WO2015099550A1

WO2015099550A1 - Magnetic liquid-liquid extraction for purification and partitioning of substances

Info

Publication number: WO2015099550A1
Application number: PCT/PT2014/000078
Authority: WO
Inventors: Vijaykumar L. DHADGE; Maria Raquel AIRES-BARROS; Ana AZEVEDO; Ana Cecília AFONSO ROQUE
Original assignee: Faculdade De Ciências E Tecnologia Da Universidade Nova De Lisboa; Instituto Superior Técnico
Priority date: 2013-12-23
Filing date: 2014-12-19
Publication date: 2015-07-02
Also published as: PT107379A

Abstract

The present invention discloses a method for the purification and partitioning of substances such as biomolecules. The process comprises the steps of mixing the functionalised superparamagnetic materials (1) with the aqueous two phase system components (3) and (5) and with the crude complex mixture (2) containing the target substance(6), either produced by fermentation or cell culture, chemically synthesized or extracted from natural sources.

Description

DESCRIPTION

"Magnetic liquid-liquid extraction for purification and partitioning of substances"

Field of the Invention

The present invention discloses a method of isolation and partitioning of substances. An object of the present invention is to provide a method for the separation and purification of the target substance, particularly a biomolecule from a crude mixture containing different types impurities including proteins, nucleic acids, small organic molecules, virus, cells and cell debris. Process comprises the steps of mixing the functionalized superparamagnetic materials with the selected aqueous two phase system along with crude mixture produced by cell fermentation, or by chemical synthesis or extracted from natural sources.

Background of the Invention

The challenging promise of new (bio) technology derived drugs for the treatment of several diseases such as cancer and auto-immune disorders has been mostly fulfilled by monoclonal antibodies (mAbs) [1, 2], although other bioprocess-derived products (e.g. gene therapy products, cells and stem cells, etc) , as well as chemical drugs have also contributed for this purpose. The traditional purification methods used in the (bio) pharmaceutical, (bio) chemical, food and environmental industry are too expensive and complex to scale up to a level sufficient to deal with the increasing product demand [2], The chromatography-based technologies now available for extraction and purification of biological and chemical materials at industrial scale are reaching a point close to their maximum capacity [3] . Downstream processing usually encompasses four stages, namely, recovery, isolation, purification and polishing. With recovery, isolation and polishing making up only 20% of the total downstream costs, the major process limitations are found in the selective purification steps, currently dominated by chromatography, which accounts for >70% of the downstream costs, mainly owing to media cost and relatively long cycle times. Current upstream titer increase is pushing chromatography beyond its physical and economic limits. Hence novel, low cost and easy to scale up non-chromatographic methods for the downstream processing of biological and chemical materials are receiving special attention from the academic and industrial communities [4] . Among these, liquid-liquid extraction, including aqueous two-phase systems (ATPS) and magnetic separation represent interesting alternatives to chromatographic methods worth exploitation. ATPS form when solutions of two incompatible polymers, or one polymer and a salt, are mixed together above certain concentrations [5] . The partition of substances between two aqueous phases is influenced by complex phenomena which involve van der Waals forces [6], hydrogen bonds, charge interactions, hydrophobic interactions and steric effects [7] . Still, ATPS represents a promising alternative for biological and chemical materials purification with tests already performed in industrial settings [11] . However, the time needed for phase separation and settling is high, the selectivity of the process is low and several unit operations can be required to achieve high purity. On the other end, the recycling of polymers and solutions is troublesome but needed due to the high costs involved. Magnetic particles (MPs) modified with ligands typically employed in chromatographic media (e.g. ion-exchange, affinity) constitute highly selective adsorbents to target biological and chemical molecules and materials [8] . In addition, the magnetic responsive nature of these particles allows their selective manipulation and separation in the presence of other suspended solids with promising applications in process integration. Magnetic separation has been tested at small-scale for immunoglobulin G (IgG) capture from cell culture supernatants, and provided similar yields and purity when compared to chromatography columns typically employed [14] . Also, the potential for in situ product recovery has been recently explored [26] . The high cost of commercially available MPs are the major challenges faced by this technology [10] . Nonetheless, both ATPS and magnetic separation are alternative technologies to chromatography, aiming at high throughput and process integration, and seeking to avoid problems associated with most chromatographic supports, such as high cost, limited capacity and diffusion limitations.

The possibility to combine magnetic particles with ATPS has been previously addressed. The article published in [11, 12] put forward a technique of accelerated phase separation using non-functionalized magnetic particles. Particles as such do not bind any solutes or materials on their surface. In a further publication [13] authors employed magnetic particles functionalized with an antibody (an extremely costly option) to enhance ATPS achieving a very poor purity level (49%) from crude samples, by using 3.5M KSCN solution as an eluent which represents an extremely harsh condition. The authors could not employ low pH for elution of the protein due to the dissolution of particles. In addition, in this work a high amount of particles were needed to achieve a very low purification level (40 mg of particles for 8 g of aqueous two-phase system; ratio of 5), with the limitation of partitioning the magnetic beads to the top phase, after one hour required for stirring all components of the mixture. Also, the amount of salt needed was high. Later, the [14] has disclosed the invention in which a surfactant was added to an ATPS together with magnetic particles which makes the process more complex and expensive and generating difficulty in removal of surfactant from final product, generating purity constraint in end use of product [15].

Summary of the Invention

A method for the purification and partitioning of substances, comprising the steps of: a) preparation of an aqueous two phase system; b) addition of the superparamagnetic materials along with the crude mixture; c) phase separation and particle removal by magnetic force; d) washing and isolation of the target substance from superparamagnetic materials.

The crude mixture consists on for example i) a cell culture obtained by fermentation or cell culture, ii) cell homogenate/lysate, iii) an extract of a natural product; iv) the products of a chemical synthesis; and it is added to the aqueous two phase system under constant agitation along with superparamagnetic materials.

The aqueous two phase system is composed by mixtures of, but not limited to, polymers, salts, sugars, amino acids, surfactants .

The substances in which the method applies comprise, but are not limited to, antibiotics, vitamins, peptides, polypeptides, proteins, antibodies, hormones, nucleic acids, nucleic acids derivatives, organelles, virus, viruslike particles, vesicles or cells.

In this method the recovery of the target substance is performed by chemical or physical means, such as, but not limited to an aqueous solution containing buffering compounds as Tris-HCl and Glycine-NaOH, or the action of temperature or pressure .

The washing referred in the step d) can be performed using for example, binding buffer before actual elution.

The concentration of wash solution and elution solution is between 10 mM to 200 mM and 0.2 M to 2 M respectively and the pH of the wash solution and the elution solution are between 2 and 12.

In another embodiment, the method can comprise a further step of recovering the superparamagnetic materials from mixture using magnetic force wherein permanent magnet or electromagnet is used for separation process followed by washing, until removal of unbound materials, and elution, wherein the wash solution is an aqueous solution.

Additionally, the method of the present invention meets the strict and demanding requirements for larger industrial scales for manufacture of therapeutic mAbs.

Detailed Description of the Invention

The present invention is based upon the integration of ATPS with magnetic separation. The invention consists of using of aqueous two phase system formed by polyethylene glycol (PEG) and Dextran along with incorporation of superparamagnetic particles functionalized with ligands displaying affinity and selectivity towards antibody molecules, such as boronic acid (APBA) or (2-(3- aminophenol ) -6- (4-amino-l-naphthol) -4-chloro-s-triazine

(ligand 22/8) . The invention relates to a method for the isolation of antibodies from a crude mixture, comprising the steps of (a) Mixing the antibody with an aqueous two phase solution comprising PEG and Dextran; (b) addition of functionalized superparamagnetic materials into the system;

(c) removal of superparamagnetic materials from the aqueous-two phase system; (d) separation and elution of antibodies from superparamagnetic materials. In accordance with the present invention, the adequate concentrations of PEG and Dextran, and any other additive as salt, play an important role in partitioning of antibody and provide isolation of antibodies with high efficiency and high performance both in terms of yield and purity. In addition, it is to be understood that the same process performance may be achieved by using alternatives to PEG or Dextran using functionalized superparamagnetic materials and are not limited to the ones disclosed in this invention.

The present technology can be applied to any situation where the partitioning of a solute between two immiscible liquid phases occurs. This involves the field of extraction and purification, detection, diagnostics, therapeutics and partitioning studies, among others. This technology can be applied in the chemical, biochemical, biotechnology, biopharmaceutical, pharmaceutical, environmental, food, textile, mining, security industries or in any other setting where partitioning of solutes between two immiscible liquid phases occurs and can be useful for a purpose.

Brief description of the figures

For an easier understanding of the embodiment the figures are attached, which represent preferred ways how to implement the procedure that, however, do not intend to limit the scope of the present invention.

FIG.l. Method's squeme, in wich:

1- Superparamagnetic materials

2- Crude mixture

3- Bottom phase

4- Magnet

5- Top phase

6- Biomolecule

Partition of pure human IgG in hybrid PEG/dextran systems supplemented with MPs - (A) GA-MP, (B) GA-APBA-MP for increasing salt concentration.

FIG.2. Partition of pure human IgG in hybrid PEG/dextran systems supplemented with magnetic particles coated with gum arabic and containing the ligand boronic acid, for increasing salt concentration, in which:

1- IgG in Top phase

2- IgG bound

3- IgG in Bottom phase

FIG.3. Pure IgG extraction parameter in PEG/Dextran systems with increasing salt concentration for EPS-22/8 coated MPs

, in which:

1- IgG in Top phase 2- IgG bound

3- IgG in Bottom phase

Example 1 :

Aqueous two-phase systems (ATPS) composed of PEG and dextran were supplemented with several surface modified superparamagnetic particles (MPs) at distinct salt concentrations. The partition of pure human IgG (hlgG) in the upper and lower phases as well as the amount adsorbed at the MPs surface was investigated, indicating that MPs coated with dextran and gum Arabic established the lowest amount of non-specific interactions. The binding capacity of gum arabic coated particles modified with aminophenyl boronic acid (GA-APBA-MP) found to be excellent in combination with ATPS, yielding high antibody recovery (92%) and purity (98%) from cell culture supernatants . The presence of MPs in the ATPS was found to speed up phase separation (from 40 to 25 min) , to consume a lower amount of MPs (half of the amount needed in magnetic fishing) and to increase the yield and purity of a mAb purified from a cell culture supernatant, when compared with ATPS or magnetic fishing processes alone.

Superparamagnetic Materials

Iron oxide particles were prepared as detailed in [16] · Particles were coated with two silica layers (thin S1O₂ layer and TEOS by a sol-gel process) to confer pH resistance. Particles were then coated with several polymers including gum Arabic (GA) , reacted with GLYMO to introduce active epoxide groups, and reacted with APBA yielding GA-APBA-MP particles.

Magnetic Extraction Studies Antibody purification study was carried out by partitioning pure human IgG in hybrid PEG/dextran systems supplemented with GA-MP and GA-APBA-MP for increasing salt concentration (Figure 2) . ATPSs were prepared by weighting the corresponding stock solutions of PEG and dextran polymers along with salt in order to achieve the desired final composition of each system. Pure hlgG extraction studies were performed by adding 1 ml of 1 g/L hlgG stock solution dissolved in PBS buffer. In hlgG extraction studies from CHO cell culture (1.16 g/L hlgG), the supernatant loading ranged from 1 to 1.5 ml. All systems were prepared in 15 ml graded test tubes to a total final weight of 5 g by adding water (milli-Q) , PEG of molecular weight 3350 Da of 40% stock solution (final 5% w/w) , dextran 500,000 Da of 20% stock solution (final 8% w/w) , MP at a final concentration of 0.02% (w/w) and NaCl in the concentration range of 100- 500 mM. The hlgG extraction studies were carried out by thoroughly mixing each system components in a vortex shaker for 15 minutes, followed by phase separation at room temperature. After phase separation, the test tubes were held on a magnetic separator and samples of each of the phases along with superparamagnetic particles were collected. The particle washing was carried out for five times, the first one with milli-Q water and then four consecutive washes with 20 mM HEPES buffer at pH 8.5 (0.5 ml volume each wash) . Elution of adsorbed hlgG was then triggered using 1.5 M Tris-HCl buffer at pH 8.5 (5 elution fractions with 0.5 ml volume each). Total protein content was quantified using the Bradford method. The amount of hlgG in both bottom and top phases and bound to MPs were quantified by HPLC on a porous protein-A affinity column. The purity of protein preparations on top and bottom phases was evaluated by SDS-PAGE. The respective gels were prepared according to a standardized protocol [16] .

Antibody Purification from CHO Crude Samples

The partition of a mAb against interleukin-8 and the protein impurities from a CHO cell supernatant in PEG/Dextran ATPS supplemented with GA-APBA-MP was evaluated. The amount of antibody present in the upper and lower phases was negligible. Partition coefficients of hlgG in ATPS showed variations with change of NaCl concentration (0-500 mM) ranging from 0.18 to 0.55. The percentage of hlgG eluted from GA-APBA-MP using various buffers at different pH conditions was tested being the best 1.5 M Tris.HCl pH 8.5. More than 90% of the mAb bound to the particles, was observed. The purity of both upper and lower phases as well as the elution supernatants from superparamagnetic nanoparticles were analyzed by SDS-PAGE, showing the high purity of the antibody eluted from the particles, and the presence of contaminants in the upper and lower phases. From the HPLC results, it was concluded that about 92% of total hlgG bound is eluted from the support at 200 mM salt concentration with a purity higher than 98%. The particles were reused for several times and their efficiency maintained.

Example 2:

The capability of EPS (exopolysaccharide) coated MPs for the covalent attachment of a synthetic affinity ligand makes these particles useful for recovery of antibodies. EPS coated MPs also shows applicability for use in an integrated process technology combining magnetic separation process with aqueous two-phase extraction for the purification of human antibodies. The ATPS process composed of 8 % PEG and 5 % dextran afforded high recovery yield in presence of EPS-22/8 coated MPs. The magnetic supports can be effectively used for five times with partial reduction in binding capacity. In multiple extraction steps, the MPs bound 92% of loaded hlgG with a final purity level of 98.5%.

Superparamagnetic Materials Functionalized With Ligand 22/8

Iron oxide magnetic particles were coated with two layers of silica and then with EPS. The particles were aminated and then the synthesis of ligand 22/8 performed in situ as detailed in [17] .

Magnetic Extraction Studies

Aqueous two phase extraction system composed of 8 % (w/w) PEG (3350) and 5 % (w/w) Dextran (500,000) was used to investigate biopolymer coated MPs performance. For preparation of ATPS (5 g) , PEG-3350 and Dextran-500000 were weighed in 15 mL graduated glass tube. The superparamagnetic particles were added to each system at a final concentration of 0.02 % (w/w) . In pure hlgG extraction studies, 1 mL of a 1 g/L hlgG stock solution was added, while in supernatant mAbs extraction studies, the supernatant loading ranged from 1 to 1.5 ml of a 1.35 g/L hlgG containing cell culture medium. Salt concentration was varied between 100 to 500 mM for all systems. The final weight of 5 g was achieved by adding water (Milli-Q) . All components were then thoroughly mixed in a vortex shaker and the system was then allowed for phase separation for 2 to 4 hours, at room temperature. After phase separation, the test tubes were positioned in a magnetic separator for the recovery of superparamagnetic particles. The two-phases were carefully removed and samples of each phase were collected for further analyses. The MPs were subsequently washed with Milli-Q water and then with 50 mM phosphate buffer of pH 8. The hlgG adsorbed to the MPs was eluted using 50 mM Glycine-NaOH buffer at pH 11. The amount of hlgG released from the MPs was further determined by Protein A HPLC.

Antibody Crude Extract Purification

For the aqueous two-phase extraction, the MPs (MP-EPS-22/8) were incubated for 40 minutes at room temperature in the ATPS. After incubation, the MPs were separated and the supernatant was carefully collected. The separated particles were then washed five times with 500 μΐ binding buffer (50 mM Phosphate, pH 8) . After washing, the bound hlgG was eluted using the elution buffer (50 mM Glycine- NaOH at pH 11) . In order to study the best elution conditions, various elution buffers like Tris-HCl of pH 8.5 having concentration 0.1 M, 0.2 M, 0.5 M, 1 M, 1.5 M, Sorbitol of concentration 0.5M, 1 M and 100 mM citrate buffer of pH 3 as well as pH 8.5 were tested. All collected samples were quantified by affinity HPLC using a porous protein A affinity column. The BCA method and SDS-PAGE (12.5% Acrylamide/Bisacrylamide) in denaturing conditions were also used for purity analysis. The SDS-PAGE gels were prepared according to a standardized protocol.

Magnetic Particles Performance in Purification

The partition of pure hlgG in PEG/dextran ATPS supplemented with EPS coated MPs was also evaluated. According to the results addition of salt affected the hlgG partitioning along with MPs. Higher hlgG concentration in upper phase was observed in presence of high concentrations of NaCl. The salt concentration was studied in the 100-500 mM of NaCl. It was observed that by increasing salt concentration there is a decrease in the concentration of hlgG in the lower dextran-rich phase and a simultaneous an increase in the concentration of hlgG in the upper PEG-rich phase (Figure 3). After aqueous two-phase extraction step, the particles were washed with washing buffer (50 mM phosphate at pH 8), whereas bound hlgG was released using an elution buffer composed by 50 mM Glycine-NaOH at pH 11. Impuritites were distributed along the upper and lower phase, whereas pure antibody (purity higher than 95%) was observed in the elution sample from the MPs.

References

[1] M. Kuczewski, E. Schirmer, B. Lain, G. Zarbis- Papastoitsis, Biotechnol. J. 6 (2011)56.

[2] L. Zhang, Y. Xu, H. Yao, L. Xie, J. Yao, H. Lu, P.

Yang, Chem. Eur. J. 15 (2009) 10158.

[3] N. Oztiirk, M. Emin Gunay, S. Akg51, A. Denizli, Biotechnol. Prog. 23 (2007) 1149.

[4] E. Watanabe, J. Fujii, K. ojima, S. Iwatsuki, M.

Inamo, H.D. Takagi, K. Ishihara, Inorg. Chem. Commun. 13 (2010) 1406.

[5] A. . Shukla, J. Th5mmes, Trends Biotechnol. 28 (2010) 253.

[6] D. Low, R. O'Learly, N.S. Pujar, J. Chromatogr. B 848 (2007) 48.

[7] A.M. Azevedo, P.A.J. Rosa, I.F. Ferreira, A.M.M.O. Pisco, J. de Vries, R. Korporaal, T.J. Visse, M.R. Aires- Barros, Sep. Purif. Technol. 65 (2009) 31-39. [8] S.S. Farid, J. Chromatogr. B 848 (2007) 8.

[9] H.Nagaoka, J. Analytical Chem. 83 (2011) 9197.

[10] L.Borlido, A. M. Azevedo, A.G. Sousa, P.H. Oliveira, A.C.A. Roque,M.R. Aires-Barros, J. Chromatogr. B 903 (2012) 163.

[11] Analytical Biochemistry. 167(2): p. 331-339.

[12] Enzyme Microb. Tech, 1990. 12: p. 95-103.

[13] M.Suzuki, M. ., T. Shiraishi, H. Takeuchi Affinity Partitioning of Protein A Using a Magnetic Aqueous Two-Phase System. J. Ferment .Bioeng. , 1995. 80: p. 78- 84.

[14] M.Franzreb, J.B., Method for magnetically supported extraction. US Patent 2010/0116747 Al, 2010.

[15] J.S. Becker a, O.R.T.T., M. Franzreb, Protein separation with magnetic adsorbents in micellar aqueous two-phase systems. Separation and Purification

Technology, 2009. 65: p. 46-53.

[16] V.L. Dhadge., A.H., A. zevedo. , M. R. Aires-Barros., A.C.A.Roque. , Boronic acid modified magnetic materials for antibody purification, in J. R. Soc. Interface 2013.

[17] Dhadge, V.L, Morgado P.I, Freitas F., Reis M.A.,

Azevedo A.M., Aires-Barros R., Roque ACA. 2014. An

Extracellular polymer at the interface of superparamagnetic bioseparation. Journal of the Royal Society Interface. 11 (100) : 20.140.743.

Claims

1. A method for the purification and partitioning of substances, comprising the steps of: a) preparation of an aqueous two phase system; b) addition of the superparamagnetic materials along with the sample to purify; c) phase separation and particle removal by magnetic force; d) washing and isolation of the target substance from superparamagnetic materials.

2. Method according to claim 1, characterized in that the substances comprise, but are not limited to, antibiotics, vitamins, peptides, polypeptides, proteins, antibodies, hormones, nucleic acids, nucleic acids derivatives, organelles, virus, virus-like particles, vesicles or cells.

3. Method according to claim lcharacterized in that superparamagnetic materials used were magnetic particles functionalized with a ligand beneficial for the purification including but not limited to, enzymes, antibodies and derived structures, peptides, polypeptides, proteins, nucleic acids, ion-exchange ligands, hydrophobic interaction ligands, mixed-mode ligands, and many more in related category.

4. Method according to claim 1, characterized in that the aqueous two phase system is composed by mixtures of, but not limited to, polymers, salts, sugars, amino acids, surfactants.

1

5. Method according to claim 1, characterized in that the crude mixture consists on, but not limited to, i) a cell broth obtained by fermentation or cell culture, ii) cell homogenate/lysate, iii) an extract of a natural product; iv) the products of a chemical synthesis .

6. Method according to claim 1, characterized in that the crude mixture is added to the aqueous two phase system under constant agitation along with superparamagnetic materials .

7. Method according to claim 1, comprising a further step of recovering the superparamagnetic materials from mixture using magnetic force.

8. Method according to claim 6, characterized in that the separated superparamagnetic materials are washed until removal of unbound materials.

9. Method according to claim 6, characterized in that the wash solution is an aqueous solution.

10. Method according to claim 1, characterized in that the recovery of the target substance is performed by chemical or physical means, such as, but not limited to an aqueous solution containing buffering compounds, or the action of temperature or pressure.

2