Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCTION PROCESS F'OR INTRAVENOOS IMMUNE SERDM
GLOBULIN AND R88QLTANT PRODUCT
BACRGROOND OF THE INVENTION
The present invention relates to an integral,
multi-step commercial process for the production of
intravenously administrable immune serum globulin
s containing IgG (y-globulin) as the main ingredient.
Various processes are known for obtaining
intravenously administrable y-globulin solutions from
starting materials resulting from Cohn fractionation
of human plasma. Certain of the Cohn fractions
~o contain higher titres of y-globulin than others.
Usual starting materials for a y-globulin solution are
Cohn Fraction II or Cohn Fraction II + III.
Although prior art processors employ various
separation and sterilization techniques, process
~s modifications are constantly sought for improving
final product purity and safety, and overall yield.
Many commercial processes employ either a
solvent/detergent step for viral inactivation, or a
heat treatment step for viral inactivation. To date,
2o the art has not provided a multi-step process
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beginning with Cohn Fraction II paste or II + III
paste including two different viral inactivation
procedures as part of an efficient, high yield y-
globulin manufacturing process.
U.S. Patent 5,151,499 by Kameyama et al. is
directed to a process for producing viral inactivated
protein compositions in which a protein composition is
subjected to a viral inactivation for envelope viruses
in a solvent/detergent treatment of the protein
composition and a viral inactivation for non-envelope
viruses in a heat treatment of the protein
composition. The X499 patent teaches that preferably
the solvent/detergent step occurs first and in the
presence of a protease inhibitor, followed by a heat
treatment. Where the heat treatment is carried out in
the liquid state, the protein is first recovered from
the solvent/detergent by adsorption onto an ionic
exchange column, prior to any heat treatment. The
liquid heat treatment can be carried out in the
zo presence of a sugar, sugar alcohol or amino acid
stabilizer. Although the X644 patent lists many
starting protein compositions including
immunoglobulin, its production examples employ
Factor IX, thrombin, fibrinogen and fibronectin.
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Removal of denatured protein produced in a heat
treatment step through fractionation is not
considered.
Certain prior art processes for production of
intravenously injectable~y-globulin solutions describe
the incorporation of a liquid heat treatment carried
out in the presence of sorbitol heat stabilizer in a
multi-step purification procedure beginning with Cohn
Fraction II + III paste. In U.S. Patent 4,845,199 by
Hirao et al., Cohn Fraction II + III is subjected to
polyethylene glycol (hereinafter "PEG") fractionation
(8% w/v PEG followed by 12% w/v PEG), then ion
exchange chromatography (DEAE-Sephadex) and removal of
human blood group antibody prior to a liquid heat
treatment in the presence of sorbitol as a protein
stabilizer. On the other hand, Example 1 of
U.S. Patent 4,876,088 by Hirao et al. describes the
preparation of intravenously injectable 'y-globulin
solution from Cohn Fraction II + III paste in which
zo the paste is suspended in water, its pH adjusted to
5.5 and centrifuged, with the supernatant then being
heat treated for viral inactivation in the presence of
33% w/v of sorbitol, followed by PEG fractionation
(6%/12%) which would remove heat denatured protein and
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then by other purification steps including DEAE-
Sephadex ion exchange chromatography.
SUl~IARY OF THE INVENTION
An object of the present invention is to provide
an integral, commercially useable process for
producing a highly purified y-globulin solution from
the Cohn fractionation process.
Another object of the present invention is to
provide very pure intravenously aamlniszrap~e -y-
globulin solution free of both envelope and non-
envelope viruses, including all heat sensitive
viruses.
A further object of the present invention is to
provide a commercial y-globulin process enabling
removal of any denatured protein produced during heat
sterilization prior to a second stage viral
inactivation.
The above and other objects which will be
apparent to the skilled artisan are provided by the
Zo present invention in which an alcoholic Cohn fraction,
which may be partially purified, but is rich in y-
4
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globulin, is heat treated in aqueous medium in the
presence of a heat stabilizer for viral inactivation
and is thereafter first subjected to PEG
fractionation, and then to a second viral inactivation
s in the presence of a solvent, preferably a solvent-
detergent mixture, for disruption of envelope viruses,
followed by separation from the solvent or solvent-
detergent mixture.
In a preferred embodiment of the present
~o invention, sorbitol is the heat stabilizer and
trialkyl phosphate is the solvent.
In another preferred embodiment of the present
invention, denatured products of the heat treatment
viral inactivation are removed by the PEG
is fractionation prior to the second viral inactivation
for providing an exceedingly pure heat treated 7-
globulin.
In another preferred embodiment of the present
invention, any particulates present are removed prior
zo to the solvent-detergent treatment.
In still another embodiment of the invention,
there is provided a heat-sterilized and solvent-
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detergent sterilized y-globulin suitable for
intravenous administration.
DETAILED DESCRIPTION OF THE INVENTION
A fraction containing immunoglobulin is used as
the starting material. This fraction is not
particularly limited in so far as it originates from
human serum and contains an immunoglobulin fraction.
Specific examples of such an immunoglobulin-containing
fraction include Fraction II + III and Fraction II
~o obtainable by ethanol fractionation of Cohn, and paste
of immunoglobulin-containing fractions equivalent
thereto. Other starting materials are Fractions I +
II + III, and Fraction II + IIIw. The starting
material may contain impurities, such as human blood-
~s group antibodies, plasminogen, plasmin, kallikrein,
prekallikrein activator, IgM, IgA, IgG polymers
(hereinafter "aggregates"), etc.
The preferred starting materials are Cohn
Fraction II or Cohn Fraction II + III. When Cohn
2o Fraction II + III paste is used, it is recommended
that it first be subjected to a preliminary washing
procedure to form Fraction II + IIIw, which is
thereafter used in the process of this invention.
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"Fraction II + IIIw" is a disodium phosphate solution-
washed Cohn Fraction II + III precipitate.
Fraction II + IIIw can be obtained by suspending
Fraction II + III precipitate in cold water for
injection in a ratio of about 1 kilogram of II + III
paste per about 20 volumes of water. A sodium
phosphate solution is added to the final concentration
of approximately 0.003M sodium phosphate for
solubilizing lipids, lipoproteins and albumin. Cold
ethanol is added to bring the final alcohol
concentration to about 20%. During the alcohol
addition, temperature is gradually lowered to -5~1°C
and pH is maintained or adjusted to 7.2~0.1, for
example by using acetate buffer or dilute sodium
~s hydroxide. The Fraction II + IIIw precipitate which
forms is recovered by centrifugation and/or filtration
while maintaining the temperature at -5~1°C.
Prior to the first viral inactivation step of the
present invention, various preliminary purification
zo and/or aggregate-reducing steps can be carried out.
For example, when Fraction II + IIIw paste is used,
typically containing about 20% alcohol and more than
70% IgG, it can be suspended in 3 to 10 volumes,
preferably 3 to 5 volumes, of cold water at a
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temperature of about 0 to 5°C and with pH being
adjusted to be between 4.5 to 6.0, preferably 5.0 to
5.5 using pH 4.0 acetate buffer or hydrochloric acid.
The mixture is agitated for about 2 to 15 hours to
s allow all of the y-globulin to go into solution.
Thereafter, undissolved protein such as albumin and a-
globulins can be removed by centrifugation and/or
filtration.
Where a different starting Cohn fraction is
employed, the initial step or steps of the process can
be appropriately selected where desired for carrying
out a preliminary purification for obtaining a
fraction of high IgG content to be further processed.
For example, where Cohn Fraction II (contains over
95% IgG) has been separated from Cohn Fraction III,
with Fraction II to be further processed, the initial
processing can be at an acid pH of 3.2 to 5.0,
preferably 3.8 to 4.2, as described by Uemura et al.
U.S. Patent 4,371,520, in order to break down immune
zo globulin aggregates present into immune globulin
monomers and dimers, since aggregates are known to
possess anti-complementary activity (ACA). As another
alternative, with Cohn Fraction II + III starting
material, the Uemura, et al. patent low pH treatment
2s can be carried out as an additional step following an
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initial purif ication step as above described and prior
to the viral inactivating heat treatment step.
For the heat sterilization step, the immune
globulin protein is dissolved in water or, if in the
form of an aqueous mixture such as the filtrate
collected from the above-described partial
purification of Fraction II + III, it can be used as
is, and a sugar, sugar alcohol and/or amino acid heat
stabilizer is added thereto. The heat stabilizer is
~o preferably sucrose, maltose, sorbitol or mannitol,
most preferably sorbitol. The sugar or sugar alcohol
is added to the immune globulin solution as a powder
or first mixed with a small volume of water and then
added, to provide a final concentration of about l0 to
50 w/v%, up to saturation. At this point, the aqueous
solution of immune globulin contains sufficient water
so that this solution contains about 1 to 6% total
protein, a typical Fraction II + III starting material
containing about 300 grams protein per kilogram paste.
zo Following addition of the heat stabilizer, the
mixture is heated at about 50-70°C for about 10-100
hours, preferably at about 60°C, for about 10 to
20 hours, for viral inactivation of heat sensitive
viruses. The heat treatment step not only inactivates
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viruses, but also through the protein denaturization
effect thereof, can preferentially reduce the amount
of certain undesirable proteins normally associated
with Cohn Fractions II + III, such as prekallikrein,
s plasmin, plasminogen and IgA.
After the heat treatment, cold water to the
extent necessary is added so that protein
concentration is maintained at about 0.3 to 2.0 %.
The solution is cooled to 0-2°C.
Next, PEG fractionation is carried out on the
heat treated solution. PEG fractionation is a well
known procedure in the art of purification of immune
globulin in order to separate the desired IgG monomer
and dimer from IgG aggregate and from other impurities
naturally occurring in the starting plasma protein
fraction. However, in the instant process, the PEG
fractionation also accomplishes a separation between
the desired IgG monomer and dimer, and unwanted
denatured protein products produced by the heat
zo treatment. These denatured protein products are
denatured prekallikrein, plasminogen, plasmin, IgA,
IgM and aggregates.
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Any of the PEG fractionation procedures
documented in the prior art can be used. In general,
two stages of PEG fractionation are carried out. In
the first stage of PEG fractionation, PEG
concentration and pH are selected so that the desired
IgG monomer and dimer remain in solution while
undesired proteins such as aggregate are precipitated
out of solution. Following centrifugation and/or
filtration, PEG concentration is increased with
adjusting the pH to cause the desired IgG monomer and
dimer to precipitate.
For example, a first stage of PEG fractionation
can be carried out at a pH of about 5.0 to 7.5,
preferably within about 6.5 to 7.5 pH when Fraction II
+ IIIw paste is used as starting material, and
preferably within about 5.5 to 6.0 pH when Fraction II
+ III paste is used as starting material, with a PEG
concentration ranging from about 4 to 8%, preferably
either 4 to 6% when Fraction II + IIIw paste is used
zo as starting material, or 6 to 8% when
Fraction II + III paste is used as starting material.
While maintaining cold temperatures of about 0-2°C,
the first stage of PEG fractionation can be carried
out for about 1 to 8 hours, after which the
z5 precipitate is removed as above-described. The
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filtrate will then have its pH adjusted to about 8.0
to 9.0, preferably about 8.5 to 8.9, and additional
PEG added for final concentration of about 10 to 15%,
preferably about 12%. The precipitate formed, which
s is purified immunoglobulin, is removed by filtration
and/or centrifugation.
Further details of PEG fractionation procedures
usable in the practice of the present invention can be
found in the above-described U.S. Patent 4,876,088 by
Hirao et al. and U.S. Patent 4,845,199 by Hirao et al.
The final essential step of the present invention
is to carry out a second viral inactivation procedure
utilizing a solvent or solvent-detergent mixture. As
described below, further purification procedures,
specifically those involving the use of ionic exchange
resins, can be carried out prior to and/or following
the solvent-detergent treatment. A particularly
advantageous procedure is to carry out an anionic
exchange treatment prior to the solvent detergent
zo viral inactivation and then a cationic exchange
treatment after the solvent detergent viral
inactivation. By this procedure, certain undesirable
protein materials (such as prekallikrein activator,
IgA, IgM and albumin) found within human plasma can be
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removed from the IgG by use of the anionic exchanger
and then further such materials (prekallikrein
activator, IgA, IgM, albumin and PEG) along with the
residual reagents used in the solvent-detergent
treatment can be removed through the cationic exchange
procedure.
If not otherwise accomplished during the overall
process the solution to be subjected to the solvent-
detergent should be treated for removal of all
particulate matter, which can include denatured
protein. Therefore, it is preferred to filter the
solution with a 1 micron or finer filter prior to
solvent-detergent addition. This will also reduce the
likelihood of virus being present within a large
particle and thereby possibly avoiding exposure to the
solvent-detergent.
Today, the preferred solvent for inactivation of
envelope viruses is trialkyl phosphate. The trialkyl
phosphate used in the present invention is not subject
Zo to particular limitation, but it is preferable to use
tri(n-butyl)phosphate (hereinafter "TNBP"). Other
usable trialkyl phosphates are the tri(ter-
butyl)phosphate, the tri(n-hexyl)phosphate, the tri(2-
ethylhexyl)phosphate, and so on. It is possible to
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use a mixture of 2 or more different trialkyl
phosphates.
The trialkyl phosphate is used in an amount of
between 0.01 to 10 (w/v)%, preferably about 0.1 to
3 (w/v)%.
The trialkyl phosphate may be used in the
presence or absence of a detergent or surfactant. It
is preferable to use trialkyl phosphate in combination
with the detergent. The detergent functions to
~o enhance the contact of the viruses in the immune
globulin composition with the trialkyl phosphate.
Examples of the detergent include polyoxyethylene
derivatives of a fatty acid, partial esters of
anhydrous sorbitol such as Polysorbate 80 (Tradename:
Tween 80, etc.) and Polysorbate 20 (Tradename: Tween
20, etc.); and nonionic oil bath rinsing agent such as
oxyethylated alkylphenol (Tradename: Triton X100,
etc.) Examples include other surfactants and
detergents such as Zwitter ionic detergents and so on.
2o When using the detergent, it is not added in a
critical amount; for example, it may be used at ratios
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between about 0.001% and about 10%, preferably between
about 0.01% and 3%.
In the present invention, the trialkyl phosphate
treatment of the immune globulin containing
s composition is carried out at about 20 to 35°C,
preferably 25 to 30°C, for more than 1 hour,
preferably about 5 to 8 hours, more preferably about
6 to 7 hours.
During the trialkyl phosphate treatment, immune
globulin is present at about a 3 to 8% protein
solution in aqueous medium.
If not carried out prior to the solvent-detergent
treatment, an anionic exchange treatment can be
carried out on the solvent detergent treated immune
~s globulin. Preferably, at least a cationic exchange
treatment is carried out on the solvent-detergent
treated product. The ionic exchange treatments are
carried out with immune globulin dissolved in an
aqueous solvent, generally having a pH of about 5-8,
Zp with where desired low ionic strength for maximum
adsorption of IgG. The protein concentration
generally is within the range of about 1-15 w/v%, more
preferably from about 3 to 10 w/v%. The ionic
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exchanger is equilibrated with the same aqueous
solvent as used, and either a batch or continuous
system can be carried out. For instance, anionic
exchange batch-wise treatment can be carried out by
s mixing the immune globulin solution with the anionic
exchanger in an amount from about 10 to 100 ml per ml
of the pretreated anionic exchanger (for example,
1 gram of DEAE Sephadex A-50 resin swells to about
20 grams wet weight in 0.4% sodium chloride solution),
~o stirring the mixture at about 0-5°C for about 0.5 to
hours, and then filtering or centrifuging at 6,000
to 8,000 rpm for 10 to 30 minutes to recover the
supernatant liquor. Continuous treatment can be
affected by passing immune globulin solution through
a column of the anionic exchanger at a rate from about
to 100 ml per ml of the ionic exchanger and
recovering the non-adsorbed fraction.
The anionic exchanger to be used, for example,
comprises anion exchanging groups bonded to an
2o insoluble carrier. The anion exchanging groups
include diethylaminoethyl (DEAE), a quaternary
aminoethyl (QAE) group, etc., and the insoluble
carrier includes agarose, cellulose, dextran,
polyacrylamide, etc.
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Usable cationic exchangers are carboxy methyl
Sephadex (CM-Sephadex) CM-cellulose, SP-Sephadex, CM-
Sepharose and S-Sepharose. 1 ml of pretreated
cationic exchanger (for example, 1 gram of CM-Sephadex
C-50 resin swells to about 30-35 grams wet weight in
0.4% sodium chloride solution) is mixed with 0.5 ml to
ml of immune globulin solution and stirred at 0-5°C
for 1-6 hours. The suspension is centrifuged or
filtered to recover the IgG adsorbed resin. Also, a
continuous process can be employed.
When the above-described conditions are used with
the cationic exchanger, the IgG will be adsorbed, and
thereafter following washing of the protein-adsorbed
cationic exchange resin, IgG can be eluted, for
example by about a 1.4 N sodium chloride solution.
Following the steps of the above process, the IgG
is clarified, diafiltered and concentrated to the
extent needed. If desired, a stabilizer such as D-
sorbitol can be added and final adjustments made to
2o yield a solution of a composition containing about 100
mg/ml IgG, and 50 mg/ml D-sorbitol, with pH being at
5.4. This solution is then sterile filtered through
sterilized bacterial retentive filters and filled into
vials.
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The following examples are set forth to
illustrate the invention but are non-limiting.
Where desired, other immune globulin purification
procedures can be appropriately combined with the
processes described herein. For example, a bentonite
clarification step, useful for reducing the levels of
kallikrein and pre-kallikrein activator can be
employed. An illustration of this is set forth in
Example 1, hereinbelow.
Example i: Heat Treated and Solvent-detergent
Treated ~y-Globulin
Six hundred and eighty five grams of Fr II + IIIw
paste was suspended in about 11.9 kg of cold water.
Sodium acetate trihydrate solution was added to the
~s suspension to a final concentration of approximately
0.04M to selectively solubilize IgG. After mixing for
about 15 minutes, pH of the suspension was adjusted to
4.8 with pH 4.0 acetate buffer. Cold alcohol (95%)
was added to the suspension to a final concentration
20 of 17%. During the alcohol addition the temperature
of the suspension was lowered gradually to about -6°C.
Three hundred and three grams of acid washed Celite
535 available from Celite Corporation was added as a
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filter aid to the suspension to a final concentration
of about 2.0%. After mixing for one hour, the Celite
and the Fraction III paste containing unwanted protein
such as plasmin, plasminogen, IgA and IgM were then
removed by filtration utilizing a filter press. The
filtrate was further clarified by 0.45 ~cm and 0.2 ~m
filters .
The pH of the Fraction II + III w clarified
solution was adjusted to 4.0 with 1.0 N hydrochloric
acid and then concentrated by ultrafiltration to about
3.4 liters (1/5th the original volume). Cold water
equal to the amount removed by the 1st ultraf filtration
was added to the concentrated solution and it was
again concentrated by ultrafiltration to about 1/5th
the original volume. At this step, protein
concentration of the solution was about 2%. The
solution was further concentrated to about 4% and
diafiltered against cold water until the conductivity
of the solution was below 300 ~,S/cm to help avoid
2o protein aggregation and denaturation during heat
treatment. The solution was further concentrated to
about 8.8% protein. D-sorbitol was added to the
solution to a ffinal concentration of about 33%. After
mixing for 30 minutes, pH of the sorbitol containing
z5 solution was adjusted to 5.5 with 0.5 N sodium
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hydroxide. The solution was then heated for 10 hours
at 60°C. 'After the heat treatment, cold water equal
to 3 times the volume of the heat treated solution was
added and the diluted solution was cooled to 0-2°C.
s The pH of the solution was adjusted to 6.9 with
0.25 N sodium hydroxide and 50% polyethylene glycol
(PEG) 3350 was added to the solution to give a final
PEG concentration of 4%. Sodium chloride
concentration of the solution was adjusted to about
8 mM to aid in precipitation of impurities and
aggregates at pH 6.9. The precipitate so formed was
removed by filtration. The pH of the filtrate was
adjusted to 4.8 with 1.0 N hydrochloric acid and
bentonite was added to a final concentration of about
~s 0.25%. The pH of the bentonite suspension was
readjusted to about 5.2 and then the suspension was
filtered to remove bentonite. The filtrate pH was
adjusted to 8.5 with 0.25 N sodium hydroxide and 50%
PEG 3350 solution was added to a final PEG
to concentration of 12%. The precipitate so formed
(purified immune globulin) was removed by
centrifugation.
About 175 grams of purified immune globulin paste
obtained as above was suspended in about 790 g of 0.3%
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sodium chloride solution. pH of the suspension was
adjusted to 5.5 and then the suspension was mixed for
2~ hours. Sixty two grams of previously equilibrated
DEAE-Sephadex A-50 resin (with 0.3% sodium chloride at
pH 5.5), was added to the solution and the suspension
was mixed for 2 hours. The suspension was then
filtered to remove the resin. After adjusting the
concentration of sodium chloride to 0.4%, tri-n-butyl
phosphate (TNBP) and Polysorbate 80 mixture was added
to the filtrate to yield a solution with final
concentration of 0.3% TNBP and 1.0% Polysorbate 80.
After overnight incubation, pH of the solution was
adjusted to 5.8 and about 860 grams of previously
equilibrated CM-Sephadex C-50 (with 0.4% sodium
chloride at pH 5.8) , was added. After mixing for 2
hours, the suspension was filtered. After washing the
CM-Sephadex resin 3 times with 0.3% sodium chloride,
adsorbed IgG was eluted with 1.4 N sodium chloride.
The eluate was clarified, diafiltered and
2o concentrated. D-sorbitol was added and final
adjustments were made to yield a solution with
composition of about 100 mg/mL IgG, 50 mg/mL D-
sorbitol at pH 5.4. The solution was then sterile
filtered through sterilized bacterial retentive filter
zs and filled into vials.
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The intermediate bentonite step in this Example
is useful for further reducing the presence of
hypotensive enzymes such as kallikrein and pre-
kallikrein activator.
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Test Results on Product from Example i
Test Parameters
Anti-Complementary
Activity (CHsou/mg IgG) 0.34
IgG Purity (%) 100
IgG Content (mg/mL) 112.7
Prekailikrein 21
(% CBER Ref#3)
Measles Antibody (% CBER
~o Ref Lot No. 176) 0.67
IgG Molecular Size
Distribution by HPLC
(%) Monomer 82.2
~5 (%) Dimer 17.4
(%) Fragments 0.10
(%) Aggregates 0.3
Hepatitis A Antibody
(titer) 1:200
Hepatitis B Antibody
20 (titer) 1:1024
IgA (1~9/~) 22
IgM (l~g/~1 16
Plasminogen (ng/mL) ND
Plasmin (ng/mL) 16
25 pH 5 . 4
ND= None Detected
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Example 2: Heat Treated and solvent-Detergent
Treated y-Globulin
One (1) kg of Fr II + III paste was suspended in
4.5 kg of cold water at 0-2°C. After mixing for
1 hour, pH of the suspension was adjusted to 5.0 with
1 N hydrochloric acid. After mixing for 3 hours at
pH 5.0, the precipitate was removed by centrifugation.
D-sorbitol was added to the centrifugate to a final
concentration of 33% and mixed for 1 hour. The pH of
to the suspension was adjusted to 5.5 and then it was
heated for 10 hours at 60°C. After the heat
treatment, cold water equal to 3 times the volume of
the heat treated solution was added and the diluted
solution was cooled to o-2°C. Fifty percent
polyethylene glycol (PEG) 3350 solution was added to
a final concentration of 6% PEG. The pH of the 6% PEG
suspension was adjusted to 5.7 with 0.5 N sodium
hydroxide and the suspension was mixed for 2 hours.
Acid wash Celite 535 was added to a final
2o concentration of 1.5% and the suspension was mixed for
1 hour. The precipitate along with the Celite was
removed by filtration. The pH of the filtrate was
adjusted to 8.8 with 0.5 N sodium hydroxide, and the
PEG concentration adjusted to 12% with the addition of
25 50% PEG solution. The pH of the 12% PEG suspension
was readjusted to 8.8, the suspension being mixed for
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1 hour and filtered to collect the purified immune
globulin paste. About 251 g of purified immune
globulin paste was recovered.
Two hundred fifty one grams of purified immune
globulin paste obtained as above was suspended in
about 1.4 kg of 0.3% sodium chloride solution. After
mixing for 1 hour, pH of the suspension was adjusted
to 6.0 with 5% acetic acid. After the paste was
dissolved completely, 104 grams of DEAE-Sephadex A-50
resin, previously equilibrated with 0.3% sodium
chloride at pH 6.0, was added to the solution and
mixed for 2 hours. The resin was removed by
filtration. The filtrate was further clarified
through 0.2 ~cm filter. Concentration of sodium
chloride in the clarified solution was increased to
0.4% by the addition of sodium chloride. Tri-n-butyl
phosphate (TNBP) and Polysorbate 80 mixture was added
to the solution to give a final concentration of 0.3%
TNBP and 1.0% Polysorbate 80. The solution was then
zo incubated for 1 hour at 27°C and left overnight in a
cold box at 4°C. Next day, pH of the solution was
adjusted to 5.8 and 1.8 kg of CM-Sephadex C-50 resin
previously equilibrated with 0.4% sodium chloride at
pH 5.8, was added. After mixing for 2 hours, the
2s resin was separated by filtration. After washing the
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CM-Sephadex resin 3 times with 0.3% sodium chloride,
adsorbed IgG was eluted with 1.4 N sodium chloride.
The eluate was clarified, diafiltered and
concentrated. D-sorbitol was added and final
adjustments were made to yield a solution with
composition of about 100 mg/mL of IgG and about 50
mg/mL D-sorbitol. The solution was split into 2
parts, part A and part B. The pH of part A was
adjusted to 5.4 and part B was adjusted to pH 4.3.
~o The solutions of part A and part B were then
individually sterile filtered through sterilized
bacterial retentive filters and filled into vials.
26
CA 02281938 1999-08-20
WO 99133484 PCT/US98/25208
Test itesults on Product from Example 2
Test Parameters
Anticomplementary
Activity (CHsou/mg IgG) <0.05
IgG Purity (%) 100
IgG Content (mg/mL) 104.2
Prekallikrein
(% of CBER Ref #3) ND
Diphtheria Antibody
(Antitoxin U/mL) 8.2
IgG Molecular Size Hp_54 uH 4.3
Distribution by HPLC
(%) Monomer 88'8 g7,5
(%) Dimer 109 2.3
(%) Fragments 03 ND
(%) Aggregates <0.3 <0.3
Hepatitis A Antibody
(titer) ~ 1:100
IgA (l~g/mL) 78
zo IgM (J~g/~) 28
Kallikrein (A~) 0.09
Plasminogen (ng/mL) <84
Plasmin (ng/mL) <8.4
ND= None Detected
2s Variations of the invention will be apparent to
the skilled artisan.
27
