Note: Descriptions are shown in the official language in which they were submitted.
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LARGE SCALE PREPARATION OF ALPHA-1 PROTEINASE INHIBITOR
AND USE THEREOF
FIELD OF THE INVENTION
The present invention relates to a process for the purification of alpha-1
proteinase
inhibitor (API) from a mixture of proteins, to compositions comprising same
and use
thereof. More particularly, the present invention relates to a process for the
large scale
purification of API from blood plasma or from plasma fractions to obtain
pharmaceutical grade API. The present invention also relates to formulations
comprising the purified API, specifically ready to use liquid formulations and
methods
of using same.
BACKGROUND OF THE INVENTION
Certain human plasma proteins useful for therapeutic purposes and other
applications can be obtained only from pooled blood donations. Recombinant
production of plasma proteins is complicated by the fact that these proteins
require
accurate glycosylation patterns in order to maintain their function and/or
half-life in the
human body. Therefore even with the attendant risks of viral or other
contamination the
only approved available source for some proteins such as alpha 1 proteinase
inhibitor is
human plasma itself.
Alpha-1 proteinase inhibitor (API) is a derivative of human plasma belonging
to
the family of serine proteinase inhibitors. It is a glycoprotein having an
average
molecular weight of 50,600 daltons, produced by the liver and secreted into
the
circulatory system. The protein is a single polypeptide chain, to which
several
oligosaccharide units are covalently bound. API has a role in controlling
tissue
destruction by endogenous serine proteinases, and is the most prevalent serine
proteinase inhibitor in blood plasma. Among others, API inhibits trypsin,
chymotrypsin,
various types of elastases, skin collagenase, renin, urokinase and proteases
of
polymorphonuclear lymphocytes.
API is currently used therapeutically for the treatment of pulmonary emphysema
in patients who have a genetic deficiency in API. Purified API has been
approved for
replacement therapy in these patients. The normal role of API is to regulate
the activity
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of leukocyte elastase, which breaks down foreign proteins present in the lung.
When
API is not present in sufficient quantities to inhibit elastase activity, the
elastase breaks
down lung tissue. In time, this imbalance results in chronic lung tissue
damage and
emphysema.
API was also proposed as a treatment for patients homozygous for the defective
cystic fibrosis (CF) transmembrane conductance regulator (CFTR) genes, who
suffer
from recurrent endobronchial infections and sinusitis, malabsorption due to
pancreatic
deficiency, obstructive hepatobiliary disease and reduced fertility. The major
cause of
morbidity and mortality among CF patients is lung diseases. CFTR regulates
transport
of water and salts in the epithelial cells which cover internal and external
surfaces of the
body. In CF patients, the CTFR protein is defective due to a mutation,
resulting in a
defective water and salt transport and the production of thick secretions in
several
organs (e.g. lung, pancreas).
The membrane defect caused by the CFTR mutation leads to chronic lung
inflammation and infection. Chronic lower respiratory infection provokes a
persistent
inflammatory response in the airway, resulting in chronic obstructive disease.
As
pulmonary reserves decrease, CF patients become prone to episodes of
exacerbation,
characterized by worsening symptoms of respiratory infection, particularly by
Peusdomonas aeruginosa, accompanied by acute decline in lung function. In
normal
individuals, elastase secreted by neutrophils in response to infection is
neutralized by
API, which is known to penetrate into pulmonary tissue. In patients with CF,
however,
the unregulated inflammatory response overwhelms the normal protease
(elastase)/
antiprotease (API) balance. The abnormal cycle is destructively self-
perpetuating and
self-expanding: increased elastase leads to the recruitment of more
neutrophils to the
lung, which in turn secrete additional proteases. This leads to the
accumulation of
elastase in the lung and ultimately to tissue damage, destruction of the lung
architecture,
severe pulmonary dysfunction and, ultimately, death. It is suggested that
supplement of
additional API may reduce the deleterious effects associated with excessive
amounts of
elastase. The demand for API already exceeds the availability of the current
supply, and
this problem may become more pronounced as research suggests additional
therapeutic
uses for API. In order to maximize the available supply of API, a process for
purifying
API from human plasma should have the highest yield possible, and alternative
sources
should be also considered. Therefore, more efficient means of isolation,
suitable for
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GMP (good manufacture practice) large-scale production, is required.
Several groups have reported production of recombinant API. (For example, G.
Wright et al., Biotechnology, Vol. 9, pp. 830-834 (1991); A. L. Archibald et
al., Proc.
Nat'l. Acad. Sci. USA., Vol.87, pp. 5178-5182 (1990)). However, at present,
human
plasma is the only approved source of therapeutic API.
Various methods of purifying API from human plasma have been described. The
majority of these methods are directed to laboratory scale isolation while
others pertain
to production on a commercial level. Several methods of isolation are
disclosed, for
example in U.S. Patent Nos. 4,379,087 and 5,610,285. Many early methods
employed
ammonium sulfate precipitation from human plasma followed by dialysis, further
employing chromatographic step on DEAE-cellulose. However, the methods
described
for dialysis are not easily applicable to large-scale purification, and are
lengthy, time-
consuming processes likely to compromise the activity of the isolated protein.
A large-scale purification of API from human plasma was disclosed by Kress et
al., (Preparative Biochem., 3:541-552, 1973)). The precipitate from the 80%
ammonium
sulfate treatment of human plasma was dialyzed and chromatographed on DEAE-
cellulose. The concentrate obtained was again dialyzed and gel filtered on
SEPHADEXTm G-100. The API-containing fractions were chromatographed twice on
DE-52 cellulose to give API.
Glaser et al., (Preparative Biochem., 5:333-348, 1975) isolated API from Cohn
Fraction IV-1 paste. In this method, dissolved IV-1 fraction was
chromatographed on
DEAE-cellulose, QAE-SEPHADEXTm, concanavalin-A-SEPHAROSETm, and
SEPHADEXTm-G-150 to give API. However, Glaser et al. achieved only a 30%
overall
yield from fraction IV-1 paste.
Podiarene et al., (Vopr. Med. Khim. 35:96-99, 1989) reported a single step
procedure for isolation of API from human plasma using affinity chromatography
with
monoclonal antibodies. API specific activity was increased 61.1 fold with a
yield of
only 20% from plasma.
Burnouf et al., (Vox. Sang. 52:291-297, 1987) starting with Cohn Fraction
effluent II+III used DEAE chromatography and size exclusion chromatography to
produce an API 80-90% pure (by SDS-PAGE) with a recovery of 65-70% from this
effluent.
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Hein et al., (Eur. Respir. J. 9:16s-20s, 1990) presented a process that
employs
Cohn Fraction IV-1 paste as the starting material and utilized fractional
precipitation
with polyethylene glycol followed by anion exchange chromatography on DEAE-
SepharoseTm. The final product has a purity of about 60% with 45% yield from
IV-1
paste.
Dubin et al., (Prep. Biochem. 20:63-70, 1990) used a two-step chromatographic
purification whereby alpha-PI, C1-inhibitor, alpha-1 antichymotrypsin, and
alpha-1
trypsin inhibitor were first eluted from Blue SepharoseTM and then API was
purified by
gel filtration. Purity and yield data were not given.
U.S. Patent No. 4,749,783 discloses a method where biologically inactive
proteins
in a preparation were removed by affinity chromatography after a viral
inactivation step.
The basis of the separation between the native and denatured forms of the
protein was
the biological activity of the native protein towards the affinity resin and
not physical
differences between the native and denatured proteins.
An integrated plasma fractionation system based on polyethylene glycol (PEG)
was disclosed by Hao et al. (Proceedings of the International Workshop on
Technology
for Protein Separation and Improvement of Blood Plasma Fractionation, Sept. 7-
9,
1977, Reston, Va). In the published method Cohn cryoprecipitate was mixed with
increasing concentrations of PEG in order to obtain four different PEG
fractions. The
four fractions obtained were 0-4% PEG precipitate, 4-10% PEG precipitate, 10-
20%
PEG precipitate and 20% PEG supernatant. The 20% PEG supernatant fraction was
dominated by albumin but also contained most of the API. However, this
fraction also
contained numerous other proteins, including all of the alpha- 1 -acid
glycoprotein,
antithrombin 111, ceruloplasmin, haptoglobin, transferrin, CI esterase
inhibitor,
prealbumin, retinol binding protein, transcortin, and angiotensinogen.
Several other groups have combined PEG precipitation with other purification
methods in an attempt to isolate API. For instance, U.S. Patent Nos.
4,379,087;
4,439,358; 4,697,003 and 4,656,254, all employ a PEG precipitation step in
processes of
isolating API. However, the disclosed methods do not attempt to separate
active from
non-active API.
Japanese Patent No. 8-99999 discloses the use of PEG precipitation in
combination with an SP-cation exchanger. The methods described therein do not
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separate fully active API from inactive API. The specific activity of fully
active API
should be 1.88 (using an Extinction coefficient 5.3), but the product achieved
by this
process only shows a relative activity of 1Ø Moreover, the best yield
achieved by
combining PEG precipitation and SP-cation exchange steps was only 50%, and
does not
appear to be easily scaled up to a commercial production level.
U.S. Patent No. 5,610,285 discloses a purification process which combines
successive anion and cation exchange chromatography steps. The initial anion
exchange
chromatography step binds API to the column; however, it also binds numerous
contaminating proteins, particularly lipoproteins. Lipoproteins are plentiful
in many of
the materials from which API is isolated (e.g. Cohn IV-1 paste), and so tend
to occlude
the column. Such occlusion requires columns of considerable size, additional
dialysis/filtration steps, and at least two cation chromatography steps. Those
requirements reduce efficiency and practicality of the method for large-scale
processes.
Further, in the '285 process all API, both inactive and active protein, bind
to the anion
exchange column. When the API is eluted from that column in accordance with
that
method, i.e., high salt phosphate buffer, both active and inactive protein
come off the
column. Thus, there is no separation of the active from the inactive protein.
U.S. Patent No. 6,093,804 discloses a method combining removal of lipoproteins
from the source material, followed by subsequent anion and cation exchange
steps,
which result in highly purified, highly active API. However, this method
proved to be
efficient for small to mid-scale production of processing source material in
the range of
few kilograms.
As mentioned above, the demand for API exceeds available supply. Thus, there
is
a great need for, and it would be highly advantageous to have a process for a
large-scale
production of API, in which quality, which refers to both purity and activity,
is not
compromised for quantity. Moreover, it would be highly beneficial to have
stable, viral-
inactivated ready to use formulations of the purified API.
SUMMARY OF THE INVENTION
The present invention relates to a process for the production of alpha-1
proteinase
inhibitor (API), suitable for processing scaled-up amounts of source material
in the
range of at least tens of kilograms, and which yields a highly purified,
highly active
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API. The present invention also relates to formulations comprising the
purified API,
specifically to liquid formulations in which the API is highly stable and
methods of
using same.
The process provided by the present invention combines removal of
contaminating substances (i.e., lipids, lipoproteins and other proteins) and
separation of
active from inactive API by sequential chromatography steps. The present
invention
discloses for the first time a process which is suitable for a large-scale
production of
API, i.e. for processing of source material amounts in the range of tens of
kilograms.
When the obtained source material is of high quality, i.e. the source material
is obtained
after filtration, the process of the present invention is suitable for
processing source
material amounts in the range of hundred of kilograms. Hitherto, processes
described to
yield API at high purity and activity were proved to be effective only for
processing
small to mid scale amounts of source material. As disclosed in the present
invention,
efficient large-scale API production is achieved by employing a combination of
two
methods for the removal of contaminating substances from an initial protein
suspension,
by the use of successive anion, cation and anion exchange resins with specific
eluants
and by meeting the GMP requirements of a large-scale production. In particular
the
methods of the present invention employ a minimum number of different buffers;
automated preparation of solutions; use of solutions which can be kept under
ambient
storage conditions; and in particular avoid buffers and reagents prone to
microbial
contaminations. The purified API according to the present invention is at
least 90%,
preferably at least 95% pure (i.e. 95% w/w of the total protein) and of the
purified API
at least 90% is active. The yield of the disclosed large-scale process is
preferably at least
50% from Cohn IV-1 paste, and typically at least 60%.
According to certain embodiments the end product of the process of the present
invention is a liquid suitable for direct use. This currently preferred
embodiment is
advantageous to the currently available end product in the form of a powder,
as, unlike
powder, the liquid preparation requires no additional drying and subsequent
reconstitution steps prior to administration. Furthermore, the API of the
present
invention is highly stable, and its formulations, including the liquid
formulation, do not
require any stabilizers.
According to one aspect, the present invention provides a process for the
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production of highly purified, active API. According to one embodiment, the
end
product of the process is in a liquid form.
According to one embodiment, the present invention provides a process for
purifying alpha-1 proteinase inhibitor (API) from an unpurified mixture of
proteins
comprising:
a. dispersing the unpurified mixture of proteins containing API in an
aqueous medium;
b. removing a portion of contaminating lipids and proteins by adding a
lipid
removal agent to the aqueous dispersion and precipitating the portion of
contaminating proteins from said aqueous dispersion;
c. loading the API-containing supernatant of step (b) on a first anion
exchange resin with a buffer solution having pH and conductivity such
that API is retained on the first anion exchange resin;
d. eluting an API-containing fraction from said first anion exchange resin
with the same type of buffer as in step (c) having adjusted pH and
conductivity;
e. loading the API-containing fraction of step (d) on a cation exchange
resin
in said same type of buffer having appropriate pH and conductivity such
that API is not retained on the cation exchange resin;
f. collecting the flow-through of step (e) that contains API;
g. loading the API-containing fraction of step (f) on a second anion
exchange
resin with said same type of buffer having appropriate pH and
conductivity such that API binds to the second anion exchange resin;
h. eluting API from said second anion exchange resin with said same type of
buffer having adjusted pH and conductivity to obtain a solution containing
purified, active API.
According to one embodiment, the process of the present invention provides
purified API comprising at least 60%, preferably at least 80%, more preferably
at least
90% and most preferably at least 95% API out of the total protein, wherein at
least 90%,
preferably 95% of the pure API is active.
Throughout the process of the present invention only one type of buffer is
used,
with adjustment of pH and conductivity as required throughout the various
process
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steps. As used herein, the term "one type" "same type" or "single type" of
buffer, used
herein interchangeably, refers to a buffer with one specific anion species.
According to one embodiment, the buffer is any suitable acid/salt combination
that provides acceptable buffer capacity in ranges of pH required throughout
the process
of the present invention. According to preferred embodiments the process uses
a buffer
other than citrate-based buffer. According to yet another embodiment, the
buffer anion
is acetate. According to a further embodiment, the buffer solution is sodium
acetate.
According to one embodiment, the process of the present invention further
comprises viral removal and/or viral inactivation steps. Methods for viral
removal and
inactivation are known in the art.
One method for viral removal is filtration, preferably nanofiltration,
removing
both enveloped and non-enveloped viruses. According to one embodiment, the
viral
removal step comprises filtration. According to another embodiment, the virus
removal
step is performed after the cation exchange chromatography. Typically, the
cation
exchange flow-through solution containing API is concentrated, and then
nanofiltered.
According to one embodiment, the method of viral inactivation employed by the
present invention comprises a solvent/detergent (S/D) treatment. The viral
inactivation
step is preferably performed prior to loading the solution on the second anion
exchange
resin. According to one embodiment, the detergent used is polysorbate and the
solvent
is Tri-n-Butyl-Phosphate (TnBP). According to another embodiment, the
polysorbate is
polysorbate 80. According to one embodiment Polysorbate 80 may be added at
from
about 0.8% to about 1.3% volume per weight (v/w) of the resulting mixture and
TnBP
may be added from about 0.2% to about 0.4% weight per weight of the resulting
mixture.
Any unpurified mixture of proteins containing a substantial amount of API may
be used as a starting material for API purification according to the process
of the present
invention. According to one embodiment, the API-containing protein mixture is
selected from plasma, particularly from plasma Cohn fractions IV paste.
According to
another embodiment, the API-containing protein mixture is Cohn fraction IV-I
paste.
According to some embodiments of the present invention the unpurified mixture
of proteins comprising API is dispersed in water, and the pH of the dispersion
is
adjusted to a pH range of from about 8.0 to about 9.5. The pH adjustment
stabilizes the
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API and promotes the dissolution of the API in the dispersion, thereby
increasing the
production yield.
According to one embodiment, the lipid removal agent utilized for the removal
of
lipids and lipoproteins from the unpurified protein suspension is silicon
dioxide
(Aerosi1114), and the contaminating proteins are precipitated from the
suspension with
polyalkylene glycol. According to one embodiment, the polyalkylene glycol is
polyethylene glycol. According to yet another embodiment, the pH of the
dispersion is
reduced before the addition of the polyalkylene glycol. According to one
currently
preferred embodiment, the pH is reduced to a pH range of from about 5.0 to
about 6.5.
The pH reduction improves the precipitation, and the lipid removal agents and
the
precipitate are removed from the suspension. Removal of the precipiate from
the
solution can be performed by various methods as is known to a person skilled
in the art,
including centrifugation and filtration, specifically filter-press filtration.
The supernatant
from this step is in a pH range suitable for the first anion exchange
chromatography (pH
from about 5.0 to about 6.5). To further prepare the supernatant for loading
on the anion
exchange resin its conductivity is adjusted to from about 0.5 to about 3.5
mS/cm.
According to certain embodiments the first and the second anion exchange resin
is
a DEAE-Sepharose resin and the cation exchange resin is Carboxymethyl-
Sepharose
resin. The chromatography sequential steps according to the process of the
present
invention are performed with a single type of buffer throughout the process.
However,
individual sequential steps are performed under different pH and conductivity
conditions, to provide the appropriate conditions required in each of those
steps. The
adjustment of the pH and conductivity in the buffer can be performed by any
suitable
method as is known to a person skilled in the art.
It has been previously shown that the separation of active from inactive API
can
be achieved by anion exchange chromatography. The cation exchange resin is
used to
further purify the API-containing fraction from substances that bind to the
cation
exchange resin, while the API passes through the resin. According to one
embodiment,
the pH of the API-containing fraction is adjusted to between 5.3 and 5.6 and
the
conductivity to from about 0.8 to about 1.1 mS/cm before loading the API-
containing
fraction on the cation exchange resin.
The present invention further comprises methods for separating active API from
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other contaminating substances, including solvent/detergent compounds used for
viral
inactivation as described herein above. According to one embodiment, this
separation is
advantageously achieved by the second anion exchange chromatography. According
to
one embodiment, before loading on the second anion exchange resin the pH of
the API-
containing fraction is adjusted to from about 6.0 to about 8.0 and the
conductivity to
from about 2.0 to about 4.0 mS/cm.
According to another embodiment, the process of the present invention further
comprises the steps of changing the ionic composition of the solution
containing
purified, active API to contain a physiologically compatible ion and
sterilizing the
resulted solution to produce a fluid pharmaceutical preparation.
According to one embodiment, the solution containing the purified active API
is
concentrated before the ion exchange. According to another embodiment, the
physiologically compatible ion is selected from the group consisting of
phosphate ion,
chloride ion and combinations thereof. Typically, the pH of the pharmaceutical
preparation is about 7Ø According to yet another embodiment, the protein
concentration of the solution containing the purified active API is adjusted
to the range
of 20-40 mg/ml prior to sterilization.
According to another aspect, the present invention provides a pharmaceutical
preparation comprising a purified active API produced by the process of the
present
invention. According to yet another aspect, the present invention provides a
pharmaceuticl preparation comprising purified, active API in the form of ready
to use
sterile solution. This solution may be used for therapeutic, diagnostic or
reagent
purposes.
According to one embodiment, the fluid pharmaceutical preparation comprises at
least 90%, preferably 95%, more preferably 99% API out of the total proteins.
According to another embodiment, at least 90% of the API is in its active
form.
Typically, the pharmaceutical preparation contains from about 1% to about 3%
API,
preferably about 2% API.
According to one embodiment, the fluid pharmaceutical preparation is devoid of
a
stabilizing agent.
As used herein, the term "stabilizing agent" refers to a compound that
stabilizes
the active ingredient (herein API) within the pharmaceutical preparation.
"Stabilization"
CA 02900719 2015-08-19
refers to the process of preventing the loss of specific activity and/or
changes in
secondary structure from the native glycoproteins. Typically, such stabilizers
include
albumin, sucrose and mannitol.
According to one embodiment, the API in the fluid pharmaceutical preparation
is
stable for at least 3 month, preferably 4 month, more preferably 6 month when
the
pharmaceutical preparation is stored in a temperature range of between 20 C to
25 C.
According to another embodiment, the API in the fluid pharmaceutical
preparation is stable for at least 12 month, preferably 24 month, more
preferably 36
month, when the pharmaceutical preparation is stored in a temperature range of
between
2 C to 8 C.
As used herein, the term "stable" refers to API activity at the end of the
storage
period that is at least 90%, preferably 95%, more preferably 100% the initial
API
activity.
According to yet another aspect, the present invention provides a
pharmaceutical
composition comprising as an active ingredient a therapeutically effective
amount of
API produced by the process of the present invention, further comprising a
pharmaceutically acceptable excipient, diluent or carrier.
According to one embodiment, the pharmaceutical composition comprises at least
90%, preferably 95%, more preferably 99% API out of the total proteins.
According to
another embodiment, at least 90% of the API is in its active form.
Pharmaceutical compositions for use in accordance with the present invention
may be formulated in conventional manner using one or more physiologically
acceptable carriers, which facilitate processing of the active compounds into
preparations which can be used pharmaceutically. Proper formulation is
dependent
upon the route of administration chosen.
According to one embodiment, the API produced by the process of the present
invention is formulated in the form selected from the group consisting of
aqueous
solution and a powder. According to another embodiment, the pharmaceutical
composition is devoid of a stabilizer.
For injection, the compounds of the invention may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as Hank's
solution,
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Ringer's solution, or physiological saline buffer.
For administration by inhalation, the fluid API produced according to the
process
of the present invention is conveniently delivered in the form of an aerosol
spray, from
a pressurized pack or a nebulizer. Aerosol spray is typically prepared with
suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-
tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the
dosage unit
may be determined by providing an apparatus comprising a valve to deliver a
metered
amount. Alternatively, lyophilized powder form of API produced by the process
of the
present invention can be mixed with a suitable powder base such as lactose or
starch to
form capsules and cartridges of, for example, gelatin for use in an inhaler or
insufflator.
According to one currently preferred embodiment, the fluid pharmaceutical
preparation is used to form an aerosol spray for inhalation.
According to yet another aspect, the present invention provides a method for
treating a subject in need thereof comprising administering a therapeutically
effective
amount of API produced according to the process of the present invention.
According to one embodiment, the method is used for treating a disease or
disorder selected from the group consisting of pulmonary emphysema, chronic
obstructive pulmonary disorder, cystic fibrosis associated lung diseases and
disorders,
psoriasis and atopic dermatitis.
According to one embodiment the method is used for treating pulmonary
emphysema.
According to another embodiment, the method is used for treating cystic
fibrosis
associated lung diseases and disorders.
According to one embodiment, the therapeutically effective amount of API is
administered intravenously or by inhalation.
The present invention is explained in greater details in the description,
figures,
and claims below.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 describes the protein profile on a native Tris-Glycine gradient gel
during the API
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WO 2005/027821 PCT/1L2004/000881
production process. Fig. 1A: Coomassie blue stained gel. Fig.1B: Ponceau-S
stained
gel. Fig. 1C: Immunoblot with Goat anti-API, HRP-conjugate antibodies. Lanes
of the
gels are as follows: 1, 2 ¨Final drug product; 3, 4 ¨ In house API standard; 5
¨
Albumin; 6 ¨ Transferrin; Anti-D (IgG); 8, 9, 10 ¨ Polymeric fraction of API.
FIG. 2 describes the protein profile on a 4%-12% gradient SDS-PAGE during the
API
production process. Fig. 2A: Coomassie blue stained gel. Fig.2B: Ponceau-S
stained
gel. Fig. 2C: Immunoblot with Goat anti-API, HRP-conjugate antibodies. Lanes
of the
gels are as follows: 1 - Sample buffer; 2, 3 - dispersion before the addition
of Aerosil; 4,
5: Dispersion before the addition of PEG; 6,7: eluate after first anion
exchange
chromatography; 8, 9: filtrate after cation exchange chromatography; 10:
eluate after
second anion exchange chromatography; 11: end product of the process (drug
substance); 12, 13: formulated API (drug product); 14, 15 ¨ commercial and in-
house
molecular weight standards, respectively.
FIG. 3 shows far (Fig. 3A) and near (Fig. 3B) UV circular dichroism Spectra of
API
Lots 6112006, 6113010, 612301 and API Primary Reference Standard.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for the production of highly pure,
active
and stable alpha-1 proteinase inhibitor (API) on a large-scale production. A
particular
advantage of the process provided by the present invention is its efficacy in
processing
source material in the range of tens to hundreds of kilograms, without
compromising
purity and activity of the API product, being at least 90% pure, of which at
least 90% is
active. The amount of source material that can be proceed according to the
teaching of
the present invention depended on the quality of the source material (higher
amounts
can be processed when the material is filtered). Furthermore, by the process
of the
present invention a ready to use liquid product could be obtained, comprising
highly
stable API.
Definitions
As used herein, the term "Alpha-1 proteinase inhibitor" (API) refers to a
glycoprotein produced by the liver and secreted into the circulatory system.
API belongs
to the Serine Proteinase Inhibitor (Serpin) family of proteolytic inhibitors.
This
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glycoprotein of MW of 50,600 Da consists of a single polypeptide chain
containing one
cysteine residue and 12-13% carbohydrates of the total molecular weight. API
has three
N-glycosylation sites at asparagine residues 46, 83 and 247, which are
occupied by
mixtures of complex bi- and triantennary glycans. This gives rise to multiple
API
isoforms, having isoelectric point in the range of 4.0 to 5Ø The glycan
monosaccharides include N-acetylglucosamine, maimose, galactose, fucose and
sialic
acid. API serves as a pseudo-substrate for elastase; elastase attacks the
reactive center
loop of the API molecule by cleaving the bond between methionine358 -
serine359
residues to form an API-elastase complex. This complex is rapidly removed from
the
blood circulation. API is also referred to as "alpha-1 antitrypsin" (AAT). The
term
"glycoprotein" as used herein refers to a protein or peptide covalently linked
to a
carbohydrate. The carbohydrate may be monomeric or composed of
oligosaccharides.
The term "cystic fibrosis" refers to an inherited autosomal recessive disorder
caused by mutations in the gene encoding the cystic fibrosis transmembrane
conductance regulator (CFTR) cr channel.
The term "Emphysema" refers to a condition in which there is a decrease in
respiratory function and often breathlessness due to over-inflation of the
alveoli in the
lungs resulting from the damage done to the walls of the alveoli by the
destructive
neutrophil elastase.
As used herein, the term "API-containing pharmaceutical preparation" or "fluid
pharmaceutical preparation" refers to the solution composition containing the
purified
API as obtained at the end of the process of the present invention. The term
"pharmaceutical composition" refers to the aforementioned pharmaceutical
preparation,
further comprising excipients, diluents or carriers. The "pharmaceutical
preparation"
according to the present invention is always in the form of a liquid; the
pharmaceutical
composition may be in any suitable administering form, as is known in the art.
According to one embodiment, the present invention provides a process for
purifying API from an unpurified mixture of proteins comprising:
a. dispersing the unpurified mixture of proteins containing API in an
aqueous medium;
b. removing a portion of contaminating lipids and proteins by adding a
lipid
removal agent to the aqueous dispersion and precipitating the portion of
14
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contaminating proteins from said aqueous dispersion;
c. loading the API-containing supernatant of step (b) containing API on
a
first anion exchange resin with a buffer solution having pH and
conductivity such that API is retained on the first anion exchange resin;
d. eluting an API-
containing fraction from said first anion exchange resin
with the same type of buffer as in step (c) having adjusted pH and
conductivity;
e. loading the API-containing fraction of step (d) on a cation exchange
resin
in said same type of buffer having appropriate pH and conductivity such
that API is not retained on the cation exchange resin;
f. collecting the flow-through of step (e) that contains API;
g. loading the API-containing fraction of step (f) on a second anion
exchange resin with said same type of buffer having appropriate pH and
conductivity such that API binds to the second anion exchange resin;
h. eluting API from
said second anion exchange resin with said same type of
buffer having adjusted pH and conductivity to obtain a solution
containing purified, active API.
This process provides API fractions of at least about 90% API out of the total
protein; often providing fractions of greater than about 95% pure API; and can
achieve
fractions of 99% pure API. Of the API, at least 90% is active. At least 95%
active API
is also achieved. Activity of API is measured by trypsin inhibition as
exemplified herein
below.
It has been previously disclosed that anion exchange chromatography is the
principle stage in which active API is separated from inactive API, (U.S.
Patent No.
6,093,804). However, the method described in said US patent, adjusted to a
small to
mini-scale production, utilizes various buffer types to achieve such
separation (loading
the anion exchange with non-citrate buffer and eluting an API-containing
fraction with
citrate-based buffer). The present invention provides a process which meets
the
requirements of large-scale production, one of them being the use of minimal
number of
different solutions. The present invention therefore discloses the use of one
type buffer
in all chromatography steps, while adjusting the pH and conductivity of the
buffer as
required throughout the various process steps.
CA 02900719 2015-08-19
According to one embodiment, the buffer is any suitable acid/salt combination
that provides acceptable buffer capacity in ranges of pH required throughout
the process
of the present invention. According to preferred embodiments the process uses
a buffer
other than citrate-based buffer. According to one embodiment, the anion buffer
is
acetate. According to another embodiment, the buffer solution is sodium
acetate.
The sterility of the preparations of the present invention is of major
concern, as
the product should be administered to humans for therapeutic purposes, in
particular by
intravenous administration or by inhalation. Although the plasma source
material is
examined for the presence of contaminating viruses, and a great effort is
taken to
exclude contaminated donor fractions, there is a need to further assure that
the end
product of the process would be virus-free.
According to one embodiment, the process of the present invention further
comprises viral removal and/or viral inactivation steps. Viral reduction can
be
accomplished by several processes, including nanofiltration; solvent/detergent
treatment; iodine inactivation, e.g., treatment with an iodinated ion exchange
matrix
material such as iodinated SEPHADEXTm (as disclosed in PCT applications WO
97/48422 and WO 97/48482); treatment with Pathogen Inactivating Compounds;
heat
inactivation; gamma irradiation; or any other suitable virucidal process.
Lipid coated viruses are effectively inactivated by treatment with non-ionic
biocompatible solvents and detergents. Methods for virus inactivation by
solvent-
detergent applications are described, for example, in EP 0131740. However, non-
lipid
coated viruses cannot be inactivated by solvent-detergent treatments, thus,
other
inactivation methodologies have to be used for their inactivation, including
eliminating
by physical means, e.g., the filtration of the preparation through very small
filter holes
so as to remove viruses by size exclusion (nanofiltration).
According to one embodiment, the viral removal step comprises filtration. Both
enveloped and non-enveloped viruses are removed by filtration, preferably by
nanofiltration, or any other filtration methods known in the art. According to
one
embodiment, a virus removal step is performed after the cation exchange
chromatography. According to one embodiment, the cation exchange flow-through
solution containing API may be concentrated by ultrafiltration. Prior to
nanofiltration,
the pH of the concentrated retentate may be adjusted to from about 6.8 to
about 7.7, and
16
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its conductivity to from about 2.5 to about 3.5 mS/cm. The filtrate
("nanofiltrate") is
collected for the subsequent step of viral inactivation.
According to one embodiment, the method of viral inactivation employed by the
present invention comprises a solvent/detergent (S/D) treatment. This step is
preferably
taken prior to loading the solution on the second anion exchange resin.
According to
one embodiment, the detergent used is a non-ionic detergent such as
polysorbate and the
solvent is TnBP. According to another embodiment, the polysorbate is
polysorbate 80.
According to one embodiment Polysorbate 80 may be added at from about 0.8% to
about 1.3% weight per weight of the resulting mixture and TnBP from about 0.2%
to
about 0.4% weight per weight of the resulting mixture. According to one
embodiment,
S/D viral inactivation is performed in a pH range of between about 6.0 and 9.0
and
conductivity range of between about 2.0 and 4.0 mS/cm.
The unpurified mixture of proteins from which the API is collected is
preferably
Cohn Fraction IV-1 paste, but can include other Cohn Fractions, separately or
in
combination, human blood plasma, plasma fractions, or any protein preparation
containing API. For instance, the present process is applicable to
purification of
recombinant human API from the milk of transgenic animals. (When milk may be
used
as starting material, an ammonium sulfate or sodium chloride precipitation
step is first
employed to separate API from caseins, and the precipitate is taken through
the present
purification process.) According to one embodiment, the unpurified mixture of
proteins
comprising API is dispersed in an aqueous medium, preferably water, at a ratio
of
between about 20 to about 35 liter per about 1 kg of source material,
specifically Cohn
Fraction IV-1 paste. The pH of the dispersion is adjusted to a pH range of
from about
8.0 to about 9.5. The pH adjustment stabilizes the API and promotes the
dissolution of
the API in the dispersion, thereby increasing the production yield. Dispersion
may take
place at elevated temperature, for further increase in API solubility.
According to one
embodiment, dispersion is performed, or the solution is heated at a
temperature of
between 35 C and 40 C.
A particular advantage of the present invention is the ready elimination of
contaminants or by-products that otherwise compromise the efficiency of API
purification processes. In particular, Cohn Fraction IV-1 paste preparations
contain a
significant amount of the lipoprotein Apo A-1, which has the effect of
inhibiting
17
CA 02900719 2015-08-19
column flow and capacity during purificition. Other non-desired proteins such
as
albumin and transferrin are also present in the paste preparation. Removing a
portion of
such contaminants is performed according to the present invention by two
sequential
steps: (a) removing contaminating lipids and lipoproteins by lipid removal
agent and (b)
precipitating a portion of contaminating protein from the API-containing
aqueous
dispersion.
According to one embodiment, the lipid removal agent is silicon dioxide
(AerosilTm). The AerosilTM is added at a ratio of 1:10 to 1:14 AerosilTM: Kg
of IV-1
paste. This step is performed at a high pH of about 9.0, and the resulting
mixture is
stirred for about 60-120 min. at a temperature of between 35 C and 40 C.
According to
one embodiment, polyalkylene glycol is used for precipitating the portion of
contaminating proteins, for example polyethylene glycol (PEG) or polypropylene
glycol
(PPG). Other alcohols known to those skilled in the art to have similar
properties may
be used. According to one embodiment, polyethylene glycol is used. According
to yet
another embodiment, the PEG used in the process of the present invention has a
molecular weight of between 2,000 and 10,000 KDa, preferably has a molecular
weight
of between 3,500 and 4,500 KDa. The PEG added to the solution is at least
about 2%
weight per volume of the mixture formed. According to one embodiment, the PEG
added is about 3% to 15% weight per volume of the mixture formed. According to
another embodiment, PEG is added at between 10 to 12% weight per volume of the
resulting mixture. Before the addition of the polyalkylene glycol the
temperature of the
mixture is adjusted to room temperature (at the range of from about 20 C to 25
C) and
the pH of the dispersion is reduced. The pH reduction improves the
precipitation and the
supernatant from this step is in a pH range suitable for the first anion
exchange
chromatography. According to one embodiment, the pH is reduced to a pH range
from
about 5.0 to about 6.5 by the addition of, for example, acetic acid. In
addition, a salt
such as sodium chloride or the like may be added to the aqueous mixture in an
amount
sufficient to achieve a conductivity of from about 0.5 to about 3.5, to
further prepare the
supernatant for loading on the anion exchange resin. The removal of
contaminating
proteins, without loss of API, enables a significant reduction in equipment
scale, e.g.,
column size.
All the above-described steps for removing contaminating substances are
performed in one container, which is highly advantageous for a commercial,
large-scale
18
CA 02900719 2015-08-19
production process.
The precipitate that forms can be separated by conventional means such as
centrifugation or filtration, and is then discarded. The supernatant is ready
for further
purification as described herein below. The above-described supernatant is
loaded on an
anion exchange resin. Various types of anion exchange resins can be used,
including
DEAE-Sephadex, QAE-Sephadex, DEAE-Sephacel, DEAE-cellulose, DEAE-Sepharose
and the like. According to one embodiment, the anion exchange resin is DEAE-
Sepharose. Variety of conditions may be used in this particular step. For best
results the
anion exchange medium is placed in a chromatographic column and the API eluted
therefrom. According to one embodiment, the anion exchange resin is first
equilibrated
with step-wise buffer application, starting with a solution having a pH of
about 3.5-4.5
and a conductivity of from about 8.0 to 12.0 mS/cm, and then with a solution
of pH
about 5.5 ¨6.5 and a conductivity of from about 2.5 to about 3.5 mS/cm. After
the resin
is equilibrated, the above-described supernatant is loaded on the first anion
exchange
resin. These conditions of pH and conductivity allow the retention of API on
the
column, while the anion exchange medium is washed. The conductivity of the
washing
buffer (at a pH of about 5.5-6.5) is increased from about 1.8-2.4 to about 2.5-
5.0 during
the washing. This increase provides suitable conditions such that the column
is loaded
to its full capacity, and yet no API is discarded in the flowthrough, to give
maximal API
yield.
The API is then eluted from the column. According to one embodiment, elution
is
performed with a buffer solution having a pH of about 5.5 to 6.5 and
conductivity of
from about 9.0 to about 11 mS/cm.
Following separation of a solution containing API from an ion exchange resin,
the
solution is treated to reduce its water content and change the ionic
composition by
conventional means such as by diafiltration, ultrafiltration, lyophilization,
etc., or
combinations thereof.
According to one embodiment, the API-containing effluent obtained after the
first
anion exchange chromatography is concentrated by ultrafiltration. The
retentate is then
diafiltered against pure water to reach conductivity within the range of from
about 3.5 to
about 4.5 mS/cm.
To further purify the API-containing solution obtained after the first anion
19
CA 02900719 2015-08-19
exchange chromatography the solution is loaded on a cation exchange resin with
the
same type of buffer used for the anion-exchange step, having appropriate pH
and
conductivity such to allow the API to pass and be washed off with the buffer
flow
through, while contaminating substances are retained on the cation exchange
resin.
According to one embodiment, the cation exchange resin is carboxymethyl-
sepharose resin, placed in a chromatography column. The cation exchange resin
is first
equilibrated with step - wise buffer applications, starting with a solution of
pH about
3.5-4.5 and a conductivity of from about 8.0 to 12.0 mS/cm, and then with a
solution of
pH about 5.5 ¨ 6.5 and a conductivity of from about 0.8 to about 1.1 mS/cm.
The API-
containing fraction is loaded on the column with the same buffer as in the
second
equilibration step (pH about 5.5 ¨ 6.5 and a conductivity of from about 0.8 to
about 1.1
mS/cm) and the flow through is collected.
Again, as disclosed herein above, the API-containing solution obtained after
the
cation exchange chromatography can be treated to reduce its water content
According
to one embodiment, the solution is concentrated by ultrafiltration.
As disclosed herein above, the anion-exchange chromatography is used
principally to separate active API from inactive API. The present invention
further
comprises methods for separating active API from other contaminating
substances,
including solvent/detergent compounds used for viral inactivation as described
herein
above.
According to one embodiment, such separation is achieved by the second anion
exchange chromatography. The present invention shows that advantageously,
contaminating substances, particularly non-ionic detergents and solvents
commonly
used for viral inactivation, are not retained in the DEAE-Sepharose anion
exchange
resin under the conditions of the present invention as detailed herein below.
The API
eluted from the second anion exchange chromatography step is therefore not
only
highly active, but also highly pure. The anion exchange resin is first
equilibrated with
step-wise buffer application, starting with a solution of pH about 3.5-4.5 and
a
conductivity of from about 8.0 to 12.0 mS/cm, and then with a solution of pH
about 5.5
¨6.5 and a conductivity of from about 2.5 to about 3.5 mS/cm. Next, the API-
containing
fraction, typically, after viral inactivation treatment, is loaded on the
second anion
exchange resin. At this stage, the pH of the loading buffer may be elevated,
and washing
CA 02900719 2015-08-19
can be performed in one step, as the solution is already purified from the
majority of
contaminating proteins. According to one embodiment, the pH of the loading
buffer is
from about 6.0 to about 8.0 and the conductivity from about 2.0 to about 4.0
mS/cm.
The pH of the washing buffer is about 5.5-6.5, and the conductivity about 2.5-
3.5
mS/cm.
The API is then eluted from the column. According to one embodiment, elution
is
performed with a buffer solution at a pH of about 5.5 to 6.5 and conductivity
of from
about 11 to about 13 mS/cm.
The solution containing active, purified API obtained after the second anion
exchange chromatography can be further processed to obtain pharmaceutical
preparation for therapeutic, diagnostic, or other uses. To prepare the product
for
therapeutic administration the process of the present invention further
comprises the
steps of changing the ionic composition of the solution containing purified,
active API
to contain a physiologically compatible ion and sterilizing the resulted
solution.
Physiologically compatible substances used in the practice of the present
invention are,
for example, sodium chloride, sodium phosphate and glycine, having a buffered
pH
compatible with physiological conditions.
According to one embodiment, the ionic composition of the solution containing
active, purified API is changed to contain the physiologically compatible
phosphate ion
by diafiltration against sodium phosphate buffer, at a physiological pH of
about 7Ø
The resulted solution is then concentrated and filter-sterilized to obtain a
fluid
pharmaceutical preparation suitable for therapeutic administration.
According to another aspect, the present invention provides a fluid
pharmaceutical
preparation comprising a purified active API produced by the process of the
present
invention. This solution may be used for therapeutic, diagnostic or reagent
purposes.
According to one embodiment, the fluid pharmaceutical preparation comprises at
least 90%, preferably 95%, more preferably 99% API out of the total proteins.
According to another embodiment, at least 90% of the API is in its active
form.
The API-containing pharmaceutical preparation of the present invention is
highly
pure. Compared to other plasma-derived approved products, the API-solution
obtained
by the methods of the present invention contains reduced amount of impurities.
Hitherto
known compositions of API include at least one protein stabilizer, including
albumin,
21
CA 02900719 2015-08-19
sucrose and marmitol. The present invention now discloses an API preparation
which is
devoid of a protein stabilizer, and yet is very stable, as exemplified herein
below. This
purity enables prescribing the API pharmaceutical preparation of the present
invention
to any subject in need thereof. For example, API preparation containing
sucrose as a
protein stabilizers are restricted from prescription to diabetes patients;
mannitol is
known to cause allergies to a certain percentage in the population, and thus
administering mannitol-containing drug to a subject may cause anaphylactic
reaction.
Protein stabilizers, specifically protein stabilizers derived from human
sources such as
albumin are undesirable in the API-pharmaceutical preparation, as such
proteins by
themselves should undergo purification processes, and the final product may
contain
further impurities.
The purified API obtained by the process of the present invention is highly
stable.
According to one embodiment, the API in the fluid pharmaceutical preparation
is stable
for at least 3 month, preferably 4 month, more preferably 6 month when the
pharmaceutical preparation is stored in a temperature range of between 20 C to
25 C.
According to another embodiment, the API in the fluid pharmaceutical
preparation is stable for at least 12 month, preferably 24 month, more
preferably 36
month, when the pharmaceutical preparation is stored in a temperature range of
between
2 C to 8 C.
The filter-sterilized API-containing solution can be used directly, and also
can be
incorporated into pharmaceutical composition which may be used for therapeutic
purposes. The term "pharmaceutical composition" is intended in a broader sense
herein
to include preparations containing a protein composition in accordance with
this
invention used not only for therapeutic purposes, but also for reagent or
diagnostic
purposes as known in the art or for tissue culture. The pharmaceutical
composition
intended for therapeutic use should contain a therapeutic amount of API, i.e.,
that
amount necessary for preventative or curative health measures. If the
pharmaceutical
preparation is to be employed as a reagent or diagnostic, then it should
contain reagent
or diagnostic amounts of API.
According to yet another aspect, the present invention provides a
pharmaceutical
composition comprising as an active ingredient a therapeutically effective
amount of
API produced by the process of the present invention, further comprising a
22
CA 02900719 2015-08-19
pharmaceutically acceptable excipient, diluent or carrier.
As used herein, the term "therapeutically effective amount" refers to an
amount of
a protein or protein formulation or composition which is effective to treat a
condition in
an living organism to whom it is administered over some period of time.
According to one embodiment, the pharmaceutical composition comprises at least
90%, preferably 95%, more preferably 99% API out of the total proteins.
According to
another embodiment, at least 90% of the API is in its active form.
Pharmaceutical compositions of the present invention may be manufactured by
processes well known in the art, e.g. by means of conventional mixing,
dissolving,
granulating, grinding, pulverizing, dragee-making, levigating, emulsifying,
encapsulating, entrapping or lyophilizing processes.
Pharmaceutical composition for use in accordance with the present invention
thus
may be formulated in conventional manner using one or more acceptable diluents
or
carriers comprising excipients and auxiliaries, which facilitate processing of
the active
compounds into preparations, which can be used pharmaceutically. Proper
formulation
is dependent on the route of administration chosen.
API formulations or compositions may be appropriate for a variety of modes of
administration. These may include oral, parenteral, subcutaneous, intravenous,
pulmonary, intralesional, or topical administration. According to certain
embodiments,
the present invention relates to pharmaceutical compositions to be
administered
parenterally and by inhalation.
Pharmaceutical compositions for parenteral administration may be formulated
for
intravenous injections, intravenous infusion, intradermal, intralesional,
intramuscular,
and subcutaneous injections or depots. The API-containing pharmaceutical
preparation
produced by the process of the present invention is advantageous over hitherto
known
API-containing preparation, as the API is highly stable also when the
preparation is kept
in a liquid from. Therefore, it is not necessary to lyophilize the API-
preparation for
stable storage in a form of a powder. Subsequently, there is no need to
reinstate the
powder to a liquid before use for parenteral administration.
As described herein above, API is used for the treatment of pulmonary
diseases.
Intravenous administered API affects such pulmonary diseases by diffusing into
the
23
CA 02900719 2015-08-19
lung and neutralizing elastase and other proteases. By examining broncho-
alveolar
lavage (BAL) specimens it has been shown that exogenous API, administered
intravenously and diffused into the lungs caused an increase of more than 3-
fold in the
pulmonary API levels, and that the anti-elastase activity doubles 6 days after
API
infusion (ATewers et al., Am. Rev. Respir. Dis. 135:539-43, 1987).
Nevertheless, when
administered intravenously most of the API never reaches the lung. It has been
estimated that only 2% of the intravenously administered dose reaches the lung
(Hubbard & Crystal, Lung (Suppl): 565-578, 1990).
Therefore, administration of API by the inhalation route may be more
beneficial
as it reaches directly the lower respiratory tract. This route should require
lower
therapeutic doses of API and thus the scarce supply of human plasma-derived
API
would be available for the treatment of more patients. This rout of
administration may
be also more effective in neutralizing neutrophil elastase. In addition,
administration by
inhalation is simpler and less stressful for the patient than the intravenous
route and
would reduce the burden on the local health care system (by requiring less
clinical
input).
Formulations of pharmaceutical compositions for administration by the rout of
inhalation are known in the art. In general, for administration by inhalation,
the active
ingredients are delivered in the form of an aerosol spray from a pressurized
pack or a
nebulizer with the use of a suitable propellant, e.g.,
dichlorodifluoromethane,
trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. The
operating
conditions for delivery of a suitable inhalation dose will vary according to
the type of
mechanical device employed. For some aerosol delivery systems, such as
nebulizers,
the frequency of administration and operating period will be dictated chiefly
by the
amount of the active composition (API according to the present invention) per
unit
volume in the aerosol. Typically, the higher the concentration of the protein
in the
nebulizer solution the shorter is the operating period. Some devices such as
metered
dose inhalers may produce higher aerosol concentrations than others and thus
will be
operated for shorter periods to give the desired result.
Other devices such as powder inhalers are designed to be used until a given
charge of active material is exhausted from the device. The charge loaded into
the
device will be formulated accordingly to contain the proper inhalation dose
amount of
24
CA 02900719 2015-08-19
API for delivery in a single administration. (See generally, Remington's
Pharmaceutical
Sciences, 18th Ed. 1990, Mack Publishing Co., Easton, Pa., Chapter 92 for
information
relating to aerosol administration).
Regardless of what device is used for aerosolization, the active ingredient
will be
in the form of a dispersion of particles. The dispersion of particles may be
in the form of
liquid droplets or in the form of powder (dry or suspended). According to one
embodiment, the API-containing fluid pharmaceutical preparation will be used
directly
for aerosolization.
According to yet another aspect, the present invention provides a method for
treating a patient in need thereof comprising administering a therapeutically
effective
amount of API produced by the process of the present invention.
According to one embodiment, the method is used for treating pulmonary
emphysema. It is known that patients with API deficiency have a low level
burden of
neutrophils in their lower respiratory tracts. Evaluation of the API levels
and anti-
neutrophil elastase defenses in such patients demonstrated that both are
markedly
reduced. Taken together, these observations support the hypothesis that a
deficiency of
API predisposes a patient to emphysema by altering the balance between
neutrophil
elastase and anti-neutrophil elastase in the lower respiratory tract. Whereas
normal
persons have an adequate anti-neutrophil elastase screen to protect the lower
respiratory
tract, those with API deficiency do not, permitting the neutrophil elastase to
destroy
lung tissue. Thus, providing patient with endogenous API- deficiency with
exogenic
API at the correct dose can overcome the deleterious effects of such
deficiency.
According to another embodiment, the method is used for treating lung diseases
and disorders associated with cystic fibrosis.
Loss of pulmonary function is a primary cause of death in patients suffering
from
cystic fibrosis. Patients with a Forced Expiratory Volume in one second (FEV1)
below
30% of their predicted value have a 2-year mortality of greater than 50%. The
current
mortality rate is 1.2 deaths per 100 patients per year; the median survival is
32 years.
Of the deaths in which a case was specified, 94% were due to cardiorespiratory
failure.
Respiratory failure is characterized by increasing dyspnea, hypoxemia and
elevation of
arterial PCO2. During their lifetime, CF patients are restricted in their day-
to-day
activities due to reduced lung function and constant pulmonary infections as a
result of
CA 02900719 2015-08-19
their condition.
One of the major side effects of chronic infection associated with CF is the
chronic presence of phagocytic neutrophils in the lungs in response to
bacterial
infections and the release of various chemoattractants. These leukocytes
secrete
elastase, which has the potential to destroy the elastic tissue of the lung.
In addition,
neutrophils of patients with CF have been shown to be in a state of increased
responsiveness and tend to degranulate more readily, releasing tissue-
destroying
elastase. Thus, patients with CF appear to have a state of unregulated
inflammatory
response, which overwhelms the normal protease (elastase)/antiprotease (API)
balance,
leading to the accumulation of elastase in the lung and ultimately to tissue
damage.
Previous studies have shown that much of the pulmonary damage in CF results
from the presence of unneutralized elastase and other proteases. The abnormal
cycle is
destructively self-perpetuating and self-expanding: increased elastase leads
to the
recruitment of more neutrophils to the lung that in turn secrete additional
proteases.
This cycle further overwhelms the natural normal protease
(elastase)/antiprotease
balance leading to destruction of the lung architecture, severe pulmonary
dysfunction
and ultimately death.
Patients with CF suffer from chronic lung infections that have to be treated
with
antibiotics. For example, azithromycin, an antibiotic recently proven to be
effective in
the treatment of Pseudomonas aeruginosa bacteria. With the increased longevity
of CF
patients, there will be associated newer infections such as B. cepacia and S.
maltophilia.
It is possible that prophylactic use of API in CF patients may improve their
pulmonary
function and thereby reduce the occurrence of such infections.
Preferably, API is administered to CF patients by the inhalation route. It has
been
previously demonstrated (McElvaney et al, 1991) that aerosolized alpha-anti-
trypsin
given to cystic fibrosis patients suppressed neutrophil elastase in the
respiratory
epithelial lining fluid (ELF), restored the anti-neutrophil elastase capacity
in the ELF
and reversed the inhibitory effect of the ELF on the ability of neutrophils to
effectively
combat Pseudomonas infection. Advantageously, aerosol formulations can be
readily
produced using the API liquid preparation of the present invention. For
aerosolized
formulation, higher API concentration in the range of 10% is required. As
exemplified
herein below, API produced by the process of the present invention is
essentially stable
26
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at this concentration for at least 8 weeks, preferably for 12 weeks, when
stored at a
temperature range of 2-8 C.
The principles of the invention, may be better understood with reference to
the
non-limiting examples below.
EXAMPLES
Measuring API activity
Concentration of active API is determined by inhibition of porcine pancreatic
elastase. In principle, the assay compares between the inhibition rate of
elastase
obtained with a test sample and the inhibition rate of elastase obtained with
API a
reference standard (Kamada, Israel), which is considered as having 100%
activity.
Elastase activity is measured by the rate of cleavage of the elastase
substrate (Succinyl-
Alanine-Alanine-Alanine-p-Nitroanilide) resulting in the release of the
cleavage
product, which absorbs at 405 nm. The decrease of elastase activity in the
presence of
API is in direct relation with the amount of active API in the reaction
mixture.
Activity of API reference standard is estimated by inhibition of trypsin
esterase
activity. Active API binds active trypsin in a stoichiometric ratio of 1 : 1
under the assay
conditions and decrease trypsin esterase activity, which is measured according
to a
method similar to the USP 25 method. The amount of trypsin active sites in the
trypsin
preparation is determined previously by titration with p-Nitrophenyl-p-
Guanidino-
Benzoate (NPGB): The increment of absorption at 402 nm of the trypsin
preparation
following addition of the titration substrate is in direct correlation with
the amount of
active trypsin molecules present in the trypsin preparation.
Example 1: Pretreatment of the source material
In a preferred embodiment, the starting material is Cohn Fraction IV-1 paste,
which is obtained by the Cohn-Oncley fractionation technique, well known to
those of
skill in the art. The preparation of an aqueous solution from the Fraction IV -
1 paste is
described below.
The IV-1 paste is dissolved in about 35 volumes of water-for-injection grade
water, (IV-1 paste weight in kg times 35). The amount of starting paste, 75-87
Kg per
purification process, is added to a jacketed stainless steel tank in portion.
The pH of the
27
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mixture is adjusted to 9.2 immediately after mixing of the first portion, and
it was
further adjusted by 0.5N NaOH until all the paste and water are added. The
solution is
mixed for approximately 10 minutes.
Fraction IV-I paste, like other plasma fractions, contains various proteins,
such as
lipoproteins, immunoglobulins, globulin, metalloproteins, etc. These proteins
must be
separated from the API to produce a liquid stable preparation, but some will
also bind to
an ion exchange resin and thereby interfere with the purification of API.
Before adding
the solution to an anion exchange resin, therefore, a portion of these
contaminating
proteins is preferably removed. According to the present invention, removing
such
contaminating fraction is performed by two steps.
Removing of lipids and lipoproteins
To the dispersion obtained above a lipid-removing agent, AerosilTM (silicon
dioxide) was added at 78-82 g/Kg paste. After the addition of Aerosil, the pH
of the
resulting mixture was adjusted to 8.8 with NaOH 0.5N. The mixture was
incubated for
90 min. at 38 C and a stirring rate of 870-1450 rpm. After 90 min. the
dispersion was
cooled to 22.5 C and the pH was adjusted to 6.1.
Precipitation of contaminating proteins
To the cooled dispersion obtained as above Polyethylene glycol (PEG) of mean
molecular weight of 4,000 KDa was added at 10.5-11.5% weight per volume of the
resulted mixture while stirring at 2660-2900 rpm. After PEG dissolution the pH
of the
dispersion was adjusted to 6.0 with 2% acetic acid. Conductivity was adjusted
with
solid NaC1 to 3.0 mS. A precipitate of contaminating proteins and viruses,
including
prion proteins, was formed. This precipitate was removed by continuous
centrifugation
(Self-desludging centrifuge, model CSA19-06-476, Westfalia) at a
centrifugation rate of
300-450 liter/hr. The sediment obtained by the centrifugation was discarded;
the
supernatant was further filtered using 1 um (nominal) cellulose fiber depth
filter and a
pressure of < 25 psi.
Example 2: First anion exchange chromatography
The resin used for the first anion exchange chromatography was DEAE-Sepharose
fast Flow, packed in a stainless steel 316-L column (CF 1000/150 SS
CHROMAFLOW,
Pharmacia), having a volume of 117 liter.
28
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The DEAE-Sepharose resin was first equilibrated step-wise with sodium acetate
buffer as follows:
a) pH 4.0, conductivity 10.0 mS/cm
b) pH 6.0, conductivity 3.0 mS/cm
Flow rate: 7-20 liter/min.
After the procedure described in example 1 the filtered supernatant obtained
was
already at the suitable pH and conductivity conditions of pH 6.0 and 3.0
mS/cm. It was
therefore directly loaded on the equilibrated DEAE-Sepharose column, at a flow
rate of
12-14 liter/min.
In such large-scale production, it is critical to adjust the washing
conditions such
that the column will be loaded at its maximal capacity, and yet that no API
would leak
out in the flow through. This was achieved by a two-step column wash, both
with
sodium acetate buffer at the following conditions: first wash - pH 6.0,
conductivity 2.1
mS/cm; second wash: pH 6.0, conductivity 3.0 mS/cm. The API is retained on the
column, and other proteins, for example albumin and transferrin are washed
out.
Elution of the API from the column was performed with a sodium acetate buffer
having a pH of 6.2 and conductivity of 10.0 mS/cm. The pressure on the column
was <
35 psi and the flow rate 12-14 liter/min. TheAPI-containing fraction was then
treated to
adjust its water contentand ion composition as described in Example 3 below.
Example 3: Adjustment of water content and ion composition
The API- containing effluerit was concentrated to 100 kg (total weight) by
ultrafiltration with polysulfone membrane with a nominal cut off of 10 kD and
total
membrane area of 12.2 m2 (UFP-10C-65) in a Hollow Fiber cartridge (Amersham
Biosciences). The retentate pressure was 15-20 psi and the flow rate 20-40
liter/min.
The filtrate was then discarded and the retentate was diafiltered against
water-for-
injection (WFI) grade water. The retentate pressure was 15-20 psi at a flow
rate of 20-
40 liter/min., and the diafiltration was continued until the solution reached
a
conductivity of 4.0 mS/min.
Example 4: Cation exchange chromatography
To further purify the API-containing fraction obtained after the procedure of
example 3 from remaining contaminating substance, the solution was subjected
to
29
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cation exchange chromatography using the same sodium acetate buffer under
conditions
which allow the retention of only the contaminating substances of the cation
resin,
while the API was washed out with the column flow through.
The cation exchange resin used was CM-Sepharose Fast Flow, packed in a
stainless steel 316-L column (CF 1000/150 SS CHROMAFLOW, Pharmacia), having a
volume of 117 liter.
The resin was equilibrated with sodium acetate buffer under the following
conditions:
a) pH 4.0, conductivity 10.0 mS/cm;
b) pH 5.35, conductivity 0.95 mS/cm
Flow rate: 7-22 liter/min.
Before loading the API-containing fraction on the column, the conductivity of
the
solution was adjusted with NaC1 to 0.95 mS/cm and the pH was adjusted with
2%acetic
acid to 5.35. The protein concentration of the solution was 0.5%, and total
protein load
was < 4 Kg. Flow rate was 18-22 liter/min at a pressure of 29 < psi. The
column flow
through containing the API was collected. The pH was adjusted to 6.75 with
0.15 M
NaOH and the conductivity to 3.0 mS/cm with solid NaCl. The resulting solution
was
ultrafiltered using polysulfone membrane with a nominal cut off of 10 Kd and
total
membrane area of 12.2 m2 (UFP-10C-65) in a Hollow Fiber cartridge (Amersham
Biosciences). The retentate was collected and the effluent discarded.
Example 5: Viral inactivation
In a preferred embodiment of the invention, the API-containing fraction
obtained
after the action exchange chromatography is subjected to viral removal and
inactivation.
According to the preferred embodiment, viral removal was performed by
nanoffltration. The API-containing solution was subjected to pre-filtration
through
polysulfone membrane with a pore size of 0.1 + 0.2 I.un, nominal surface area
of 0.6 m2
(5441358 K-1 SS, Sartorius), at a pressure of <30 psi. The retentate was
collected in
600 liters stainless steel 316-L jacketed and pressurized container equipped
with a
marine type stirrer. The retentate was diluted with WFI to protein
concentration of 6.0-
8.5 mg/ml, and pH and conductivity was re-adjusted to 7.25 with 0.15 M NaOH/2%
acetic acid and 3.0 mS/cm with solid NaCI, respectively. The temperature of
the
CA 02900719 2015-08-19
container was kept at 22.5 C.
Nanofiltration was performed with Planova 15N filter (15N1-000;Asahi Kasei
Corporation) having a nominal surface are of 1.0 m2. The operating pressure
obtained
by N2 was 13.8 psi. Volume transferred per m2 of filter was more than 250
liters.
Further to virus removal, the API-containing solution was subjected to viral
inactivation using the solvent/detergent method. Polysorbate 80 was added to
0.95-
1.25% w/w final concentration and TnBP to a final concentration of 0.28-0.33%
v/w.
The mixture was stirred for about 4.5-5.5 hrs. at 30-50 rpm.
Example 6: Second anion exchange chromatography
Second anion exchange chromatography is employed to further purify the API-
containing solution obtained after viral inactivation from the employed
solvent and
detergent. As during the steps taken after the first anion exchange
chromatography some
of the active API may have become inactive, inactive AP is also removed. Thus,
the
resulting solution after this step contains highly purified, active API.
The resin used for the second anion exchange chromatography was DEAE-
Sepharose Fast Flow, packed in a stainless steel 316-L column (BPSS 800/150
SS,
Pharmacia), having a volume of 75 liter.
The DEAE-Sepharose resin was first equilibrated step-wise with sodium acetate
buffer as follows:
a) pH 4.0, conductivity 10.0 mS/cm
b) pH 6.0, conductivity 3.0 mS/cm
As the API-containing fraction was now partially purified, a faster flow rate
of 18-
22 liter/min. was employed. Of the 'same reason, the pH of this fraction could
be
elevated to 7.5 and the column could be washed with one set of buffer
conditions. The
conductivity of the loaded solution was 3.0 mS/cm, and the total protein load
about 2.5
Kg. The pH of the sodium acetate washing buffer was 6.0 and its conductivity
3.0
mS/cm. Total wash volume was 1125-1200 liter at a flow rate of 18-22
liter/min.
Elution of the API from the column was performed with a sodium acetate buffer
having a pH of 6.0 and conductivity of 12.0 mS/cm. The eluate of the second
anion
exchange column contained 96% pure API, of which more than 90% were active.
31
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Example 7: Purity of API through the process
Samples from different steps of the process were analyzed by techniques of
protein separation and detection. Fig. 1 describes the protein profile of API
in the
pharmaceutical preparation produced by the process of the present invention;
standard
API prepared by Kamda, Israel; and polymeric API on native Tris-Glycine 8% to
16%
gradient gels. Under native conditions, proteins are separated according to
both,
molecular weight and structure. Non-denatured samples were loaded in a sample
buffer
(Invitrogen TM LC2673) on the gels in the amounts detailed in table 1.
Polymeric forms
of API were derived from a purified API (lot No. 6110003, Kamada) stored at 35
C for
6 months that was subjected to gel permeation chromatography using Sephacryl
200HR
column. Three fractions (lanes 8-10) were collected for analysis on the native
gels. The
gels were run in a Tris-Glycine running buffer (10 x Invitrogen TM LC2672).
The gels
were stained by Commassie blue (Fig. 1A), Ponceau-S (Fig.1B) or blotted onto a
nitrocellulose membrane (BioRad Trans Blot). Inununobloting was then performed
using anti-human API antibodies, conjugated with horseradish peroxidase
(HRP,ICN/Cappel, 55236), used at a dilution of 1:400 (Fig. 1C).
Table 1: Samples loaded onto the native gels
Lane(s) Sample Protein loaded ( g/lane)
A
1/2 Final drug product 2.5/5 1/2
3/4 In-house API standard 2.5/5 1/2
5 Albumin 3.5 1.4
6 Transferrin 3.5 1.4
7 Anti-D (IgG) 3.5 1.4
8 Polymeric fraction of API 3.6 1.44
9 Polymeric fraction of API 3.6 1.49
10 Polymeric fraction of API 3.6 1.32
Samples obtained during the purification process were separated by 4-12% SDS-
PAGE. Gels were stained with Cornmassie Blue (Fig. 2A), Ponceau-S staining
(Fig. 2B)
or immunoblotted with Goat anti-API, HRP-conjugate antibodies. Samples are
detailed
in table 2.
32
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Table 2: Samples loaded onto the SDS-PAGE gels
Lane(s) Sample Protein loaded (gg/lane)
A
1 Sample buffer
2, 3 Pre-Aerosil 15 3
4, 5 Pre-PEG 15 3
6, 7 First DEAE eluate 4.0 0.8
8, 9 Ultrafiltration after cation exchange 2.0 0.4
Second DEAE eluate 2.0 0.4
11 Drug substance 2.0 0.4
12, 13 Drug product 2.0 0.4
14 MW size marker- commercial 100 per lane
MW in-house marker 4.6 2.3
Figures 1 and 2 clearly show that most contaminating proteins are removed
during
the process of the present invention. The final drug substance shows only one
sharp and
=
5 clean band.
Example 8: Further purification of API
After the second anion exchange chromatography described in Example 6, the
fraction containing the active, pure API was concentrated by ultrafiltration
(UFP-10C-
10 65: polysulfone membrane with nominal cut off of 10 KDa and total
nominal membrane
area of 12.2 m2). The filtrate was discarded and the retentate, concentrated
to 60 kg was
further subjected to diafiltration. Diafiltration was performed to replace the
acetate ion
by physiologically acceptable ion, specifically phosphate ion. The
diafiltration
conditions were filtration against sodium phosphate buffer, 18-22 nM in 0.6-
0.8% NaC1,
15 pH 7.0, retentate pressure of 15-20 psi. Final pH of the retentate was a
physiological pH
of 7Ø After second ultrafiltration, the protein concentration was brought to
22-24
mg/ml. The preparation was filter sterilized using two polysulfone filters of
0.45 and
0.2 gm (Sartorius, 5101507H9-B) in a serial placement. Filtration was
performed under
a pressure of < 25 psi and the sterile filtrate was collected in pre steam-
sterilized
container. The final API- containing preparation comprised 22-24 mg/ml protein
at a pH
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of 7.0 0.1, NaC1 at a concentration of 6-8 mg/ml and phosphate concentration
of 1 8-
22 mM. This solution is a pharmaceutical grade, ready to use preparation, and
is
therefore designated as pharmaceutical preparation.
Example 9: Characterization of Physico-chemical and Structural Properties of
API
Characterization studies of three lots of the API containing pharmaceutical
preparation produced by the process of the present invention by the Kamada
(Israel):
were performed to obtain information on physico-chemical and structural
properties of
the API molecule. Three its were examined: lots 6112006, 6113010 and 6123010.
The
analyses of lot #6112006 (manufacturing: June 2002) were performed after 12-20
months storage of the lot at 2-8 C, whereas the analyses of lots #6112010 and
#6123010
(manufacturing: October 2003) were initiated immediately after the production
and
were completed within 3 months in which the lots were stored at 2-8 C.
The characterization studies were performed in three laboratories, at the
Kamada
Analytical R&D and Validation Laboratory (Israel), at M-Scan Limited (UK) and
at the
Weiz,mann Institute of Science (Israel).
Mass-Spectrometry of Intact Protein
Delayed Extraction-Matrix Assisted Laser Desorption Ionization-Time of Flight
(MALDI-TOF MS) Mass Spectrometry was used for the determination of the intact
molecular weight of API.
MALDI-TOF MS is a technique in which a co-precipitate of a UV-light absorbing
matrix and a protein molecule are irradiated by a nanosecond laser pulse. Most
of the
laser energy is absorbed by the matrix, which prevents unwanted fragmentation
of the
molecule. The ionized protein molecules are accelerated in an electric field
and enter the
flight tube. During the flight in this tube different molecules are separated
according to
their mass to charge ratio and reach the detector at different times. In this
way each
molecule yields a distinct signal. This method allows accurate measurement of
intact
molecular weight of biopolymers from 400 up to 500,000 Da.
All the tree lots examined had a comparable intact molecular weight, as shown
in
Table 3 below:
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Table 3: Molecular weight of intact API
API Lot # Intact Molecular Weight, Da
6112006 50,600 Da + 50 Da
6113010 50,550 Da 50 Da
6123010 50,500 Da 50 Da
Amino Acid Composition Analysis, and Determination of the Extinction
Coefficient
Amino acid composition is an important characteristic of proteins. The
combination of the amino acid analysis with spectrophotometric determination
of the
absorbance at 280 nm allowed the determination of the molar extinction
coefficient of
API. Dividing the molar extinction coefficient of the protein by its intact
molecular
weight results in the extinction coefficient of a 0.1% protein solution.
For amino acid analysis, the API lot samples were hydrolysed with constant
boiling HC1, derivatized using Phenylisothiocyanate and analyzed by Reverse
Phase
(RP)-HPLC. API in lots 6112006, 6113010 and 6123010 had comparable amino acid
compositions (See Table 4 below). The slight differences in the content of
aspartic and
glutamic acids between API lot 6112006 and API lots 6113010 and 6123010 are
related
to the duration of hydrolysis; 24h for lot 6112006 compared tol 6 h for lots
6113010 and
6123010.
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Table 4: Amino Acid Composition
API Lot API Lot API Lot
E #6112006, #6113010,
#61213010,
Amino acid xpected Calculated ratio Calculated
ratio Calculated ratio
ratio
relative to Leu = relative to Leu = relative to Leu =
45 45 45
Aspartic acid 43 28.5 16.9
16.0
Glutamic acid 50 42.5 27.4
26.1
Serine 21 19.9 20.2
20.9
Glycine 22 21.6 20.9
21.6
Histidine 13 13.9 12.9
12.9
Arginine 7 7.6 7.0 7.0
Threonine 30 25.5 29.5
28.7
Alanine 24 23.4 23.3
23.4
Proline 17 16.9 16.5
17.2
Tyrosine 6 5.7 5.8 5.8
Valine 24 21.8 21.7
21.3
Methionine 9 9.3 8.9 9.2
Isoleucine 19 17.4 17.0
16.7
Leucine 45 45.0 45.0
45.0 .
Phenylalanine 27 27.0 26.7
26.5
Lysine 34 35.5 30.7
30.2
Tryptophan 2 ND ND ND
Cystein 1 ND ND ND
ND=not detected
The extinction coefficient of API was measured for lot #6113010 and #6123010
which had comparable extinction coefficients, as is shown in Table 5.
Table 5: API Extinction Coefficient
API Lot # Molar Extinction Extinction Coefficient (0.1%)
at 280
Coefficient, L.mor I = cm-I nm
6113010 24,618 0.49
6123010 22,908 0.45
Peptide Mapping
Peptide Mapping was performed following Cyanogen bromide (CNBr) digestion,
using MALDI-MS and on-line LC-Electrospray (ES) MS. The concept of the chosen
method of mapping is generation of peptides by chemical digestion,
determination of
their molecular weights and accurate determination of the presence or absence
of the
36
i
CA 02900719 2015-08-19
components parts of the known protein sequence. The techniques of ES-MS using
an
atmospheric pressure ionisation source and MALDI-MS create the possibility of
accurately measuring the intact molecular weights of biopolymers up to 130 kDa
and
500 kDa respectively. The coupling of on-line microbore and nano-capillary
HPLC
directly with Electrospray Mass Spectrometric detection of the separated
digest products
increases the power of the technique. A definite advantage of this mapping
procedure is
that there is an equal probability of observing the C-terminal as well as the
N-terminal
regions of the protein.
Peptide mapping profile of all three lot examined, lots #6112006, #6113010 and
#6123010 is presented in Table 6, showing that all three lots have a
comparable profile.
Table 6: Peptide Mapping by Cyanogen Bromide (CNBr) Digestion
Item Lot #6112006, Lot #6113010, Lot #6123010,
Molecular Mass Molecular Mass Molecular Mass
of Peptide/ of Peptide / of Peptide /
Glycopeptide Glycopeptide Glycopeptide
Glycopeptide 243-351 residue
(homoserine) plus 13,967 13,971 13,971
NeuAc2Hex5HexNAc4
N-terminal peptide residues 1-63
(homoserine) plus 9184 9185 9185
NeuAc2Hex5HexNAc4
Fucosylated N-terminal
glycopeptide residues 1-63 9331 9331 9331
(homoserine) plus
NeuAc2Hex5HexNAc4Fuc
Glycopeptide residue 64-220
(homoserine) plus 20,170.5 20,174 20,175
NeuAc2Hex5HexNAc4
The 24aa signal peptide ND ND ND
Peptides 1-63, 243-351 and 64-2 ND ND ND
in non-glycosylated forms
C-terminal peptide residues 386- 970.5 970.7 970.8
394 486.0 (Doubly 486.0 (Doubly 486.4 (Doubly
charged) charged) charged)
Confirmation of the sequence 100% 100% 100%
N-Linked Oligosaccharide Population Analysis
The purpose of the test was to obtain the profile of the oligosaccharides
bound to
the API and determine the API glycosylation sites, i.e. the points of binding
of
37
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oligosaccharides to the polypeptide. Only N-linked oligosaccharides are
evaluated
because 0-linked oligosaccharides are not known to exist in API.
Oligosaccharide analysis included enzymatic digestion of API samples with
chymotrypsin and subsequent digestion with peptide N-glycosidase F (PNGase F),
separation of the released carbohydrates from peptides using C18 Sep-pak
cartridge,
permethylation of the glycans and finally their analysis by Fast Atom
Bombardment
(FAB)-MS and MALDI-TOF MS. The results are presented in table 7 below.
Table 7: N-Linked Oligosaccharide Population
Glycans Lot #6112006 Lot #6113010, Lot #6123010,
m/z Signal m/z Signal m/z Signal
NeuAc2 Hex5.HexNAc4 279 (Major 279 (Major 2793 (Major
Signal) Signal) Signal)
NeuAc3.Hex6.HexNAc5 3604 (Minor 3604 (Minor 3605 (Minor
Signal) Signal) Signal)
NeuAc2Hex5.HexNAc4Ft 2969 (Minor 2969 (Minor 2968 (Minor
Signal) Signal) Signal)
NeuAc3J-lex6.HexNAc5.Fi 3779 (Minor 3779 (Minor 3779 (Minor
Signal) Signal) Signal)
N-Glycosylation Sites Asn-46, Asn-83 Asn-46, Asn- Asn-46, Asn-83
and Asn-247 83 and and Asn-247
Asn-247
Monosacccharide Composition Analysis
The monosacccharide composition of a glycoprotein is a basic characteristic of
its
oligosaccharide part. For monosaccharide analysis, the samples of the API lots
were
methanolysed, derivatised and analyzed by chromatography/mass spectrometry
(GC/MS). The method allowed the estimation of the monosaccharides per mole of
glycoprotein and, thus, the approximation of the total oligosaccharide
percent. The
monosaccharide composition of the three lots examined is shown in Table 8
below.
38
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Table 8: Monosaccharide Composition
Mono- Lot #6112006 Lot #6113010 Lot #6123010
saccharides Mono- Ratio Mono- Ratio Mono- Ratio
saccharide (Mannose saccharide (Mannose saccharide (Mannose
nmoles =3.0) nmoles =3.0) nmoles =3.0)
present/mg present/mg present/mg
of protein of protein of protein
Fuc 7.7 0.1 12.0 0.2 10.4 0.2
Man 197 3.0 200 3.0 204 3.0
Gal 182 2.8 208 3.1 216 3.2
GalNAc ND ND ND
GleNAc = 125 1.9 222 3.3 242 3.6
NeuAc 93 1.4 128 1.9 98 1.4
Circular Dichroism
The purpose of the Circular Dichroism (CD) spectroscopy measurements was to
characterize the secondary and tertiary structures of the API obtained by the
process of
the present invention. The CD of the API was measured in two regions: the near
UV,
320-260 nm, and far UV, 260-180 nm. Far UV CD spectrum was used to determine
CL-
helices, and near UV CD was used to characterize the tertiary structure.
CD spectroscopy measures differences in the absorption of the left-handed
polarized light versus right-handed polarized light, which arise due to
structural
asymmetry. The absence of regular structure results in zero CD intensity,
while an
ordered structure results in a spectrum, which can contain both positive and
negative
signals. Far and near UV CD Spectra of API Lots 6112006, 6113010, 612301 and
API
Primary Reference Standard #02/07 are shown in Figure 3A and B, respectively.
Example 10: API stability
A. Stability of typical API pharmaceutical preparation comprising 2% API
Materials and methods
The stability of API in the pharmaceutical preparation obtained by the process
of
the present invention was measured up to 36 months at storage temperature of 5
3 C,
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according to the following parameters as described in Table 9 below:
appearance;
content of active API; distribution of molecular size (concentration of API
monomers
and concentration of API aggregates. API aggregates are inactive); and pH.
API activity was assayed by the elastase inhibition assay as described above,
and
by the presence of API aggregates analyzed by analytical liquid chromatography
[Size
exclusion (SEC) HPLC] using a Zorbax GF-250 column. Total protein
concentration
was determined by absorbance at 280 nm.
Results
Stability of a typical API pharmaceutical preparation produced by the process
of
the present invention, containing 2% API, is shown in Table 9 below.
Table 9: Stability of API stored at 5 3 C
Test Appearance Active
Distribution of Molecular pH
API size
content
Specification The solution 2.0-2.4
Polymer & Monomer>90% 6.8-
is clear and g/100m1 aggregate
7.2
colorless <10%
Start Pass 2.2 0.0 98.5
6.8
(8.5.2000)
1 Month Up* Pass 2.2 0.3 98.3
6.8 .
(8.6.2000) Inv.** Pass 2.3 0.3 98.1
6.8 _
2 Months Up* Pass 2.1 0.4 98.9
6.8
(8.7.2000) Inv.** Pass 2.1 0.4 98.8
6.8
3 Months Up* Pass 2.0 0.4 98.7
6.8
(8.8.2000) Inv.** Pass 2.0 0.4 97.8
6.9
6 Months Up* Pass 2.0 0.8 98.3
6.9
(8.11.2000) Inv.** Pass 2.1 0.8 98.2
6.8
9 Months Up* Pass 2.0 1.0 99.0
6.8
(9.2.2001) Inv.** Pass 2.0 1.3 98.1
6.8
12 Months Up* Pass 2.0 1.0 98.5
6.8
(8.5.2001) Inv.** Pass 2.1 1.3 98.0
6.8 _
18 Months Up* Pass 2.2 1.5 98.0
6.8
(8.11.2001) Inv.** Pass 2.2 1.2 98.8
6.8
24 Months Up* Pass 1.9 9.3 90.7
6.8
_
(8.5.2002) Inv.** Pass 1.9 9.4 90.6
6.8
36 Months Up* Pass 2.0 11.0 89.0
6.8
(8.5.2003) Inv.** Pass 2.0 11.0 89.0
6.8
*Up: Upright position
**Inv.: Inverted position
,
CA 02900719 2015-08-19
As shown in table 9, the API obtained by the process of the present invention
is
highly stable. As described herein above, a specific advantage of the
pharmaceutical
preparation of the present invention is that the highly purified, active API
is also highly
stable, in a ready to use liquid solution and without the presence of any
protein
stabilizer.
B. Stability of concentrated API pharmaceutical preparation comprising 5-20%
API
The purpose of the study was to determine API stability under conditions
favorable for Aerosol production. These conditions include increased API
concentration
to about 10%, and preferably up to 20%. The stability of the concentrated
solutions was
determined in the cold (2-8 C) and at ambient temperature (-25 C). Stability
was
examined by the formation of aggregates (aggregate forms of API are inactive)
and by
measuring API activity as described herein above.
Materials and methods
API solutions in a protein concentration range of 5% - 20% were prepared from
a
2% API preparation (lot no. 6013009, Kamada, Israel). The solutions were
incubated in
the absence of Tween 80 and in the presence of Tween 80 at concentrations of
0%,
0.01%, 0.05% and 0.1%; one set was kept under refrigeration at 2 C-8 C and the
other
set was kept at 20 C-25 C. The samples of both sets were analyzed at times
zero, 4
weeks, 8 weeks and 12 weeks for protein content, API activity, concentration
of API
monomers and concentration of API aggregates.
The 2% API solution was concentrated from 2% API to ¨ 20% and higher, by an
A.G. Technology NMWC 10000 ultrafilter (UFP-10C-4X2A). Based on the starting
volume and API concentration, aliquots of concentrated solution were withdrawn
at
various stages during ultrafiltration. The following API concentrates were
obtained:
6.34%, 12.2%, 18.1%, and 22.84%. These solutions served for the preparation of
the
final solutions used in the study, containing 5%, 10%, 15%, and 20% protein.
Results
Stability of concentrated API preparations prepared as described below and
kept
at 20-25 C is shown in Table 10 below.
41
CA 02900719 2015-08-19
Table 10: Stability of concentrated API preparations stored at 20-25 C.
Group Sample Protein API Specific HPLC % HPLC
%
Description I.D. (mg/mL) (mg/mL) API API
API
(*) Activity Monomer
Aggregates
50 a 51.92 48.8 0.94 96.8
3.23
5% API 530 a T-1 49.06 , 50.4 1.03
96.54 3.48
No Tween 560 a T-1 N.D. N.D. N.D.
N.D. N.D.
590 a T-1 50.9 45.6 0.90 95.01 4.95
50b 50.93 48.2 0.95 97.1
2.81
5% API 530 b T-1 49.45 50.3 1.02
94.82 5.19
0.01%Tween 560b T-1 N.D. N.D. N.D. N.D. N.D.
5901) T-1 51.6 46.8 0.91 94.43 5.42
5o c 51.87 49.3 0.95 96.7
3.30
5% API 530 c T-1 50.49 53.3 1.05
95.41 4.60
0.05%Tween 560 c T-1 48.9 46.9 0.96 93.82 6.10
590 c T-1 51.3 44.0 0.86 92.28 7.56
50d 51.81 47.8 0.92 96.4
3.62
5% API 530 d T-1 48.90 51.5 1.05
96.40 3.60
O. 1%Tween 560 d T-1 48.9 44.3 0.90 93.59 6.29
590 d T-1 51.0 42.0 0.82 91.04 8.90
10o a 95.48 95.3 1.00 96.4
3.56
10% API 103o a T-1 97.18 102.6 1.05
95.22 4.78
No Tween 1060a T-1 96.7 90.2 0.93
93.89 6.08
1090a T-1 93.8 90.5 0.97 92.72 7.21
100 b 95.76 95.0 0.99 96.4
3.62
10% API 1030 b T-1 97.40 106.4 1.09
95.11 4.89
0.01%Tween 1060 b T-1 96.2 90.9 0.94 93.98 5.98
10901) T-1 95.3 89.0 0.93 92.07 7.85 ,
100c 97.08 93.6 0.96 96.5
3.55
10% API 1030 c T-1 98.17 106.2 1.08
94.35 5.66
0.05%Tween 1060c T-1 97.9 86.7 0.88 92.36 7.65
1090c T-1 96.5 89.6 0.93 90.72 9.22
100 d 98.01 96.4 0.98 96.2
3.81
10% API 1030 d T-1 97.29 102.7 1.05 ,
93.61 6.39
0. 1%Tween 1060d T-1 96.3 91.7 0.95 92.39 7.61
1090d T-1 96.9 85.3 0.88 89.43 10.49
42
,
CA 02900719 2015-08-19
Table 10 (cont.)
Group Sample Protein API
Specific HPLC (% HPLC (%
Description I.D. (mg/nil) (mg/ml) API API API
(I) Activity
Mono.) Aggreg.)
150 a 147.50 147.2 1.00 96.1 3.90
15% API 1530 a T-1 148.60 139.2 0.94 93.00 7.01
No Tween 1560 a T-1 153.5 145.9 0.95 92.29 7.67
1590a T-1 151.2 134.2 0.89 89.56 10.36
150b 148.60 151.6 1.02 95.9 3.80
15% API 1530 b T-1 152.00 139.2 0.92 92.95 7.06
0.01%Tween 1560 b T-1 152.0 144.5 0.95 91.58 8.40
1590b T-1 151.1 134.6 0.89 88.46 11.39
150c 150.10 149.4 0.99 96.0 4.00
15% API 1530 c T-1 150.20 137.6 0.92 92.07 7.94
0.05%Tween 156o c T-1 154.6 143.9 0.93 , 90.05 9.82
1590c T-1 148.4 145.4 0.98 86.50 13.37
150d 149.00 154.7 1.04 96.2 3.74
15% API 1530 d T-1 152.60 142.4 0.93 91.80 8.21
0.1%Tween 1560 d T-1 154.4 143.0 0.93 89.27 10.65
1590d T-1 147.7 140.5 0.95 _ 85.37 14.38
200a 186.8 192.3 1.03 95.6 4.44
20% API 2030 a T-1 190.7 169.0 0.89 90.95 8.98
NoTween 2060 a T-1 197.8 159.0 0.80 89.19 10.76
2090a T-1 193.1 159.0 0.82 84.93 14.98
20013 191.9 186.5 0.97 95.4 4.59
20% API 2030 b T-1 202.9 175.1 0.86 90.03 9.60
0.01%Tween 2060 b T-1 194.7 159.1 0.82 85.86 14.04
2090b T-1 192.1 161.1 - 0.84 85.19 14.70
20o c 194.6 192.2 0.99 95.7 4.34
20% API 2030 c T-1 186.4 169.7 0.91 89.85
10.06
0.05%Tween 2060 c T-1 187.0 160.1 0.86 87.02 12.98
2090 c T-1 195.4 160.9 0.82 82.17 17.75
200 d _ 192.3 190.0 0.99 95.4 4.63
20% API 2030 d T-1 199.2 171.8 0.86 88.83
11.10
0. 1%Tween 2060d T-1 196.5 160.8 0.82 88.20 11.75
2090d T-1 189.0 167.8 0.89 81.39 18.58
*Sample Codes:
I. % - 20 ,15 ,10 ,5API
11. Subscripts: 0-time zero, 30-4 weeks, 60-8 weeks, 90-12 weeks
III. a: no Tween, b: 0.01% Tween, c: 0.05% Tween, d: 0.1% Tween
IV. T-1: 25 C, T-2: 2-8 C
Stability of concentrated API preparations prepared as described below and
kept
in the cold at 2-8 C is shown in Table 11 below.
43
,
CA 02900719 2015-08-19
Table 11: Stability of concentrated API preparations stored at 2-8 C.
Group Sample Protein API Specific HPLC
(% HPLC (%
Description I.D. (mg/mL) (mg/mL) API API Mono.)
API
(I) Activity Aggreg.)
5% API 50a 51.92 48.8 0.94 96.8 3.23
No Tween 530 a T-2 48.73 50.3 1.03
96.85 3.15
_ 560 a T-2 49.2 50.4 1.02 96.46 3.55
590 a T-2 50.4 48.6 0.97 96.14 3.87
5% API 50b 50.93 48.2 0.95 97.1 2.81
0.01%Tween 530 b T-2 49.12 52.1 1.06 96.58 3.43
560 b T-2 49.4 48.6 0.98 95.83 4.18
590 b T-2 50.8 45.2 0.89 95.55 4.46
500 51.87 49.3 0.95 96.7 3.30
5% API 530 c T-2 49.67 52.1 1.05 95.60 4.41
0.05%Tween 560 c T-2 49.3 48.4 0.98 94.89 5.12
590 c T-2 49.4 48.6 0.98 93.56 6.45
50 d 51.81 47.8 0.92 96.4 3.62
5% API 530d T-2 49.06 52.8 1.08 94.52 5.35
0. 1%Tween 560d T-2 49.4 47.2 0.96 92.94 7.07
590 d T-2 49.9 46.2 0.93 91.68 8.32
100 a 95.48 95.3 1.00 96.4 3.56
10% API 1030 a T-2 96.58 103.9 1.08 96.18 3.82
No Tween 1060 a T-2 96.9 94.1 0.97
95.53 4.48
1090 a T-2 96.6 99.7 1.03 95.03 4.89
10013 95.76 95.0 0.99 96.4 3.62
10% API 1030 b T-2 97.02 108.0 1.11 96.06 3.95
0.01%Tween 1060 b T-2 96.6 95.7 0.99 95.36 4.65
1090 b T-2 97.3 97.4 1.00 94.15 5.82
100c 97.08 93.6 0.96 96.5 3.55
10% API 1030c T-2 97.73 98.40 1.01 95.18 4.72
0.05%Tween 1060c T-2 97.5 96.5 0.99 94.82 5.19
1090 c T-2 97.3 97.9 1.01 93.32 6.64
100 d 98.01 96.4 0.98 96.2 3.81
10% API 1030d T-2 96.96 106.7 1.10 94.59 5.41
O. 1%Tween 1060d T-2 99.7 97.5 0.98 93.66 6.35
1090 d T-2 96.9 93.9 0.97 91.37 8.51
150 a 147.50 147.2 1.00 96.1 3.90
15% API 1530 a T-2 150.00 150.7 1.00 94.95 5.06
No Tween 1560 a T-2 150.6 145.5 0.97 95.17 4.85
1590 a T-2 146.7 149.7 1.02 93.88 6.06
150b 148.60 151.6 1.02 95.9 3.80
15% API 1530 b T-2 150.40 145.0 0.96 94.90 5.21
0.01%Tween 1560 b T-2 149.1 149.3 1.00 94.26 5.75
1590 b T-2 146.6 153.0 1.04 93.11 6.90
44
-
,
CA 02900719 2015-08-19
Table 11 (Cont.)
Group Sample Protein API
Specific HPLC (% HPLC (%
Description I.D. (mg/mL) (mg/mL) API API Mono.) API
(*) _ Activity Aggreg.)
150c 150.10 149.4 0.99 96.0
4.00
15% API 1530 c T-2- 150.20 138.9 0.92 95.00
= 4.84
0.05%Tween 1560c T-2 152.1 150.1 0.99 93.67 6.33
1590 c T-2 146.7 149.5 1.02 92.24 7.77
150d 149.00 154.7 1.04 96.2
3.74
15% API 1530 d T-2 149.00 141.8 0.95
94.90 5.08
0.1%Tween 1560 d T-2 152.5 146.8 0.96 93.18 6.83
1590 d T-2 146.9 _ 148.0 1.01 91.10 8.92
200 a _ 186.8 192.3 1.03 95.6 4.44
20% API 2030 a T-2 194.7 185.8 0.95 94.03
5.99
NoTween 2060a T-2 194.5 190.9 0.98 93.90
6.11
2090 a T-2 190.3 194.8 1.02 92.33 7.47
200b 191.9 186.5 0.97 95.4
4.59
20% API 2030 b T-2 200.3 189.9 0.95 94.03
5.97
0.01%Tween 2060 b T-2 192.9 194.9 1.01 93.55 6.46
2090 b T-2 189.8 188.8 0.99 91.85 7.87
200 c 194.6 192.2 0.99 95.7
4.34
20% API 2030 c T-2 207.7 193.6 0.93 93.75
6.26
0.05%Tween 2060 c T-2 189.7 202.4 1.07 92.36 7.65
2090 c T-2 196.0 189.9 0.97 91.25 8.57
200d 192.3 190.0 0.99 95.4
4.63
20% API 2030d T-2 198.2 190.3 0.96 93.33
6.67
O. 1%Tween 2060d T-2 190.2 202.0 1.06 92.69 7.32
2090 d T-2 196.4 185.8 0.95 90.10 9.53
*Sample Codes:
I. 5, 10, 15, 20 - % API
11. Subscripts: 0-time zero, 30-4 weeks, 60-8 weeks, 90-12 weeks
III. a: no Tween, b: 0.01% Tween, c: 0.05% Tween, d: 0.1% Tween
Iv. T-1: 25 C, T-2: 2-8 C
The data shown in the above tables demonstrate that the decline in the
percentage
of the active monomeric form of API is not always reflected by the activity
data
obtained by the elastase assay. Therefore, API activity was evaluated by the
percentage
of API monomers measured by HPLC.
Graphic presentation (not shown) was used to estimate the stability of API
stored
in the above-described conditions after 12 and 24. The results are summarized
in Table
12 below.
CA 02900719 2015-08-19
Table 12: Expected percentage of API monomers after storage for 12 month at 2-
8 C.
Concentration of Concentration of Percent of API Monomer
API, % Tween 80, % Time 12 24
zero months months
0 97.0 94.1 91.3
0.01 90.2 83.1
0.05 84.3 71.7
0.1 77.0 57.4
0 96.6 90.9 85.2
0.01 87.7 78.7
0.05 84.6 72.6
0.1 78.0 59.4
0 96.2 88.3 80.4
0.01 84.1 72.0
0.05 80.6 65.0
0.1 75.4 54.6
0 95.6 84.2 72.8
0.01 82.2 70.0
0.05 78.3 61.0
0.1 76.6 57.6
Conclusions
5 The use of
ultrafiltration for concentrating the 2% solution obtained by the
process of the present caused some generation of aggregates. Thus, the
concentrated
solutions already contained higher aggregate concentration at the beginning of
the assay
compared to the aggregate concentration in the initial 2% solution. The
presence of
Tween 80 in the concentrated solution caused an increase in aggregation
through the
10 storage
period. This effect is concentration dependent. In addition, increase in the
initial
concentration of API caused an increase in the formation of aggregates. This
phenomenon was observed throughout the assay, in both temperature conditions
and
with or without Tween 80. However, general analyses of all the data obtained
clearly
show that concentrated solutions of API stored at 2-8 C are essentially stable
for at least
15 3 months,
and will maintain a good potency at this temperature for one year, even in the
presence of some Tween 80. Therefore, the ready to use API-containing fluid
preparation produced by the process of the present invention is highly
suitable for the
preparation of pharmaceutical compositions to be administered parenterally as
well as
by inhalation.
46
CA 02900719 2015-08-19
The foregoing description of the specific embodiments will so fully reveal the
general nature of the invention that others can, by applying current
knowledge, readily
modify and/or adapt for various applications such specific embodiments without
undue
experimentation and without departing from the generic concept, and,
therefore, such
adaptations and modifications should and are intended to be comprehended
within the
meaning and range of equivalents of the disclosed embodiments. It is to be
understood
that the phraseology or terminology employed herein is for the purpose of
description
and not of limitation. The means, materials, and steps for carrying out
various disclosed
chemical structures and functions may take a variety of alternative forms
without
departing from the invention.
47
