WO2012045331A1 - Hybrid process for the purification of biomolecules - Google Patents
Hybrid process for the purification of biomolecules Download PDFInfo
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- WO2012045331A1 WO2012045331A1 PCT/EP2010/064754 EP2010064754W WO2012045331A1 WO 2012045331 A1 WO2012045331 A1 WO 2012045331A1 EP 2010064754 W EP2010064754 W EP 2010064754W WO 2012045331 A1 WO2012045331 A1 WO 2012045331A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39591—Stabilisation, fragmentation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
Definitions
- the present invention relates to the purification of biomolecules, especially to the purification of peptides, proteins and their derivatives.
- biotechno logical synthesis techniques such as fermentation in large scale
- biotechno logical production process The purification of biomolecules obtained from e.g. biotechno logical synthesis techniques such as fermentation in large scale is a factor which up to date largely determines the usability of a biotechno logical production process.
- a purification process for biomolecules comprising the steps of a) performing at least one aqueous two phase extraction, thereby generating at least one product stream and at least one waste stream b) performing at least one affinity membrane adsorption purification step with the waste stream(s) of step a)
- aqueous two phase extraction in the sense of the present invention especially means and/or includes a process in which two immiscible phases are formed based on the incompatibility of two polymers or one polymer and one salt, dissolved in water respectively.
- the high water content in some applications 55-90 weight%) in both phases allows for partitioning applications involving biomolecules such as proteins, without denaturing their structures.
- WO 2009/112149 A2 for further information which is hereby incorporated by reference.
- product stream in the sense of the present invention especially means and/or includes a solution and/or suspension that contains a greater amount (>80%) of the initial amount of the target-biomolecules.
- waste stream in the sense of the present invention especially means and/or includes a solution and/or suspension from which the product stream was separated and which (if it were a one-step procedure) would be discarded.
- affinity membrane adsorption purification in the sense of the present invention especially means and/or includes a process where the biospecific affinity of a ligand immobilized in an adsorption membrane towards the target molecule is used to purify a crude solution.
- the biomolecules are essentially all organic structures that satisfy the following two conditions: a) An aqueous two phase extraction can be applied to partition the molecular content of the biomolecule of interest into a product stream and a waste stream. b) A molecule exists that binds to the targeted biomolecule and that varies in binding strength according to external conditions such as environmental pH. On top of that, it must be possible that such a molecule can be immobilized in the pores of the membrane adsorber.
- biomolecules which are target of the purification process comprise >95 wt-%, preferably >97 wt-%, more preferred >99 wt-% and most preferred >99,5 wt-% out of proteins.
- proteins in the sense of the present invention is to be understood in the broadest meaning and includes proteins, peptides, protein and peptide derivatives such as (but not limited to) proteins and proteins including non-natural amino acids, ⁇ and/or ⁇ - amino acids (both natural and non-natural), glycosylated amino acid residues, substituted residues with halogens, antibodies, catalytic active proteins and peptides, proteins and peptides including covalently and non-covalently bound metals and metal ions.
- proteins in the sense of the present invention is meant and understood to include all these molecules although in some areas in the field the term “proteins” is lesser or not used for such molecules and is used for a better readability of this text rather than being intended to be limiting.
- the initial concentration of NaCl (Sodium chloride) in step b) is >0 to ⁇ 20 wt%. It has been found out that surprisingly at such a high salt concentration for most application step b) is still performed at an acceptable rate. Since a higher salt concentration is advantageous for step a) the overall process is improved largely.
- the initial concentration of NaCl (Sodium chloride) in step b) is >3 to ⁇ 18% wt%, most preferred >8 wt% to ⁇ 16 wt%.
- the initial concentration of phosphate in step b) is >5 to ⁇ 30 wt%. It has been found out that surprisingly at such a high salt concentration for most application step b) is still performed at an acceptable rate.
- the initial concentration of phosphate in step b) is >8 to ⁇ 25% wt%, most preferred >10 wt% to ⁇ 20 wt%.
- the initial concentration of PEG (Polyethylene glycol) in step b) is >0 and ⁇ 4 wt%.
- the initial concentration of PEG (Polyethylene glycol) in step b) is ⁇ 2 wt%, most preferred ⁇ 1 wt%.
- the initial concentration of the biomolecules to be purified in step b) is >0.01 g/L and ⁇ 4 g/L. This has shown to increase the overall recovery rate for many applications.
- the initial concentration of the biomolecules to be purified in step b) is >0.05 g/L and ⁇ 2 g/L, more preferred >0.1 g/L and ⁇ 1 g/L.
- the initial concentration of the biomolecules to be purified in step b) is ⁇ 30 % of the initial (molar) concentration before performing step a). This has shown to increase the overall recovery rate for many
- the initial concentration of the biomolecules to be purified in step b) is ⁇ 25 % of the initial concentration more preferred ⁇ 15 %.
- Appropriate stock solutions 50wt% PEG in water and 40wt% phosphate in water
- the desired amount of solid NaCl, IgG stock solution and stock solutions are added to a test tube, before the mixture is diluted with water to the desired initial concentration.
- the samples are vortexed until the solid NaCl is dissolved completely.
- the single-step extraction of IgG was done using an ATPS (Aqueous two phase system) in the following concentration range:
- the waste stream (bottom phase) was analyzed before the Second Step (i.e. affinity membrane adsorption) was performed.
- the analysis of the waste stream gave the following exemplary composition:
- IgG Gammanorm ® by Octapharma has been used.
- the buffer compositions were used as listed below
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Engineering & Computer Science (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a purification process of biomolecules, especially proteins and includes an aqueous two phase extraction and a membrane affinity adsorption of the waste stream of the aqueous two phase extraction
Description
Hybrid Process for the Purification of Biomolecules
The present invention relates to the purification of biomolecules, especially to the purification of peptides, proteins and their derivatives.
The purification of biomolecules obtained from e.g. biotechno logical synthesis techniques such as fermentation in large scale is a factor which up to date largely determines the usability of a biotechno logical production process.
Especially the purification of biomolecules based on amino acids, such as peptides, proteins and their derivatives has been shown for many applications to be quite tedious and with inacceptable great losses of product during the purification step.
There is therefore a constant need for novel purification procedures which can be conducted on a large-scale basis and with a higher recovery rate of the biomolecules to be purified.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a purification process for biomolecules which is at least partly able to overcome the disadvantages of the prior art and to provide a large-scale process for the purification of biomolecules with a high purity and recovery rate.
This object is solved by a process according to Claim 1 of the present invention.
Accordingly, a purification process for biomolecules is provided comprising the steps of a) performing at least one aqueous two phase extraction, thereby generating at least one product stream and at least one waste stream b) performing at least one affinity membrane adsorption purification step with the waste stream(s) of step a)
Surprisingly, it has been found that using such a "hybrid" or "two-step" process for many applications the recovery rate of biomolecules can greatly be increased whilst maintaining an excellent purity.
In this regard it should be noted that the nature of the proposed two-stage process allows for decoupling and thereby "separation" of the two steps regardless of time and place, respectively.
The term "aqueous two phase extraction" in the sense of the present invention especially means and/or includes a process in which two immiscible phases are formed based on the incompatibility of two polymers or one polymer and one salt, dissolved in water respectively. Besides the low interfacial stress between the two aqueous phases the high water content ( in some applications 55-90 weight%) in both phases allows for partitioning applications involving biomolecules such as proteins, without denaturing their structures. In this regard it is referred to the WO 2009/112149 A2 for further information which is hereby incorporated by reference.
The term "product stream" in the sense of the present invention especially means and/or includes a solution and/or suspension that contains a greater amount (>80%) of the initial amount of the target-biomolecules.
The term "waste stream" in the sense of the present invention especially means and/or includes a solution and/or suspension from which the product stream was separated and which (if it were a one-step procedure) would be discarded.
The term "affinity membrane adsorption purification" in the sense of the present invention especially means and/or includes a process where the biospecific affinity of a ligand immobilized in an adsorption membrane towards the target molecule is used to purify a crude solution.
According to a preferred embodiment of the present invention, the biomolecules are essentially all organic structures that satisfy the following two conditions: a) An aqueous two phase extraction can be applied to partition the molecular content of the biomolecule of interest into a product stream and a waste stream. b) A molecule exists that binds to the targeted biomolecule and that varies in binding strength according to external conditions such as environmental pH. On top of that, it must be possible that such a molecule can be immobilized in the pores of the membrane adsorber.
The term "essentially" means and/or includes that the biomolecules which are target of the purification process comprise >95 wt-%, preferably >97 wt-%, more preferred >99 wt-% and most preferred >99,5 wt-% out of proteins. The term "proteins" in the sense of the present invention is to be understood in the broadest meaning and includes proteins, peptides, protein and peptide derivatives such as (but not limited to) proteins and proteins including non-natural amino acids, β and/or γ- amino acids (both natural and non-natural), glycosylated amino acid residues, substituted residues with
halogens, antibodies, catalytic active proteins and peptides, proteins and peptides including covalently and non-covalently bound metals and metal ions. The term "proteins" in the sense of the present invention is meant and understood to include all these molecules although in some areas in the field the term "proteins" is lesser or not used for such molecules and is used for a better readability of this text rather than being intended to be limiting.
According to a preferred embodiment of the present invention, the initial concentration of NaCl (Sodium chloride) in step b) is >0 to <20 wt%. It has been found out that surprisingly at such a high salt concentration for most application step b) is still performed at an acceptable rate. Since a higher salt concentration is advantageous for step a) the overall process is improved largely.
Preferably the initial concentration of NaCl (Sodium chloride) in step b) is >3 to <18% wt%, most preferred >8 wt% to <16 wt%.
According to a preferred embodiment of the present invention, the initial concentration of phosphate in step b) is >5 to <30 wt%. It has been found out that surprisingly at such a high salt concentration for most application step b) is still performed at an acceptable rate. Preferably the initial concentration of phosphate in step b) is >8 to <25% wt%, most preferred >10 wt% to <20 wt%.
According to a preferred embodiment of the present invention, the initial concentration of PEG (Polyethylene glycol) in step b) is >0 and <4 wt%.
Preferably the initial concentration of PEG (Polyethylene glycol) in step b) is < 2 wt%, most preferred < 1 wt%.
According to a preferred embodiment of the present invention, the initial concentration of the biomolecules to be purified in step b) is >0.01 g/L and <4 g/L. This has shown to increase the overall recovery rate for many applications. Preferably the initial concentration of the biomolecules to be purified in step b) is >0.05 g/L and <2 g/L, more preferred >0.1 g/L and <1 g/L.
According to a preferred embodiment of the present invention, the initial concentration of the biomolecules to be purified in step b) is <30 % of the initial (molar) concentration before performing step a). This has shown to increase the overall recovery rate for many
applications, the initial concentration of the biomolecules to be purified in step b) is <25 % of the initial concentration more preferred <15 %.
The aforementioned components, as well as the claimed components and the components to be used in accordance with the invention in the described embodiments, are not subject to any special exceptions with respect to their size, shape, material selection and technical concept such that the selection criteria known in the pertinent field can be applied without limitations.
Additional details, characteristics and advantages of the object of the invention are disclosed in the subclaims and the following description of the respective figures—which in an exemplary fashion—show one preferred example of purification process according to the invention.
The proposed invention has been tested for the target molecule ImmunoglobulinG (IgG). First Step: Aqueous Two-Phase Extraction
The First Step has been performed according to the diploma thesis of Omer Yildirim
"Experimental investigation and mathematical modelling of monoclonal antibody liquid-
liquid equilibrium in aqueous two-phase systems'" (TU Dortmund, incorporated by reference) unless as otherwise set forth below.
Appropriate stock solutions (50wt% PEG in water and 40wt% phosphate in water) were prepared. The desired amount of solid NaCl, IgG stock solution and stock solutions are added to a test tube, before the mixture is diluted with water to the desired initial concentration. The samples are vortexed until the solid NaCl is dissolved completely. The single-step extraction of IgG was done using an ATPS (Aqueous two phase system) in the following concentration range:
TABLE I
In accordance with Markus Meier's diploma thesis "Experimented und theoretische Untersuchung des Fliissig-Fliissig-Gleichgewichts wassriger Polyethyleneglykol-Salzsysteme zur Extraktion von monoklonalen Antikorpern" (TU Dortmund, incorporated by reference) it is found that such initial concentrations of PEG and phosphate leads to an ATPS with approximate salt concentrations as follows:
TABLE II
After the First step was completed, the waste stream (bottom phase) was analyzed before the Second Step (i.e. affinity membrane adsorption) was performed. The analysis of the waste stream gave the following exemplary composition:
TABLE III
Further investigations concerning the second step were also performed using a "stock waste solution" having the following salt concentrations:
TABLE IV
Component c c
[wt%] [M]
NaH2P04 23 0.22
K2HP04 11.1 0.73
NaCl 7.2 1.42
pH 6 was found for this
solution
Second Step: Membrane affinity adsorption
The membrane adsorber Sartobincf1 Protein A 2 mL by Sartorius Stedim™ installed in an AKTA purifier 100™ was used for all experiments. The concentration of IgG was assessed using the UV detector (wavelength 280nm). For the target molecule IgG Gammanorm® by Octapharma has been used. The buffer compositions were used as listed below
TABLE V
Step Buffer pH Component c
[mM]
Equilibration/ PBS 7.4 Na2HP04 10
Washing KH2P04 1.5
NaCl 140
KC1 3
Elution Glycine 2.8 NH2CH2COOH 100
10.0-
Regeneration NaOH 11.0 NaOH 50
NaCl 1000
In order to test the validity of the method, before performing the Second Step, the initial IgG concentration was measured and several purifications were performed for different concentrations of IgG:
TABLE IV
c (IgG) mads IMloss,wash meiu recovery rate
[g/i] [mg] [mg] [mg] [%]
1.33 22.90 4.66 23.55 85
1.32 24.83 7.23 25.16 78
0.96 26.38 5.69 26.40 82
0.94 25.73 5.54 26.21 84
0.29 25.74 2.44 26.19 93
0.29 24.77 2.38 26.06 96
0.11 24.35 3.27 25.88 94
c - concentration of IgG,
- adsorbed mass of IgG during loading, loss (wash) - massioss,wash - mass of IgG lost during washing, meiu - eluted mass of IgG, recovery rate = meiu/(mads + mi0ss,wash)
The results reveal that a high recovery rate is achieved regardless of the initial IgG
concentration. It is expected that upon optimization of the experimental setup, for similar systems recovery rates of 99% can be achieved.
The particular combinations of elements and features in the above detailed embodiments are exemplary only; the interchanging and substitution of these teachings with other teachings in this and the patents/applications incorporated by reference are also expressly contemplated. As those skilled in the art will recognize, variations, modifications, and other implementations of what is described herein can occur to those of ordinary skill in the art without departing
from the spirit and the scope of the invention as claimed. Accordingly, the foregoing description is by way of example only and is not intended as limiting. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measured cannot be used to advantage. The invention's scope is defined in the following claims and the equivalents thereto. Furthermore, reference signs used in the description and claims do not limit the scope of the invention as claimed.
Claims
1. A purification process for biomolecules comprising the steps of a) performing at least one aqueous two phase extraction, thereby generating at least one product stream and at least one waste stream
b) performing at least one affinity membrane adsorption purification step with the waste stream(s) of step a) 2. The process of Claim 1 whereby the biomolecules are essentially proteins
3. The process of Claim 1 or 2, whereby the initial concentration of PEG (Polyethylene glycol) in step b) is >0 to <4 wt% 4. The process of any of the Claims 1 to 3, whereby the initial concentration of NaCl
(Sodium chloride) in step b) is >0 to <20 wt%.
5. The process of any of the Claims 1 to 4, whereby , the initial concentration of phosphate in step b) is >5 to <30 wt%
6. The process of any of the claims 1 to 5, whereby the initial concentration of the
biomolecules to be purified in step b) is >0.01 to <4 g/L
7. The process of any of the claims 1 to 4, whereby the initial concentration of the
biomolecules to be purified in step b) is <30 % of the initial concentration before performing step a)
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Citations (3)
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|---|---|---|---|---|
| WO2009112149A2 (en) | 2008-03-10 | 2009-09-17 | Bayer Technology Services Gmbh | Method for purifying therapeutic proteins |
| WO2010030393A1 (en) * | 2008-09-15 | 2010-03-18 | Millipore Corporation | Methods for quantifying protein leakage from protein based affinity chromatography resins |
| WO2010062244A1 (en) * | 2008-11-25 | 2010-06-03 | Ge Healthcare Bio-Sciences Ab | Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins |
-
2010
- 2010-10-04 WO PCT/EP2010/064754 patent/WO2012045331A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009112149A2 (en) | 2008-03-10 | 2009-09-17 | Bayer Technology Services Gmbh | Method for purifying therapeutic proteins |
| WO2010030393A1 (en) * | 2008-09-15 | 2010-03-18 | Millipore Corporation | Methods for quantifying protein leakage from protein based affinity chromatography resins |
| WO2010062244A1 (en) * | 2008-11-25 | 2010-06-03 | Ge Healthcare Bio-Sciences Ab | Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins |
Non-Patent Citations (8)
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| AZEVEDO ANA M ET AL: "Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography", JOURNAL OF CHROMATOGRAPHY B, vol. 877, no. 1-2, January 2009 (2009-01-01), pages 50 - 58, XP002638887, ISSN: 1570-0232 * |
| AZEVEDO ET AL: "Optimisation of aqueous two-phase extraction of human antibodies", JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 132, no. 2, 22 October 2007 (2007-10-22), pages 209 - 217, XP022308446, ISSN: 0168-1656, DOI: DOI:10.1016/J.JBIOTEC.2007.04.002 * |
| BOI ET AL: "Membrane adsorbers as purification tools for monoclonal antibody purification", JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL SCIENCES & APPLICATIONS, ELSEVIER, AMSTERDAM, NL, vol. 848, no. 1, 12 March 2007 (2007-03-12), pages 19 - 27, XP005922824, ISSN: 1570-0232, DOI: DOI:10.1016/J.JCHROMB.2006.08.044 * |
| DIAMOND A D ET AL: "AQUEOUS TWO-PHASE SYSTEMS FOR BIOMOLECULE SEPARATION", ADVANCES IN BIOCHEMICAL ENGINEERING, BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 47, 1 January 1992 (1992-01-01), pages 89 - 135, XP002930072, ISSN: 0724-6145 * |
| MAO LISONG NATHAN ET AL: "Downstream antibody purification using aqueous two-phase extraction.", BIOTECHNOLOGY PROGRESS 2010 NOV-DEC LNKD- DOI:10.1002/BTPR.477 PUBMED:20853347, vol. 26, no. 6, November 2010 (2010-11-01), pages 1662 - 1670, XP002638888, ISSN: 1520-6033 * |
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| ROSA ET AL: "Affinity partitioning of human antibodies in aqueous two-phase systems", JOURNAL OF CHROMATOGRAPHY, ELSEVIER SCIENCE PUBLISHERS B.V, NL, vol. 1162, no. 1, 31 July 2007 (2007-07-31), pages 103 - 113, XP022182050, ISSN: 0021-9673, DOI: DOI:10.1016/J.CHROMA.2007.03.067 * |
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