U.S. patent application number 09/973141 was filed with the patent office on 2002-11-28 for chromatographic method for high yield purification and viral inactivation of antibodies.
Invention is credited to Alred, Patricia, Lebing, Wytold, Lee, Douglas C., Paul, Hanns-Ingolf.
Application Number | 20020177693 09/973141 |
Document ID | / |
Family ID | 25374006 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177693 |
Kind Code |
A1 |
Lebing, Wytold ; et
al. |
November 28, 2002 |
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 effect 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, respectively. IgA and IgM may be
eluted in high yield and purity.
Inventors: |
Lebing, Wytold; (Clayton,
NC) ; Alred, Patricia; (New Market, MD) ; Lee,
Douglas C.; (Apex, NC) ; Paul, Hanns-Ingolf;
(Cary, NC) |
Correspondence
Address: |
WOMBLE CARLYLE SANDRIDGE & RICE
P.O. BOX 7037
ATLANTA
GA
30357-0037
US
|
Family ID: |
25374006 |
Appl. No.: |
09/973141 |
Filed: |
October 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09973141 |
Oct 9, 2001 |
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09270724 |
Mar 17, 1999 |
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09270724 |
Mar 17, 1999 |
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08879362 |
Jun 20, 1997 |
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Current U.S.
Class: |
530/390.1 ;
530/387.1; 530/388.1; 530/390.5; 530/412; 530/416 |
Current CPC
Class: |
A61L 2202/22 20130101;
A61L 2/0088 20130101; C07K 16/065 20130101; A61L 2/23 20130101;
A61L 2/18 20130101; A61L 2/0023 20130101; A61L 2/0088 20130101;
A61L 2/23 20130101; A61L 2/18 20130101 |
Class at
Publication: |
530/390.1 ;
530/388.1; 530/387.1; 530/390.5; 530/412; 530/416 |
International
Class: |
C07K 016/00; C12P
021/08; A23J 001/00; C07K 001/00; C07K 014/00; C07K 017/00 |
Claims
What is claimed is:
1. 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
sequential steps a) through e) of a) adjusting the pH of the
starting solution to be within a range of from about 3.8 to about
4.5 to form an intermediate solution comprising dissolved
antibodies, b) adding a source of caprylate ions to the
intermediate solution of step a) and adjusting the pH of the
intermediate solution to be within a range of from about 5.0 to
about 5.2 to form a precipitate and a supernatant solution
comprising dissolved antibodies, c) incubating the supernatant
solution under conditions of time, temperature, and caprylate ion
concentration to inactivate substantially all enveloped viruses, d)
contacting the supernatant solution with at least one ion exchange
resin under conditions that allow binding of at least some of the
other substances including IgA or IgM to the resin while not
allowing binding of the antibodies including IgG to the resin, and
e) collecting the IgG antibodies, the method further comprising the
non-sequential step f) of, f) separating the IgA or IgM from the
ion exchange resin column by elution; and g) precipitating IgA or
IgM from eluate of step f) to result in a significant reduction of
non-enveloped viruses.
2. The method of claim 1, wherein the starting solution comprises
plasma-derived antibodies.
3. The method of claim 1, wherein the starting solution is obtained
from a mammalian cell culture medium.
4. A method of preparing a purified, virally inactivated
immunoglobulin A preparation from a starting material 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 none of the immunoglobulin G is bound to the
first resin but immunoglobulin A and other substances are bound to
the first resin, g) separating a fraction containing substantially
all of the immunoglobulins other than A from the result of step f),
h) eluating 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; and i) separating
the eluated immunoglobulin A to botain a purified, virally
inactivated immunoglobulin A preparation.
5. A method of preparing a purified, virally inactivated
immunoglobulin M preparation from a starting material 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 inmmunoglobulins 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 none of the immunoglobulin G is bound to the
first resin but immunoglobulin A and other substances are bound to
the first resin, g) separating a fraction containing substantially
all of the immunoglobulin G from the result of step f), h)
contacting the fraction of step g) with a second anion exchange
resin under conditions of pH and ionic strength such that
substantially none of the immunoglobulin G is bound to the second
resin but immunoglobulin M and other substances are bound to the
second resin, i) eluating IgM from the second 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; and
i) separating the eluated immunoglobulin M to obtain a purified,
virally inactivated immunoglobulin A preparation.
Description
[0001] This is a continuation-in-part of U.S. Ser. No. 08/879,362
filed Jun. 20, 1997.
BACKGROUND OF THE INVENTION
[0002] 1. Field
[0003] This disclosure is generally concerned with protein
purification and virus inactivation I removal and specifically with
an improved process for the purification of gamma globulins from
blood plasma and other sources.
[0004] 2. Background
[0005] 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
[0006] 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).
[0007] 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.
[0008] 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).
[0009] 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.
[0010] A two step purification of immunoglobulins from mammalian
sera and ascites fluid has been described (McKinney et al., 1987).
First albumin and other non-IgC proteins were precipitated using
caprylic acid, and then ammonium sulfate was added to the
supernatant to precipitate the IgG.
[0011] 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, vasoactive
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.
[0012] 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).
[0013] Tenold (1996) shows the use of caprylate as a partitioning
agent for the isolation of albumin from Cohn fraction II+III or
IV-1 effluent. Again the sodium caprylate is used to denature (and
precipitate) globulins.
Viral Inactivation
[0014] 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.
[0015] 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
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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
[0020] 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 our
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.
[0021] This method differs from the prior art since it 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.
[0022] 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
[0023] FIG. 1 is a flow chart describing the process of the
invention.
[0024] FIG. 2 is a flow chart showing the prior art process for
isolating antibodies.
SPECIFIC EMBODIMENTS
Materials and Methods
[0025] Adjustments of pH were done with 1 M acetic acid, 2 M acetic
acid, 6% NaOH, 1 M NaOH, or 1 M HCI. 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.
[0026] The procedure is robust and simple. (See FIG. 1.) 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 since
immunoglobulin demand generally far exceeds supply.
[0027] 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.
[0028] The primary steps of the process are defined as:
[0029] 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.
[0030] 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.
[0031] 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 2 mM, and adjusting the pH up to 5.0 to 5.2, preferably
5.1, with a base (such as 1 M NaOH).
[0032] 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.
[0033] 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).
[0034] 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.
[0035] 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.
[0036] 8) The clarified solution is diluted with purified water to
reduce conductivity between 1-8 mS/cm, preferably less than 5
mS/cm.
[0037] 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.
[0038] 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.
[0039] 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 since reduced purity is
observed as ionic strength is increased during the
chromatography.
[0040] 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.
[0041] 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.
[0042] Surprisingly, 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 form the eluate in the customary
manner.
[0043] 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.
[0044] As used herein, percent values for concentrations are
determined on a weight/volume basis.
[0045] 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.
[0046] 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
[0047] Purification of IgG from Cohn Fraction II+III Paste
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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 IgC flowed
through the column while impurities (including the caprylate) were
bound to the two anion columns.
[0052] 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.
[0053] 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%). (See FIG.
2, which outlines the prior process.)
1TABLE 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
[0054] 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.
[0055] 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
Purification of IgA and IgM from Cohn Fraction II+III Paste
[0056] 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. The serendipitous discovery was made when cleaning the
columns with a one (1) molar sodium chloride solution. However, it
is believed that a buffered solution having at least the
conductivity of 100 mM sodium chloride would provide similar
results. As used herein any solution having such conductivity is
considered an equivalent elution solution. An eluation solution
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.
Discussion
[0057] 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.
[0058] In addition to yield decrease, classical precipitation
requires the use of ethanol. Since 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.
[0059] 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%).
[0060] 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
[0061] 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).
[0062] The process is also designed to minimize loss of IgC. 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.
[0063] 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.
[0064] 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.
REFERENCES
[0065] Bloom, James W., et al., Removal of protein A from antibody
preparations, U.S. Pat. No. 4,983,722 (Jan. 8, 1991).
[0066] Condie, Richard M., Preparation of intravenous human and
animal gamma globulins and isolation of albumin, U.S. Pat. No.
4,136,094 (1979).
[0067] Friesen, Albert D., Process for preparing purified immune
globulin (IgG), Canadian Pat. No. 1,201,063 (1986).
[0068] Habeeb, A. F. S. A., et al., Preparation of human
immunoglobulin by caprylic acid precipitation, Prep. Biochem. 14:
1-17 (1984).
[0069] Kimura, Tokosuke, et al., Method of preparing gamma globulin
suitable for intravenous administration, U.S. Pat. No. 4,476,109
(1984).
[0070] Kothe, Norbert, et al., Manufacturing intravenous tolerable
immunoglobulin-G preparation, U.S. Pat. No. 5,164,487 (1992).
[0071] Lebing, Wytold R., et al., A highly purified antithrombin
III concentrate prepared from human plasma fraction IV-1 by
affinity chromatography, Vox Sang. 67: 117-24 (1994).
[0072] Lembach, Kenneth J., Protein compositions substantially free
from infectious agents, U.S. Pat. No. 4,534,972 (1985).
[0073] McKinney, Michella M., et al., A simple, non-chromatographic
procedure to purify immunoglobulins from serum and ascites fluid,
J. Immunol. Meth. 96: 271-78 (1987).
[0074] Moller, et al., Unmodified Intravenously Administered
Immunoglobulin Preparations Containing Immunoglobulin M and/or A,
U.S. Pat. No. 5,410,025 (1995).
[0075] Neurath, Alexander R., et al., Undenatured virus-free
biologically active protein derivatives, U.S. Pat. No. 4,540,573
(1985).
[0076] Pajaudier, L., et al., Preparation of human IgA as
by-product of routine fractionation, Vox. Sang. 23: 165-75
(1972).
[0077] Plan, Robert A.M., et al., Process for the preparation of
purified albumin by thermocoagulation and albumin obtained by said
process, U.S. Pat. No. 3,992,367 (1976).
[0078] Russo, C., et al., Immunol. Meth. 65: 269-71 (1983).
[0079] Schneider, et al., Process for isolating albumin from the
blood, U.S. Pat. No. 4,156,681 (1979).
[0080] Seng, Richard L., et al., Viral inactivation process, U.S.
Pat. No. 4,939,176 (1990).
[0081] Steinbuch, M., et al., The isolation of IgG from mammalian
sera with the aid of caprylic acid, Arch. Biochem. Biophys. 134:
279-94 (1969).
[0082] Steinbuch, M., et al., Preparation of an IgM and IgA
enriched fraction for clinical use, Prep. Biochem. 3: 363-73
(1973).
[0083] Tenold , Robert, Intravenously injectable immune serum
globulin, U.S. Pat. No. 4,396,608 (1983).
[0084] Tenold, Robert, Low temperature albumin fractionation using
sodium caprylate as a partitioning agent, U.S. Pat. No. 5,561,115
(Oct. 1, 1996).
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