U.S. patent number RE43,655 [Application Number 11/873,878] was granted by the patent office on 2012-09-11 for chromatographic method for high yield purification and viral inactivation of antibodies.
This patent grant is currently assigned to Bayer HealthCare LLC. Invention is credited to Patricia Alred, Scott A. Cook, Wytold R. Lebing, Douglas C. Lee, Hanns-Ingolf Paul, Klaus-Peter Radtke.
United States Patent |
RE43,655 |
Lebing , et al. |
September 11, 2012 |
Chromatographic method for high yield purification and viral
inactivation of antibodies
Abstract
An improved process for the purification of antibodies from
human plasma or other sources is disclosed. The process involves
suspension of the antibodies at pH 3.8 to 4.5 followed by addition
of caprylic acid and a pH shift to pH 5.0 to 5.2. A precipitate of
contaminating proteins, lipids and caprylate forms and is removed,
while the majority of the antibodies remain in solution. Sodium
caprylate is again added to a final concentration of not less than
about 15 mM. This solution is incubated for 1 hour at 25.degree. C.
to affect viral inactivation. A precipitate (mainly caprylate) is
removed and the clear solution is diluted with purified water to
reduce ionic strength. Anion exchange chromatography using two
different resins is utilized to obtain an exceptionally pure IgG
with subclass distribution similar to the starting distribution.
The method maximizes yield and produces a gamma globulin with
greater than 99% purity. The resin columns used to obtain a high
yield of IgG retain IgM and IgA. IgA and IgM may be eluted from
these resins in high yield and purity.
Inventors: |
Lebing; Wytold R. (Seattle,
WA), Lee; Douglas C. (Apex, NC), Radtke; Klaus-Peter
(Apex, NC), Cook; Scott A. (Ballwin, MO), Paul;
Hanns-Ingolf (Leverkusen, DE), Alred; Patricia
(Downington, PA) |
Assignee: |
Bayer HealthCare LLC
(Tarrytown, NY)
|
Family
ID: |
25374006 |
Appl.
No.: |
11/873,878 |
Filed: |
October 17, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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09973141 |
Oct 9, 2001 |
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09270724 |
Mar 17, 1999 |
6307028 |
|
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08879362 |
Jun 20, 1997 |
5886154 |
|
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Reissue of: |
10270918 |
Oct 15, 2002 |
6955917 |
Oct 18, 2005 |
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Current U.S.
Class: |
530/387.1;
435/417; 530/413; 435/413; 435/412; 530/417; 530/418; 530/412;
435/418; 435/419; 530/419; 530/390.5 |
Current CPC
Class: |
A61L
2/0088 (20130101); A61L 2/23 (20130101); A61L
2/0023 (20130101); C07K 16/065 (20130101); A61L
2/18 (20130101); A61L 2/0088 (20130101); A61L
2/23 (20130101); A61L 2/18 (20130101); A61L
2202/22 (20130101) |
Current International
Class: |
C07K
16/00 (20060101); C07K 1/14 (20060101); C07K
1/30 (20060101); C07K 1/36 (20060101) |
References Cited
[Referenced By]
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1201063 |
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39 27 112 |
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0 374 625 |
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Dec 1989 |
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EP |
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0 440 483 |
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Aug 1991 |
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EP |
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0 447 585 |
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Sep 1991 |
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EP |
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Primary Examiner: Mosher; Mary E
Attorney, Agent or Firm: Connolly Bove Lodge & Hutz,
LLP
Parent Case Text
This application is a continuation-in-part of U.S. application Ser.
No. 09/973,141, filed Oct. 9, 2001 now abandoned, which is a
continuation of U.S. application Ser. No. 09/270,724, filed Mar.
17, 1999 (now U.S. Pat. No. 6,307,028), which is a
continuation-in-part of U.S. application Ser. No. 08/879,362 filed
Jun. 20, 1997 (now U.S. Pat. No. 5,886,154).
Claims
What is claimed is:
1. A method of obtaining purified IgA from a solution comprising
monomeric IgA at from about 8 mg/ml to about 15 mg/ml and at from
about 35% to about 55% purity, and dimeric IgA IgM, IgG, and
albumin contaminants, the method comprising: passing the solution
over a size exclusion chromatographic resin; collecting a first
IgA-containing fraction substantially free of contaminants other
than IgG and dimeric IgA; passing the first IgA-containing fraction
over a protein G-affinity chromatographic resin; collecting a
second IgA-containing fraction.
2. A method of preparing a purified IgA preparation from a starting
solution comprising immunoglobulins and other substances, the
method comprising: adjusting the pH of the starting material to
allow formation of an intermediate solution comprising dissolved
immunoglobulins; adjusting the intermediate solution to conditions
of pH, temperature, and caprylate concentration such that a first
precipitate and a first supernatant comprising immunoglobulins are
formed; separating the first supernatant from the first
precipitate; incubating the first supernatant under conditions of
time, pH, temperature and caprylate concentration such that a
second precipitate and a second supernatant comprising
immunoglobulins are formed; separating the second supernatant from
the second precipitate; contacting the second supernatant with an
anion exchange resin under conditions of pH and ionic strength such
that IgA binds to the anion exchange resin; separating a fraction
containing at least a portion of contaminants and immunoglobulins
other than IgA from the IgA bound to the anion exchange resin;
eluting IgA from the first anion exchange resin column with a
buffered solution having a conductivity in the range of that found
in a solution of at least 100 mM sodium chloride; collecting the
eluted IgA to obtain a purified, IgA preparation; passing the IgA
preparation over a size exclusion chromatographic resin; collecting
a first IgA-containing fraction substantially free of contaminants
other than IgG and dimeric IgA; passing the first IgA-containing
fraction over a protein G-affinity chromatographic resin; and
collecting a second IgA-containing fraction.
3. A method of claim 2, wherein the first IgA-containing fraction
is substantially free of contaminants other than IgG.
4. A method of claim 2, wherein the starting solution comprises IgA
at from about 35% to about 55%.
5. A method of claim 2, wherein the starting solution comprises
dimeric IgA and the second IgA-containing fraction is substantially
free of dimeric IgA.
6. A method of claim 2, wherein the starting solution comprises
from about 8 mg/ml to about 15 mg/ml IgA.
7. A method of preparing a purified IgA preparation from a starting
solution comprising immunoglobulins and other substances, the
method comprising the steps of: a) adjusting the pH of the starting
material to be within a range of from about 3.8 to about 4.5 to
form an intermediate solution comprising dissolved immunoglobulins;
b) adjusting the intermediate solution of step a) to conditions of
pH, temperature, and caprylate concentration such that a first
precipitate and a first supernatant comprising immunoglobulins are
formed, wherein the conditions under which the first precipitate
and first supernatant form comprise a pH within a range of from
about 5.0 to about 5.2 and a caprylate concentration within a range
of from about 15 mM to about 25 mM; c) separating the first
supernatant from the first precipitate; d) incubating the first
supernatant under conditions of time, pH, temperature and caprylate
concentration such that a second precipitate and a second
supernatant comprising immunoglobulins are formed, wherein the
conditions under which the second precipitate and second
supernatant form comprise a pH within a range of about 5.0 to about
5.2 and a caprylate concentration within a range of about 15 mM to
about 40 mM; e) separating the second supernatant from the second
precipitate; f) contacting the second supernatant with a first
anion exchange resin under conditions of pH and ionic strength such
that substantially no IgG or IgM is bound to the first resin but
IgA and other substances are bound to the first resin; g)
separating a fraction containing substantially all of the
immunoglobulins other than IgA from the result of step f); h)
eluting IgA from the first anion exchange resin column with a
buffered solution having a conductivity in the range of that found
in a solution of at least 100 mM sodium chloride; i) separating the
eluted IgA to obtain a purified IgA preparation; j) passing the IgA
preparation over a size exclusion chromatographic resin; k)
collecting a first IgA-containing fraction substantially free of
contaminants other than IgG and dimeric IgA; l) passing the first
IgA-containing fraction over a protein G-affinity chromatographic
resin; and m) collecting a second IgA-containing fraction.
.Iadd.8. A method of preparing a purified, virally inactivated
antibody preparation from a starting solution comprising antibodies
and other substances at an initial pH, the method comprising the
steps of: (a) adding sodium caprylate to the starting solution to
obtain a sodium caprylate concentration within a range of from
about 15 mM to about 25 mM and adjusting the pH to within a range
of from about 5.0 to about 5.2 to form a precipitate and a
supernatant solution comprising antibodies, and (b) incubating the
supernatant solution under conditions of time, pH, temperature, and
caprylate ion concentration to inactivate substantially all
enveloped viruses to produce a purified, virally inactivated
antibody preparation. .Iaddend.
.Iadd.9. A method of preparing a purified, virally inactivated
antibody preparation from a starting solution comprising antibodies
and other substances at an initial pH, the method comprising the
steps of: (a) adding sodium caprylate to the starting solution and
adjusting the pH to form a precipitate and a supernatant solution
comprising antibodies, and (b) incubating the supernatant solution
under conditions of time, pH, temperature, and caprylate ion
concentration to inactivate substantially all enveloped viruses to
produce a purified, virally inactivated antibody preparation,
wherein the conditions under which substantially all enveloped
viruses are inactivated comprise a temperature of about
25-35.degree. C., a period of time of about 15 minutes to about 6
hours, pH of the supernatant solution of 5.0 to 5.2 and caprylate
ion concentration of about 15 mM to about 60 mM. .Iaddend.
.Iadd.10. A method of preparing a purified, virally inactivated
immunoglobulin preparation from a starting material comprising
immunoglobulin and other substances, the method comprising the
steps of (a) adjusting the starting material to conditions of pH,
temperature, and sodium caprylate concentration such that a first
precipitate and a first supernatant comprising immunoglobulin are
formed, (b) separating the first supernatant from the first
precipitate, (c) incubating the first supernatant under conditions
of time, pH, temperature, and caprylate concentration to inactivate
substantially all enveloped viruses, and such that a second
precipitate and a second supernatant comprising immunoglobulin are
formed, and (d) separating the second supernatant from the second
precipitate to produce a purified, virally inactivated
immunoglobulin preparation. .Iaddend.
.Iadd.11. The method of claim 10 wherein the conditions under which
said first precipitate and first supernatant form comprise a pH
within a range of from about 5.0 to about 5.2 and a sodium
caprylate concentration within a range of about 15 mM to about 25
mM. .Iaddend.
.Iadd.12. The method of claim 10 wherein the conditions under which
substantially all enveloped viruses are inactivated and the second
precipitate and second supernatant form comprise a pH within a
range of about 5.0 to about 5.2, a caprylate concentration within a
range of about 15 mM to about 40 mM, a temperature of about
25-35.degree. C., and a period of time of about 15 minutes to about
6 hours. .Iaddend.
Description
BACKGROUND OF THE INVENTION
1. Field
This disclosure is generally concerned with protein purification
and virus inactivation/removal and specifically with an improved
process for the purification of gamma globulins from blood plasma
and other sources.
2. Background
Carboxylic acids such as caprylic acid have been used in both
preparation of plasma products (precipitation of proteins) and
inactivation of viruses. See, for example, the summary of such use
in Seng et al (1990).
Fractionation Using Caprylate:
During human immunoglobulin preparation caprylic acid is generally
recognized as an effective precipitating agent for most plasma
proteins at pH 4.8, so long as parameters such as temperature and
ionic strength are optimized. Steinbuch et al. (1969) have
described the precipitation of the bulk of the plasma proteins with
caprylic acid without affecting IgG, ceruloplasmin and IgA.
Steinbuch et al. isolated IgG from mammalian sera using caprylic
acid and reported that extensive non-immunoglobulin precipitation
was best obtained at slightly acidic pH, but not below pH 4.5.
Plasma was diluted 2:1 with 0.06 M acetate buffer, pH 4.8, and then
treated with 2.5 wt. % caprylate to initiate precipitation. Batch
adsorption of the supernatant on DEAE-cellulose was used to clear
additional impurities from the isolated IgG fraction. Later work by
Steinbuch et al. showed the use of caprylic acid to precipitate
most proteins and lipoproteins (other than the immunoglobulins)
present in Cohn ethanol Fraction III. (Steinbuch et al., 1973).
The method of Steinbuch, supra, was applied to cell culture medium
and ascites fluid from mice, using 0.86 wt. % caprylic acid for
recovery of IgG. (Russo et al., 1983). The same method was applied
to diluted human plasma using 2.16 wt. % caprylate. (Habeeb et al.,
1984). Habeeb et al. followed the caprylic acid precipitation with
fractionation on DEAE cellulose. The resulting plasma-derived IgG
was free of aggregates, plasmin and plasminogen. In addition, the
IgG obtained was low in anticomplement activity and relatively
stable during storage.
As a result of these studies, scientists further developed several
techniques for purifying IgA, IgG, alpha-1 acid glycoprotein, and
prealbumin, concluding concurrently that the precipitation reaction
was highly temperature and pH dependent. (Steinbuch et al., 1969;
Steinbuch et al., 1973; see also Tenold, 1996).
As an example, IgA has been prepared as a routine fractionation
by-product from Cohn fraction III, based on IgA solubility with
caprylic acid present at pH 4.8. (Pejaudier et al., 1972). IgA
isolated from cold ethanol Fraction III by DEAE-cellulose
adsorption and elution was further purified by caprylic acid
precipitation. Conditions for precipitation were 1.5-2% protein
concentration, 0.9% sodium chloride, pH 5.0, 1.12 wt. % caprylic
acid.
A two step purification of immunoglobulins from mammalian sera and
ascites fluid has been described (McKinney et al., 1987). First
albumin and other non-IgG proteins were precipitated using caprylic
acid, and then ammonium sulfate was added to the supernatant to
precipitate the IgG.
U.S. Pat. No. 5,164,487 to Kothe et al. (1992) concerns the use of
caprylic acid for the manufacture of an intravenously tolerable IgG
preparation free from aggregates, vaso-active substances and
proteolytic enzymes. The method includes contacting the starting
material containing IgG with 0.4% to 1.5% caprylic acid before
chromatographic purification with an ion exchange or hydrophobic
matrix.
Sodium caprylate has also been used to purify albumin. According to
these methods, sodium caprylate is added to process plasma, and
protects the albumin when the process stream is exposed to high
temperatures. Extreme temperatures not only denature process stream
globulins, but may also generate contaminant neo-antigens,
(Schneider et al., 1979; Condie, 1979; see also Plan, 1976).
Tenold (1996) shows the use of caprylate as a partitioning agent
for the isolation of albumin from Cohn fraction II+III or IV-I
effluent. Again, the sodium caprylate is used to denature (and
precipitate) globulins.
Viral Inactivation:
U.S. Pat. No. 4,939,176 to Seng et al. (1990) reports a process for
inactivating viruses in solutions of biologically active proteins
by contacting the solutions with caprylic acid. The preferred
conditions recited for the process were pH 4 to pH 8, and 0.07% to
0.001% of the non-ionized form of caprylic acid.
Other methods of viral inactivation through the use of chemical
agents are known. U.S. Pat. No. 4,540,573 to Neurath (1985) teaches
the use of di-or tri-alkyl phosphates as antiviral agents. U.S.
Pat. No. 4,534,972 to Lembach (1985) describes a method of
rendering solutions of therapeutically or immunologically active
proteins substantially free of infectious agents. In Lembach's
method a solution of protein is contacted with a transition metal
complex, e.g. copper phenanthroline, and a reducing agent to effect
inactivation of viruses without substantially affecting the
activity of the protein.
Anion Exchange Chromatography:
Bloom et al. (1991) gives an example of the use of anion exchange
chromatography to purify antibody preparations. Their method
includes contacting a solution containing antibodies and
contaminating protein A with an anion exchange resin and then
eluting the antibodies from the resin under conditions of
increasing ionic strength.
Canadian Patent 1,201,063 to Friesen teaches the preparation of an
IgG suitable for intravenous use by subjecting a plasma fraction to
a two stage separation process using two different anion exchange
resins. In each stage the buffer that is used to equilibrate the
anion exchange resin is also used to elute the IgG-containing
fraction from the resin.
A method of isolating a human IgG- and albumin-containing
composition for intravenous administration has been described by
Kimura et al. (1984). The method involves precipitation steps under
controlled conditions of pH, ethanol concentration, ionic strength
and temperature.
U.S. Pat. No. 5,410,025 to Moller et al. discloses a process of
preparing a polyclonal chemically unmodified immunoglobulin
preparation by anion exchange chromatography, where at least 5% by
weight of all the immunoglobulin it contains is IgM.
SUMMARY OF THE INVENTION
The invention is an improved process for the purification of
antibodies (especially of the IgG type) from human plasma and other
sources. The process involves suspension of the antibodies at pH
3.8 to 4.5 followed by addition of caprylic acid (or other source
of caprylate) and a pH shift to pH 5.0 to 5.2. A precipitate of
contaminating proteins, lipids and caprylate forms and is removed,
while the majority of the antibodies remain in solution. Sodium
caprylate is again added to a final concentration of not less than
about 15 mM. This solution is incubated under conditions sufficient
to substantially reduce the titer of active virus (e.g., for 1 hour
at 25.degree. C.). A precipitate (mainly caprylate) is removed and
the clear solution is diluted with purified water to reduce ionic
strength. Anion exchange chromatography using two different resins
is utilized to obtain an exceptionally pure antibody preparation
with antibody subclass distribution similar to the starting
distribution.
This method combines virus inactivation and removal as an integral
part of the processing scheme and minimizes post virus treatment
manipulation of the gamma globulin solution. By integrating virus
treatment into the processing scheme, the method maximizes yield
and produces a gamma globulin with greater than 99% purity.
Further, it has now been found that when two resin columns are used
in series, such columns retain IgA and IgM respectively, and that
subsequent elution of each column with a buffered solution having a
conductivity at least about that of a 100 mM sodium chloride
solution, frees the retained IgA and IgM fractions from,the columns
in high yield and purity.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows a chromatogram showing that a size exclusion
chromatographic step results in a purified fraction containing
monomeric IgA contaminated with IgG. IgM, dimeric IgA, albumin and
other contaminants were substantially eliminated by size exclusion
chromatography. The box outline shows the fraction (peak 2) used
for protein G SEPHAROSE experiments to remove IgG (see FIG. 2).
FIG. 2 shows a chromatograph of the protein G affinity
chromatography of peak 2 as illustrated in FIG. 1.
DETAILED DESCRIPTION OF THE INVENTION
Materials and Methods
Adjustments of pH were done with 1 M acetic acid, 2 M acetic acid,
6% NaOH, 1 M NaOH, or 1 M HCl. Sodium caprylate stock solution was
made by dissolving 30% sodium caprylate in water for injection by
mixing. Human plasma fraction II+III was produced as described by
Lebing et al. (1994). All reagents were USP grade or better.
Nephelometry was done using a Beckman Array 360 Nephelometer and
Beckman kits. Analytical HPLC was done using HP 1050 systems with
Tosohaas G3000SW and G4000SW SEC columns. Protein was determined
using the Biuret method.
The procedure is robust and simple. The process begins by
redissolving precipitated antibodies in purified water at a pH
around 4.2. In practice, increasing the amount of water per unit of
paste results in increased yield. However, when processing hundreds
of kilograms of paste it is practical to sacrifice some yield in
order to keep vessel and column scale within workable limits.
Yields across the dissolving step, viral inactivation, and
chromatography are relatively important because immunoglobulin
demand generally far exceeds supply.
Inactivation of enveloped viruses requires that the bulk of the pH
sensitive precipitate be removed prior to the inactivation step. In
addition, sodium caprylate content should be 15-60 mM during the
25.degree. C. hold to achieve complete inactivation of enveloped
viruses. Virus inactivation studies have confirmed that caprylate
at 16 mM or 18 mM inactivates over 4 log units of Bovine Viral
Diarrhea Virus and Pseudorabies virus (both enveloped viruses) in
30 minutes at 24.degree. C. This additional chemical virus
inactivation supplements the virus inactvation of a pH 4.25 hold
step also incorporated into the manufacturing process.
The primary steps of the process are defined as
1) Suspending a composition containing precipitated immunoglobulins
in purified water for injection (WFI) at 5.degree. C. with vigorous
mixing. In a preferred embodiment fraction II+III paste is used,
but other sources may also be used, such as ascites fluid, tissue
culture media containing antibodies, other human plasma fractions,
or animal plasma fractions. 2) Dissolving immunoglobulins into
solution by lowering the mixture to pH 3.8 to 4.5, preferably 4.2,
by the addition of acid, preferably acetic acid, with further
vigorous mixing. 3) Adding a source of caprylate ions (e.g., 40%
w/v sodium caprylate in water) to a final concentration of 15 mM to
25 mM, preferably 20 mM, and adjusting the pH up to 5.0 to 5.2,
preferably 5.1, with a base (such as 1 M NaOH). 4) Removal of
precipitated proteins, lipids, and caprylate by filtration at
ambient temperature (e.g., 5-25.degree. C.). The filtration
requires addition of filter aid (for example, in this case the
filter aid is 2% to 5% diatomaceous earth). The solution is
filtered using normal flow filtration. This step results in
significant reduction of non-enveloped virus. Centrifugation may be
substituted for filtration. 5) Addition of further caprylate to
adjust the concentration back up to about 15 mM to about 60 mM,
preferably 20 mM, while pH is held at 5.0-5.2, preferably 5.1, by
the addition of acid (e.g. 1 M acetic acid). 6) The temperature is
increased to about 25-35.degree. C., preferably 25.degree. C., and
held for a period of about 15 minutes to about 6 hours, preferably
about one hour. Longer incubation times may be used with some
sacrifice in yield. A precipitate of principally caprylate and some
additional protein is formed during this step. 7) Filter aid
(diatomaceous earth) is added and precipitate is removed by normal
flow filtration. Enveloped viruses are inactivated by the caprylate
hold, and non-enveloped viruses are captured on the filter pad. 8)
The clarified solution is diluted with purified water to reduce
conductivity between 1-8 mS/cm, preferably less than 5 mS/cm. 9)
Passing the solution through two anion exchange chromatography
columns linked in series. The anion exchangers are chosen for
ability to remove IgA, IgM, albumin and other remaining protein
impurities. After loading, the columns are washed with
equilibration buffer. The flow through and wash fraction are
collected as purified IgG. Both columns are equilibrated with the
same buffer and at the same pH.
Several anion exchange resin combinations may be utilized depending
on selectivity of the resins. The anion exchange resins are chosen
for their ability to selectively remove the impurities found in
alcohol/pH precipitated plasma fractions. In developing this
method, satisfactory purifications were obtained with combinations
of PHARMACIA BIOTECH Q & ANX resins and E. MERCK TMAE
FRACTOGEL.
Conditions described for the chromatography generally range from pH
5.0 to 5.2. At pH<5.0 impurities pass through the columns. At
pH>5.2 yield is sacrificed. Ionic strength during the
chromatography is relatively important because reduced purity is
observed as ionic strength is increased during the
chromatography.
In preferred embodiments, the solution is applied directly to the
first anion exchanger which has been equilibrated with 20 mM sodium
acetate at pH 5.1. This is followed by applying the non-binding
fraction (the flow through) from the first anion exchange column
directly onto the second anion exchange column. This column has
also been equilibrated with 20 mM acetate buffer at pH 5.1. The
protein solution is typically loaded onto the first column at a
ratio of 50-110 mg IgG/ml packed resin. The protein solution is
typically loaded onto the second column at a ratio of 75-95 mg
IgG/ml packed resin. The protein to resin ratios can also be
adjusted beyond these limits, but doing so will have an impact on
yield and purity. The protein solution is followed by approximately
2 column volumes of the equilibration buffer, which washes any
non-bound IgG off of the columns. The unbound fraction is collected
as highly purified IgG, which is then diafiltered and the protein
is concentrated to final formulation values.
The preferred conditions for final product are chosen based on
patents held by this manufacturer. These conditions (low pH and low
salt) would, in theory, benefit any IgG product. The collected
protein is adjusted to pH 4.2. It is ultrafiltered to a
concentration of approximately 5% (w/v). It is then diafiltered
with purified water.
The purified IgG is either concentrated to a stable liquid
formulation (as described by Tenold, 1983) or other appropriate
final formulation (e.g. a freeze dried formulation). For a liquid
formulation the purified IgG is concentrated to yield either 5% or
10% IgG (w/v) following sterile filtration. Prior to filtration,
the pH is adjusted to 3.80 to 4.25 and maltose or glycine is added
to adjust osmolarity to be compatible for intravenous injection.
The sterile bulk is then held for not less than 21 days to reduce
anti-complement activity and to inactivate enveloped viruses.
It was found that IgA and IgM could be obtained from the resin
columns at significant yield and purity. IgA is obtained by eluting
the first anion resin exchange column with a buffered solution
having at least the conductivity of a 100 mM sodium chloride
solution. The preferred range is believed to be in the conductivity
range of about 100 mM to 250 mM sodium chloride. However, a
significant yield has been obtained with elution by 1 Molar sodium
chloride. IgM is obtained in about 90% purity from the second anion
resin column with the same procedure. The products are separated
from the eluate in the customary manner.
In one embodiment of the invention, IgA from the bound fraction of
the first anion exchange column of the IgG purification process, as
described above, is further purified by size exclusion
chromatography. The first anion exchange eluate (eluted using a
buffered solution having at least the conductivity of a 100 mM
sodium chloride solution) was applied to a gel filtration column.
The elution profile had five protein peaks identified by
immunonephelometry as IgM, dimeric IgA, monomeric IgA, IgG, and
albumin, respectively. Rechromatography of the monomeric IgA peak
on a gel filtration column resulted in one major protein peak and
two small shoulders containing monomeric IgA, dimeric IgA, and IgG,
respectively. Fractions containing monomeric IgA were pooled,
eliminating those that contained dimeric IgA and IgG.
Several preparations of highly purified IgA migrated as a 160,000
Da band on non-reduced polyacrylamide gels. A minor band of
slightly lower molecular weight was interpreted as a form having a
different glycosylation pattern. The identity of IgA was further
confirmed by SDS-PAGE and western blots comparing non-reduced and
reduced forms of IgG, IgM, and IgA.
IgA dissociated into two fragments of 20,000 Da and 60,000 Da on a
reducing SDS-PAGE, consistent with the molecular weight of the IgA
light chain and the .alpha.-heavy chain of the molecule. In western
blots, IgA, but not IgM or IgG, was detected by alkaline
phosphatase-labeled, antihuman IgA under non-reducing and reducing
conditions. Endotoxin levels in the final preparations of IgA were
determined to be <0.05 U/ml. This value was deemed acceptable
for in vivo experiments based on the release criterion for
intravenously administered immunoglobulins (0.5 U/ml).
In yet another embodiment of the invention, IgA from a high salt
eluate of the first anion exchange column is further purified using
size exclusion chromatography and subsequent protein G affinity
chromatography. The purified IgA can also be passed through a
nanofiltration device, demonstrating a pathogen clearance mechanism
for the IgA preparation.
The size exclusion chromatographic step results in a purified
fraction containing monomeric IgA contaminated with IgG. IgM,
dimeric IgA, albumin and other contaminants were substantially
eliminated by size exclusion chromatography. See FIG. 1 and Table 2
below. The contaminating IgG was substantially eliminated by a
protein affinity chromatographic step, as determined by
immunonephelometry. See FIG. 2 and Table 4. SDS-PAGE confirmed that
this process was capable of yielding IgA in the flow-through
fraction of greater than 95% purity.
Purified IgA was also subjected to nanofiltration, demonstrating
feasibility of this method for viral clearance using IgA prepared
according to the methods of the present invention.
As used herein, percent values for concentrations are determined on
a weight/volume basis.
As used herein, to substantially reduce the titer of active virus
means to reduce the titer of active virus by at least about 2 log
units, more preferably at least about 3 log units, and most
preferably at least about 4 log units.
As used herein, substantially all of a protein means at least about
90% of the protein. Substantially none of a protein means less than
about 5% of the protein.
EXAMPLE 1
Purification of IgG from Cohn Fraction II+III Paste
Fraction II+III paste was solubilized in 12 volumes of 5.degree. C.
purified water. The mixture pH was adjusted to pH 4.2 with acetic
acid, and mixed for 1 hour. This step put the IgG into
solution.
The mixture pH was then adjusted up to pH 5.2 with NaOH and sodium
caprylate (the "pH swing"). Proteins and lipids were precipitated.
The mixture was clarified by filtration to remove precipitate which
would interfere with virus inactivation. The caprylate
concentration was adjusted to 20 mM at pH 5.1, and the mixture was
incubated for 1 hour at 25.degree. C. to effect enveloped virus
inactivation.
The mixture was filtered to produce a clear solution for
chromatography. The solution conductivity was adjusted to between
2.0 and 3.0 mS/cm using purified water. The pH of the solution was
adjusted to 5.0 to 5.2 following the conductivity adjustment.
The solution was then applied directly to two anion exchange
columns (a strong anion exchanger followed by a weak anion
exchanger). The two columns were linked in series. The IgG flowed
through the column while impurities (including the caprylate) were
bound to the two anion columns.
The pH of the collected flow through from the chromatography was
adjusted to 3.8 to 4.0 using acetic acid. It was diafiltered with
seven exchanges of buffer (purified water). It was then
concentrated and final formulated at pH 4.2.
The overall yield from paste dissolving to final product was 69%
(see the table). This was a significant improvement over the prior
process yield using the alcohol process wash (48%).
TABLE-US-00001 TABLE 1 Yield Summary Starting Recovery Recovery
Process Amount g/liter plasma % Process New Chromatography Process
7.0 kg Starting II + III paste 6.5 Post CIM Treatment 5.45 84% Post
Chromatography 5.0 77% Final Container 4.5 69% Old Production
Process 7.0 kg Starting II + III paste 6.5 Effluent III Filtrate
III Final Container 3.1 48%
EXAMPLE 2
Purification of IgG from Cell Culture Medium
Cell line growth media containing secreted monoclonal antibodies is
first adjusted to the proper pH and conductivity. This accomplished
by diafiltering against purified water while adjusting the pH to
4.2 with acetic acid. The conductivity should be less than 1.0
mS.
Purification of the monoclonal antibody is achieved by following
the steps above. The purified monoclonal antibody is then
concentrated and final formulated to a pH of 4.2 using glycine,
maltose, or other suitable excipients. By formulating at pH 4.2 a
liquid solution stable for 2 years at 5.degree. C. can be achieved.
This is highly desirable from a commercial standpoint.
EXAMPLE 3
Recovery of IgA and IgM from Cohn Fraction II+III Paste
The process described in Example 1 was followed and IgG obtained in
high yield and purity as described. However, subsequent
experimentation revealed that IgA could be eluated from the first
anion exchange resin column with a high concentration salt
solution, and that IgM could be eluated from the second anion
exchange resin column with a similar high concentration salt
solution. It is believed that a buffered solution having at least
the conductivity of 100 mM sodium chloride would provide similar
results. An eluant would have a preferred range of conductivity
equivalent to that of 100 to 250 mM sodium chloride. The first
anion exchange column is preferably a strong anion exchange resin
such as Pharmacia Biotech Q and the second anion exchange column is
preferably a weak anion exchange resin such as Pharmacia Biotech
ANX.
EXAMPLE 4
Purification of IgA by Size Exclusion Chromatography
Monomeric IgA from human plasma was purified by two consecutive
size-exclusion chromatography steps on SUPERDEX 200 using a
BIOLOGIC chromatography station (BioRad, Richmond, Calif.). The
high-salt eluate of the Q-SEPHAROSE chromatography step (see
Example 3) was used as starting material and contained 13 mg/ml of
IgA having a purity of 45% by immunonephelometry and total protein
determination.
Briefly, 20-ml of starting material were applied to a prepacked,
2-liter 200 XK 50/100 column (5 cm.times.93 cm, Pharmacia, Upsala,
Sweden) and run at 10 ml/min in Tris Buffered Saline (TES) buffer
(Sigma, St. Louis, Mo.). Fractions were analyzed by nephelometry,
SDS-PAGE, and size exclusion-FPLC, Fractions positive for monomeric
IgA were pooled, concentrated to a final volume of 10 ml with an
AMICON concentration chamber (YM 10 membrane, room
temperature)(Amicon, Beverly, Mass.), and reapplied to the SUPERDEX
200 column using the same running conditions as in the first run.
The final product was formulated in 0.2 M glycine, pH 4.25 and
sterile filtered using a 0.2 .mu.m membrane.
EXAMPLE 5
Characterization of IgA Purified by Size Exclusion
Chromatography
Immunonephelometry was used to quantify IgA in different fractions
from the purification process of Example 4. A BECKMAN COULTER ARRAY
360 System was used along with goat anti-human IgA as per
manufacture directions (Beckman Coulter, Brea, Calif.). A reference
plasma was used for calibration.
SDS-polyacrylamide gel electrophoresis (reducing and non-reducing)
was performed with a PHAST System (Pharmacia, Upsala, Sweden),
precast 4-15% gradient gels, and SDS buffer strips (1% SDS).
Samples were reduced with 1% 2-mercaptoethanol (Sigma, St. Louis,
Mo.) and heated at 100.degree. C. for 1 min. Gels were developed by
Fast Coomassie staining using technique file No. 200
(Pharmacia).
Purified monomeric IgA was analyzed by size exclusion FPLC using a
SUPERDEX 200 HR 10/30 column (1 cm.times.30 cm, Pharmacia, Upsala,
Sweden) and a BIOLOGIC chromatography system (BioRad, Richmond,
Calif.). Fifty microliters of final product were applied to the
column and run at 0.75 ml/min in TBS buffer (Sigma, St. Louis,
Mo.).
Protein bands were transferred from SDS-PAGE to nitro-cellulose
membranes using the PHAST System transfer kit. IgA was detected
using alkaline phosphatase-labeled goat anti human IgA antibody
(Jackson Immuno Research, West Grove, Pa.). [Bayer Notebook No. CRB
9926-845 pp. 22-30]
Analysis of purified IgA samples was carried out using LAL COATEST
Endotoxin Assay (Chomogenix, Molndol, Sweden).
Total protein concentration was determined by A.sub.280 using an
extinction coefficient of 1 for protein solutions and 1.37 for
IgA.
EXAMPLE 6
Purification of IgA by Size Exclusion and Affinity
Chromatography
Q-SEPHAROSE eluates as described in Example 3 (BIOTECH Q) were
pooled and determined to have a final volume of 41.86 L. The IgA
concentration in the pool was determined to be 4.95 g/L by
nephelometry.
The pooled eluate was applied over a 2.times.0.5-m.sup.2 PELLICON
30K (Millipore P2B030A05; Millipore Corporation, Bedford, Mass.)
membrane and concentrated 2.5-fold from an A.sub.280 of 11.94 AU/ml
(41.86 L) to an A.sub.280 of 29.30 AU/ml (16.36 L). This target
A.sub.280 was determined by comparison to the Q-SEPHAROSE elute lot
used as described in Example 4 above.
Size Exclusion Chromatography
Nephelometry testing indicated that the concentrated final pool had
an [IgA]=11.68 g/L, a 2.4-fold increase over the 4.95 g/L in the
unconcentrated pool. Seventy-three ml of pooled Q-SEPHAROSE eluate
was loaded on a 10 cm.times.93 cm SUPERDEX 200 column equilibrated
in TBS, pH 7.4. The column was isocratically eluted with TBS at
26.75 cm/hr and fractions collected manually in sterile bottles.
These fractions were analyzed by immunonephelometry. The
chromatogram is shown in FIG. 1.
As shown in FIG. 1, peak 1 typically contained IgM and dimeric IgA,
peak 2 contained monomeric IgA and IgG, and peak 3 contained
albumin. Peak 4 absorbed at A.sub.280 but had no detectable levels
of IgG, IgA, IgM, or albumin by immunonephelometry. Peak 2
contained IgA and IgG (outlined with a rectangle on the
chromatogram of FIG. 1). This fraction was used for protein
G-SEPHAROSE experiments for removal of the IgG. By
immunonephelometry, peak 2 yielded 500 mg of IgA, corresponding to
a yield of 59%. The amounts of each protein, as determined by
nephelometry, were as indicated in Table 2. Table 3 below reports
the various mass balances and IgA yield.
TABLE-US-00002 TABLE 2 Immunonephelometry of SUPERDEX 200 peaks
Fraction Albumin (mg) IgA (mg) IgG (mg) IgM (mg) Load 109 862 320
131 1 0 437 49 120 2 0 376 232 0 3 97 0 4.9 0 4 0 0 0 0
TABLE-US-00003 TABLE 3 Mass balances and IgA yield A280 IgG* IgA*
IgM* Albumin* Mass Balance 8% 89% 94% 89% 92% Yield N.A. N.A. 59%
N.A. N.A. *concentrations determined by immunonephelometry
Protein G-Sepharose Affinity Chromatography
For further purification of IgA, a PHARMACIA PROTEIN G-SEPHAROSE
FAST FLOW column (0.5 cm.times.5.9 cm) was equilibrated with EQ
buffer (1.times.TBS; 20 mM Tris-HCl, 0.9% NaCl, pH 7.4) and the
sample (SUPERDEX 200 IgA/IgG peak) was loaded in the same EQ
Buffer. The column was run at a linear velocity of 200 cm/hr. The
flow through was pooled for analysis. After unbound sample was
washed out with EQ buffer, bound protein was eluted using 100 mM
glycine, pH 2.5. The eluate was also pooled for analysis. The resin
was then re-equilibrated with consecutive washes using 5.times.TBS
followed by 1.times.TBS. The A.sub.280 chromatogram trace for the
PHARMACIA PROTEIN G-SEPHAROSE FAST FLOW chromatography is shown in
FIG. 2.
Analysis of the load, flow through and eluate fractions included
A.sub.280 measurements, nephelometry and SDS-PAGE, with results
described below.
TABLE-US-00004 TABLE 4 A.sub.280 and Nephelometry of PROTEIN
G-SEPHAROSE Fractions Protein G-SEPHAROSE Volume A.sub.280/mL IgA
Nephel. IgG Nephel. Fraction Description (ml) (AU/ml) (mg/ml)
(mg/ml) Sample Load 33.14 1.12 0.58 0.33 Flow-Through 51.0 0.41
0.35 <0.01 Eluate 10.0 1.42 0.01 0.77
It is clear that the flow through fraction contains IgA with little
or no IgG present. The data for the eluate fraction show that the
PROTEIN G-SEPHAROSE resin binds almost exclusively IgG and minimal
IgA. Accordingly, protein G is an effective means of separating IgA
and IgG. SDS-PAGE analysis supported these findings. IgA was found
to be >95% pure by reducing SDS-PAGE.
Mass balance for the PROTEIN G-SEPHAROSE FAST FLOW chromatography
was calculated for both IgA and IgG using both A.sub.280
measurements and nephelometry. Overall mass balance on the basis of
A.sub.280 was 94.6%. On the basis of nephelometry, IgA mass balance
was 93.8%, while IgG mass balance was 75.2%. IgA yield across this
step was 93.2%.
EXAMPLE 7
Nanofiltration of Purified IgA
IgA generated according to Example 5 was used to demonstrate
feasibility of viral clearance using nanofiltration technology.
Vmax trials using VIRESOLVE NFP (Millipore Corporation, Bedford,
Mass.) were performed to demonstrate feasibility in a non-virally
challenged system. Two IgA concentrations were used, 0.373 mg/ml
and 2.24 mg/ml (both in TBS, pH 7.5; equivalent buffer conditions
for the Protein G-SEPHAROSE flow thru fractions which contain
>95% pure IgA). Both IgA concentrations demonstrated 98% IgA
recovery based on A.sub.280. Using flow rate, the data suggested to
process 100 L in 2 hours, 4.times.10'' cartridges would be required
for IgA at 0.373 mg/ml and 10.times.10'' cartridges needed for IgA
at 2.24 mg/ml.
DISCUSSION
Immunoglobulins precipitate with the II+III fraction during the
Cohn alcohol fractionation. Precipitation relies on the overall
charge of the protein surface and its interaction with the solvent.
Exacting salt, alcohol, and pH ranges can somewhat limit the range
at which proteins precipitate. However, most proteins precipitate
across a wide range of pH and alcohol concentration (as much as 1.5
pH units and 10% alcohol). Thus precipitation ranges of proteins
tend to overlap. All three major immunoglobulin types, IgG, IgA,
and IgM, are coprecipitated due to the similarity of their
isoelectric points. Further separation of the immunoglobulin is
complicated by this similarity. Therefore, production schemes which
utilize precipitation require that a significant amount of the IgG
is coprecipitated with the IgA and IgM.
In addition to yield decrease, classical precipitation requires the
use of ethanol. Because ethanol destabilizes the proteins, reduced
temperatures (typically -5.degree. C.) are required during
processing to increase protein stability. Chromatography can avoid
problems of protein denaturation that commonly arise in
precipitation strategies. The protein chromatography steps
generally can be done under conditions which favor protein
stability. Another disadvantage of ethanol fractionation is that
due to its chemical nature alcohol is a potential explosion hazard
which requires explosion proof facilities and special handling
protocols. This fact significantly increases the cost of the
fractionation process, a drawback which does not exist with
conventional chromatographic methods.
Ion exchange chromatography takes advantage of surface distribution
and charge density on both the protein and the ion exchange media.
The anion exchange resin presents a positively charged surface. The
charge density is specific to the resin and generally is
independent of pH (within the working range of the resin). A
typical anion exchanger will bind proteins which have a net
negative charge (i.e. when the pH of the solution is above the
isoelectric point of the protein). In reality, the surface of a
protein does not present a singular charge; rather it is a mosaic
of positive, negative, and neutral charges. Surface structure is
specific to a given protein and will be affected by solution
conditions such as ionic strength and pH. This uniqueness can be
exploited to establish specific conditions where individual
proteins will bind or release from the anion exchange resin. By
establishing these conditions, proteins with only slightly
differing surface or charge properties can be effectively separated
with high yield (>95%).
Improvements in the structure of chromatography resin supports have
made large scale chromatography a practical alternative to more
conventional purification methods. Rigid resins allow large volumes
to be processed rapidly (<5 hours), and high ligand density
gives the increased capacity necessary for large volume processing.
These factors coupled with high yields, product purity and process
simplicity favor the use of chromatography in large scale
manufacturing.
CONCLUSION
The chromatography process described herein takes advantage of the
high specificity of chromatography resins. Two anion exchangers are
used to selectively remove protein contaminants and the viral
inactivation agent. The resulting product is of >99% purity when
assayed by either nephelometry or size exclusion chromatography
(SEC-HPLC).
The process is also designed to minimize loss of IgG. Virus
inactivation and removal has been carefully integrated into the
dissolving and chromatography steps, therefore increasing the
process efficiency. The overall yield from paste dissolving to
final product is 69% (see the table). This is a significant
improvement over the current process yield using the alcohol
process wash (48%). While the process minimizes the loss of IgG, it
also provides a new and efficient method to obtain IgM and IgA in
good yield and purity.
The process was performed on human Cohn fraction II+III paste in
example 1. However, it is anticipated that the process may be used
with equivalent results on plasma fractions isolated from non-human
animals as well.
The above examples are intended to illustrate the invention and it
is thought variations will occur to those skilled in the art.
Accordingly, it is intended that the scope of the invention should
be limited only by the claims below.
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