U.S. patent application number 12/355686 was filed with the patent office on 2009-07-23 for enhanced purification of phosphorylated and nonphosphorylated biomolecules by apatite chromatography.
Invention is credited to Peter S. Gagnon.
Application Number | 20090186396 12/355686 |
Document ID | / |
Family ID | 40876787 |
Filed Date | 2009-07-23 |
United States Patent
Application |
20090186396 |
Kind Code |
A1 |
Gagnon; Peter S. |
July 23, 2009 |
ENHANCED PURIFICATION OF PHOSPHORYLATED AND NONPHOSPHORYLATED
BIOMOLECULES BY APATITE CHROMATOGRAPHY
Abstract
Methods are disclosed for the use of apatite chromatography,
particularly without reliance upon phosphate gradients, for
fractionation or separation of phosphorylated and nonphosphorylated
biomolecules. Integration of such methods into multi-step
procedures, with other fractionation methods are additionally
disclosed.
Inventors: |
Gagnon; Peter S.; (San
Clemente, CA) |
Correspondence
Address: |
PILLSBURY WINTHROP SHAW PITTMAN LLP
ATTENTION: DOCKETING DEPARTMENT, P.O BOX 10500
McLean
VA
22102
US
|
Family ID: |
40876787 |
Appl. No.: |
12/355686 |
Filed: |
January 16, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61011513 |
Jan 18, 2008 |
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61062663 |
Jan 28, 2008 |
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61069859 |
Mar 19, 2008 |
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61070841 |
Mar 27, 2008 |
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61135787 |
Jul 24, 2008 |
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61189467 |
Aug 20, 2008 |
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Current U.S.
Class: |
435/235.1 ;
536/23.1; 536/25.4 |
Current CPC
Class: |
C07K 1/18 20130101; C07K
1/165 20130101; C07K 1/16 20130101 |
Class at
Publication: |
435/235.1 ;
536/25.4; 536/23.1 |
International
Class: |
C12N 7/02 20060101
C12N007/02; C07H 21/04 20060101 C07H021/04 |
Claims
1. A method for purifying at least one non-aggregated biomolecule
from an impure preparation containing said biomolecule comprising
the steps of (a) contacting the impure preparation with an apatite
chromatography support and (b) conducting elution in the presence
of an ionic species selected from the group consisting of borate,
sulfate, monocarboxylates, and monocarboxylic zwitterions.
2. The method of claim 1 wherein the ionic species is borate or
sulfate.
3. The method of claim 1 wherein the ionic species is a
monocarboxylate selected from the group consisting of acetate,
proprionate, lactate, pyruvate, gluconate, and glucuronate.
4. The method of claim 3, wherein the monocarboxylate is sodium or
potassium lactate.
5. The method of claim 1 wherein the ionic species is a
monocarboxylic zwitterion selected from the group consisting of
glycine, proline, lysine, and histidine.
6. The method of claim 1, wherein the monocarboxylic zwitterion
possesses a pKa suitable for buffering in the pH range selected for
the particular purification, is used as the primary buffering
species, and is present at a concentration greater than 50 mM.
7. The method of claim 2, wherein the ionic species is borate and
the borate is present at a pH where the borate lacks significant
buffer capacity.
8. The method of claim 7, wherein the ionic species is borate and
the borate is supplied as sodium borate or potassium borate and is
present at a pH of 8.7 or less.
9. The method of claim 2, wherein the ionic species is borate and
the borate is supplied at a pH where the borate has significant
buffer capacity, and the borate is present at a concentration
greater than 50 mM.
10. The method of claim 9, wherein the borate is sodium borate or
potassium borate and is present at a pH of 8.7 or greater.
11. The method of claim 2 wherein the ionic species is borate and
the borate is the primary eluting ion.
12. The method of claim 2 wherein the ionic species is sulfate and
the sulfate is the primary eluting ion.
13. The method of claim 3 wherein the monocarboxylate is the
primary eluting ion.
14. The method of claim 5 wherein the monocarboxylic zwitterion is
the primary eluting ion.
15. The method of claim 1, wherein the apatite chromatography
support is hydroxyapatite.
16. The method of claim 1, wherein the apatite chromatography
support is fluorapatite.
17. The method of claim 1, wherein the apatite chromatography
support is in its native form.
18. The method of claim 1, wherein the apatite chromatography
support is in a calcium-derivatized form.
19. The method of claim 1, wherein the apatite chromatography
support is converted to its calcium derivatized form after the step
of contacting the impure preparation with the apatite
chromatography support.
20. The method of claim 1, wherein the apatite chromatography
support is in equilibrium between its native form and a
calcium-derivatized form.
21. The method of claim 1, wherein the elution is conducted in the
presence of one or more of an additional salt not comprising the
ionic species, glycine, arginine, urea, or a nonionic organic
polymer.
22. The method of claim 1, wherein the biomolecule is not an
antibody or immunoreactive antibody fragment.
23. The method of claim 1 wherein, the biomolecule fails to bind
the calcium-derivatized form of the apatite chromatography
support.
24. The method of claim 23, wherein the biomolecule of interest has
a molecular weight greater than 15,000 daltons and an isoelectric
point less than 9.5.
25. The method of claim 1 wherein the biomolecule is a
phosphorylated biomolecule to be purified from non-phosphorylated
biomolecules.
26. The method of claim 25 wherein the biomolecule is a
polynucleotide, vaccine, or lipid enveloped virus.
27. The method of claim 26 wherein the biomolecule is a nucleic
acid.
28. A method for purifying at least one non-aggregated biomolecule
from an impure preparation containing said biomolecule comprising
the steps of (a) contacting the impure preparation with an apatite
chromatography support, wherein the apatite chromatography support
is in a calcium-derivatized form when it is contacted with the
biomolecule and (b) substantially converting the
calcium-derivatized apatite chromatography support to its native
form prior to eluting the biomolecule.
29. The method of claim 28, wherein the converted native form
chromatography apatite support is eluted with phosphate as the
primary eluting ion.
30. The method of claim 29, wherein the biomolecule is not an
antibody or immunoreactive antibody fragment.
31. A method for purifying at least one non-aggregated biomolecule
from an impure preparation containing said biomolecule comprising
the steps of (a) contacting the impure preparation with an apatite
chromatography support, wherein the apatite chromatography support
is in native form when it is contacted with the biomolecule and (b)
substantially converting the native form apatite chromatography
support to a calcium-derivatized form prior to eluting the
biomolecule.
32. The method of claim 31, wherein conversion of the apatite
chromatography support to the calcium derivatized form causes
elution of the biomolecule of interest.
33. The method of claim 31, wherein the biomolecule is not an
antibody or immunoreactive antibody fragment.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
applications Ser. No. 61/011,513 filed Jan. 18, 2008; 61/062,663
filed Jan. 28, 2008; 61/069,859 filed Mar. 19, 2008; 61/070,841
filed Mar. 27, 2008; 61/135,787 filed Jul. 24, 2008; 61/189,467
filed Aug. 20, 2008, each of which are expressly incorporated
herein by reference in their entireties.
FIELD OF THE INVENTION
[0002] This invention relates in certain embodiments to methods for
enhancing the fractionation or purification of phosphorylated and
nonphosphorylated biomolecules by apatite chromatography in the
presence of one or more of borate compounds, sulfate compounds,
monocarboxylate compounds, and/or in the presence of calcium
compounds. In certain embodiments, the invention may permit more
effective removal of phosphorylated contaminants from
nonphosphorylated products. In other embodiments, the invention may
permit more effective removal of nonphosphorylated contaminants
from phosphorylated products. In these or other embodiments, the
invention may improve pH control during fractionation.
BACKGROUND OF THE INVENTION
[0003] Hydroxyapatite [HA] is a crystalline mineral of calcium
phosphate with a structural formula of
Ca.sub.10(PO.sub.4).sub.6(OH).sub.2. Fluorapatite may be prepared
by fluoridating hydroxyapatite, creating a mineral with the
structural formula Ca.sub.10(PO.sub.4).sub.6F.sub.2.
Protein-reactive sites on both minerals include pairs of positively
charged calcium ions (C-sites) and triplets of negatively charged
phosphate groups (P-sites). C-sites interact with proteins via HA
calcium chelation by protein carboxyl clusters. C-sites interact
with phosphorylated solutes such as DNA, endotoxin,
phosphoproteins, and lipid enveloped viruses via HA calcium
coordination by solute phosphate residues. Calcium chelation and
coordination are sometimes referred to as calcium affinity. P-sites
interact with proteins via phosphoryl cation exchange with
positively charged protein amino acid residues (Gorbunoff,
Analytical Biochemistry 136 425 (1984); Kawasaki, J. Chromatography
152 361 (1985)). Hydroxyapatite is most commonly eluted with
phosphate gradients. The strong calcium affinity of phosphate
suspends calcium chelation and coordination interactions, while its
ionic character suspends phosphoryl cation exchange interactions.
Some applications elute hydroxyapatite with combinations of
phosphate and chloride salts. Chlorides preferentially elute the
phosphoryl cation exchange interaction while having relatively
little effect on calcium affinity interactions. (Gagnon et al,
Bioprocess International, 4(2) 50 (2006)).
[0004] Native hydroxyapatite and fluorapatite can be converted to
calcium-derivatized forms by exposure to soluble calcium in the
absence of phosphate. (Gorbunoff, Anal. Biochem., 136 425 (1984)).
This converts P-sites into secondary C-sites, abolishing phosphoryl
cation exchange interactions, increasing the number of C-sites, and
fundamentally altering the selectivity of the apatite support.
Small alkaline proteins typified by lysozyme (13.7-14.7 Kda, pI
10.7) and ribonuclease (14.7 kDa, pI 9.5-9.8) fail to bind to
calcium-derivatized apatites, but most other proteins bind so
strongly that even 3 M calcium chloride is inadequate to achieve
elution (Gorbunoff). Other chloride salts also fail to achieve
elution. Calcium-derivatized apatites are restored to their native
forms by exposure to phosphate buffer, at which point they may be
eluted by methods commonly applied for elution of native apatite
supports.
[0005] The effects of different salts on the selectivity of a given
apatite are unpredictable. For example, in the absence of
phosphate, sodium chloride is unable to elute most IgG monoclonal
antibodies from native hydroxyapatite, even at concentrations in
excess of 4 moles per liter (Gagnon et al, 2006, Bioprocess
International, 4(2)50). This implies extremely strong binding. In
exclusively phosphate gradients, IgG is typically one of the latest
eluting proteins, usually requiring 100-150 mM phosphate. This also
implies strong binding. When eluted with a combination of lower
concentrations of both salts, such as 0.25 M sodium chloride and 50
mM phosphate however, IgG is one of the earliest eluting proteins.
Other paradoxes reinforce the point: increasing the sodium chloride
concentration in the presence of phosphate, which causes IgG to
bind less strongly, has the opposite effect on DNA (Gagnon et al,
2005, Bioprocess International, 3(7) 52-55). Additionally, Bovine
serum albumin (BSA) elutes at about 100 mM phosphate without
respect to sodium chloride concentration; and lysozyme elutes at a
higher phosphate concentration than BSA in the absence of sodium
chloride but fails to bind in the presence of 1 M sodium
chloride.
[0006] Ammonium sulfate, sodium sulfate, and other sulfate salts
are commonly used for precipitation of proteins, or to cause
proteins to bind to hydrophobic interaction chromatography media.
They can also be used to enhance binding with biological affinity
chromatography media such as protein A, and have even been reported
to cause proteins to bind to ion exchangers (Gagnon, 1996,
Purification Tools for Monoclonal Antibodies, ISBN 0-9653515-9-9;
Mevarech et al, 1976, Biochemistry, 15, 2383-2387; Leicht et al,
1981, Anal. Biochem., 114, 186-192; Arakawa et al, 2007, J.
Biochem. Biophys. Met., 70, 493-498). Sulfates have occasionally
been reported for elution of ion exchangers at low concentrations
for research applications but are seldom exploited in preparative
applications due to concerns over protein precipitation
(Kopaciewicz et al, 1983, J. Chromatogr., 266 3-21; Gooding et al,
1984, J. Chromatogr., 296, 321-328; Rounds et al, 1984, J.
Chromatogr., 283 37-45). None of these methods is an appropriate
model for apatites because none of them exploits calcium affinity
for binding.
[0007] Several authors have concluded that, "The presence of . . .
(NH.sub.4).sub.2SO.sub.4 seems not to affect the elution [of
hydroxyapatite]." (Karlsson et al, 1989, in Protein Purification:
Principles, High Resolution Methods, and Applications, Chapter 4,
ISBN 0-89573-122-3). Even this reference mentions the application
of sulfate strictly in the context of phosphate gradients. In the
rare cases where alternatives to phosphate as a primary eluting
salt have been discussed in the literature, suggestions have
included calcium chloride, citrate and fluoride salts, but without
mention of sulfates (Gagnon, 1996; Karlsson et al, 1989;
Gorbunoff). Other publications indicate that sulfate salts in
particular should be unsuitable as primary eluting agents for
hydroxyapatite because " . . . SO.sub.3H do[es] not form complexes
with calcium." (Gorbunoff).
[0008] Borate salts have been likewise overlooked. Borate is
occasionally used in the field of chromatography as a buffering
agent at pH values from about 8.8 to 9.8 (pK .about.9.24). It is
also used infrequently at alkaline pH to modify the charge
characteristics of cis-diol compounds to selectively enhance their
retention on anion exchangers. In contrast to phosphates,
chlorides, and sulfates, all of which exhibit molar conductivities
of about 90 mS/cm, a 1 M solution of borate at pH 7 has a molar
conductivity of about 9 mS.
[0009] Acetates have been compared to chlorides for hydroxyapatite
separation of IgG from aggregates and were found to support
inferior fractionation (Gagnon et al, Practical issues in the
industrial use of hydroxyapatite for purification of monoclonal
antibodies, Poster, 22.sup.nd national meeting of the American
Chemical Society, San Francisco, Sep. 10-14, 2006
<http://www.validated.com/revalbio/pdffiles/ACS_CHT.sub.--02.pdf>.
Monocarboxylic acid salts have been otherwise neglected, and the
elution potential of monocarboxylic zwitterions totally so.
[0010] Hydroxyapatite is used for purification of a wide variety of
biomolecules, including proteins, phosphoproteins, carbohydrates,
polynucleotides, and viral particles. The column is usually
equilibrated and the sample applied in a buffer that contains a low
concentration of phosphate. Adsorbed biomolecules are usually
eluted in an increasing gradient of phosphate salts. Alternatively,
some biomolecules may be eluted in an increasing gradient of
chloride salts, but both elution formats impose disadvantages on
purification procedures. The high phosphate concentration in which
antibodies elute in phosphate gradients has strong buffer capacity
that may interfere with subsequent purification steps. The high
conductivity at which some biomolecules elute in chloride gradients
may also interfere with downstream steps. Both situations require
either that the eluted biomolecule be diluted extensively, or that
it be buffer-exchanged, for example by diafiltration, in order to
modify the conditions to render the preparation suitable for
application to a subsequent purification step. Dilution and buffer
exchange have a negative impact on process economics. As a result,
apatite chromatography steps are often placed at the end of a
purification process. This tends to eliminate them from
consideration as capture steps. It also discourages the use of HA
as an intermediate step. A further disadvantage of chloride
gradients is that the application of chloride to hydroxyapatite
causes an uncontrolled reduction of pH. Acidic pH causes
destruction of hydroxyapatite and risks adverse affects to
biomolecules bound to it.
[0011] Another limitation of hydroxyapatite with biomolecule
purification is that the binding capacity for some biomolecules is
reduced at elevated conductivity values. This strongly reduces its
versatility since the salt concentration of cell culture
supernatants and biomolecule-containing fractions from purification
methods such as ion exchange and hydrophobic interaction
chromatography, confers sufficient conductivity to reduce the
binding capacity of hydroxyapatite to such an extent that it may
not be useful for a particular application. This disadvantage can
be overcome by diafiltration or dilution of the sample prior to its
application to the hydroxyapatite column, but as noted above, these
operations increase the expense of the overall purification
process. Alternatively, the disadvantage can be ameliorated by
using a larger volume of hydroxyapatite, but this increases process
expense by requiring larger columns and larger buffer volumes. It
also causes the antibody to elute in a larger volume of buffer,
which increases overall process time in the subsequent purification
step.
SUMMARY OF THE INVENTION
[0012] The present invention in certain embodiments relates to
methods of fractionating or purifying a desired biomolecule from an
impure preparation by contacting said preparation with a native or
calcium-derivatized apatite chromatography support, then eluting
the support in the presence of an ionic species which is a sulfate,
borate, monocarboxylic organic acid salt or monocarboxylic
zwitterion. In certain embodiments the ionic species is the primary
eluting ion in the eluent. In certain embodiments the eluent is
substantially free of phosphate as an eluting ion.
[0013] In certain embodiments of the inventions, a method for
purifying a biomolecule from an impure preparation is provided
wherein the impure preparation is contacted with an apatite
chromatography support in either the calcium derivatized form or in
its native form and the apatite support is converted to the other
form prior to elution of the biomolecule.
[0014] In certain embodiments of the invention, the desired
biomolecule to be fractionated or purified is a biomolecule other
than an antibody or antibody fragment.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Advantages of some embodiments of the invention include the
following: 1) Calcium-derivatized apatites support higher binding
capacity than native hydroxyapatite for most biomolecules, even at
high conductivity values, thereby making apatite chromatography
more effective as a capture method, or as an intermediate
fractionation step following high-salt elution from another
fractionation step such as ion exchange or hydrophobic interaction
chromatography; 2) Calcium-derivatized apatites also produce unique
selectivities that may enable effective biomolecule fractionation,
including removal of aggregates, in situations where native
apatites fail to do so; 3) biomolecules may be bound to a native
apatite support which is then converted to the calcium-derivatized
form to achieve a particular selectivity for elution or; 4)
biomolecules may be bound to a calcium-derivatized apatite support
which is them converted to the native form for elution. 5) Sulfate,
borate, and certain monocarboxylic acids or zwitterions are able to
elute biomolecules from apatite supports in the absence of
phosphate; 6) Elution in the presence of sulfate, borate, and
certain monocarboxylic acids or zwitterions produces unique
selectivities that permit effective fractionation of biomolecules
that may not be adequately served by elution with phosphate or by
combinations of phosphate and chloride; 7) Borate permits elution
of biomolecules at low conductivity values, and does so without
imposing significant buffer capacity at neutral pH, thereby
facilitating use of the eluted biomolecule in subsequent ion
exchange chromatography steps without the necessity for intervening
steps such as diafiltration; 8) Borate and certain monocarboxylic
acids or zwitterions create an increase in pH on contact with
apatites which can be used to counteract the effect of chlorides on
pH, thereby attenuating or eliminating the pH reduction that
otherwise accompanies the introduction of chlorides; 9) Sulfate
differentially enhances the retention of phosphorylated
biomolecules, thereby enhancing their separation from
non-phosphorylated biomolecules.
[0016] In certain embodiments the ionic species is borate. In
certain embodiments the borate is sodium borate or potassium
borate. In certain such embodiments the primary eluting ion is
borate. In certain embodiments the borate is present at a pH where
the borate lacks substantial buffering capacity; in certain such
embodiments the pH is less than 8.7. In certain other embodiments
the borate is present at greater than 50 mM and at a pH where the
borate has substantial buffering capacity; in certain such
embodiments the pH is 8.7 or greater.
[0017] In certain embodiments the ionic species is sulfate. In
certain embodiments the sulfate is sodium or potassium sulfate. In
certain embodiments the sulfate is the primary eluting ion.
[0018] In certain embodiments the ionic species is a monocarboxylic
acid salt. In certain such embodiments the monocarboxylate acid
anion is formate, acetate, lactate, succinate, pyruvate, gluconate,
glucuronate or proprionate. In certain embodiments the
monocarboxylate is the primary eluting ion.
[0019] In still other embodiments the ionic species is a
monocarboxylic zwitterion. In certain such embodiments the
monocarboxylate zwitterion is glycine, proline, lysine or
histidine.
[0020] In some embodiments, the biomolecule preparation may be
applied to the apatite chromatography support under conditions that
permit binding of the desired biomolecule and contaminants, with
purification being achieved subsequently by application of an
elution gradient. This mode of chromatography is often referred to
as bind-elute mode.
[0021] In some embodiments, the impure biomolecule preparation may
be applied to the apatite chromatography support under conditions
that prevent binding of the desired biomolecule, while binding
contaminants. This mode of application is often referred to as
flow-though mode. Bound contaminants may be removed subsequently
from the column by means of a cleaning step.
[0022] Suitable apatite chromatography supports include native
hydroxyapatite, calcium-derivatized hydroxyapatite, native
fluorapatite, and calcium-derivatized fluorapatite.
[0023] In certain embodiments, elution may be achieved exclusively
by means of increasing the concentration of the ionic species such
as borate, sulfate, or monocarboxylic acids or zwitterions. In
certain of such embodiments such elution is achieved with a single
ionic species as the eluting ion, e.g., borate or sulfate.
[0024] In some embodiments, elution may be achieved by borate in
combination with calcium, magnesium, phosphate, sulfate, chloride,
monocarboxylic acids or zwitterions, arginine, glycine, urea, or
nonionic organic polymers.
[0025] In some embodiments, elution may be achieved by sulfate in
combination with calcium, magnesium, phosphate, borate, chloride,
monocarboxylic acids or zwitterions, arginine, glycine, urea, or
nonionic organic polymers.
[0026] In some embodiments, elution may be achieved by
monocarboxylic acids or zwitterions in combination with calcium,
magnesium, phosphate, borate, sulfate, chloride, arginine, glycine,
urea, or nonionic organic polymers.
[0027] In certain embodiments, the method for purifying a
biomolecule from an impure preparation containing said biomolecule
includes the steps of (a) contacting the impure preparation with an
apatite chromatography support, wherein the apatite chromatography
support is in a calcium-derivatized form when it is contacted with
the biomolecule and (b) substantially converting the
calcium-derivatized apatite chromatography support to its native
form prior to eluting the biomolecule. In certain such embodiments
the biomolecule is eluted with phosphate as the primary eluting
ion.
[0028] In certain embodiments, the method for purifying a
non-aggregated biomolecule from an impure preparation containing
said biomolecule involves the steps of (a) contacting the impure
preparation with an apatite chromatography support, wherein the
apatite chromatography support is in its native form when it is
contacted with the biomolecule and (b) substantially converting the
native form apatite chromatography support to a calcium-derivatized
form prior to eluting the biomolecule. In certain such embodiments
the conversion of the apatite chromatography support to the calcium
derivatized form causes elution of the biomolecule of interest.
[0029] In certain embodiments, phosphorylated biomolecules of
interest are separated by a method of the invention from
non-phosphorylated biomolecules. In other embodiments,
non-phosphorylated biomolecules of interest are separated by a
method of the invention from phosphorylated biomolecules.
[0030] Embodiments of the invention may be practiced in combination
with one or more other purification methods, including but not
limited to size exclusion chromatography, protein A and other forms
of affinity chromatography, anion exchange chromatography, cation
exchange chromatography, hydrophobic interaction chromatography,
mixed mode chromatography, and various filtration methods. It is
within the ability of a person of ordinary skill in the art to
develop appropriate conditions for these methods and integrate them
with the invention herein to achieve purification of a particular
antibody or antibody fragment.
[0031] Terms are defined so that the invention may be understood
more readily. Additional definitions are set forth throughout this
disclosure.
[0032] "Apatite chromatography support" refers to a mineral of
calcium and phosphate in a physical form suitable for the
performance of chromatography. Examples include but are not limited
to hydroxyapatite and fluorapatite. This definition is understood
to include both the native and calcium-derivatized forms of an
apatite chromatography support.
[0033] "Salt" refers to an aqueous-soluble ionic compound formed by
the combination of negatively charged anions and positively charged
cations. The anion or cation may be of organic or inorganic origin.
Anions of organic origin include but are not limited to acetate,
lactate, malate, and succinate. Anions of inorganic origin include
but are not limited to chloride, borate, sulfate, and phosphate.
Cations of organic origin include but are not limited to arginine
and lysine. Cations of inorganic origin include but are not limited
to sodium, potassium, calcium, magnesium, and iron.
[0034] "Borate" refers to ionic compounds of boron and oxygen such
as, but not limited to boric acid, sodium borate, and potassium
borate.
[0035] "Phosphate" refers to salts based on phosphorus (V) oxoacids
such as, but not limited to, sodium phosphate and potassium
phosphate.
[0036] "Sulfate" refers to salts based on sulfur (VI) oxoacids such
as, but not limited to sodium sulfate and ammonium sulfate.
[0037] "Chloride" refers to salts such as, but not limited to
sodium chloride and potassium chloride.
[0038] "Monocarboxylic acid salt" or "Monocarboxylate" refers to
organic acid salts having a single carboxylic acid moiety including
but not limited to the sodium or potassium salts of formic, acetic,
propionic, lactic, pyruvic, gluconic, or glucuronic acid.
[0039] "Monocarboxylic zwitterion" refers to a molecule containing
a single carboxyl moiety and at least one moiety with a positive
charge. Suitable examples include but are not limited to the amino
acids glycine, proline, lysine, and histidine.
[0040] "Nonionic organic polymer" refers to any uncharged linear or
branched polymer of organic composition. Examples include, but are
not limited to, dextrans, starches, celluloses,
polyvinylpyrrolidones, polypropylene glycols, and polyethylene
glycols of various molecular weights. Polyethylene glycol has a
structural formula HO--(CH.sub.2--CH.sub.2--O).sub.n--H. Examples
include, but are not limited to, compositions with an average
polymer molecular weight ranging from 100 to 10,000 daltons. The
average molecular weight of commercial PEG preparations is
typically indicated by a hyphenated suffix. For example, PEG-600
refers to a preparation with an average molecular weight of about
600 daltons.
[0041] "Buffering compound" refers to a chemical compound employed
for the purpose of stabilizing the pH of an aqueous solution within
a specified range. Phosphate is one example of a buffering
compound. Other common examples include but are not limited to
compounds such as acetate, morpholinoethanesulfonic acid (MES),
Tris-hydroxyaminomethane (Tris), and hydroxyethylpiperazinesulfonic
acid (HEPES).
[0042] "Buffer" refers to an aqueous formulation comprising a
buffering compound and other components required to establish a
specified set of conditions to mediate control of a chromatography
support. The term "equilibration buffer" refers to a buffer
formulated to create the initial operating conditions for a
chromatographic operation. "Wash buffer" refers to a buffer
formulated to displace unbound contaminants from a chromatography
support. "Elution buffer" refers to a buffer formulated to displace
the one or more biomolecules from the chromatography support.
[0043] "Biomolecule" refers to any molecule of biological origin,
composite, or fragmentary form thereof.
[0044] "Phosphorylated biomolecule" refers to any biomolecule,
composite or fragmentary form thereof that includes at least one
phosphate residue. Phosphorylation may be natural or induced by
chemical modification. Examples include but are not limited to
nucleotides, polynucleotides, DNA, RNA, endotoxins, lipid enveloped
virus, phosphoproteins, phosphopeptides, phosphorylated amino
acids, lipoproteins (where the lipid moiety is phosphorylated),
phospholipids, glycophosphates, and glycophospholipids.
[0045] "Nonphosphorylated biomolecule" refers to any biomolecule,
composite or fragmentary form thereof that is devoid of phosphate
residues. Examples include but are not limited to proteins,
peptides, amino acids, lipids, and carbohydrates. Examples of
proteins include but are not limited to antibodies, enzymes, growth
regulators, and clotting factors.
[0046] "Biomolecule preparation" refers to any composition
containing a biomolecule which is desired to be fractionated from
contaminants.
[0047] "Antibody" refers to any immunoglobulin or composite form
thereof. The term may include, but is not limited to polyclonal or
monoclonal antibodies of the classes IgA, IgD, IgE, IgG, and IgM,
derived from human or other mammalian cell lines, including natural
or genetically modified forms such as humanized, human,
single-chain, chimeric, synthetic, recombinant, hybrid, mutated,
grafted, and in vitro generated antibodies. "Antibodies" may also
include composite forms including but not limited to fusion
proteins containing an immunoglobulin moiety.
[0048] "Antibody fragment" refers to any antibody fragment such as
Fab, F(ab').sub.2, Fv, scFv, Fd, mAb, dAb or other compositions
that retain antigen-binding function. Antibody fragments may be
derived from human or other mammalian cell lines, including natural
or genetically modified forms such as humanized, human, chimeric,
synthetic, recombinant, hybrid, mutated, grafted, and in vitro
generated, from sources including but not limited to bacterial cell
lines, insect cell lines, plant cell lines, yeast cell lines, or
cell lines of other origin. Antibody fragments may also be derived
by controlled lysis of purified antibody with enzymes such as, but
not limited to ficin, papain, or pepsin.
[0049] As it relates to the invention herein, the term "bind-elute
mode" refers to an operational approach to chromatography in which
the buffer conditions are established so that the desired
biomolecule and contaminants bind to the column upon application,
with fractionation being achieved subsequently by modification of
the buffer conditions.
[0050] As it relates to the invention herein, the term
"flow-through mode" refers to an operational approach to
chromatography in which the buffer conditions are established so
that the desired biomolecule flows through the column upon
application while contaminants are selectively retained, thus
achieving their removal.
[0051] "Analytical application" refers to a situation in which the
invention is practiced for the purpose of identifying and or
determining the quantity of the desired molecule in particular
preparation, in order to obtain information pertinent to research,
diagnosis, or therapy.
[0052] "Preparative application" refers to a situation in which the
invention is practiced for the purpose of purifying intact
non-aggregated antibody for research, diagnostic, or therapeutic
applications. Such applications may be practiced at any scale,
ranging from milligrams to kilograms of antibody per batch.
Materials
[0053] 1. Apatite Chromatography Support
[0054] Various apatite chromatography supports are available
commercially, any of which can be used in the practice of this
invention. These include but are not limited to hydroxyapatite and
fluorapatite. "Ceramic" hydroxyapatite (CHT.TM.) or "ceramic"
fluorapatite (CFT.TM.) refer to forms of the respective minerals in
which nanocrystals are aggregated into particles and fused at high
temperature to create stable ceramic microspheres suitable for
chromatography applications. Commercial examples of ceramic
hydroxyapatite include, but are not limited to CHT Type I and CHT
Type II. Commercial examples of fluorapatite include, but are not
limited to CFT Type II. Unless specified, CHT and CFT refer to
roughly spherical particles of any diameter, including but not
limited to 10, 20, 40, and 80 micron. HA Ultrogel.TM. refers to a
product comprising microfragments of non-ceramic hydroxyapatite
embedded in porous agarose microspheres.
[0055] The choice of hydroxyapatite or fluorapatite, the type, and
average particle diameter suitable for a particular fractionation
can be determined through experimentation by the skilled
artisan.
[0056] The invention may be practiced in a packed bed column, a
fluidized/expanded bed column containing the hydroxyapatite or
fluorapatite, and/or a batch operation where the hydroxyapatite or
fluorapatite is mixed with the solution for a certain time.
[0057] Certain embodiments employ CHT or CFT packed in a
column.
[0058] Certain embodiments employ CHT or CFT, packed in a column of
about 5 mm internal diameter and a height of about 50 mm, for
evaluating the effects of various buffer conditions on the binding
and elution characteristics of a particular antibody preparation of
antibody fragment preparation.
[0059] Certain embodiments employ CHT or CFT, packed in columns of
any dimensions required to support preparative applications. Column
diameter may range from 1 cm to more than 1 meter, and column
height may range from 5 cm to more than 30 cm depending on the
requirements of a particular application.
[0060] Appropriate column dimensions can be determined by the
skilled artisan.
[0061] 2. Biomolecule Preparations
[0062] Biomolecule preparations to which the invention can be
applied may include unpurified or partially purified biomolecules
from natural, synthetic, or recombinant sources. Unpurified
preparations may come from various sources including, but not
limited to, plasma, serum, ascites fluid, milk, plant extracts,
bacterial lysates, yeast lysates, or conditioned cell culture
media. Partially purified preparations may come from unpurified
preparations that have been processed by at least one
chromatography, precipitation, other fractionation step, or any
combination of the foregoing. The chromatography step or steps may
employ any method, including but not limited to size exclusion,
affinity, anion exchange, cation exchange, protein A affinity,
hydrophobic interaction, immobilized metal affinity chromatography,
or mixed-mode chromatography. The precipitation step or steps may
include salt or PEG precipitation, or precipitation with organic
acids, organic bases, or other agents. Other fractionation steps
may include but are not limited to crystallization, liquid:liquid
partitioning, or membrane filtration.
B. Description of the Method
[0063] In preparation for contacting the biomolecule preparation
with the apatite support, it is usually necessary to equilibrate
the chemical environment inside the column. This is accomplished by
flowing an equilibration buffer through the column to establish the
appropriate pH, conductivity, concentration of salts; and/or the
identity, molecular weight, and concentration of nonionic organic
polymer.
[0064] The equilibration buffer for applications conducted in
bind-elute mode may include phosphate salts at a concentration of
about 5-50 mM, or calcium salts at a concentration of about 2-5 mM,
but not mixtures of phosphate and calcium. It may optionally
include a nonionic organic polymer at a concentration of about
0.01-50%, and a buffering compound to confer adequate pH control.
Buffering compounds may include but are not limited to MES, HEPES,
BICINE, imidazole, and Tris. The pH of the equilibration buffer for
hydroxyapatite may range from about pH 6.5 to pH 9.0. The pH of the
equilibration buffer for fluorapatite may range from about pH 5.0
to 9.0.
[0065] In one embodiment, the equilibration buffer contains sodium
phosphate at a concentration of about 5 mM at a pH of 6.7, in the
presence or absence of MES or Hepes at a concentration of about
20-50 mM.
[0066] In one embodiment, the equilibration buffer contains a
calcium salt at a concentration of about 2.5 mM, in the presence of
Hepes at a concentration of about 20-50 mM and a pH of about
7.0.
[0067] The biomolecule preparation may also be equilibrated to
conditions compatible with the column equilibration buffer before
the invention is practiced. This consists of adjusting the pH,
concentration of salts, and other compounds.
[0068] After the column and biomolecule preparation have been
equilibrated, the biomolecule preparation may be contacted with the
column. Said preparation may be applied at a linear flow velocity
in the range of, but not limited to, about 50-600 cm/hr.
Appropriate flow velocity can be determined by the skilled
artisan.
[0069] In one embodiment of the bind-elute mode, a column
equilibrated in phosphate to obtain a particular binding
selectivity during column loading may be switched to calcium to
obtain a particular elution selectivity. Or the opposite may be
performed, with a column equilibrated to calcium to obtain a
particular binding selectivity, and then switched to phosphate to
obtain a particular elution selectivity.
[0070] In one embodiment of the flow-through mode, non-aggregated
biomolecule flows through the column and is collected, while
aggregated biomolecule binds to the column. The biomolecule
preparation is followed with a wash buffer, usually of the same
composition as the equilibration buffer. This displaces remaining
non-aggregated biomolecule from the column so that it can be
collected. Retained aggregates may optionally be removed from the
column with a cleaning buffer of about 500 mM sodium phosphate,
among others.
[0071] In one embodiment of an application conducted in bind-elute
mode, some combination of unwanted biomolecules, intact
non-aggregated biomolecule, and aggregated biomolecule bind to the
column. The biomolecule preparation is followed with a wash buffer,
usually of the same composition as the equilibration buffer. This
removes unretained contaminants from the column. Unwanted
biomolecule fragments may be selectively displaced by a wash buffer
that removes fragments without removing intact non-aggregated
biomolecule. Intact non-aggregated biomolecule is then eluted from
the column under conditions that leave aggregated biomolecule bound
to the column. Retained aggregates may optionally be removed from
the column with a cleaning buffer of about 500 mM sodium phosphate,
among others.
[0072] In one embodiment of the bind-elute mode, the wash buffer
may have a formulation different than the equilibration buffer.
[0073] After use, the apatite column may optionally be cleaned,
sanitized, and stored in an appropriate agent.
[0074] The invention may be practiced in combination with other
purification methods to achieve the desired level of biomolecule
purity. The invention may be practiced at any point in a sequence
of 2 or more purification methods.
C. EXAMPLES
[0075] Considerable variation in chromatographic behavior is
encountered from one biomolecule preparation to another. This
includes variation in the composition and proportion of undesired
biomolecule contaminants, intact biomolecule, biomolecule
fragments, and biomolecule aggregates, as well as variation in the
individual retention characteristics of the various constituents.
This makes it necessary to customize the buffer conditions to apply
the invention to its best advantage in each situation. This may
involve adjustment of pH, the concentration salts, the
concentration of buffering components, and the content of nonionic
organic polymer. Appropriate levels for the various parameters and
components can be determined systematically by a variety of
approaches. The following examples are offered for illustrative
purposes only.
[0076] Example 1 . Dynamic binding capacity comparison of native
and calcium-derivatized hydroxyapatite. A column of hydroxyapatite,
CHT Type II, 40 micron, 5 mm diameter, 50 mm height, was
equilibrated at a linear flow rate of 300 cm/hr with 20 mM Hepes, 3
mM CaCl.sub.2, pH 6.7. A sample of protein A purified IgG
monoclonal antibody was applied to the column by in-line dilution
at a proportion of 1 part antibody to 4 parts equilibration buffer.
Dynamic breakthrough capacity at 5% breakthrough was 114 mg/mL of
hydroxyapatite. The experiment was repeated with an equilibration
buffer of 20 mM Hepes, 3 mM CaCl.sub.2, 1 M NaCl, pH 6.7. Dynamic
capacity at 5% breakthrough was 43 mg/mL. The experiment was
repeated with an equilibration buffer of 5 mM sodium phosphate, pH
6.7. Dynamic capacity at 5% breakthrough was 29 mg/mL. The
experiment was repeated with an equilibration buffer of 5 mM sodium
phosphate, 1 M NaCl, pH 6.7. Dynamic capacity at 5% breakthrough
was 3 mg/mL. This example illustrates the dramatic improvement in
antibody binding capacity that is achieved by calcium derivatized
apatite. It will be recognized by the skilled practitioner that a
similar benefit may be obtained by substituting magnesium for
calcium.
[0077] Example 2. Purification of a biomolecule from cell culture
supernatant on native hydroxyapatite, eluted with a borate
gradient. A column of hydroxyapatite, CHT Type I, 40 micron, 8 mm
diameter, 50 mm height, was equilibrated at a linear flow rate of
300 cm/hr with 5 mM sodium phosphate, 20 mM Hepes, pH 7.0. A
monoclonal antibody preparation consisting of a mammalian cell
culture supernatant previously filtered through a membrane with
porosity of about 0.22 .mu.m, and diafiltered to about the same
conditions as the equilibration buffer was applied to the column.
The column was eluted with a linear gradient to 1 M sodium borate,
5 mM sodium phosphate, pH 7.0. The majority of contaminating
proteins eluted before the antibody. Non-aggregated antibody eluted
at an average conductivity of about 5 mS/cm. Aggregates eluted
later. The column was cleaned with 500 mM sodium phosphate, pH 7.0.
It will be recognized by the person of ordinary skill in the art
that eluted antibody may be further purified by additional
purification methods, and that the low conductivity and buffer
capacity of the eluted antibody fraction will facilitate such
methods.
[0078] Example 3. Purification of an biomolecule from cell culture
supernatant on native hydroxyapatite, eluted with a monocarboxylic
acid (lactate) gradient. A column of hydroxyapatite, CHT Type I, 40
micron, 5 mm diameter, 50 mm height, was equilibrated at a linear
flow rate of 600 cm/hr with 5 mM sodium phosphate, 20 mM Hepes, pH
7.0. 100 microliters of a monoclonal antibody preparation
consisting of a mammalian cell culture supernatant previously
filtered through a membrane with porosity of about 0.22 .mu.m, was
injected onto the column and the column washed with 2 column
volumes of equilibration buffer. The column was eluted with a 20
column volume linear gradient to 1 M sodium lactate, 20 mM Hepes,
pH 7.0. The majority of contaminating proteins eluted before the
antibody and most of the remainder eluted later. Non-aggregated
antibody eluted at an average conductivity of about 20 mS/cm.
Aggregates eluted later. The column was cleaned with 500 mM sodium
phosphate, pH 7.0.
[0079] Example 4. Purification of a biomolecule from cell culture
supernatant on native hydroxyapatite, eluted with a borate
gradient. The same column was prepared with the same buffers but
with a different IgG monoclonal antibody. The majority of
contaminating proteins eluted as previously but only about 70% of
the antibody eluted within the gradient, with the remainder eluting
with the aggregate in the 500 mM phosphate cleaning step. The run
was repeated but with 20 mM phosphate in the equilibration and
elution buffers. Under these conditions, more than 80% of the
antibody eluted within the gradient with the remainder eluting with
the aggregate in the 500 mM phosphate cleaning step. The run was
repeated but with 30 mM phosphate in the equilibration and elution
buffers. Under these conditions, more than 90% of the antibody
eluted within the gradient, while a small amount of antibody eluted
with aggregates in the 500 mM phosphate cleaning step. This example
illustrates one way to adapt the procedure to antibodies that may
not elute fully within the gradient in the absence of phosphate, or
at low phosphate concentrations. The phosphate concentration may be
increased more if necessary. Alternatively or additionally, the
borate concentration and/or pH of the eluting buffer may be
increased. It will be recognized by the skilled practitioner that
the low conductivity and buffering capacity of the borate-eluted
product make it better suited for subsequent purification by cation
exchange chromatography than elution in a sodium chloride gradient.
It will be equally recognized that the substitution of borate with
monocarboxylic acids or zwitterions with molar conductivities lower
than sodium chloride may confer a similar benefit.
[0080] Example 5. Purification of a biomolecule from cell culture
supernatant on calcium derivatized hydroxyapatite, eluted with a
borate gradient. A column of hydroxyapatite, CHT Type I, 40 micron,
8 mm diameter, 50 mm height, was equilibrated at a linear flow rate
of 300 cm/hr with 2.5 mM calcium chloride, 20 mM Hepes, pH 7.0. A
monoclonal antibody preparation consisting of cell culture
supernatant previously filtered through a membrane with porosity of
about 0.22 .mu.m and diafiltered to about the same conditions as
the equilibration buffer was applied to the column. The column was
eluted with a linear gradient to 1 M sodium borate, 2.5 mM calcium
chloride, 10% PEG-600, pH 7.0. The majority of contaminating
proteins eluted before the antibody. Antibody aggregate eluted
after non-aggregated antibody. The column was cleaned with 500 mM
sodium phosphate, pH 7.0. PEG is known to have the general effect
of enhancing the separation between fragments, intact antibody, and
aggregates on hydroxyapatite. The skilled practitioner will
recognize how to adjust the PEG concentration to optimize the
results.
[0081] Example 6. Biomolecule capture on calcium-derivatized
hydroxyapatite and elution in a sulfate gradient. A column of
hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mm
height, was equilibrated at a linear flow rate of 300 cm/hr with 20
mM Hepes, 3 mM CaCl.sub.2, pH 6.7. Cell culture supernatant
containing approximately 60 mg monoclonal IgG was equilibrated to 5
mM calcium by addition of 1 M calcium chloride at a proportion of
0.5%, then filtered to 0.22 microns. The sample was applied to the
column. No antibody was detected in the flow-through. The column
was washed with equilibration buffer, then eluted with a 20 column
volume (CV) linear gradient to 20 mM Hepes, 3 mM CaCl.sub.2, 0.5 M
sodium sulfate, pH 6.7. The antibody eluted in a single peak at
about 0.25 M sodium sulfate.
[0082] Example 7. Biomolecule capture on calcium-derivatized
hydroxyapatite, conversion to native hydroxyapatite, and elution in
a phosphate gradient. A column of hydroxyapatite, CHT Type II, 40
micron, 5 mm diameter, 50 mm height, was equilibrated at a linear
flow rate of 300 cm/hr with 20 mM Hepes, 3 mM CaCl.sub.2, pH 6.7.
Cell culture supernatant containing monoclonal approximately 40 mg
IgG was equilibrated to 5 mM calcium by addition of 1 M calcium
chloride at a proportion of 0.5%, then filtered to 0.22 microns.
The sample was applied to the column. No antibody was detected in
the flow-through. The column was washed with 5 mM sodium phosphate,
20 mM MES, pH 6.7, then eluted with a 20 CV linear gradient to 300
mM phosphate, pH 6.7. The antibody eluted in a single peak at about
165 mM sodium phosphate. This example illustrates the use of
calcium-derivatized hydroxyapatite to obtain high binding capacity,
followed by conversion to and elution from native
hydroxyapatite.
[0083] Example 8. Intermediate purification of a biomolecule by
binding in the presence of calcium, conversion to native apatite,
and elution in a sodium chloride gradient. A column of
hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mm
height, was equilibrated at a linear flow rate of 300 cm/hr with 20
mM Hepes, 3 mM CaCl.sub.2, pH 6.7. Approximately 50 mg of protein A
purified monoclonal IgG was equilibrated to 5 mM calcium by
addition of 1 M calcium chloride at a proportion of 0.5%, then
filtered to 0.22 microns. The sample was applied to the column. No
antibody was detected in the flow-through. The column was washed
with 20 mM Hepes, 10 mM sodium phosphate, pH 6.7, then eluted with
a 20 CV linear gradient to 20 mM Hepes, 10 mM phosphate, 1 M sodium
chloride, pH 6.7. The antibody eluted in a single peak at 0.6 M
sodium chloride, followed by a well-separated aggregate peak.
[0084] Example 9. Unwanted fragment and aggregate removal from a
partially purified biomolecule on native hydroxyapatite, eluted
with a borate gradient. A column of hydroxyapatite, CHT Type I, 40
micron, 8 mm diameter, 50 mm height, was equilibrated at a linear
flow rate of 300 cm/hr with 5 mM sodium phosphate, 20 mM Hepes, pH
7.0. A monoclonal antibody preparation previously purified by
protein A affinity chromatography was applied to the column. The
column was eluted with a linear gradient to 1 M sodium borate, 5 mM
sodium phosphate, 20 mM Hepes, pH 7.0. The majority of fragments
eluted before the antibody. Antibody aggregates and other
contaminating proteins eluted after non-aggregated antibody. The
column was cleaned with 500 mM sodium phosphate, pH 7.0.
[0085] Example 10. Bind-elute mode, comparison of biomolecule
elution in phosphate and sulfate gradients. A column of
hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50 mm
height, was equilibrated at a linear flow rate of 200 cm/hr with 20
mM Hepes, 3 mM CaCl.sub.2, pH 6.7. Cell supernatant containing a
monoclonal IgM antibody was applied to the column. The column was
eluted with a 20 CV linear gradient to 20 mM Hepes, 3 mM
CaCl.sub.2, 1.0 M sodium sulfate, pH 6.7. The center of the IgM
peak eluted about 415 mM sodium sulfate. DNA eluted at 855 mM
sulfate under these conditions. IgM aggregates did not elute within
the sulfate gradient and were removed in a subsequent wash step
with 500 mM phosphate. The experiment was repeated except that the
column was equilibrated with 10 mM sodium phosphate pH 6.7 and
eluted with a 20 CV linear gradient to 500 mM sodium phosphate, pH
6.7. The center of the IgM peak eluted at about 207 mM phosphate,
essentially co-eluting with DNA as revealed by its elution at 205
mM phosphate. IgM aggregates were only partially eliminated. This
example again illustrates the dramatic difference of selectivity
between sulfate and phosphate gradients, specifically and
dramatically highlights how sulfate gradients are more effective
for removal of DNA from IgM preparations, and specifically
illustrates the superior ability of sulfate gradients to eliminate
aggregates. It will also be apparent to the skilled practitioner
that these results illustrate the ability of the method to
eliminate nonphosphorylated contaminants, such as proteins, from a
preparation of a phosphorylated biomolecule, such as DNA.
[0086] Example 11. Improved pH control by the application of
borate. A column of hydroxyapatite was equilibrated to 5 mM sodium
phosphate, pH 7.0. A gradient step of 0.5 M sodium chloride, 5 mM
sodium phosphate, pH 7.0 was applied to the column. This caused the
pH to drop to about pH 5.9. The column was re-equilibrated to 5 mM
phosphate pH 7.0. A gradient step of 0.5 mM sodium chloride, 5 mM
sodium phosphate, 50 mM sodium borate, pH 7.0 was applied to the
column. Column pH dropped only to pH 6.7. It will be understood by
the skilled practitioner that the same approach can be used to
control pH in any situation where the introduction of an eluting
agent causes an unacceptable reduction of pH, and that the borate
concentration can be adjusted to achieve the desired degree of pH
control. Like borate, the application of lactate to an equilibrated
apatite support causes an increase in pH, which can likewise be
exploited to manage uncontrolled pH reduction caused by chlorides.
The skilled practitioner will recognize that other monocarboxylic
acids or zwitterions may be substituted to produce a similar
effect.
[0087] Example 12. Biomolecule fractionation with a borate
gradient. A column of hydroxyapatite, CHT Type I, 40 micron, 8 mm
diameter, 50 mm height, was equilibrated at a linear flow rate of
300 cm/hr with 5 mM sodium phosphate, 20 mM Hepes, pH 7.0. A Fab
preparation from papain digestion of an IgG monoclonal antibody was
applied to the column. The column was eluted with a linear gradient
to 1 M sodium borate, 5 mM sodium phosphate, 20 mM Hepes, pH 7.0.
The majority of contaminating Fc fragments eluted before the Fab.
Intact antibody eluted after the Fab. The column was cleaned with
500 mM sodium phosphate, pH 7.0.
[0088] Example 13. Flow-through purification of a biomolecule on
calcium-derivatized apatite. A column of hydroxyapatite, CHT Type
I, 20 micron, 5 mm diameter, 50 mm height, was equilibrated with 10
mM sodium Hepes, 2.5 mM calcium chloride, pH 7.0 at a linear flow
rate of 300 cm/hr. Calcium chloride was added to a Fab digest to a
final concentration of 2.5 mM, then loaded onto the column. The Fab
was unretained and flowed through the column at a purity of about
95%.
[0089] Example 14. Biomolecule purification by application to
native apatite, eluted by conversion to calcium-derivatized
apatite. A column of hydroxyapatite, CHT Type I, 20 micron, 5 mm
diameter, 50 mm height, was equilibrated with 5 mM sodium
phosphate, 10 mM Hepes, pH 7.0 at a linear flow rate of 300 cm/hr.
A Fab preparation was titrated to 5 mM phosphate and loaded onto
the hydroxyapatite column. The Fab was retained and eluted with a
step to 10 mM Hepes, 2.5 mM calcium chloride, pH 7.0. Purity was
greater than 95%.
[0090] Example 15. Bind-elute mode, comparison of elution of a
phosphorylated biomolecule in phosphate and sulfate gradients. A
column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50
mm height, was equilibrated at a linear flow rate of 300 cm/hr with
20 mM Hepes, 3 mM CaCl.sub.2, pH 6.7. A sample of DNA isolated from
salmon sperm was applied to the column. The column was eluted with
a 20 CV linear gradient to 20 mM Hepes, 3 mM CaCl.sub.2, 1.0 M
sodium sulfate, pH 6.7. The center of the DNA peak eluted at about
855 mM sodium sulfate. The experiment was repeated except that the
column was equilibrated with 10 mM sodium phosphate pH 6.7 and
eluted with a 20 CV linear gradient to 500 mM sodium phosphate, pH
6.7. The center of the DNA peak eluted at about 205 mM sodium
phosphate. This example illustrates the dramatic difference between
selectivity of sulfate and phosphate gradients. It will be apparent
to the skilled practitioner that these results also show the
ability of sulfate gradients to achieve more effective removal of
DNA than phosphate gradients.
[0091] Example 16. Bind-elute mode, comparison of elution of a
phosphorylated biomolecule in phosphate and sulfate gradients. A
column of hydroxyapatite, CHT Type II, 40 micron, 5 mm diameter, 50
mm height, was equilibrated at a linear flow rate of 300 cm/hr with
20 mM Hepes, 3 mM CaCl.sub.2, pH 6.7. A sample of endotoxin
prepared by phenol extraction from Salmonella enterica serotype
typhimurium was applied to the column. The column was eluted with a
20 column volume (CV) linear gradient to 20 mM Hepes, 3 mM
CaCl.sub.2, 1.0 M sodium sulfate, pH 6.7. A minor fraction of
endotoxin eluted early in the gradient, followed by a DNA
contaminant peak at 855 mM sodium sulfate. The majority of the
endotoxin failed to elute and was removed from the column by
cleaning it with 500 mM sodium phosphate, pH 6.7. The experiment
was repeated except that the column was equilibrated with 10 mM
sodium phosphate pH 6.7 and eluted with a 20 CV linear gradient to
500 mM sodium phosphate, pH 6.7. A minor fraction of the endotoxin,
corresponding to the early eluting population in the sulfate
gradient, failed to bind in phosphate and flowed through the column
immediately upon application. The center of the primary endotoxin
peak eluted at 85 mM sodium phosphate. This example illustrates the
dramatic difference between selectivity of sulfate and phosphate
gradients in general, specifically illustrates the ability of
sulfate gradients to achieve unique separations among
differentially phosphorylated biomolecules, and specifically
illustrates that some phosphorylated biomolecules do not elute from
at least some apatite chromatography supports in sulfate gradients
conducted in the absence of phosphate. It will be apparent to the
skilled practitioner that these results also show the ability of
sulfate gradients to achieve more effective removal of endotoxin
than phosphate gradients.
[0092] Example 17. Enhanced fractionation of a phosphorylated
protein from a nonphosphorylated protein by differential
enhancement of the phosphorylated protein in a sulfate gradient. A
column of hydroxyapatite, CHT Type I, 40 micron, 5 mm diameter, 50
mm height, was equilibrated at a linear flow rate of 600 cm/hr with
20 mM Hepes, pH 7.0. A purified monoclonal antibody
(unphosphorylated) and a purified alpha-casein (polyphosphorylated)
were applied, and the column was eluted in a 20 column volume
linear gradient to 500 mM phosphate. The antibody eluted at 156 mM
phosphate. Alpha-casein eluted at 223 mM phosphate. The experiment
was repeated, except eluting the column with a 20 column volume
linear gradient to 1 M ammonium sulfate, 20 mM Hepes, pH 7.0. The
antibody eluted at 308 mM sulfate. Alpha-casein did not elute
within the sulfate gradient. This shows that retention of the
unphosphorylated protein was increased by about 97%, while the
retention of alpha-casein was increased by at least 350%.
[0093] It will be understood by the person of ordinary skill in the
art how to optimize and scale up the results from experiments such
as those described in the above examples. It will also be
understood by such persons that other approaches to method
development, such as but not limited to high-throughput robotic
systems, can be employed to determine the conditions that most
effectively embody the invention for a particular antibody.
D. Additional Optional Steps
[0094] The present invention may be combined with other
purification methods to achieve higher levels of purification, if
necessary. Examples include, but are not limited to, other methods
commonly used for purification of biomolecules, such as size
exclusion chromatography, protein A and other forms of affinity
chromatography, anion exchange chromatography, cation exchange
chromatography, hydrophobic interaction chromatography, immobilized
metal affinity chromatography, mixed mode chromatography,
precipitation, crystallization, liquid:liquid partitioning, and
various filtration methods. It is within the purview of one of
ordinary skill in the art to develop appropriate conditions for the
various methods and integrate them with the invention herein to
achieve the necessary purification of a particular antibody.
[0095] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes. To the extent
publications and patents or patent applications incorporated by
reference contradict the disclosure contained in the specification,
the specification is intended to supersede and/or take precedence
over any such contradictory material.
[0096] All numbers expressing quantities of ingredients,
chromatography conditions, and so forth used in the specification
and claims are to be understood as being modified in all instances
by the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired performance sought to be obtained by the present
invention.
[0097] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only and are not
meant to be limiting in any way. It is intended that the
specification and examples be considered as exemplary only, with
the true scope and spirit of the invention being indicated by the
following claims.
* * * * *
References