococcus pneumonia.
 LytA amidase enables the attachment of this enzyme to the
phosphocholine residues present on the teichoic acids of the cell
wall surface.
 Structurally, C-LytA is built up from six conserved b-hairpins
that form four choline-binding sites.
 This module’s affinity for choline and choline structural
analogs (e.g. ternary amines) has been used for the purification
of C-LytA tagged proteins by adsorption to amine-based
chromatographic resins (e.g. DEAE cellulose) followed by
elution with choline.

11. Z domain
Z-domain is a synthetic IgG-binding peptide based on the
B-domain of staphylococcal protein A.
In this paper, C-LytA was fused to the Z domain and used
as a dual affinity ligand for mAbs purification from
complex media through aqueous two-phase extraction.
Once the two-phase system is generated, it is expected to
obtain LYTAG-Z-antibody complexes in the PEG rich-
phase, while cells and other impurities partition to the
opposite phase.

12. Experiments
1. CHEMICALS
Monobasic and dibasic potassium phosphate
sodium chloride
PEG 3350
dextran 500,000
polyacrylic acid sodium salt (NaPAA) 8000
Ethylene oxide/propylene oxide co-polymers (EOPO)
2. BIOLOGICS
Human immunoglobulin was obtained from Octapharma (Lachen, Switzerland)
as a commercially available solution for therapeutic administration
(Gammanorm), in which the active substance has a concentration of 165 mg/mL,
and at least 95% corresponds to immunoglobulin G (IgG).

13. CHO cell supernatant was produced and delivered by IcoSagen (Tartumaa, Estonia). This
supernatant contains a humanized monoclonal antibody, from IgG1 sub-class.
Hybridoma cells were grown in a mixture of two different media, composed of 25% (v/v)
of Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Gibco) and 75% (v/v) of
CD Hybridoma medium (Gibco). The IgG produced is a mouse anti-human CD34+
antibody.
3. LYTAG-Z
The LYTAG-Z fusion protein (245 aa, 28.39 kDa, theoretical pI 4.88) was expressed in
Escherichia coli and purified using CascadeTM/LYTAG technologies.
4. ATPS formulation
Four different types of ATPS were formulated:
1. PEG/phosphate,
2. PEG/dextran,
3. EOPO/dextran and
4. EOPO/NaPAA.

14. Each ATPS system was thoroughly studied using a factorial design and the best
formulation found for each ATPS was:
1. 12.5% (w/w) PEG and 12.5% (w/w) potassium phosphate;
2. 7% (w/w) PEG and 6% (w/w) dextran;
3. 10% (w/w) EOPO and 10% (w/w) dextran;
4. 10% (w/w) EOPO and 10% (w/w) NaPAA.
The corresponding biological sample (pure human serum antibodies, clarified
CHO cell culture supernatant or unclarified Hybridoma cell culture media) was
loaded at 20% (w/w).
The LYTAG-Z ligand (600 mg/L) was also loaded at 20% (w/w) of the system,
to assure a molar ratio of antibody to LYTAG-Z of 1:10. The system was
completed to 2 g with milli-Q water.

15. The ATPS were afterward mixed by vortex and
phases coalescence was achieved letting the
systems settle for 30 min.
Control systems were prepared without LYTAG-Z
and used to assess the effectiveness of the dual
ligand.
Blank systems were prepared without biological
sample and LYTAG-Z and used to evaluate and
discount the interference of the phase forming
components in the analytical methods.

16. 5. Cell counting
The total number of cells present in the top and bottom phases was counted using a
hemocytometer by trypan blue dye exclusion method.
6. Protein G affinity chromatography
IgG concentration was determined by affinity chromatography.
POROS protein G affinity column was used.
The column was equilibrated with an adsorption buffer composed by 50 mM
NaH2PO4, 150 mM NaCl at pH 7.4.
Samples were diluted ten times in the adsorption buffer.
Elution was achieved with 12 mM HCl with 150 mM NaCl at pH 2–3.
A calibration curve from 0.2 to 20 mg/L was made using Gammanorm IgG.
Absorbance was monitored at 215 nm.

17. 7. Total protein concentration
Total protein concentrations were determined using the Bradford method .
Briefly, 50 lL of sample, previously 4 times diluted, were mixed with 250
µL of Bradford reagent in a 96-well microplate.
The microplate was incubated at room temperature for 10 min.
Absorbance was measured at 595 nm.
A calibration curve from 5 to 400 mg/L was made using BSA.
Samples containing biological product were read against blanks prepared
with the same ATPS composition.

18. 8. Extraction performance parameters
To evaluate the performance of the ligand in the different systems, the partition
coefficient, the recovery yield and the purity of IgG were calculated.
Kp = IgG concentration in the top phase
IgG concentration in the bottom phase.
Recovery yield (%)=the mass of IgG in the top (or bottom) phase
the total mass of IgG added to the system
Purity(%) =IgG concentration in the top (or bottom) phase
to total protein concentrations in the top (or bottom) phase

19. Results and discussion
Selection of the ATPS based on its structural analogy to choline
Choline is an amino alcohol that seems to play an essential role in the protection
of the hydrophobic zones present in the choline binding sites of C-LytA.
Choline also induces dimerization of the monomer low-active form into a
homodimer fully active form, resulting also in a significant increase in the stability
of the protein.
Choline binding occurs through hydrophobic and cation–Π interactions.
It has been suggested that PEG interacts with the choline-binding site through
hydrophobic and CH-Π interactions that mimic the cation-Π.

20. Affinity extraction studies
The partitioning of pure human serum antibodies was studied within eight
different ATPS:
1. PEG/ phosphate,
2. PEG/dextran,
3. Three different EOPO/dextran and
4. Three EOPO/NaPAA systems.
To assess the effect of LYTAG-Z on IgG partition behavior, the partition
coefficient (Kp) was determined.
As seen in Fig. 1A, the presence of the ligand affected the partition of the
antibody towards the top phase.

21. Fig. 1. Effect of LYTAG-Z on the partitioning (log Kp) of (A) pure human serum antibodies and (B) antibodies from CHO
cell clarified supernatant in different ATPS: ( ) in the absence and ( ) in the presence of 20% LYTAG-Z.
The following systems composition as used: 12.5% PEG and 12.5% phosphate; 7% PEG and 6% dextran; 10% EOPO and
10% dextran; 10% EOPO and 10% NaPAA.
All systems contained 20% of sample loading.

22. Despite the substantial increase in the Kp values noticed in the presence of the ligand,
the log Kp of IgG remained negative, which meant that although the concentration of
IgG in the top phase had considerably increased, the concentration of IgG in the bottom
phase was still very high (although smaller) than in the top phase.
The electrostatic interaction between the positively charged IgG and the negatively
charged phosphate ions enhance the partition of IgG to the bottom phosphate-rich phase,
even in the presence of LYTAG-Z.
Regarding the EOPO/NaPAA systems, as NaPAA is a negatively charged polymer,
electrostatic interactions between NaPAA and the positively charged IgG will also favor
the partition of the antibody molecules to the NaPAA-rich (bottom) phase.
(Fig. 1B)For the remaining systems an increase in the log Kp is observed but once
again the effect of the ligand it is not enough to pull all the antibody molecules to the top
phase and the log Kp remains negative.

23. Clarification of animal cell cultures
The possibility to treat unclarified antibody crude stocks and to integrate in one
single operation clarification and partial purification without requiring a cell
removal step upstream is one of the major advantages of ATPS.
Fig. 2 shows microscopy images of the top, interface and bottom phase from the
system, confirming that Hybridoma cells partition preferentially towards the
interface or to the bottom phase. Less than 5% of the cells were found in the top
phase, which allowed a clarification higher than 95%.

24. Based on these partitioning studies, PEG/dextran ATPS showed to be a promising
system for mAbs recovery from a complex non-clarified cell culture, taking
advantage of the dual ligand.
It is important to note that the cell culture has an initial IgG purity of around 25%,
which means that with this single ATPS step we achieved not only the clarification
and capture of the antibody but also an almost 2-fold increase in the purity.

25. Conclusion
The dual ligand LYTAG-Z has shown to enhance the partition of antibodies
towards the choline structural analogue rich-phase (PEG and EOPO).
In the absence of LYTAG-Z, antibodies shown to preferentially partition to the
bottom phases, presenting a negative log Kp, and
In the presence of the ligand this value increased for all the tested systems.
The successfully partition of mAbs to the PEG rich-phase was accomplished
using 7% PEG 3350 and 6% Dextran that allows an increase in the partition
coefficient of one order of magnitude.
An IgG recovery yield of 89% was obtained with a 42% of purity, from a cell
containing medium.
The possibility of recovering the ligand for further processes is also a great
advantage of this approach.

26. References
http://www.topbritishinnovations.org/pastinnovations/monoclonalantibodies
http://www.rcsb.org/pdb/explore/explore.do?structureId=4IWT
http://www.rcsb.org/pdb/explore/explore.do?structureId=1BDC
https://www.ncbi.nlm.nih.gov/pubmed/24138575
https://en.wikipedia.org/wiki/Mixer-settler
Campos-Pinto, I., Espitia-Saloma, E., Rosa, S. A., Rito-Palomares, M., Aguilar, O.,
Arévalo-Rodríguez, M., ... & Azevedo, A. M. (2017). Integration of cell harvest with
affinity-enhanced purification of monoclonal antibodies using aqueous two-phase
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Azevedo, A. M., Rosa, P. A., Ferreira, I. F., & Aires-Barros, M. R. (2009).
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