U.S. patent application number 14/323776 was filed with the patent office on 2015-07-02 for high pressure refolding of monoclonal antibody aggregates.
The applicant listed for this patent is BAROFOLD, INC.. Invention is credited to Lyndal K. Hesterberg, Robert Nelson, Theodore W. Randolph, Matthew B. Seefeldt.
Application Number | 20150183876 14/323776 |
Document ID | / |
Family ID | 40090365 |
Filed Date | 2015-07-02 |
United States Patent
Application |
20150183876 |
Kind Code |
A1 |
Seefeldt; Matthew B. ; et
al. |
July 2, 2015 |
HIGH PRESSURE REFOLDING OF MONOCLONAL ANTIBODY AGGREGATES
Abstract
Methods for refolding antibodies, particularly monoclonal
antibodies, from aggregated and/or denatured preparations by
subjecting the antibody preparation to high hydrostatic pressure
are provided. Refolded preparations of antibodies produced by the
methods described herein are also provided.
Inventors: |
Seefeldt; Matthew B.;
(Denver, CO) ; Nelson; Robert; (Arvada, CO)
; Randolph; Theodore W.; (Niwot, CO) ; Hesterberg;
Lyndal K.; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAROFOLD, INC. |
Aurora |
CO |
US |
|
|
Family ID: |
40090365 |
Appl. No.: |
14/323776 |
Filed: |
July 3, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13405573 |
Feb 27, 2012 |
8802828 |
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14323776 |
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12208193 |
Sep 10, 2008 |
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13405573 |
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60971223 |
Sep 10, 2007 |
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Current U.S.
Class: |
530/387.3 |
Current CPC
Class: |
C07K 1/1136 20130101;
C07K 16/2827 20130101; C07K 2319/33 20130101; C07K 14/70521
20130101; C07K 2319/30 20130101; C07K 1/113 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 1/113 20060101 C07K001/113; C07K 14/705 20060101
C07K014/705 |
Claims
1. A method for refolding a sample of an antibody, comprising the
steps of: obtaining an antibody sample comprising a solution of
antibody exposed to low pH; adjusting the pH of the solution to
above pH 5.0 if necessary; exposing the antibody sample to high
hydrostatic pressure for a period of time; and reducing the
hydrostatic pressure to atmospheric pressure; wherein the antibody
sample after pressure exposure has a higher content of monomeric
antibody, a higher content of properly folded antibody, or a lower
content of aggregated antibody than the antibody sample prior to
the pressure exposure.
2. The method of claim 1, wherein the antibody is monoclonal.
3. The method of claim 2, wherein the content of monomeric or
properly folded antibody is measured by a method selected from a
binding assay specific for the antibody, analytical
ultracentrifugation, size exclusion chromatography, field flow
fractionation, light scattering analysis, light obscuration
analysis, fluorescence spectroscopy, gel electrophoresis, GEMMA,
nuclear magnetic resonance spectroscopy, electron paramagnetic
resonance spectroscopy, or reverse-phase chromatography.
4. The method of claim 2, wherein the high hydrostatic pressure is
between about 1500 bar and a value about 50 bar below the
denaturation pressure of the native antibody.
5. The method of claim 4, wherein the high hydrostatic pressure is
between about 1500 bar and about 3000 bar.
6. The method of claim 2, wherein the ionic strength of the
antibody sample comprising an antibody solution is less than a
value about 25 mM below the denaturation point of the native
antibody.
7. The method of claim 6, wherein the ionic strength of the
antibody sample comprising an antibody solution is less than about
200 mM.
8. The method of claim 2, wherein the antibody is abatacept.
9. The method of claim 8, wherein the pH of the abatacept antibody
solution is at or above about 7.
10. The method of claim 9, wherein sucrose is present in the
antibody solution at a concentration of about 5% to about 15%.
11. The method of claim 10, wherein sucrose is present in the
antibody solution at a concentration of about 10%.
12. The method of claim 7, wherein the ionic strength of the
antibody solution is less than about 100 mM.
13. The method of claim 8, wherein the amount of aggregated
antibody present in the antibody sample after pressure exposure is
decreased by at least about 20% as compared to the amount of
aggregated antibody present in the antibody sample before pressure
exposure.
14. The method of claim 8, wherein the amount of aggregated
antibody present in the antibody sample after pressure exposure is
less than about 1%.
15. A preparation of abatacept antibody comprising less than about
1.0% aggregated antibody.
16. A preparation of abatacept antibody comprising about 0.8% or
less aggregated antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of U.S. Provisional
Patent Application No. 60/971,223, filed Sep. 10, 2007. The entire
contents of that application are hereby incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] This invention relates to the high pressure refolding of
antibody aggregates, and in particular monoclonal antibody
aggregates.
BACKGROUND OF THE INVENTION
[0003] Many proteins are valuable as therapeutic agents. Protein
therapeutics are often produced using recombinant DNA technology,
which can enable production of higher amounts of protein than can
be isolated from naturally-occurring sources, and which avoids
contamination that often occurs with proteins isolated from
naturally-occurring sources.
[0004] Proper folding of a protein is essential to the normal
functioning of the protein. Improperly folded proteins are believed
to contribute to the pathology of several diseases, including
Alzheimer's disease, bovine spongiform encephalopathy (BSE, or "mad
cow" disease) and human Creutzfeldt-Jakob disease (CJD), and
Parkinson's disease; these diseases serve to illustrate the
importance of proper protein folding.
[0005] Proteins of therapeutic value in humans can be expressed in
bacteria, yeast, and other microorganisms. While large amounts of
proteins can be produced in such systems, the proteins are often
misfolded, and often aggregate together in large clumps called
inclusion bodies. The proteins cannot be used in the misfolded,
aggregated state. Accordingly, methods of disaggregating and
properly refolding such proteins have been the subject of much
investigation.
[0006] One method of refolding proteins uses high pressure on
solutions of proteins in order to disaggregate, unfold, and
properly refold proteins. Such methods are described in U.S. Pat.
No. 6,489,450, U.S. Pat. No. 7,064,192, U.S. Patent Application
Publication No. 2004/0038333, and International Patent Application
WO 02/062827. Those disclosures indicated that certain
high-pressure treatments of aggregated proteins or misfolded
proteins resulted in recovery of disaggregated protein retaining
biological activity (i.e., the protein was properly folded, as is
required for biological activity) in good yields. U.S. Pat. No.
6,489,450, U.S. Pat. No. 7,064,192, U.S. 2004/0038333, and WO
02/062827 are incorporated by reference herein in their
entireties.
[0007] Certain devices have also been developed which are
particularly suitable for refolding of proteins under high
pressure; see International Patent Application Publication No. WO
2007/062174, which is incorporated by reference herein in its
entirety.
[0008] Several monoclonal antibodies are currently in use as
therapeutic agents, for example, Herceptin.RTM. (Trastuzumab)
(Herceptin.RTM. is a registered trademark of Genentech, Inc., South
San Francisco, Calif., for a monoclonal antibody useful in treating
breast cancer) and Remicade.RTM. (Infliximab) (Remicade.RTM. is a
registered trademark of Centocor, Inc., Malvern, Pa., for a
monoclonal antibody useful in treating inflammatory disorders
involving the immune system such as rheumatoid arthritis).
Unfortunately, some of the most widely used processing steps for
monoclonal antibody production, such as Protein A/G affinity
purification and/or viral inactivation steps, require use of
solutions at pH levels as low as approximately pH 3.0 during
typical pharmaceutical protein manufacturing (Ejima et al.,
Proteins, 66:954-62 (2007)). Monoclonal antibodies readily
aggregate during treatment at pH 3.0, possibly due to
destabilization of the Fc domain. These aggregates can be difficult
to remove and result in increased production costs. See, Thommes,
J. and M. Etzel, Biotechnology Progress 23(1): 42-45 (2007) for a
discussion of these issues. High pressure refolding provides a
viable method for alleviating aggregation of monoclonal antibodies
induced by manufacturing processes.
[0009] The effect of aggregate formation conditions on the
pressure-modulated refolding yield is currently unknown. An earlier
report (St. John, R. J., J. F. Carpenter, et al., Journal of
Biological Chemistry 276(50): 46856-46863 (2001)) showed that the
refolding yields of recombinant human growth hormone from two
different insoluble aggregates contained different secondary
structures and resulted in different refolding kinetics and yields.
Thus, protein aggregates produced by different stresses exhibited
different refolding behaviors. Consequently, the specific
conditions required for refolding mAb aggregates formed after
incubation at pH 3.0 may be unpredictable.
[0010] The instant invention provides methods useful in refolding
monoclonal antibody aggregates produced after exposure to low pH,
for example, approximately pH 3.0, as well as preparations
containing such monoclonal antibody aggregates.
SUMMARY OF THE INVENTION
[0011] The present invention provides particularly effective and
efficient methods for refolding antibodies, particularly monoclonal
antibodies, using high-pressure techniques (high hydrostatic
pressure), as well as preparations of refolded monoclonal
antibodies refolded using such high-pressure techniques. More
specifically, the present invention is directed to high pressure
refolding of monoclonal antibody aggregates produced after exposure
to low pH. The methods provide routes for overcoming the
difficulties in protein therapeutic processing, by employing the
use of high pressure techniques. These methods allow for the
disaggregation, refolding, and production of high quality
antibodies, while circumventing problems that would otherwise be
associated with therapeutic protein production. The methods
advantageously provide processing benefits associated with the use
of high pressure refolding of protein aggregates.
[0012] The basic method involves obtaining an antibody sample
subsequent to incubation at low pH, for example, approximately pH
3.0 comprising a solution of antibody, exposing the antibody sample
to high hydrostatic pressure for a period of time, and then
reducing the hydrostatic pressure to atmospheric pressure,
resulting in an antibody sample with a higher content of monomeric
or properly refolded antibody than prior to the pressure
exposure.
[0013] In one embodiment, the invention embraces a method for
refolding a sample of an antibody, where the antibody sample
comprises a solution of antibody exposed to low pH (such as pH 3).
Such an antibody sample may still be at a solution condition of low
pH, in which case, the pH of the solution is adjusted to above pH
5.0. The antibody sample is then exposed to high hydrostatic
pressure for a period of time; subsequently, the pressure is
reduced to atmospheric pressure. The antibody sample, after such
pressure exposure, has a higher content of monomeric antibody, a
higher content of properly folded antibody, or a lower content of
aggregated antibody than the antibody sample prior to the pressure
exposure. In one embodiment, the antibody sample after pressure
exposure has a higher content of monomeric antibody than prior to
pressure exposure. In one embodiment, the antibody sample after
pressure exposure has a higher content of properly refolded
antibody than prior to pressure exposure. In one embodiment, the
antibody sample after pressure exposure has a lower content of
aggregated antibody than prior to pressure exposure.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 depicts high pressure refolding of pH 3 induced
aggregates of CTLA-4Ig (0.5 mg/ml) as a function of pH.
[0015] FIG. 2 depicts the effect of excipients on the refolding of
CTLA-4Ig low pH induced aggregates.
[0016] FIG. 3 depicts the effect of pressure on the refolding of
CTLA-4Ig low pH induced aggregates.
[0017] FIG. 4 depicts high pressure refolding of pH 3 induced
aggregates of Alliance mAb as a function of pressure.
[0018] FIG. 5 depicts the effect of excipients on the pressure
refolding of the Alliance mAb acid-induced aggregates.
[0019] FIG. 6 depicts the effect of temperature on refolding of the
Alliance mAb aggregated by exposure to pH 3.0.
DETAILED DESCRIPTION OF THE INVENTION
[0020] All publications and patents mentions herein are hereby
incorporated by reference in their respective entireties. The
publications and patents disclosed herein are provided solely for
their disclosure. Nothing herein is to be construed as an admission
that the inventors are not entitled to antedate any publication
and/or patent, including any publication and/or patent cited
herein. U.S. Pat. Nos. 6,489,450 and 7,064,192, United States
Patent Application Publication Nos. 2004/0038333 and 2006/0188970,
and International Patent Application Publication No. WO 2007/062174
are specifically incorporated herein by reference in their
entirety. In particular, the experimental techniques for refolding
found in those documents are incorporated by reference herein.
[0021] The embodiments of the present invention described below are
not intended to be exhaustive or to limit the invention to the
precise forms disclosed in the following detailed description.
Rather, the embodiments are chosen and described so that others
skilled in the art can appreciated and understand the principles
and practices of the present invention.
[0022] The methods of the present invention can be used to refold
monoclonal antibodies, and are especially useful for the refolding
of antibody aggregates produced after exposure to low pH. Unless
otherwise stated, the following terms used in the specification and
claims have the meaning(s) provided herein.
[0023] By "low pH" is meant solution conditions of about pH 1.0 to
5.0, preferably about pH 2.0 to about 4.0, more preferably about
2.5 to about 3.5, more preferably about 2.8 to about 3.2, more
preferably about 3.0.
[0024] As used herein, a "protein aggregate" is defined as being
composed of a multiplicity of protein molecules wherein non-native
noncovalent interactions and/or non-native covalent bonds (such as
non-native intermolecular disulfide bonds) hold the protein
molecules together. Typically, but not always, an aggregate
contains sufficient molecules so that it is insoluble; such
aggregates are insoluble aggregates. There are also oligomeric
proteins which occur in aggregates in solution; such aggregates are
soluble aggregates. In addition, there is typically (but not
always) a display of at least one epitope or region on the
aggregate surface which is not displayed on the surface of native,
non-aggregated protein. "Inclusion bodies" are a type of aggregate
of particular interest to which the present invention is
applicable. Other protein aggregates include, but are not limited
to, soluble and insoluble precipitates, soluble non-native
oligomers, gels, fibrils, films, filaments, protofibrils, amyloid
deposits, plaques, and dispersed non-native intracellular
oligomers.
[0025] "Atmospheric," "ambient," or "standard" pressure is defined
as approximately 15 pounds per square inch (psi) or approximately 1
bar or approximately 100,000 Pascals.
[0026] "Biological activity" of a protein or polypeptide as used
herein, means that the protein or polypeptide retains at least
about 10% of maximal known specific activity as measured in an
assay that is generally accepted in the art to be correlated with
the known or intended utility of the protein. For proteins or
polypeptides intended for therapeutic use, the assay of choice is
one accepted by a regulatory agency to which data on safety and
efficacy of the protein or polypeptide must be submitted. In some
embodiments, a protein or polypeptide having at least about 10% of
maximal known specific activity or of the non-denatured molecule is
"biologically active" for the purposes of the invention. In some
embodiments, the biological activity is at least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least
about 40%, at least about 50%, at least about 75%, or at least
about 90% of maximal known specific activity or of the
non-denatured molecule.
[0027] "Denatured," as applied to a protein in the present context,
means that native secondary, tertiary, and/or quaternary structure
is disrupted to an extent that the protein does not have biological
activity.
[0028] The "native conformation" of a protein refers to the
secondary, tertiary and/or quaternary structures of a protein in
its biologically active state.
[0029] "Refolding" in the present context means the process by
which a fully or partially denatured polypeptide adopts secondary,
tertiary and quaternary structure like that of the cognate native
molecule. A properly refolded polypeptide has biological activity
that is at least about 10% of the non-denatured molecule,
preferably biological activity that is substantially that of the
non-denatured molecule. In some embodiments, the biological
activity is at least about 15%, at least about 20%, at least about
25%, at least about 30%, at least about 40%, at least about 50%, at
least about 75%, or at least about 90% of the non-denatured
molecule. Where the native polypeptide has disulfide bonds,
oxidation to form native disulfide bonds is a desired component of
the refolding process.
[0030] Antibodies which can be refolded with the methods of the
invention include a wide variety of polyclonal or monoclonal
preparations; monoclonal antibodies are the preferred antibody
embodiment for refolding with the methods of the invention. The
methods of the present invention are applicable to an antibody
prepared via a typical process that may include exposure to low pH,
e.g., pH 3.0, during preparation. Examples of antibodies which can
be refolded with the methods of the invention, along with their
indications, include, but are not limited to: Avastin.RTM.
(bevacizumab, Genentech, Inc. South San Francisco, Calif.) for
treatment of metastatic colorectal cancer and non-small cell lung
cancer; Bexxar.RTM. (tositumomab, Smithkline Beecham Corp.,
Philadelphia Pa.) for treatment of non-Hodgkin's lymphoma;
Campath.RTM. (alemtuzumab, Genzyme Corporation, Cambridge Mass.)
for treatment of B-cell chronic lymphocytic leukemia; Erbitux.RTM.
(cetuximab, ImClone Systems Inc., New York, N.Y.) for treatment of
colorectal cancer; Herceptin.RTM. (trastuzumab, Genentech, Inc.
South San Francisco, Calif.) for treatment of breast cancer;
Humira.RTM. (adalimumab, Abbott Biotechnology Ltd., Hamilton
Bermuda) for treatment of rheumatoid arthritis, psoriatic
arthritis, ankylosing spondylitis, and Crohn's disease;
Lucentis.RTM. (ranibizumab, Genentech, Inc. South San Francisco,
Calif.) for treatment of wet age-related macular degeneration;
Mylotarg.RTM. (gemtuzumab ozogamicin (antibody conjugated to
antibiotic calicheamicin), Wyeth Corp., Madison, N.J.) for
treatment of acute leukemia; Orencia.RTM. (abatacept; Bristol-Myers
Squibb Co., New York, N.Y.) for treatment of rheumatoid arthritis;
Orthoclone OKT3.RTM. (muromonab-CD3, Johnson & Johnson Corp.,
New Brunswick, N.J. for treatment of transplant rejection;
Raptiva.RTM. (efalizumab, Genentech, Inc. South San Francisco,
Calif.) for treatment of plaque psoriasis; Remicade.RTM.
(infliximab, Centocor, Inc., Malvern, Pa.) for treatment of
rheumatoid arthritis, Crohn's disease, ankylosing spondylitis,
psoriatic arthritis, plaque psoriasis, ulcerative colitis;
ReoPro.RTM. (abciximab, Eli Lilly and Co., Indianapolis Ind.) as an
adjunct to percutaneous coronary intervention; Rituxan.RTM.
(rituximab, Biogen IDEC Inc., Cambridge Mass. and Genentech, Inc.
South San Francisco, Calif.) for treatment of non-Hodgkin's
lymphoma and rheumatoid arthritis; Simulect.RTM. (basiliximab,
Novartis AG, Basel, Switzerland) for treatment of acute organ
rejection; Soliris.RTM. (eculizumab, Alexion Pharmaceuticals, Inc.,
Cheshire, Conn.) for treatment of paroxysmal nocturnal
hemoglobinuria; Synagis.RTM. (palivizumab, Medlmmune, Inc.,
Gaithersburg, Md.) for treatment of respiratory syncytial virus;
Tysabri.RTM. (natalizumab, Elan Pharmaceuticals, Inc., South San
Francisco, Calif.) for treatment of multiple sclerosis;
Vectibix.RTM. (panitumumab, Immunex Corp., Thousand Oaks, Calif.)
for treatment of metastatic colorectal cancer; Xolair.RTM.
(omalizumab, Novartis AG, Basel, Switzerland) for treatment of
allergic asthma; Zenapax.RTM. (daclizumab, Roche Inc., Nutley,
N.J.) for treatment of acute organ rejection; and Zevalin.RTM.
(ibritumomab tiuxetan, Biogen IDEC Inc., Cambridge Mass.) for
treatment of B-cell non-Hodgkin's lymphoma.
[0031] A wide variety of techniques are known in the art for
protein separation and purification, such as affinity
chromatography, high-pressure liquid chromatography (HPLC),
dialysis, ion exchange chromatography, size exclusion
chromatography, reverse-phase chromatography, ammonium sulfate
precipitation, or electrophoresis. Several conditions for HPLC can
be varied for enhancing separation, such as the stationary and
mobile phases. HPLC can be used with ion-exchange columns,
reverse-phase columns, affinity columns, size-exclusion columns,
and other types of columns. FPLC, or "Fast Performance Liquid
Chromatography," can also be used. Gel-filtration chromatography
can be used at low solvent pressures. Removal of small molecules
(such as chaotropes, kosmotropes, surfactants, detergents, reducing
agents, oxidizing agents, or small molecule binding partners) from
protein solutions can be achieved via diafiltration,
ultrafiltration, or dialysis.
[0032] There are several assay methods for analyzing the
monomeric/refolded content of antibody in a sample. A specific
binding assay can be employed, to determine the degree of binding
of the antibody to a specific binding partner. A binding assay
measures the amount of active protein and is thus quite useful for
determining the amount of functional protein.
[0033] Several methods based on physical parameters are available
for analyzing and quantitating aggregated proteins, such as
antibodies, and determining amounts of aggregated proteins and
monomeric proteins. An excellent overview of several methods of
analysis of macromolecules is found in Cantor, C. R. and P. R.
Schimmel, Biophysical Chemistry Part II: Techniques for the Study
of Biological Structure and Function, W.H. Freeman & Co., New
York: 1980. Other general techniques are described in US Patent
Application Publication No. 2003/0022243.
[0034] The use of analytical ultracentrifugation for
characterization of aggregation of protein therapeutics is
specifically discussed in Philo, J. S., American Biotechnology
Laboratory, page 22, October 2003. Experiments that can be
performed using analytical ultracentrifugation include
sedimentation velocity and sedimentation equilibrium experiments,
which can be performed to determine whether multiple solutes exist
in a solution (e.g., monomer, dimer, trimer, etc.) and provide an
estimate of molecular weights for the solutes.
[0035] Size-exclusion chromatography and gel permeation
chromatography can be used to estimate molecular weights and
aggregation numbers of proteins, as well as for separation of
different aggregates. See references such as Wu, C.-S. (editor),
Handbook of Size Exclusion Chromatography and Related Techniques,
Second Edition (Chromatographic Science), Marcel Dekker: New York,
2004 (particularly chapter 15 at pages 439-462 by Baker et al.,
"Size Exclusion Chromatography of Proteins") and Wu, C.-S.
(editor), Column Handbook for Size Exclusion Chromatography, San
Diego: Academic Press, 1999 (particularly Chapters 2 and 18).
[0036] Field flow fractionation, which relies on a field
perpendicular to a liquid stream of molecules, can also be used to
analyze and separate aggregated proteins such as protein monomers,
dimers, trimers, etc. See Zhu et al., Anal. Chem. 77:4581 (2005);
Litzen et al., Anal. Biochem. 212:469 (1993); and Reschiglian et
al., Trends Biotechnol. 23:475 (2005).
[0037] Light scattering methods, such as methods using laser light
scattering (often in conjunction with size-exclusion chromatography
or other methods) can also be used to estimate the molecular weight
of proteins, including protein aggregates; see, for example,
Mogridge, J., Methods Mol Biol. 261:113 (2004) and Ye, H.,
Analytical Biochem. 356:76 (2006). Dynamic light scattering
techniques are discussed in Pecora, R., ed., Dynamic Light
Scattering: Applications of Photon Correlation Spectroscopy, New
York: Springer Verlag, 2003 and Berne, B. J. and Pecora, R.,
Dynamic Light Scattering: With Applications to Chemistry, Biology,
and Physics, Mineola, N.Y.: Dover Publications, 2000. Laser light
scattering is discussed in Johnson, C. S. and Gabriel, D. A., Laser
Light Scattering, Mineola, N.Y.: Dover Publications, 1995, and
other light scattering techniques which can be applied to determine
protein aggregation are discussed in Kratochvil, P., Classical
Light Scattering from Polymer Solutions, Amsterdam: Elsevier,
1987.
[0038] Light obscuration can also be used to measure protein
aggregation; see Seefeldt et al., Protein Sci. 14:2258 (2005); Kim
et al., J. Biol. Chem. 276: 1626 (2001); and Kim et al., J. Biol.
Chem. 277: 27240 (2002).
[0039] Fluorescence spectroscopy, such as fluorescence anisotropy
spectroscopy, can be used to determine the presence of protein
aggregates. Fluorescence probes (dyes) can be covalently or
non-covalently bound to the aggregate to aid in analysis of
aggregates (see, e.g., Lindgren et al., Biophys. J. 88: 4200
(2005)), US Patent Application Publication 2003/0203403), or Royer,
C.A., Methods Mol. Biol. 40:65 (1995). Internal tryptophan residues
can also be used to detect protein aggregation; see, e.g., Dusa et
al., Biochemistry 45:2752 (2006).
[0040] Many methods of gel electrophoresis can be employed to
analyze proteins and protein aggregation. One of the most common
methods of gel electrophoresis is polyacrylamide gel
electrophoresis (PAGE). If an aggregate is covalently linked,
denaturing PAGE (using, e.g., sodium dodecyl sulfate) can be
employed. Native PAGE (non-denaturing PAGE) can be used to study
non-covalently linked aggregates. See, e.g., Hermeling et al. J.
Phar. Sci. 95:1084-1096 (2006); Kilic et al., Protein Sci. 12:1663
(2003); Westermeier, R., Electrophoresis in Practice: A Guide to
Methods and Applications of DNA and Protein Separations 4.sup.th
edition, New York: John Wiley & Sons, 2005; and Hames, B. D.
(Ed.), Gel Electrophoresis of Proteins: A Practical Approach,
3.sup.rd edition, New York: Oxford University Press, USA, 1998.
[0041] Gas-phase electrophoretic mobility molecular analysis
(GEMMA) (see Bacher et al., J. Mass Spectrom. 36:1038 (2001),
Kaufman et al., Anal. Chem. 68:1895 (1996) and Kaufman et al.,
Anal. Biochem. 259:195 (1998)), a combination of electrophoresis in
the gas phase and mass spectrometry, provides another method of
analyzing protein complexes and aggregates.
[0042] Nuclear magnetic resonance spectroscopic techniques can be
used to estimate hydrodynamic parameters related to protein
aggregation. See, for example, James, T. L. (ed.), Nuclear Magnetic
Resonance of Biological Macromolecules, Part C, Volume 394: Methods
in Enzymology, San Diego: Academic Press, 2005; James, T. L.,
Dotsch, V. and Schmitz, U. (eds.), Nuclear Magnetic Resonance of
Biological Macromolecules, Part A (Methods in Enzymology, Volume
338) and Nuclear Magnetic Resonance of Biological Macromolecules,
Part B (Methods in Enzymology, Volume 339), San Diego: Academic
Press, 2001, and Mansfield, S. L. et al., J. Phys. Chem. B,
103:2262 (1999). Linewidths, correlation times, and relaxation
times are among the parameters that can be measured to estimate
tumbling time in solution, which can then be correlated with the
state of protein aggregation. Electron paramagnetic resonance (EPR
or ESR) can also be used to determine aggregation states; see,
e.g., Squier et al., J. Biol. Chem. 263:9162 (1988).
[0043] Reverse-phase high-pressure liquid chromatography (RP-HPLC)
can also be used to determine monomer/aggregate content of protein
preparations, although this method must be used cautiously, as the
conditions used for RP-HPLC may alter the amount of aggregate
present in the original sample.
[0044] In one embodiment of the invention, analytical
ultracentrifugation is used for the comparison of aggregates in
pressure-treated and untreated samples. In another embodiment of
the invention, size exclusion chromatography is used for the
comparison of aggregates in pressure-treated and untreated samples.
In another embodiment of the invention, field flow fractionation is
used for the comparison of aggregates in pressure-treated and
untreated samples. In another embodiment of the invention, light
scattering analysis is used for the comparison of aggregates in
pressure-treated and untreated samples. In another embodiment of
the invention, light obscuration analysis is used for the
comparison of aggregates in pressure-treated and untreated samples.
In another embodiment of the invention, fluorescence spectroscopy
is used for the comparison of aggregates in pressure-treated and
untreated samples. In another embodiment of the invention, gel
electrophoresis is used for the comparison of aggregates in
pressure-treated and untreated samples. In another embodiment of
the invention, GEMMA is used for the comparison of aggregates in
pressure-treated and untreated samples. In another embodiment of
the invention, nuclear magnetic resonance spectroscopy is used for
the comparison of aggregates in pressure-treated and untreated
samples. In another embodiment of the invention, electron
paramagnetic resonance spectroscopy is used for the comparison of
aggregates in pressure-treated and untreated samples. In another
embodiment of the invention, reverse-phase chromatography is used
for the comparison of aggregates in pressure-treated and untreated
samples.
[0045] Several conditions can be adjusted for optimal protein
refolding: protein concentration; agitation; temperature; reduction
of pressure and combinations thereof.
[0046] The concentration of protein can be adjusted for optimal
protein refolding. One advantage of high-pressure protein refolding
is that much higher concentrations of protein can be used as
compared to chemical refolding techniques. Protein concentrations
of at least about 0.1 mg/ml, at least about 1.0 mg/ml, at least
about 5.0 mg/ml, at least about 10 mg/ml, or at least about 20
mg/ml can be used. Protein in the sample may be present in a
concentration of from about 0.001 mg/ml to about 300 mg/ml. Thus,
in some embodiments the protein is present in a concentration of
from about 0.001 mg/ml to about 250 mg/ml, from about 0.001 mg/ml
to about 200 mg/ml, from about 0.001 mg/ml to about 150 mg/ml, from
about 0.001 mg/ml to about 100 mg/ml, from about 0.001 mg/ml to
about 50 mg/ml, from about 0.001 mg/ml to about 30 mg/ml, from
about 0.05 mg/ml to about 300 mg/ml, from about 0.05 mg/ml to about
250 mg/ml, from about 0.05 mg/ml to about 200 mg/ml, from about
0.05 mg/ml to about 150 mg/ml, from about 0.05 mg/ml to about 100
mg/ml, from about 0.05 mg/ml to about 50 mg/ml, from about 0.05
mg/ml to about 30 mg/ml, from about 10 mg/ml to about 300 mg/ml,
from about 10 mg/ml to about 250 mg/ml, from about 10 mg/ml to
about 200 mg/ml, from about 10 mg/ml to about 150 mg/ml, from about
10 mg/ml to about 100 mg/ml, from about 10 mg/ml to about 50 mg/ml,
from about 10 mg/ml to about 30 mg/ml, from about 0.1 mg/ml to
about 100 mg/ml, from about 0.1 mg/ml to about 10 mg/ml, from about
1 mg/ml to about 100 mg/ml, from about 1 mg/ml to about 10 mg/ml,
from about 10 mg/ml to about 100 mg/ml, or from about 50 mg/ml to
about 100 mg/ml can be used.
[0047] As used in the present context the phrase "a period of time
sufficient to form biologically active protein" and cognates
thereof refer to the time needed for the protein aggregates to be
disaggregated and to adopt a conformation where the protein is
biologically active. Typically, the time sufficient for
solubilization is about 15 minutes to about 50 hours, or possibly
longer depending on the particular protein, (e.g., as long as
necessary for the protein; for example, up to about 1 week, about 5
days, about 4 days, about 3 days, etc.). Thus, in some embodiments
of the methods, the time sufficient for formation of biologically
active protein may be from about 2 to about 30 hours, from about 2
to about 24 hours, from about 2 to about 18 hours, from about 1 to
about 10 hours, from about 1 to about 8 hours, from about 1 to
about 6 hours, from about 2 to about 10 hours, from about 2 to
about 8 hours, from about 2 to about 6 hours, or about 2 hours,
about 6 hours, about 10 hours, about 16 hours, about 20 hours, or
about 30 hours, from about 2 to about 10 hours, from about 2 to
about 8 hours, from about 2 to about 6 hours, from about 12 to
about 18 hours, or from about 10 to about 20 hours.
[0048] The sample comprising protein aggregates or denatured
protein is typically an aqueous solution or aqueous suspension. The
sample may also include other components. These additional
components may be one or more additional agents including: one or
more stabilizing agents, one or more buffering agents, one or more
surfactants, one or more salts, one or more chaotropes, or
combinations of two or more of the foregoing.
[0049] The amounts of the additional agents will vary depending on
the selection of the protein, however, the effect of the presence
(and amount) or absence of each additional agent or combinations of
agents can be determined and optimized using the teachings provided
herein.
[0050] Exemplary additional agents include, but are not limited to,
buffers (examples include, but are not limited to, phosphate
buffer, borate buffer, carbonate buffer, citrate buffer, HEPES,
MEPS), salts (examples include, but are not limited to, the
chloride, sulfate, and carbonate salts of sodium, zinc, calcium,
ammonium and potassium), chaotropes (examples include, but are not
limited to, urea, guanidine hydrochloride, guanidine sulfate and
sarcosine), and stabilizing agents (e.g., preferential excluding
compounds, etc.).
[0051] Non-specific protein stabilizing agents act to favor the
most compact conformation of a protein. Such agents include, but
are not limited to, one or more free amino acids, one or more
preferentially excluding compounds, kosmotropes, trimethylamine
oxide, cyclodextrans, molecular chaperones, and combinations of two
or more of the foregoing.
[0052] Amino acids can be used to prevent reaggregation and
facilitate the dissociation of hydrogen bonds. Typical amino acids
that can be used, but not limited to, are arginine, lysine,
proline, glycine, histidine, and glutamine or combinations of two
or more of the foregoing. In some embodiments, the free amino
acid(s) is present in a concentration of about 0.1 mM to about the
solubility limited of the amino acid, and in some variations from
about 0.1 mM to about 2 M. The optimal concentration is a function
of the desired protein and should favor the native
conformation.
[0053] Preferentially excluding compounds can be used to stabilize
the native confirmation of the protein of interest. Possible
preferentially excluding compounds include, but are not limited to,
sucrose, hexylene glycol, sugars (e.g., sucrose, trehalose,
dextrose, mannose), and glycerol. The range of concentrations that
can be use are from 0.1 mM to the maximum concentration at the
solubility limit of the specific compound. The optimum preferential
excluding concentration is a function of the protein of
interest.
[0054] In particular embodiments, the preferentially excluding
compound is one or more sugars (e.g., sucrose, trehalose, dextrose,
mannose or combinations of two or more of the foregoing). In some
embodiments, the sugar(s) is present in a concentration of about
0.1 mM to about the solubility limit of the particular compound. In
some embodiments, the concentration is from about 0.1 mM to about 2
M, from about 0.1 mM to about 1.5 M, from about 0.1 mM to about 1
M, from about 0.1 mM to about 0.5 M, from about 0.1 mM to about 0.3
M, from about 0.1 mM to about 0.2 M, from about 0.1 mM to about 0.1
mM, from about 0.1 mM to about 50 mM, from about 0.1 mM to about 25
mM, or from about 0.1 mM to about 10 mM. When present as a
percentage of solution (w/w and/or w/v), the concentration can be
about 0.1% to about 20%, about 1% to about 20%, about 1% to about
15%, about 5% to about 20%, about 5% to about 15%, about 8% to
about 12%, or about 10%.
[0055] In some embodiments, the stabilizing agent is one or more of
sucrose, trehalose, glycerol, betaine, amino acid(s), or
trimethylamine oxide.
[0056] In certain embodiments, the stabilizing agent is a
cyclodextran. In some embodiments, the cyclodextran is present in a
concentration of about 0.1 mM to about the solubility limit of the
cyclodextran. In some variations, the cyclodextran is present in a
concentration from about 0.1 mM to about 2 M.
[0057] In certain embodiments, the stabilizing agent is a molecular
chaperone. In some embodiments, the molecular chaperone is present
in a concentration of about 0.01 mg/ml to 10 mg/ml.
[0058] A single stabilizing agent maybe be used or a combination of
two or more stabilizing agents (e.g., at least two, at least three,
or 2 or 3 or 4 stabilizing agents). Where more than one stabilizing
agent is used, the stabilizing agents may be of different types,
for example, at least one preferentially excluding compound and at
least one free amino acid, at least one preferentially excluding
compound and betaine, etc.
[0059] Buffering agents may be present to maintain a desired pH
value or pH range. Numerous suitable buffering agents are known to
the skilled artisan and should be selected based on the pH that
favors (or which does not disfavor) the native conformation of the
protein of interest. Either inorganic or organic buffering agents
may be used. Suitable concentrations are known to the skilled
artisan and should be optimized for the methods as described herein
according to the teaching provided based on the characteristics of
the desired protein.
[0060] Thus, in some embodiments, at least one inorganic buffering
agent is used (e.g., phosphate, carbonate, etc.). In certain
embodiments, at least one organic buffering agent is used (e.g.,
citrate, acetate, Tris, MOPS, MES, HEPES, etc.) Additional organic
and inorganic buffering agents are well known to the art.
[0061] In some embodiments, the one or more buffering agents is
phosphate buffer, borate buffer, carbonate buffer, citrate buffer,
HEPES, MEPS, MOPS, MES, or acetate buffer.
[0062] In some embodiments, the one or more buffering agents is
phosphate buffers, carbonate buffers, citrate, Tris, MOPS, MES,
acetate or HEPES.
[0063] A single buffering agent maybe be used or a combination of
two or more buffering agents (e.g., at least two, at least 3, or 2
or 3 or 4 buffering agents).
[0064] A "surfactant" as used in the present context is a surface
active compound which reduces the surface tension of water.
[0065] Surfactants are used to improve the solubility of certain
proteins. Surfactants should generally be used at concentrations
above or below their critical micelle concentration (CMC), for
example, from about 5% to about 20% above or below the CMC.
However, these values will vary dependent upon the surfactant
chosen, for example, surfactants such as,
beta-octylgluco-pyranoside may be effective at lower concentrations
than, for example, surfactants such as TWEEN-20 (polysorbate 20).
The optimal concentration is a function of each surfactant, which
has its own CMC.
[0066] Useful surfactants include nonionic (including, but not
limited to, t-octylphenoxypolyethoxy-ethanol and polyoxyethylene
sorbitan), anionic (e.g., sodium dodecyl sulfate) and cationic
(e.g., cetylpyridinium chloride) and amphoteric agents. Suitable
surfactants include, but are not limited to deoxycholate, sodium
octyl sulfate, sodium tetradecyl sulfate, polyoxyethylene ethers,
sodium cholate, octylthioglucopyranoside, n-octylglucopyranoside,
alkyltrimethylammonium bromides, alkyltrimethyl ammonium chlorides,
and sodium bis (2 ethylhexyl) sulfosuccinate. In some embodiments
the surfactant may be polysorbate 80, polysorbate 20, sarcosyl,
Triton X-100, .beta.-octyl-gluco-pyranoside, or Brij 35.
[0067] In some embodiments the one or more surfactant may be a
polysorbate, polyoxyethylene ether, alkyltrimethylammonium bromide,
pyranosides or combination of two or more of the foregoing. In
certain embodiments, the one or more surfactant may be
.beta.-octyl-gluco-pyranoside, Brij 35, or a polysorbate.
[0068] In certain embodiments the one or more surfactant may be
octyl phenol ethoxylate, .beta.-octyl-gluco-pyranoside,
polyoxyethyleneglycol dodecyl ether, sarcosyl, sodium dodecyl
sulfate, polyethoxysorbitan, deoxycholate, sodium octyl sulfate,
sodium tetradecyl sulfate, sodium cholate,
octylthioglucopyranoside, n-octylglucopyranoside, sodium bis
(2-ethylhexyl) sulfosuccinate or combinations of two or more of the
foregoing. A single surfactant maybe be used or a combination of
two or more surfactants (e.g., at least two, at least 3, or 2 or 3
or 4 surfactants).
[0069] Chaotropic agents (also referred to as a "chaotrope") are
compounds, including, without limitation, guanidine, guanidine
hydrochloride (guanidinium hydrochloride, GdmHCl), guanidine
sulfate, urea, sodium thiocyanate, and/or other compounds which
disrupt the noncovalent intermolecular bonding within the protein,
permitting the polypeptide chain to assume a substantially random
conformation
[0070] Chaotropic agents may be used in concentration of from about
10 mM to about 8 M. The optimal concentration of the chaotropic
agent will depend on the desired protein as well as on the
particular chaotropes selected. The choice of particular chaotropic
agent and determination of optimal concentration can be optimized
by the skilled artisan in view of the teachings provided
herein.
[0071] In some embodiments, the concentration of the chaotropic
agent will be, for example, from about 10 mM to about 8 M, from
about 10 mM to about 7 M, from about 10 mM to about 6 M, from about
0.1 M to about 8 M, from about 0.1 M to about 7 M, from about 0.1 M
to about 6 M, from about 0.1 M to about 5 M, from about 0.1 M to
about 4 M, from about 0.1 M to about 3 M, from about 0.1 M to about
2 M, from about 0.1 M to about 1 M, from about 10 mM to about 4 M,
from about 10 mM to about 3 M, from about 10 mM to about 2 M, from
about 10 mM to about 1 M, or about, 10 mM, about 50 mM, about 75
mM, about 0.1 M, about 0.5 M, about 0.8 M, about 1 M, about 2 M,
about 3 M, about 4 M, about 5 M, about 6 M, about 7 M, or about 8
M.
[0072] When used in the present methods, it is often advantageous
to use chaotropic agents in non-denaturing concentrations to
facilitate the dissociation of non-covalent interactions. While a
non-denaturing concentration will vary depending on the desired
protein, the range of non-denaturing concentrations is typically
from about 0.1 to about 4 M. In some embodiments the concentration
is from about 0.1 M to about 2 M.
[0073] In certain embodiments, guanidine hydrochloride or urea are
the chaotropic agents.
[0074] A single chaotropic agent maybe be used or a combination of
two or more chaotropic agents (e.g., at least two, at least 3, or 2
or 3 or 4 chaotropic agents).
[0075] Agitation is another aspect that may be manipulated. Protein
solutions can be agitated before and/or during refolding. Agitation
can be performed by methods including, but not limited to,
ultrasound energy (sonication), mechanical stirring, mechanical
shaking, pumping through mixers, or via cascading solutions.
Agitation may be performed for any length of time, such as the
entire period of high-pressure treatment, or for one or more
periods of about 1 to about 60 minutes during high-pressure
treatment, such as about 1 minute, about 5 minutes, about 10
minutes, about 20 minutes, about 30 minutes, about 45 minutes, or
about 60 minutes.
[0076] Yet another aspect that may be manipulated is temperature.
The methods described herein can be performed at a range of
temperature values, depending on the particular protein of
interest. The optimal temperature, in concert with other factors,
can be optimized as described herein. Proteins can be refolded at
various temperatures, including at about room temperature, about
20.degree. C., about 25.degree. C., about 30.degree. C., about
37.degree. C., about 50.degree. C., about 75.degree. C., about
100.degree. C., about 125.degree. C., or ranges of from about 20 to
about 125.degree. C., about 25 to about 125.degree. C., about 25 to
about 100.degree. C., about 25 to about 75.degree. C., about 25 to
about 50.degree. C., about 50 to about 125.degree. C., about 50 to
about 100.degree. C., about 50 to about 75.degree. C., about 75 to
about 125.degree. C., about 5 to about 100.degree. C., or about 100
to about 125.degree. C.
[0077] In some embodiments of the methods, the temperature can
range from about 0.degree. C. to about 100.degree. C. without
adversely affecting the protein of interest. Thus in certain
embodiments, the temperature may be from about 0.degree. C. to
about 75.degree. C., from about 0.degree. C. to about 55.degree.
C., from about 0.degree. C. to about 35.degree. C., from about
0.degree. C. to about 25.degree. C., from about 20.degree. C. to
about 75.degree. C., from about 20.degree. C. to about 65.degree.
C., from about 20.degree. C. to about 35.degree. C., or from about
20.degree. C. to about 25.degree. C.
[0078] Although increased temperatures are often used to cause
aggregation of proteins, when coupled with increased hydrostatic
pressure it has been found that increased temperatures can enhance
refolding recoveries effected by high pressure treatment, provided
that the temperatures are not so high as to cause irreversible
denaturation. Generally, the increased temperature for refolding
should be about 20.degree. C. lower than the temperatures at which
irreversible loss of activity occurs. Relatively high temperatures
(for example, about 60.degree. C. to about 125.degree. C., about
80.degree. C. to about 110.degree. C., including about 100.degree.
C., about 105.degree. C., about 110.degree. C., about 115.degree.
C., about 120.degree. C. and about 125.degree. C.) may be used
while the solution is under pressure, as long as the temperature is
reduced to a suitably low temperature before depressurizing. Such a
suitably low temperature is defined as one below which
thermally-induced denaturation or aggregation occurs at atmospheric
conditions.
[0079] "High pressure" or "high hydrostatic pressure," for the
purposes of the invention is defined as pressures of from about 500
bar to about 10,000 bar.
[0080] In some embodiments, the increased hydrostatic pressure may
be from about 500 bar to about 5000 bar, from about 500 bar to
about 4000 bar, from about 500 bar to about 2000 bar, from about
500 bar to about 2500 bar, from about 500 bar to about 3000 bar,
from about 500 bar to about 6000 bar, from about 1000 bar to about
5000 bar, from about 1000 bar to about 4000 bar, from about 1000
bar to about 2000 bar, from about 1000 bar to about 2500 bar, from
about 1000 bar to about 3000 bar, from about 1000 bar to about 6000
bar, from about 1500 bar to about 5000 bar, from about 1500 bar to
about 3000 bar, from about 1500 bar to about 4000 bar, from about
1500 bar to about 2000 bar, from about 2000 bar to about 5000 bar,
from about 2000 bar to about 4000 bar, from about 2000 bar to about
3000 bar, or about 1000 bar, about 1500 bar, about 2000 bar, about
2500 bar, about 3000 bar, about 3500 bar, about 4000 bar, about
5000 bar, about 6000 bar, about 7000 bar, about 8000 bar, or about
9000 bar.
[0081] Reduction of pressure is another parameter that can be
manipulated. Where the reduction in pressure is performed in a
continuous manner, the rate of pressure reduction can be constant
or can be increased or decreased during the period in which the
pressure is reduced. In some variations, the rate of pressure
reduction is from about 5000 bar/1 sec to about 5000 bar/4 days (or
about 3 days, about 2 days, or about 1 day). Thus in some
variations the rate of pressure reduction can be performed at a
rate of from about 5000 bar/1 sec to about 5000 bar/80 hours, from
about 5000 bar/1 sec to about 5000 bar/72 hours, from about 5000
bar/1 sec to about 5000 bar/60 hours, from about 5000 bar/1 sec to
about 5000 bar/50 hours, from about 5000 bar/1 sec to about 5000
bar/48 hours, from about 5000 bar/1 sec to about 5000 bar/32 hours,
from about 5000 bar/1 sec to about 5000 bar/24 hours, from about
5000 bar/1 sec to about 5000 bar/20 hours, from about 5000 bar/1
sec to about 5000 bar/18 hours, from about 5000 bar/1 sec to about
5000 bar/16 hours, from about 5000 bar/1 sec to about 5000 bar/12
hours, from about 5000 bar/1 sec to about 5000 bar/8 hours, from
about 5000 bar/1 sec to about 5000 bar/4 hours, from about 5000
bar/1 sec to about 5000 bar/2 hours, from about 5000 bar/1 sec to
about 5000 bar/1 hour, from about 5000 bar/1 sec to about 1000
bar/min, about 5000 bar/1 sec to about 500 bar/min, about 5000
bar/1 sec to about 300 bar/min, about 5000 bar/1 sec to about 250
bar/min, about 5000 bar/1 sec to about 200 bar/min, about 5000
bar/1 sec to about 150 bar/min, about 5000 bar/1 sec to about 100,
about 5000 bar/1 sec to about 80 bar/min, about 5000 bar/1 sec to
about 50 bar/min, or about 5000 bar/1 sec to about 10 bar/min. For
example, about 10 bar/min, about 250 bar/5 minute, about 500 bar/5
minutes, about 1000 bar/5 minutes, about 250 bar/5 minutes, 2000
bar/50 hours, 3000 bar/50 hours, 40000 bar/50 hours, etc. In some
embodiments, the pressure reduction may be approximately
instantaneous, as in where pressure is released by simply opening
the device in which the sample is contained and immediately
releasing the pressure.
[0082] Where the reduction in pressure is performed in a stepwise
manner, the process comprises dropping the pressure from the
highest pressure used to at least a secondary level that is
intermediate between the highest level and atmospheric pressure.
The goal is to provide a hold period at or about this intermediate
pressure zone that permits a protein to adopt a desired
conformation.
[0083] In some embodiments, where there are at least two stepwise
pressure reductions there may be a hold period at a constant
pressure between intervening steps. The hold period may be from
about 10 minutes to about 50 hours (or longer, depending on the
nature of the protein of interest). In some embodiments, the hold
period may be from about 2 to about 30 hours, from about 2 to about
24 hours, from about 2 to about 18 hours, from about 1 to about 10
hours, from about 1 to about 8 hours, from about 1 to about 6
hours, from about 2 to about 10 hours, from about 2 to about 8
hours, from about 2 to about 6 hours, or about 2 hours, about 6
hours, about 10 hours, about 20 hours, or about 30 hours, from
about 2 to about 10 hours, from about 2 to about 8 hours, or from
about 2 to about 6 hours.
[0084] In some variations, the pressure reduction includes at least
2 stepwise reductions of pressure (e.g., highest pressure reduced
to a second pressure reduced atmospheric pressure would be two
stepwise reductions). In other embodiments the pressure reduction
includes more than 2 stepwise pressure reductions (e.g., 3, 4, 5,
6, etc.). In some embodiments, there is at least 1 hold period. In
certain embodiments there is more than one hold period (e.g., at
least 2, at least 3, at least 4, at least 5 hold periods).
[0085] In some variations of the methods the constant pressure
after an initial stepwise reduction may be at a hydrostatic
pressure of from about 500 bar to about 5000 bar, from about 500
bar to about 4000 bar, from about 500 bar to about 2000 bar, from
about 1000 bar to about 4000 bar, from about 1000 bar to about 3000
bar, from about 1000 bar to about 2000 bar, from about 1500 bar to
about 4000 bar, from about 1500 bar to about 3000 bar, from about
2000 bar to about 4000 bar, or from about 2000 bar to about 3000
bar.
[0086] In particular variations, constant pressure after the
stepwise reduction is from about four-fifths of the pressure
immediately prior to the stepwise pressure reduction to about
one-tenth of prior to the stepwise pressure reduction. For example,
constant pressure is at a pressure of from about four-fifths to
about one-fifth, from about two-thirds to about one-tenth, from
about two-thirds to about one-fifth, from about two-thirds to about
one-third, about one-half, or about one-quarter of the pressure
immediately prior to the stepwise pressure reduction. Where there
is more than one stepwise pressure reduction step, the pressure
referred to is the pressure immediately before the last pressure
reduction (e.g., where 2000 bar is reduced to 1000 bar is reduced
to 500 bar, the pressure of 500 bar is one-half of the pressure
immediately preceding the previous reduction (1000 bar)).
[0087] Where the pressure is reduced in a stepwise manner, the rate
of pressure reduction (e.g., the period of pressure reduction prior
to and after the hold period) may be in the same range as that rate
of pressure reduction described for continuous reduction (e.g., in
a non-stepwise manner). In essence, stepwise pressure reduction is
the reduction of pressure in a continuous manner to an intermediate
constant pressure, followed by a hold period and then a further
reduction of pressure in a continuous manner. The periods of
continuous pressure reduction prior to and after each hold period
may be the same continuous rate for each period of continuous
pressure reduction or each period may have a different reduction
rate. In some variations, there are two periods of continuous
pressure reduction and a hold period. In certain embodiments, each
continuous pressure reduction period has the same rate of pressure
reduction. In other embodiments, each period has a different rate
of pressure reduction. In particular embodiments, the hold period
is from about 8 to about 24 hours. In some embodiments, the hold
period is from about 12 to about 18 hours. In particular
embodiments, the hold period is about 16 hours.
[0088] Various combinations and permutations of the condition
above, such as agitation of the protein under high pressure at an
elevated temperature in the presence of chaotropes and redox
reagents, can be employed as desired for optimization of refolding
yields.
[0089] Optimization of reaction conditions for solubilization and
refolding in the context of the methods described herein are a
function of the characteristics of both the target antibody and any
other components or parameters of the solution. In standard
optimization experiments, the influence of pressure, pH,
temperature, ionic strength, surfactants, chaotropes, stabilizing
agents, and refolding time on refolding should be tested. Once the
key process parameters are identified, a central composite design
can be used to optimize the appropriate conditions for each
parameter. Guidance regarding typical ranges for the various
parameters is provided in more detail below.
[0090] Initial studies can be conducted to screen the effect of
solution conditions, solution pH, and high pressure treatment on
the solubilization and/or refolding of proteins. Screening studies
are typically conducted, but not limited to, empirical screens that
examine step-wise the effect of processing conditions on yields.
Synergistic effects between different parameters are not examined
in these screening studies. Exemplary screening studies that can be
conducted are as described for the cases of recombinant placental
bikunin, recombinant growth hormone, and malaria pfs48 (see e.g.,
Seefeldt et al. Protein Science, v13 (10), 2639-2650 2004, St. John
et al., Journal of Biological Chemistry, v276 (50), 46856-46864,
2001, Seefeldt, "High pressure refolding of protein aggregates:
efficacy and thermodynamics," Dept. of Chemical and Biological
Engineering Thesis, (2004), the disclosures of which are herein
incorporated by reference in their entirety, particularly with
respect to the screening studies described therein). High pressure
refolding studies of bikunin and growth hormone demonstrate the
step-wise screening process for solution conditions (pH 5-9),
temperature (0-60.degree. C.), ionic strength (0-160 mM NaCl),
non-denaturing concentrations of chaotropes (0-1.0 M urea or 0-2.0
M guanidine) and refolding time (0-24 hours). Studies can be
conducted at about 2000 bar, about 2100 bar, about 2150 bar, etc.
and compared to samples treated at atmospheric pressure. Other
parameters, including those described herein, that can be screened
include, but are not limited to, the presence and amount of
stabilizing agents, surfactants, salts, etc., as described herein.
It should be noted that statistical analysis of variance (ANOVA's)
can be used to rapidly screen which solution parameters affect
refolding yields. In addition to the teaching provided herein, U.S.
2004/0038333, Seefeldt et al. Protein Science, v13 (10), 2639-2650
2004, and St. John et al., Biotechnology Progress, v18, (3),
565-571, 2002 (incorporated herein by reference in their entirety)
also provide guidance regarding empirical screening procedures for
determining the optimal solubilization and refolding
conditions.
[0091] In this manner, the skilled artisan can determine the effect
of processing conditions on the refolding of protein aggregates
through the use of high pressure. It has been shown in the
literature that refolding reactions can have interactions between
the process conditions, which prevents single-variable screening
from effectively optimizing the process. For instance, pH affects
protein conformation stability, protein colloidal stability, and
disulfide bond formation kinetics. To effectively optimize the
effect of pH, or any other process parameter, studies need to be
conducted to account for interactions. In these instances,
statistical experimental designs need to be employed. As described
herein, solubilization is also examined as a function of urea, by
step-wise analysis in a range from 0-4.5 M urea at pH 8.0. Once the
significant parameters are identified, a face-centered statistical
designed experiment is used to optimize the refolding conditions,
taking into account interactions.
[0092] After initial optimization studies are performed for the
protein of interest, more granular optimization can be used to
determine the optimal conditions for performing the solubilization
and refolding processes. This process can generally be described as
an experimental optimization that takes into account synergistic
interactions between the critical parameters identified in the
initial step-wise studies. An effective method for conducting these
studies involves using a three or five level central composite
statistical analysis, which takes into account interactions between
the reaction parameters while minimizing the required number of
experiments.
[0093] Another useful aid for optimizing conditions and/or
monitoring solubilization or refolding is in situ spectroscopic
measurement of samples under pressure, a well-known process for
examining polypeptide stability under pressure. Using high pressure
spectroscopic techniques to observe aggregate dissolution under
pressure will help determine the optimal pressure ranges for
recovering proteins from aggregates. Custom made high pressure
cells have been routinely used for high pressure unfolding studies
and can be adapted for use in high pressure disaggregation and
refolding. Additional guidance for the skilled artisan may also be
found in Paladini and Weber, Biochemistry, 20 (9), 2587-2593 (1981)
and Seefeldt et al. Protein Science, 13 (10), 2639-2650 (2004),
incorporated by reference herein in their entirety.
[0094] Methods that can be employed to monitor the optimization of
various parameters include Fourier Transform Infrared Spectroscopy
(FTIR), circular dichroism (CD) spectroscopy (far and/or near UV),
UV spectroscopy, measurement of total protein concentrations (e.g.,
BCA assay method (Pierce Chemical Co., Rockford, Ill.), etc),
activity assays to measure the activity of the target polypeptide,
electrophoretic gels with molecular weight markers to visualize the
appearance of native protein under various conditions, HPLC
analysis of soluble polypeptide fractions, etc.
[0095] Suitable devices for performing high pressure spectroscopy
can be obtained commercially (e.g., such as fluorescence cells
available from ISS Inc., Champaign, Ill. or
fluorescence/ultraviolet absorbance cells available from BaroFold
Inc., Boulder, Colo.) or can be fabricated by the skilled artisan.
For example, Randolph et al., U.S. Patent Application Publication
No. 2004/0038333, incorporated by reference herein in its entirety,
described a high-pressure W spectroscopy cell made of stainless
steel, sealed with Btma-N 90 durometer o-rings and with an optical
port diameter of 6 mm and pathlength of 7.65 mm. The cell utilized
cylindrical sapphire windows (16 mm diameter, 5.1 mm thick) and was
capable of experiments up to 250 MPa. Separation of the sample from
the pressure transmitting fluid was facilitated by a piston device
external to the cell.
[0096] Commercially available high pressure devices and reaction
vessels, such as those described in the examples, may be used to
achieve the hydrostatic pressures in accordance with the methods
described herein (see BaroFold Inc., Boulder, Colo.). Additionally
devices, vessels and other materials for carrying out the methods
described herein, as well as guidance regarding the performing
increased pressure methods, are described in detail in U.S. Pat.
No. 6,489,450, which is incorporated herein in its entirety. The
skilled artisan is particularly directed to column 9, lines 39-62
and Examples 2-4. International Pat. App. Pub. No. WO 02/062827,
incorporated herein in its entirety, also provides the skilled
artisan with detailed teachings regarding devices and use thereof
for high hydrostatic pressure solubilization of aggregates
throughout the specification. Particular devices and teachings
regarding the use of high pressure devices are also provided in
International Patent Application Publication No. WO 2007/062174,
which is incorporated by reference herein in its entirety.
[0097] Multiple-well sample holders may be used and can be
conveniently sealed using self-adhesive plastic covers. The
containers, or the entire multiple-well sample holder, may then be
placed in a pressure vessel, such as those commercially available
from the Flow International Corp. or High Pressure Equipment Co.
The remainder of the interior volume of the high-pressure vessel
may than be filled with water or other pressure transmitting
fluid.
[0098] Mechanically, there are two primary methods of high-pressure
processing: batch and continuous. Batch processes simply involve
filling a specified chamber, pressurizing the chamber for a period
of time, and depressurizing the batch. In contrast, continuous
processes constantly feed aggregates into a pressure chamber and
soluble, refolded proteins move out of the pressure chamber. In
both set ups, good temperature and pressure control is essential,
as fluctuations in these parameters can cause inconsistencies in
yields. Both temperature and pressure should be measured inside the
pressure chamber and properly controlled.
[0099] There are many methods for handling batch samples depending
upon the specific stability issues of each target protein. Samples
can be loaded directly into a pressure chamber, in which case the
aqueous solution and/or suspension would be used as the pressure
medium.
[0100] Alternately, samples can be loaded into any variety of
sealed, flexible containers, including those described herein. This
allows for greater flexibility in the pressure medium, as well as
the surfaces to which the sample is exposed. Sample vessels could
conceivably even act to protect the desired protein from chemical
degradation (e.g., oxygen scavenging plastics are available).
[0101] With continuous processing, small volumes under pressure can
be used to refold large volumes of the sample. In addition, using
an appropriate filter on the outlet of a continuous process will
selectively release soluble desired protein from the chamber while
retaining both soluble and insoluble aggregates.
[0102] Pressurization is a process of increasing the pressure
(usually from atmospheric or ambient pressure) to a higher
pressure. Pressurization takes place over a predetermined period of
time, ranging from 0.1 second to 10 hours. Such times include 1
second, 2 seconds, 5 seconds, 10 seconds, 20 seconds, 1 minute, 2
minutes, 5 minutes, to minutes, 30 minutes, 60 minutes, 2 hours, 3
hours, 4 hours, and 5 hours.
[0103] Depressurization is a process of decreasing the pressure,
from a high pressure, to a lower pressure (usually atmospheric or
ambient pressure). Depressurization takes place over a
predetermined period of time, ranging from 10 seconds to 10 hours,
and may be interrupted at one or more points to permit optimal
refolding at intermediate (but still increased 30 compared to
ambient) pressure levels. The depressurization or interruptions may
be 1 second, 2 seconds, 5 seconds, 10 seconds, 20 seconds, 1
minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2
hours, 3 hours, 4 hours, and 5 hours.
[0104] Degassing is the removal of gases dissolved in solutions and
is often advantageous in the practice of the methods described
herein. Gas is much more soluble in liquids at high pressure as
compared to atmospheric pressure and, consequently, any gas
headspace in a sample will be driven into solution upon
pressurization. The consequences are two-fold: the additional
oxygen in solution may chemically degrade the protein product, and
gas exiting solution upon repressurization may cause additional
aggregation. Thus, samples should be prepared with degassed
solutions and all headspace should be filled with liquid prior to
pressurization.
EXAMPLES
Example 1
Use of High Pressure to Disaggregate and Refold Aggregates of
Orencia.RTM. (CTLA-4Ig; abatacept)
[0105] Aggregation states of Orencia.RTM. (Orencia.RTM. is a
registered trademark of Bristol-Myers Squibb Co., New York, N.Y.,
for pharmaceutical preparations for the treatment and prevention of
auto-immune diseases such as rheumatoid arthritis), also known as
abatacept or CTLA-4Ig, were monitored in both commercial
formulations and after exposure to pH 3 for 3 hours. Orencia.RTM.
is a soluble fusion protein, consisting of the extracellular domain
of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) fused
to the modified Fc (hinge, CH2, and CH3 domains) portion of human
immunoglobulin G1 (IgG1). Aggregates of CTLA-4Ig were pressure
treated as a function of pH, pressure and additives as described
below.
[0106] For sample preparation, aggregates were generated in 10 mM
phosphate buffer at a pH of 3.0 at a protein concentration of 12
mg/ml. The samples were treated at these conditions until an
aggregation extent of .about.20% was achieved. The samples were
then diluted to a concentration of 0.5 mg/ml in the appropriate
solution conditions. The samples were placed into sealed syringes
and pressure treated. Atmospheric controls were prepared under
identical conditions and also stored in sealed syringes.
[0107] Pressurization was increased at a rate of 500 bar/minute
until the desired pressure was achieved. During refolding, the
temperature was maintained at 22 C (R.T.). The samples were held
under pressure for approximately 16 hours and then were
depressurized at a rate of 500 bar/five minutes. The samples were
immediately prepared for SEC after depressurization.
[0108] Size Exclusion Analysis--High Pressure Liquid Chromatography
(SEC) analysis of protein fractions was conducted on a Beckman Gold
HPLC system (Beckman Coulter, Fullerton, Calif.) equipped with a
TSK G3000 SW.sub.XL size exclusion column (Tosohaas). A filtered
mobile phase of PBS (pH 7.2) at a rate of 1.0 ml/min was used, with
an 10-25 ug protein sample injection from a Beckman 507e
autosampler. Absorbance was monitored at 215 nm.
[0109] Aggregation of CTLA-4Ig at low pH was achieved by diluting
commercial formulations to a protein concentration of 12 mg/ml and
treated at pH 3 for 3 hours at 23.degree. C. to induce aggregation.
Two runs resulted in final aggregate concentrations of 15 and 21
percent respectively (Labeled Init % Agg.--FIG. 1). Aggregate
analysis was quantified by SEC. High pressure treatment at pH 7 and
higher resulted in .about.95% refolding yields and aggregate levels
lower than commercial CTLA-4Ig preparations (Orencia.RTM.).
[0110] High pressure refolding of CTLA-4Ig as a function of pH is
exemplified by aggregates (0.5 mg/ml) forming after exposure to pH
3, pressure treatment at 2000 bar for sixteen hours at 25.degree.
C. and compared to atmospheric controls (FIG. 1). Buffer
concentrations were 10 mM to maintain a low ionic strength. Samples
pressure treated in solutions of pH 7 or higher resulted in
.about.95% refolding yields, with a final aggregate concentration
of 0.8%. The final aggregate concentration of 0.8% after pressure
treatment is lower than what was present in the starting material
prior to pH 3 induced aggregate (1.2%).
[0111] Atmospheric controls treated at identical temperature and
solution conditions refolded to significantly lower levels relative
to samples treated at high pressure. The basis for the decreased
refolding at lower pHs is unknown. Error bars depict 95% confidence
intervals. Throughout the entire analysis, total protein area was
monitored to ensure that protein adsorption across the SEC column
was minimal and the sizing method was quantifying aggregate levels
accurately. Refolding at 5 mg/ml at pH 7.0 also resulted in
refolding, with similar yields to the 0.5 mg/ml samples (data not
shown).
[0112] Studies were conducted to examine high pressure refolding of
CTLA-4Ig aggregates at pH 7.0 as a function of excipients; more
specifically, to examine the effect of excipients (250 mM arginine,
10% (w/v) sucrose, and 0.01% (w/v) Tween 20) on the refolding yield
of CTLA-4Ig acid-induced aggregates (0.5 mg/ml) at pH 7 (10 mM
buffer) (FIG. 2). Excipients did not significantly increase the
refolding yield over samples that were refolded in buffer alone
(low ionic strength solution conditions) (FIG. 1). The presence of
arginine decreased refolding yields; this data is consistent with
an ionic-strength dependent effect. It was previously observed that
high pressure treatment in the presence of 250 mM NaCl at pH 7
resulted in the aggregation of native CTLA-4Ig (data not
shown).
[0113] To illustrate high pressure refolding of CTLA-4Ig aggregates
at pH 7.0 as a function of pressure, aggregates of CTLA-4Ig (0.5
mg/ml) were treated at pH 7.0 (10 mM Buffer) as a function of
pressure (0-3 kbar) at 25.degree. C. for 16 hours. Pressures of
2000 bar were sufficient for the refolding of the aggregate with
yields of .about.95% relative to the initial aggregate
concentration; increased pressures did not provide any increased
yields (see FIG. 3).
[0114] Aggregation states of CTLA-4Ig were monitored in both
commercial formulations and after exposure to pH 3 for 3 hours.
Aggregates were pressure treated as a function of pH, pressure and
additives. This illustrative study showed: high pressure refolded
pH 3 induced aggregates of CTLA-4Ig (0.5 mg/ml), reducing aggregate
levels from 18% to 0.8% (95% yield); high pressure refolding
reduced the aggregate levels below those originally found in
commercial formulations of CTLA-4Ig (Orencia.RTM.) being 0.8% vs.
1.3%, respectively; existing native protein is preserved during
high pressure refolding in the appropriate solution conditions,
however high pressure treatment in solutions of high ionic strength
induced aggregation; refolding yields are not affected by moderate
differences of initial aggregate concentrations (21% vs. 15%);
refolding yields are pH dependent below pH 7; and, refolding
pressures of 2000 to 3000 bar produce equivalent results
Example 2
Use of High Pressure to Disaggregate and Refold Aggregates of the
"Alliance mAb"
[0115] Aggregation states of a monoclonal antibody which was a gift
of Alliance Laboratories were monitored after exposure to pH 3. The
sequence and properties of this aggregate were unknown. Aggregates
of the monoclonal antibody were pressure treated at a pH of 7.2 as
a function of pressure and additives, as described below.
[0116] Aggregate samples were generated by dialysis into buffer at
a pH of 3.0 at a protein concentration of 37 mg/ml. The samples
were dialyzed in the buffer for six hours and then maintained at
room temperature for six additional hours. The samples were then
diluted to a concentration of either 0.3 or 5 mg/ml in the
appropriate solution conditions. Buffer and ionic strength
concentrations were 50 mM TES, pH 7.2, 150 mM NaCl. The samples
were placed into sealed syringes and pressure treated. Atmospheric
controls were prepared under identical conditions and also stored
in sealed syringes.
[0117] Pressure was increased at a rate of 500 bar/minute until the
desired pressure was achieved. During refolding, the temperature
was maintained at 22 degrees C. (room temperature). The samples
were held under pressure for approximately 16 hours and then were
depressurized at a rate of 500 bar/five minutes. The samples were
immediately prepared for SEC after depressurization.
[0118] Size Exclusion Analysis--High Pressure Liquid Chromatography
(SEC) analysis of protein fractions was conducted on a Beckman Gold
HPLC system (Beckman Coulter, Fullerton, Calif.) equipped with a
TSK G3000 SW.sub.XL size exclusion column (Tosohaas). A filtered
mobile phase of PBS (pH 7.2) at a rate of 1.0 ml/min was used from
a Beckman 507e autosampler. Absorbance was monitored at 215 nm.
[0119] Aggregation of the Alliance mAb at low pH was induced by
diluting 50 mg/ml formulations to a protein concentration of 33.5
mg/ml and exposure to pH 3 for 6 hours at 25.degree. C. The final
aggregate content was estimated to be 32% percent. Aggregate
analysis was quantified by SEC.
[0120] To illustrate high pressure refolding of Alliance mAb
aggregates as a function of pressure, aggregates of the Alliance
mAb (5 mg/ml) formed after exposure to pH 3 were pressure treated
at 2000 bar for sixteen hours at 25.degree. C. at a pH of 7.2.
Pressures of 1 bar, 1000 bar, 1500 bar, 2000 bar and 3000 bar were
tested. Of the samples tested, only aggregates pressure treated at
1000 to 2000 bar resulted in increased monomer content (FIG. 4).
Aggregates pressure treated to 3000 bar resulted in increased
aggregation, demonstrating the importance of the refolding window.
Atmospheric controls treated at identical temperature and solution
conditions also had lower monomer content. High pressure treatment
at pressures of 1000-2000 bar resulted in the desired
refolding.
[0121] Studies were conducted to examine high pressure refolding of
Alliance mAb aggregates at pH7.2 as a function of excipients. In
order to examine the effect of excipients (250 mM arginine, 10%
(w/v) sucrose, and 0.01% (w/v) Tween 80) on the refolding yield of
Alliance mAb acid-induced aggregates (5 mg/ml) at pH 7.2 (FIG. 5).
Excipients did not significantly increase the refolding yield over
samples that were refolded in buffer alone, i.e., at pH 7.2 without
aggregates present (FIG. 5).
[0122] To illustrate the high pressure refolding of the aggregates
of the Alliance mAb, aggregates (0.3 mg/ml) were incubated at pH
7.2 as a function of temperature (25-37 degrees C.) for 16 hours.
Pressures of 2000 bar at 25 degrees C. were sufficient for
increasing the amount of monomer and increased temperature did not
provide any increased yields. Increased temperature did not
increase monomer peak area. (see FIG. 6).
[0123] Aggregation states of the Alliance mAb were monitored after
exposure to pH 3.0. Aggregates of the mAb were pressure treated at
a pH of 7.2 as a function of pressure and additives. This
illustrative study showed: high pressure treatment of pH 3-induced
aggregates at a pH of 7.2 resulted in increased monomer peak area
as analyzed by SEC in comparison to atmospheric conditions, having
values of 1.6 million and 1.8 million, respectively; addition of
additives such as 250 mM arginine, 0.05% Tween 80, and 10% sucrose
did not significantly effect the refolding yield of the Alliance
mAb; elevated temperatures of 37.degree. C. did not significantly
increase the refolding yield of Alliance mAb aggregates at pressure
treated to 2000 bar at pH 7.2 at 25.degree. C.; and, pressures of
3000 bar induced aggregation of native mAb. Ongoing analytical
ultracentrifugation (AUC) studies in order to quantify the
refolding yields more accurately, as analysis of total peak area
suggests that some protein adsorption may occur during SEC
analysis.
[0124] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is apparent to those skilled in the art that
certain minor changes and modifications will be practiced.
Therefore, the description and examples should not be construed as
limiting the scope of the invention.
* * * * *