Note: Descriptions are shown in the official language in which they were submitted.
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Production of protein preparations with a reduced
aggregate content
Description
The invention concerns a process for the production of
protein preparations with a reduced aggregate content
and in particular for the production of medical
preparations of blood plasma proteins such as albumin.
Human plasma proteins have been purified on a large-
scale for several decades and have been available for
therapy and prophylaxis. Various methods can be used to
prepare pure proteins or protein fractions from a
complex plasma mixture such as fractionation by means of
selective precipitation or separation of the protein
mixtures by means of chromatographic methods such as ion
exchange chromatography, gel filtration and affinity
chromatography. These methods are also very often
combined in order to obtain optimal results.
Precipitation methods with ethanol have also been
successfully used for a long time to fractionate plasma
proteins on a large-scale (Cohn et al., J. Am. Chem.
Soc. 68 (1946), 459-475; Kistler and Nitschmann, Vox
Sang. 7 (1962), 414-424). This method is still used
nowadays worldwide to isolate large amounts of plasma
proteins, in particular albumin, immunoglobulins and
coagulation factors and also other proteins from human
plasma especially for medical applications. However,
ethanol fractionation of plasma proteins has some
disadvantages. Thus a partial denaturation of sensitive
proteins can occur especially at high alcohol
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concentrations or/and high temperatures. Such a
der.aturation can lead to the partial or complete loss of
the physiological function or to structural changes
which can manifest themselves as activation of
proenzymes or formation of new antigen determinants or
protein aggregates.
In order to inactivate potential infectious contaminants
e.g. viruses or other pathogens, it is possible to
subject blood plasma preparations to a final
pasteurisation step. Albumin can for example be
pasteurised by heating to 60 to 64°C for 10 h in the
presence of stabilizers such as N-acetyl tryptophanate
or sodium caprylate (Gellis et al., J. Clin. Invest. 27
(1948), 239-244). Other pasteurisation methods are
disclosed for example in the US patents 2,897,123;
3,227,626; 4,379,085; 4,440,679; 4,623,717 and
4,803,073. Pasteurised preparations of plasma proteins
have proven to be very safe with regard to the
transmission of viruses and pathogens. An additional
advantage of pasteurisation is that it is not necessary
to add toxic additives to the protein preparation and
thus in many cases an inactivation of pathogens is
possible in the final container. However, a disadvantage
of pasteurisation is that there is often a considerable
increase of aggregate formation.
However, this aggregate formation which leads to the
formation of visual or subvisual particles sometimes in
high amounts, is extremely undesirable especially for
medical and pharmaceutical applications. Thus large
particles can directly impair the function of capillary
vessels. Undesired side-effects of subvisual particles
or protein aggregates are known and have been described
for the various protein formulations. Hence, most of the
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approval authorities insist on limits for the aggregate
content of for example albumin or immunoglobulin
solutions. Aggregates in immunoglobulin solutions can
for example trigger an uncontrolled activation of the
complement system and lead to serious side-effects. It
has been described that albumin aggregates disappear
very rapidly from the circulation, probably block the
RES system and sensitize the organism for states of
shock. Hence aggregates in protein solutions can lead in
extreme cases to life-threatening situations which have
to be avoided under all circumstances.
Due to the very broad range of applications of plasma
proteins there is a major need for processes which can
improve the safety, tolerance and biological activity of
the proteins. In addition such methods should be
economic and simple to use.
According to the present invention it was surprisingly
found that protein preparations with a reduced aggregate
content can be produced by improving a thermal treatment
for example to eliminate potential infectious
contaminants, by a prior or subsequent separation step
such that denaturation or/and aggregate formation is
significantly reduced or prevented in the final product.
The desired active and native ingredient is enriched by
the separation step in a protein or protein mixture
which is subsequently to be thermally treated. Hence a
subject matter of the present invention is a process for
the production of protein preparations with a reduced
aggregate content comprising a thermal treatment and the
process is characterized in that aggregates, denatured
proteins or/and contaminants present in the protein
preparation are separated before or/and after the
thermal treatment.
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The process according to the invention is particularly
suitable for the production of preparations of blood
plasma proteins but is not limited thereto. The blood
plasma proteins are preferably selected from the group
comprising plasminogen, albumin, factor VIII, factor IX,
fibronectin, immunoglobulins, kininogen, antithrombin
III, a-1-antitrypsin, pre-kallikrein, fibrinogen,
thrombin, (apo)-lipoproteins and blood plasma protein
fractions which contain one or several of these
proteins. It is particularly preferred to produce a
preparation of albumin, immunoglobulins or (apo)-lipo-
proteins and most preferably an albumin preparation.
An albumin preparation with a reduced aggregate content
produced by the process according to the invention is
considerably more suitable for medical applications than
preparations of the prior art. The albumin preparations
according to the invention can be used as plasma
expanders and also to treat burns. In addition other
applications are also possible such as ultrasound
imaging or as a stabilizing additive to protein
solutions especially if they are highly active proteins
or peptides at low concentrations such as hormones,
chemokines, cytokines or enzymes which nowadays are
often produced using recombinant technology.
The process according to the invention is also
particularly suitable for the production of preparations
of chemically modified proteins in which case the
functional groups of the proteins, in particular
functional side chains, are modified. Chemically
modified proteins, e.g. albumin, can for example be used
as carriers of functional molecules which in vivo do not
reach the desired compartments of the organism or which
do not have the desired activity in a free form. The
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chemical modification is preferably selected from the
group comprising polyethylene glycol modification,
iodination, acylation e.g. acetylation, oxidation e.g.
with peroxides, nitroxylation and cross-linking e.g.
with bifunctional linkers.
The essential step in the process according to the
invention i.e. an at least partial separation of
aggregates, denatured proteins or/and contaminants
before or/and after a thermal treatment can comprise a
precipitation e.g. with ammonium sulfate, ethanol or
polyethylene glycol combined with a separation of the
precipitated aggregates by known methods e.g. by
centrifugation. In addition the separation can also
include a gel filtration e.g. with Fractogel EMD Bio SEC
(Merck) or with other gel filtration media such as
Sephadex or Sepharose (Pharmacia). However, the
separation is preferably carried out by membrane
filtration using membranes of a suitable pore size e.g.
with an exclusion size of 30 to 1000 kD and in
particular of 100 to 500 kD which separates undesired
components with a molecular weight above the exclusion
size. The separation can also additionally include a
nanofiltration such as that which is used to remove
viruses e.g. using the filtration materials DV20 or DV50
(Pall Corp., New York, USA) or Planova 15N (Asaki,
Tokyo, JP).
The thermal treatment which is carried out before or
preferably after the separation preferably comprises a
temperature increase for a long period e.g. a
pasteurisation step which can be carried out in a known
manner. The thermal treatment usually comprises heating
the protein solution to at least 55°C for an adequate
period in order to at least substantially inactivate
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infectious contaminants. The duration of the heating is
preferably at least 5 h, particularly preferably ca. 10 h.
The temperature during the heating is preferably in a
range between 60 and 65°C. In order to prevent
inactivation of the proteins, the thermal treatment is
preferably carried out in the presence of known
stabilizers such as N-acetyl tryptophanate or sodium
caprylate in the case of albumin.
Whereas the aforementioned separation step already
adequately reduces the formation of aggregates,
denatured proteins or/and contaminants for many protein
mixtures, in other cases the desired results can only be
obtained by carrying out a pretreatment step before the
separation in which components in the protein
preparation that can aggregate or/and denature are
converted into a state that can be separated. The
pretreatment step preferably comprises changing the
physicochemical parameters in the protein preparation,
in particular a stress treatment in order to aggregate
denatured, partially denatured or sensitive molecules or
contaminants that are already present in the preparation
or to convert them otherwise into a state which can be
removed in the subsequent separation step. This change
of physicochemical parameters can for example be a
change of the pH, a change and in particular an
elevation of the temperature, a change in the ionic
strength or the dielectric constant, the application of
shear forces or a combination of two or several of these
measures.
This pretreatment step particularly preferably comprises
an elevation of the temperature of the protein
preparation. The extent and duration of this temperature
increase depend on the respective protein preparation or
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its sensitivity. On the one hand the temperature
increase and its duration should be sufficient to
convert the largest possible proportion of aggregatable
components into a state which can be separated; on the
other hand the selected conditions should be not too
drastic in order to largely avoid inactivation of the
desired proteins in the preparation. It has proven to be
advantageous for many proteins such as albumin to heat
the protein preparation for a period of 30 min to 4 h,
in particular for 1 h to 2.5 h to a temperature of 40 to
70°C, in particular of 45 to 65°C.
The process according to the invention is particularly
suitable for the production of medical protein
preparations with a reduced aggregate content. Compared
to an otherwise identical protein preparation without
the separation step, the aggregate content is preferably
reduced by at least 10 %, particularly preferably by at
least 30 % and most preferably by at least 50 %. In some
cases the aggregate content is even reduced by 90 % or
more.
Finally the invention also concerns a protein
preparation with a reduced aggregate content which has
been produced by the process according to the invention.
This protein preparation is considerably more suitable
especially for medical applications than preparations of
the prior art since it causes considerably fewer
intolerance reactions when used therapeutically.
Finally the invention is also elucidated by the
following examples.
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Examples
Example 1
An albumin solution (10 % in 10 mmol/1 NaCl) was
incubated for 90 min at 45°C. Subsequently the solution
was diafiltered through a cassette with a molecular
weight cut off of 300 kD. The ultrafiltrate which mainly
contains monomeric albumin was pasteurised for 10 h at
60°C in the presence of stabilizers (16 mmol/1 Na
caprylate, 16 mmol/1 acetyl tryptophan).
Results:
The aggregate content was 2 % after the preincubation,
> 0.1 % in the ultrafiltrate after the ultrafiltration
and 0.6 % after pasteurisation. A strong accumulation of
aggregates was measured (> 80 %) in the retentate of the
ultrafiltration. An aggregate content of 6 % was
measured after the pasteurisation without preincubation
and subsequent diafiltration. The results were not
significantly changed by adding stabilizers (N-acetyl
tryptophanate and sodium caprylate) during the
preincubation of the albumin.
An analogous experiment was carried out with a
commercially available albumin solution (20 % in
140 mmol/1 NaCl, 12 mmol/1 N-acetyltryptophanate and Na
caprylate). The behaviour of the solutions during the
experiment and the results were essentially identical to
those of the 10 % albumin solution as a starting
material.
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Determination of the agqreaate content in solutions:
1 mg protein was separated by means of high performance
gel filtration on a Superose HR 10/30 column (Pharmacia)
in 70 mmol/1 potassium phosphate buffer, pH 7.0 at a
flow of 1.0 ml/min. The detection was carried out by
determining the absorbance at 280 nm. The monomer and
aggregate content of the samples was calculated from the
areas of the protein peaks.
Example 2
An albumin solution (10 % containing stabilizers 8 mmol/1
in each case) was adjusted to pH 10.5 with 1 mol/1 NaOH
and subsequently heated for 2 h at 65°C. Afterwards the
solution was cooled to room temperature and the pH value
was back titrated to 7 with 1 mol/1 HC1.
The aggregates that had formed were removed by selective
precipitation as follows:
(a) Ammonium sulfate was added to the solution up to a
saturation of 25 %, 30 % and 35 %. The turbidity
which subsequently occurred was removed by
centrifugation and the supernatant was re-
equilibrated by means of gel filtration on Sephadex
G-25 (PD-10, Pharmacia) in 150 mmol/1 NaCl.
Subsequently it was pasteurised for 10 h at 60°C in
the presence of a stabilizer (Na caprylate, N-
acetyltryptophanate, 8 mmol/1 in each case).
(b) Polyethylene glycol (PEG) 3000 was added to the
solution up to a final concentration of 6.1 %, 7.1 %
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and 8.3 % and the turbidity which this caused was
removed by means of centrifugation. The supernatant
was subsequently re-equilibrated as described in (a)
and pasteurised in the presence of stabilizers to
obtain the final product.
Results:
% aggregate % Decrease of
content in the aggregate formation
final product relative to a
control
Albumin without
pretreatment 2 2 . 9
(control)
Ammonium sulfate 2 5 15.8 3 1
% I
30 3.7 84
%
35 2.8 88
%
pEa 6 . 2 0 . 5 10
1
%
7.1 16.8 27
%
I 8.3 9.3 59
%
Example 3
Activation of PEG:
5.5 g cyanuric chloride was dissolved in 400 ml
anhydrous benzene containing 10 g sodium carbonate. 19 g
PEG 1900 was added to the mixture and it was stirred
overnight at room temperature. The solution was filtered
and 600 ml petroleum ether was added slowly. The
suspension was filtered and the precipitate was
dissolved in 400 ml benzene. The precipitation and
filtration process was repeated several times in order
to remove free cyanuric chloride.
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Bindinct of the activated PEG to albumin:
1 g albumin was dissolved in 100 ml 0.1 mol/1 sodium
tetraborate, pH 9.2. 8 g activated PEG was added at 4°C
and the pH was kept at 9.2 for one hour. In this manner
80 to 90 % of the primary amino groups were modified
with PEG. After the reaction, excess PEG was removed by
diafiltration (10 kD cut off membrane) and the solution
was rebuffered against 10 mmol/1 NaCl. The solution was
subsequently preincubated at 45°C as described in
example 1, diafiltered through a 300 kD cassette and
pasteurised for 10 h at 60°C in the presence of
stabilizers.
Results:
An aggregate content of 10 % was measured in PEG-
modified albumin without preincubation. It was possible
to reduce the aggregate content to 4.5 % by
preincubation and subsequent diafiltration.
Example 4
A 20 % albumin solution was diluted with 0.1 mol/1
borate buffer pH 9.5 to 10 % and cooled to 0°C.
Subsequently 20 % (v/v) cold KI3 solution was added and
it was incubated for 30 min at 0°C. The reaction was
stopped by addition of a few drops of NaS03 (1 mol/1),
the protein solution was incubated for 2 h at 60°C and
cooled. An aliquot was incubated for 8 h at 60°C (to
assess the aggregate formation).
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The remainder of the solution was diafiltered over a
300 kD membrane against 4.5 volumes of 140 mmol/1 NaCl.
Finally the retentate was concentrated to a protein
concentration of 20 %. The ultrafiltrate was rebuffered
by means of 10 kD diafiltration: diafiltration against
volumes 140 mmol/1 NaCl and subsequent concentration
on the 10 kDa diafiltration membrane to a protein
content of 15 to 20 %. The resulting protein solution
was pasteurised as in example 1 in the presence of
stabilizers.
An aggregate content of 5.2 % was achieved in the
iodated albumin with pretreatment (preincubation, 300 kD
diafiltration); an aggregate content of 12.7 % was
measured without pretreatment i.e. a reduction of 59 %
was achieved.
Example 5
Albumin (20 %) was diluted 1:2 with saturated Na acetate
solution pH 7.5 and cooled to 0°C. Acetic anhydride was
metered in portions within 1 h (same weight as the added
protein). Subsequently the pH was adjusted to 7.5, the
sample was filtered through a 1.2 ~,m filter and
rebuffered by means of 10 kD diafiltration (against 30
volumes 140 mmol/1 NaCl).
The protein solution was incubated for 2 h at 60°C, then
cooled and diafiltered over a 300 kD membrane against 4
volumes 140 mmol/1 NaCl. Finally the retentate was
concentrated to ca. 20 %. The ultrafiltrate was
rebuffered by means of 10 kD diafiltration
(diafiltration against 10 volumes 150 mmol/1 NaCl) and
concentrated on the same membrane to a protein content
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of 15 - 20 %. The solution of acetylated albumin was
subsequently pasteurised in the presence of stabilizers
as described in example 1.
An aggregate content of 62 % was measured in the final
product with pretreatment (preincubation, 300 kD,
diafiltration); the acetylated product gelled without
the pretreatment i.e. soluble protein was no longer
present.
Example 6
An albumin solution (10 %) containing 0.5 mmol/1 EDTA
was adjusted to pH 3.2 with 0.1 M perchloric acid and
heated to 30°C. H202 was added to the protein solution
to a final concentration of 0.5 mmol/1. Subsequently it
was incubated for 2 h at 30°C. After the incubation a
kD diafiltration was carried out against 4 volumes
140 mmol/1 NaCl. The retentate was diafiltered over a
300 kD membrane against 2 volumes 140 mmol/1 NaCl, and
finally the retentate was concentrated to a protein
concentration of 15 to 20 %.
The ultrafiltrate was rebuffered by means of 10 kD
diafiltration (10 volumes 140 mmol/1 NaCl) and
concentrated on the same membrane to a content of 10 to
% protein. The solution of oxidized albumin was
subsequently pasteurised in the presence of stabilizers
as described in example 1.
An aggregate content of 1.1 % was measured in the final
product with pretreatment (300 kD diafiltration).
Without pretreatment the oxidized albumin had an
aggregate content of 11.1 %. The aggregate content could
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hence be reduced by 90 %.
Example 7
An albumin solution (10 %, 20 ml) was adjusted to pH 8.5
to 9.0 with NaOH. 50 mg dimethyl suberimidate suspended
in 2 ml triethanolamine at pH 9.7 was added to this and
it was incubated for 2 h at room temperature. The
reaction was stopped by addition of 10 % (v/v) 0.1 mol/1
Tris pH 8.5. The protein solution was subsequently
incubated for 2 h at 60°C and then cooled. 15 ml of this
solution was diafiltered over a 300 kD membrane (against
12 volumes 140 mmol/1 NaCl). Finally the retentate was
concentrated as strongly as possible.
The ultrafiltrate was rebuffered by means of 10 kD
diafiltration against 30 volumes 140 mmol/1 NaCl and
subsequently concentrated to a content of 15 to 20 %
protein by means of vacuum dialysis against 140 mmol/1
NaCl. The solution of albumin modified by the cross
linker was subsequently pasteurised in the presence of
stabilizers as described in example 1.
An aggregate content of 2.5 % was measured in the final
product with pretreatment (300 kD diafiltration).
Without pretreatment the modified albumin had an
aggregate content of 20.5 %. The aggregate content could
hence be reduced by 88 %.
Example 8
4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidin-1-oxyl
(BrAcTPO; dissolved in ethanol, 500 mg/ml) was added to
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an albumin solution (10 % protein, in 10 % ethanol)
(0.177 g BrAcTPO/g protein). The solution was heated to
45°C, the pH was adjusted to 9.5 with NaOH and kept at
this pH for 2 to 3 h by the continuous addition of lye.
Subsequently the pH was back titrated to 7.2 with HC1 and
the solution was heated for 90 min to 60°C. Afterwards a
300 kD diafiltration with 15 volumes 140 mmol/1 NaCl was
carried out. The ultrafiltrate was diafiltered with a
kD membrane, 5 to 10 volumes 140 mmol/1 NaCl and
finally concentrated on the same membrane to a protein
content of 20 %. The solution was pasteurised in the
presence of stabilizers as described in example 1.
An aggregate content of 0.1 % was measured in the
polynitroxylated albumin with pretreatment (incubation,
300 kD diafiltration). Without pretreatment the modified
albumin had an aggregate content of 5.5 %. The aggregate
content could hence be reduced by 90 %.
Example 9
A solution of apolipoprotein A-I (10 g/1 in 10 mmol/1
NaCl) was incubated for 2 h at pH 5.0 and 60°C.
Subsequently the pH was adjusted to 7.5 and guanidine
HC1 was added to a concentration of 2 mol/1 and
incubated for 2 h at 45°C. This solution was diafiltered
over a 300 kD membrane (10 vol 10 mmol/1 NaCl) and
subsequently diafiltered with a 10 kD membrane and
concentrated to 10 g/ml.
Aggregate contents of 2.4 % to 5.4 % were measured in
the apolipoprotein A-I solutions without pretreatment (4
experiments); the aggregate content was reduced to 0.4
to 0.8 % with pretreatment (3 experiments).
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Example 10
A solution of immunoglobulin G (20 g/1, < 3 mmol NaCl/1)
was incubated for 12 h at pH 7.0 and 45°C. The content
of aggregates increased during the pretreatment from
< 0.1 to 1.0 %. It was subsequently gel filtered over an
acrylic gel (Sepharyl S-300 HR), the pH was adjusted to
5.3 with HC1 and it was concentrated with a 10 kD
membrane to 120 g/1. The aggregate content was 0.2 % and
remained stable during storage for 6 months at 37°C. The
content increased to 0.8 % under the same conditions in
a non-treated reference solution.
