Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SYSTEM AND PROCESS FOR PRODUCING MULTI-COMPONENT
BIOPHARMACEUTICALS
FIELD OF STUDY
This disclosure relates to devices and methods for preparing multi-component
biopharmaceutical formulations within a closed manufacturing system.
BACKGROUND
Biopharmaceutical formulations often consist of multiple active ingredients
within a single composition. Vaccines are one of the most familiar product
types that
comprise multiple biological and non-biological components in single
formulation.
Those skilled in the art often encounter challenges in preparing such
formulations
including system clogging, inaccuracies, and low binding of active
ingredients. Such
problems may be overcome by using a closed, disposable system (e.g., "single
use").
Thus, there is a recognized need in the art for such a system. Single-use
processing has
major advantages over conventional methods, such as a lower potential for
contamination
(e.g., particulates and bioburden), reduced capital expenditure, and
elimination of in-
house cleaning and sterilization steps. Exemplary systems are described below.
SUMMARY OF THE DISCLOSURE
Described herein are sterile, closed, disposable systems for formulating a
biopharmaceutical composition comprising multiple active agents. The system
typically
includes multiple components (or parts) linked in series, the components
typically being
one or more buffer reservoirs; multiple reservoirs of active agents, each
reservoir
containing a different active agent or combination of active agents; one or
more pumps;
one or more sterilizing filters; and, a station for mixing the formulations
with one
another, the station typically including at least one intermediate formulation
reservoir
corresponding to each reservoirs in (b), optionally at least one auxiliary
reservoir
containing one or more additional components, and at least one pump for
combining the
contents of each load cell and the auxiliary reservoir in a final bulk
formulation reservoir.
In certain embodiments, one or more additional components may also be added to
form a
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final formulation. In some embodiments, the active agent may be an antigen and
/ or the
one or more additional components may be one or more adjuvants. Other
embodiments
are described in and / or may be derived from the description provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Exemplary sterile, closed, disposable system.
Figure 2. Exemplary system for blending intermediate formulations into a final
formulated bulk bag.
Figure 3. Exemplary sterile, closed, disposable system.
Figure 4. Exemplary system for blending intermediate formulations into a final
formulated bulk bag.
Figure 5. Prior art process, "Scenario 2", where proteins are filtered while
being added
to the formulation bag, diluted and alum-adjuvanted.
Figure 6. New process, "Scenario 1, Part A", where proteins are first
individually
adjuvanted.
Figure 7. New process, "Scenario 1, Part B", where proteins are filtered,
individually
adjuvanted and diluted.
Figure 8. Exemplary filtration assembly.
Figure 9. Study CA-08-077, Scenario 1, Part A: Formulation and Sampling
Assembly.
Figure 10. Study CA-08-077, Scenario 1, Part B: Trivalent (adj) Formulation
and
Sampling Assembly.
Figure 11. Study CA-08-077-C, Scenario 2: (Unadjuvanted) Proteins added to
formulation system while filtered.
Figure 12. Multivalent Broth Formulation Assembly. The manifold on the left
represents
the lines from each antigen, buffer and excipient used in the process.
Figure 13. Exemplary sterile, closed, disposable system showing case (A)
holding pinch
valve assemblies, the control system (B) for reading inputs and feed outputs,
and the load
cell control panel and display (C).
Figure 14. Exemplary sterile, closed, disposable system with pinch valves (IA,
1B, 1C,
2A, 2B, 2C, 3A, 3B, 3C, 4A, 4B, 4Q.
Figure 15. Exemplary container.
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Figure 16. Exemplary system.
DETAILED DESCRIPTION
A sterile, closed, disposable system for formulating biopharmaceutical
compositions containing multiple active agents is described herein. The system
typically
includes multiple components (or parts) linked in series, the components
typically being
one or more buffer reservoirs; multiple reservoirs of active agents, each
reservoir
containing a different active agent or combination of active agents; one or
more pumps;
one or more sterilizing filters; and, a station for mixing the formulations
with one
another, the station typically including at least one intermediate formulation
reservoir
corresponding to each reservoirs in (b), optionally at least one auxiliary
reservoir
containing one or more additional components, and at least one pump for
combining the
contents of each load cell and the auxiliary reservoir in a final bulk
formulation reservoir.
In certain embodiments, the system includes a buffer reservoir; multiple
reservoirs of
active agents, each reservoir containing a different active agent or
combination of active
agents; one or more pumps; one or more sterilizing filters; multiple single-
use, pre-
sterilized bags, each bag containing a formulation of an active agent or
combination of
active agents corresponding to those in the reservoirs; and, a station for
mixing the
formulations contained within the bags with one another to produce a final
formulation,
where these parts are operably linked to one another in series. The system may
also
comprise one or more reservoirs for waste materials. Any or all of these parts
may
comprise the system described herein. In certain embodiments, one or more
additional
components (e.g, an adjuvant) may also be added to form a final formulation.
In some
embodiments, the active agent may be an antigen and / or the one or more
additional
components may be one or more adjuvants. Other embodiments are described in
and / or
may be derived from the description provided herein.
The process typically begins with a concentrated, purified active agent (e.g.,
protein) and ends with a sterile, filtered, final formulated bulk ready for
sterile connection
to a filling line (e.g., for a vaccine). The process may include, for example,
mixing a
purified protein with a buffer and / or excipient, filtering the mixture and
optionally
adding adjuvant to form an intermediate stock solution, optionally adding
additional
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buffer and / or excipient as needed, and blending the intermediate stock
solutions to form
a final bulk formulation. Thus, in some embodiments, a pre-filter integrity
test, a double
(e.g., 2X) sterile filtration of concentrated purified protein(s) (e.g.,
active agent), and the
addition of one or more buffers (and optionally one or more excipients) is
performed.
Each protein may be added to an intermediate bag following a thorough flushing
of the
lines with buffer. Proteins may be adjuvanted in these bags, with one
intermediate bag
dedicated to each protein, and mixed to ensure adequate binding activities.
For instance,
proteins may be adsorbed to an aluminum adjuvant (e.g., aluminium phosphate
(ALPO4),
aluminum hydroxide (A1OOH), phosphate treated ALOOH) to nearly 100% or 100%. A
post-filter integrity test may then be performed on the final filter. The next
step may
involve combining the individual adjuvanted proteins from the intermediate
bags into a
final 5L formulation bag. The blended formulation may then be further diluted
with
adjuvant top-up to achieve desired concentrations within the multi-component
biopharmaceutical formulation. In one embodiment, the process provides for a
vaccine
formulation comprising multiple antigens, at least one adjuvant, and buffers
and / or
excipients in the final product. The stages of the process preferably include,
for example,
filtration, intermediate formulation, final formulation, and blending (Figs. 1
- 4). Other
embodiments are also contemplated.
The parts of the system described herein are typically operably linked to one
another in series to provide a closed system for producing a multi-component
biopharmaceutical formulation. For example, the system may include a buffer
reservoir
linked to multiple reservoirs of active agents, each reservoir containing a
different active
agent or combination of active agents, which may be driven through a
sterilizing filter
using a pump, and then into one or more optional single-use, pre-sterilized
bags that may
optionally contain additional components (e.g., one or more adjuvants) that
may each
containing a formulation of an active agent or combination of active agents
with or
without one or more additional components, and then into a single container
(which may
contain one or more additional components (e.g., one or more adjuvants))
linked to a
station for mixing the formulations contained within the bags with one
another. In this
way, multiple active agents and / or additional components may be mixed into a
single
formulation. The system is useful for preparing a wide variety of multi-
component
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biopharmaceutical products (e.g., containing multiple active agents). This
system may
combine, for example, ingredient addition (e.g., buffers, active agents,
additional
components such as adjuvants), filtering, and blending into sterile, operably
linked
processing lines and, in particular, assemblies. A particular advantage of
this system is
that the process is contained to maintain sterility. The process is based on
displacement of
fluid in the lines, in-process measurement in the bags during addition, and
filtration of
multiple components through the same filtration assembly before mixing.
Another
advantage is that the reservoirs, bags, tubing and other materials may be
disposable. The
reservoirs, bags and tubing and other materials that contact stock solutions
(e.g.,
containing active agents) are typically manufactured of a material that in not
reactive
with the active agent such that the active agent maintains its integrity when
stored
therein.
The system described herein also typically contains one or more pumps. For
example, the system may include one or more peristaltic pumps. Suitable pumps
include
but are not limited to Masterflex or Watson-Marlow pumps or any other pump
known to
one skilled in the art. As for the reservoirs described above, the single-use,
pre-sterilized
bags for containing a formulation of an active agent or combination of active
agents
corresponding to those in the reservoirs are typically manufactured of a
material that in
not reactive with the active agent such that the active agent maintains its
integrity when
stored therein. Exemplary materials are readily available in the art.
The disposable items are preferably gamma sterilized and assembled using a
sterile connection device such as a tube welder or Kleenpak connector. To
prevent
multiple sterile filter integrity testing, taking up to 15 minutes per filter,
a 2X sterile
filtration assembly has been designed and may be utilized to filter all
filterable
components in one closed, single-use process. Using hanging load cell
technology and
movement of fluid in the lines by displacement, components may be dispensed
accurately
into the bags. The dispensing volumes by weight may be calculated using known
concentration values, specific gravity, and an expected final formulation
volume.
It is preferred that the system or parts of the system are maintained in a
sterile,
closed environment without direct contact with the formulation unless the
system or part
of the system ' is also sterile, preferably single use, and maintains the
sterile liquid
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pathway of the closed system assembly. A single-use system offers flexibility
to such
changes, allows faster scale-up compared with standard technologies (stainless
steel
counterparts) (Cardona and Allen, 2006), and provides cost advantages.
Exemplary
single-use assemblies (e.g., as shown in the Examples) consist of two (2) and
three (3) D
bags connected to a manifold of tubing, connectors, and filters but variations
are also
possible. The bags used in the Examples were custom-made by the bag
manufacturer,
assembled, sealed into bags, and gamma-irradiated using a validated
sterilization method.
The films and tubings used in the systems described herein preferably exhibit
inert
compatibility properties, gamma-irradiation stability, quality testing,
biological safety
testing, and low leachables / extractables profile (Cardona and Allen, 2006).
The films
and tubings utilized in the system are preferably consistent for each
component. The
bags, tubing and filters are supported by stands and holding apparatuses
assuring proper
alignment and dispensing control for the connections. The system also
typically includes
reinforced tubing to meet pressure requirements for inline filter integrity
testing of the
liquid sterilizing grade filters (Cardona and Allen, 2006). In both pre- and
post- integrity
testing, after flushing the final filter with buffer, compressed air may be
applied from the
filter tester, connected in-line, through a 0.2- m vent filter just upstream
of the final
filter.
For a disposables filtration and formulation design, the user will typically
consider and adjust as necessary any of chemical composition of the active
agent or other
components utilized, the concentration thereof, pH, viscosity, solubility,
particle size,
osmolarity, ionic strength, surfactant addition, shear sensitivity, specific
gravity, product
internal reactions (desired or undesired), and inter-component compatibility
prior to
manufacturing (Cardona and Allen, 2006; Motzkau and Okhio, 2005; Luckiewicz,
2004).
Dispensing volumes by weight may be calculated using known concentration
values,
specific gravities, and an expected final formulation volume. Setup is
typically
performed with processing liquids that have fluid properties similar to water
including
density, viscosity, and pH (physiological). Once the proteins and other
constituents are
primed to the main processing line, the line may be flushed with buffer to an
in-line
waste bag. Addition of other components may be performed after zeroing a
connected
formulation bag on a hanging load cell and peristaltic pumping the desired
amount of
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volume by weight. Protein and buffer solutions may be passed through a closed,
disposable sterilizing grade filtration assembly into a sterile bag where
additional
components (e.g., adjuvant) may be directly added. For vaccines, at this time,
a
technology does not exist to sterile filter the aluminum-based adjuvant due to
the
aluminum particle size; however, such filtration is contemplated herein. At
this time,
adjuvant is added directly to the formulation intermediates. This allows pre-
adsorption of
the antigens onto the aluminum-based adjuvant, a requirement for the
processing of some
vaccine products. The ingredient lines are connected to the disposable,
sterile assemblies
using a sterile connection device, such as a tube welder or sterile connector.
A standard sterilizing filtration has four process stages:
preparation/flushing, pre-
integrity testing, filtration, and post-integrity testing (Baumfalk and
Finazzo, 2006). To
prevent multiple sterile filter integrity tests for single component
filtration, each taking up
to 15 minutes per filter, an assembly was designed to sterile filter
components in one
closed, single-use process. A second filter may be added as a redundant step
to satisfy
regulatory expectations. In-line bioburden sampling also preferably supports
no more
than 10 colony-forming units / 100 mL of product to be filtered. The system
described
herein also contains one or more sterilizing filters, having a pore size at
least 0.5 m, and
more preferably 0.1-0.45 m in size. Filters are typically included as well.
Filters may
be of any suitable pore size, but are typically from about 0.2 to 0.7 m.
Other suitable
pore sizes include, for example, about 0.22, 0.45, 0.5, and / or 0.65 m.
Suitable filter
materials include but are not limited to, for example, polyvinylidene fluoride
(PVDF) and
polyethersulphone (PES), or combinations thereof (PVDF/PES). Suitable filters
include,
for example, Millipore Millipak 20, Sartorius Sartopore 2, Pall EBV, Pall EKV
and / or
Pall EDF. Where more than one filter is used in the system, the filters may be
the same
or different according to either brand or pore size. For instance, where two
filters are
utilized, the first may have a pore size of about 0.2 to 0.7 m (e.g., about
0.22, 0.45, 0.5,
and / or 0.65 m) and second a pore size of about 0.2 to 0.7 m (e.g., about
0.22, 0.45,
0.5, and / or 0.65 m). In certain embodiments, the first filter has a pore
size of about
0.22, 0.45, 0.5, and / or 0.65 m and the second a pore size of about 0.22 m.
A number
of filtration studies may be carried out to demonstrate that process outputs
fall within
expected error ranges or satisfy pre-determined criteria for successful
multivalent
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filtration and formulation. These processes are typically carried out at
ambient
temperature, although other temperatures may also be utilized. Other suitable
filters and
filtration systems may also be suitable as would be understood by one of skill
in the art.
For example, a sterile, closed, disposable system includes, for example, one
or
5, more single-use, pre-sterilized bags, filters, connectors, and/or tubing
assemblies as
shown in Figure 1. A preliminary step in using the system may include flushing
of the
lines and an inline, pre-filter integrity test. Fig. 2 illustrates an
exemplary system for
blending intermediate formulations into a final formulated bulk bag. Dilution
and
addition of other components (e.g., adjuvant(s)) may take place at this step
to achieve the
desired final concentrations. Bulk product may be tube sealed from the line
for mixing
prior to filling. Individual formulations of a single active agent (e.g., an
antigen) may
also be diluted from the original bulk concentration and adjuvanted for
individual pre-
adsorption. Downstream of the final sterility filter, the system may be
considered
"closed" from the surrounding environment, eliminating the need for ISO Class
5/Grade
A clean room or isolator conditions, increasing sterility assurance, and
reducing cleaning
steps, cost and energy.
The system may involve passing individual or combined components (e.g., active
agent(s), buffer(s), and / or surfactant(s)) of the multi-component
biopharmaceutical
formulation solutions through a closed, disposable sterilizing grade
filtration assembly
into a sterile bag where one or more additional components (e.g., one or more
adjuvants)
are directly added to the formulation. Thus, proteins (e.g., antigens) may be
individually
(optionally) combined with other components (e.g., adjuvant(s)) and diluted in
intermediate bags. For vaccines, this allows pre-adsorption of the antigens
onto the
adjuvant (e.g., an aluminum-based adjuvant) as a formulated intermediate stock
of each
antigen prior to final blending into the final formulation, and may be
referred to as an
intermediate stock antigen formulations.
Multiple active agents (e.g., antigens) may be filtered through the same dual
filter
assembly with buffer flushing through the filter between each protein
filtration to remove
the residual proteins from the filter for formulation of intermediate
individual stock
antigen formulations. The intermediate formulations serve three purposes (with
respect to
vaccines):
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= pre-adsorption of an individual antigen onto an adjuvant to better control
cross-interactions;
= dilution from a highly concentrated bulk (up to 60 times higher than the
final
formulation concentrations), which allows processing a greater volume of
lower concentrate in the lines and into the final formulated bulk; and,
= ability to store and re-purpose the intermediate stock concentrates for
other
similar formulations (e.g., bivalent, trivalent, quadrivalent, pentavalent) or
doses.
Instead of blending all proteins together prior to filtration, the system
described
herein allows for controlled, successive protein filtration, preventing
potential unwanted
interactions at filter face (e.g., binding, clogging). Important parameters
involved in filter
selection include materials, compatibility, wettability, sterilization,
adsorption, structure,
and membrane pore size, distribution and thickness (Cardona and Inseal, 2006;
Motzkau
and Okhio, 2005). In addition, to compare the performance of these filters
further,
throughput per square meter of the filters can be measured; though one must
consider the
geometry and effective filtration area to avoid non-linear calculations
(Priebe and Jornitz,
2006). The effluent should be tested to ensure minimal protein loss (Cordona
and Inseal,
2006). Lower flushing volumes reduce waste and time of processing while still
maintaining a high quality of filtrate. Once the desired filtration system has
been fully
developed, additional performance testing including microbial retention,
integrity and
extractables/leachables should be initiated (Motzkau and Okhio, 2005). Further
adsorption studies are necessary at time of process validation (Motzkau and
Okhio,
2005).
In the system described herein, ingredient addition may be based on product
specific gravity, desired volumes by weight, and zeroing of bag weight in-line
prior to
addition. Small bags (1L) in series, such as those containing intermediates,
are prone to
moving around on scales or balances, leading to inaccuracies when attempting
to measure
weight in bags. Accordingly, load cells may be supported by a post and
bracketing
assembly designed to weigh suspended bags during addition. These may be
selected for
their ability to withstand measurement disturbances from side loads (bag
swaying) and
they have moveable load points, making it convenient to hang bags of different
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configurations. In addition, these should have high individual accuracy with a
combined
error of 0.02 % and repeatability of 0.01 %, and be designed to discount
measurements due
to thermal or vibration interference. Bags may be primed, tared, and weighed
using the
device with a microprocessor-based control with display. Consideration should
also be
made for flow into intermediate and final formulation bags as they are
suspended while
ingredients are pumped into these bags. Pumping activities must be properly
sequenced
with opening and closing of the lines. For small scale, this may be
accomplished
manually. Readings from the load cells once ingredients may be pumped into the
hanging
bags preferably have an average percentage difference of 0.15% (n = 35,
practical
minimum and maximum weights applied) compared with target weight.
In addition to weighing, manufacturing of multi-component biopharmaceutical
compositions in disposables can require a variety of processes that occur in
parallel or
immediately following component addition, including:
= Suspension of ingredients prior to and during dispensing;
= Blending of intermediates or final formulated bulks after ingredient
addition;
= Dissolving;
= Storage;
= Heating/cooling;
= Suspension of final formulation prior to and during filling
Mechanical attributes to interface these processes should also be considered,
including
but not limited to:
= Type of mixing (e.g., wave, impeller, paddle) ;
= Agitator location, shape, and size relative to vessel, as applicable;
= Mixing system parameters (e.g., speed, pitch);
= Processing line length and diameter;
= Bag and holding vessel size, shape, rigidity, placement;
= Bag assembly suitable processing and storage temperature ranges; and,
= Bag internal/external accessories (e.g., tubing, baffles, jacketing) and
sterilization of product contact mixing components.
During a final blending step or a fill, material may need to stay suspended in
the
bag while dispensed either into another container or directly to the final
filled container
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(e.g., vial, syringe). The location of the outlet line is critical in that the
line needs to be
pumped out of the container during mixing without addition of air in the
lines. A dip tube
or bottom drain is ideal for preventing air from getting into the lines,
otherwise air must
be evacuated from the bags. "Scaling up" this system may require a rigid
container to
hold the bags in the assembly and inverting load cells, as well as larger
capacity bags,
filters, connectors, tubing, pumps, mixing, weighing and controls to reduce
processing
steps.
Common quantitative indicators used to measure mixing studies are typically
those that test a variable as a function of mixing time (Tin), such as when
turbidity, pH,
or conductivity reaches steady state or homogeneity at Tin. When mixing
systems are
engineered around a multivalent product, chemical and physical characteristics
(e.g.,
foaming-prone excipients, aggregation-prone proteins and heavy mineral-based
suspensions), ingredients, stage of manufacturing and target volumes and
concentrations
must be considered. While the above-mentioned test methods support certain
aspects of
efficient mixing, for a multivalent formulation, the product and adjuvant
concentrations,
for vaccines, antigen adsorption to adjuvant and other exchanges must also be
tested as a
function of Tin or predetermined mixing parameters. Additional studies may be
performed to quantify protein loss across the filtration assembly,
identification of
leachables from disposable assemblies with surfactants and adjuvants,
determination if
order of component addition induces unwanted aggregation and characterization
of
process conditions.
A "station" is also typically provided for mixing the formulations contained
within the bags with one another to produce a final formulation. This station
typically
includes the devices needed to combine the formulations together to produce a
sterile,
homogenous mixture thereof. For instance, the station may comprise a container
for the
formulation that is compatible with a device or system for mixing or
homogenizing the
formulation without disrupting the integrity of the active agents contained
therein. For
instance, the mixing or homogenizing system may include a magnetic stir bar
and a stir
plate including a source of magnetic energy for rotating the magnetic stir bar
that is
contained within the bag. Alternatively, a pump may be utilized.
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Buffers may be used to maintain the stability and otherwise support the
integrity
of the components forming the biopharmaceutical formulation. A suitable buffer
is any
that exerts a desired effect upon the formulation. For instance, a buffer may
be used to
provide, stabilize, and / or maintain the pH of the formulation. Exemplary
buffers that
may be used as described herein include but are not limited to, for example,
TAPS (3-
{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid, pH 7.7-9.1), bicine
(N,N-
bis(2-hydroxyethyl)glycine, pH 7.6-9.0), tris (tris(hydroxymethyl)methylamine,
pH 7.5-
9.0; e.g, Tris-HC1), HEPES (4-2-hydroxyethyl-l-piperazineethanesulfonic acid,
pH 6.8-
8.2), TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid, pH. 6.8-
8.2),
MOPS (3-(N-morpholino)propanesulfonic acid, pH 6.5-7.9), PIPES (piperazine-
N,N'-
bis(2-ethanesulfonic acid), pH 6.1-7.5), cacodylate (dimethylarsinic acid, pH
5.0-7.4),
and MES (2-(N-morpholino)ethanesulfonic acid, pH 5.5-6.7), among others. These
buffers are typically contained within individual reservoirs of the system but
may also be
part of the composition comprising a stock solution of active agent. Many
other suitable
buffers are known to those of skill in the art.
The system described herein also typically contains more than one reservoir
containing one or more active agents. Active agents may include any that
provide a
desired effect (e.g., a therapeutic effect) of the biopharmaceutical
formulation upon a host
(e.g., human, animal) to whom or to which it is administered. Active agents
may be
contained within reservoirs alone or in combination with other active agents.
Active
agents may also be contained within reservoirs with "inactive agents" such as,
for
example, buffers or other components that are not necessarily active agents.
Active
agents may include antigens, antibodies, hormones, and / or growth factors,
and may be
combined with additional components such as adjuvants, any of which may be in
purified
form, and may be used alone or in combination with one another.
In some embodiments, the antigens may include one or more "immunogens" for
inducing or enhancing an immune response that is beneficial to the host. An
immunogen
may be a moiety (e.g., polypeptide, peptide or nucleic acid) that induces or
enhances the
immune response of a host to whom or to which the immunogen is administered.
An
immune response may be induced or enhanced by either increasing or decreasing
the
frequency, amount, or half-life of a particular immune modulator (e.g, the
expression of a
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cytokine, chemokine, co-stimulatory molecule). This may be directly observed
within a
host cell or within a nearby cell or tissue (e.g., indirectly). The immune
response is
typically directed against a target antigen. For example, an immune response
may result
from expression of an immunogen in a host following administration thereof to
the host.
The immune response may result in one or more of an effect (e.g., maturation,
proliferation, direct- or cross-presentation of antigen, gene expression
profile) on cells of
either the innate or adaptive immune system. For example, the immune response
may
involve, effect, or be detected in innate immune cells such as, for example,
dendritic
cells, monocytes, macrophages, natural killer cells, and / or granulocytes
(e.g.,
neutrophils, basophils or eosinophils). The immune response may also involve,
effect, or
be detected in adaptive immune cells including, for example, lymphocytes
(e.g., T cells
and / or B cells). The immune response may be observed by detecting such
involvement
or effects including, for example, the presence, absence, or altered (e.g.,
increased or
decreased) expression or activity of one or more immunomodulators such as a
hormone,
cytokine, interleukin (e.g., any of IL-1 through IL-35), interferon (e.g., any
of IFN-I
(IFN-a, IFN-(3, IFN-E, IFN-K, IFN-T, IFN-c, IFN-(o), IFN-II (e.g., IFN-y), IFN-
III (IFN-
X1, IFN- A2, IFN- X3)), chemokine (e.g., any CC cytokine (e.g., any of CCL1
through
CCL28), any CXC chemokine (e.g., any of CXCL1 through CXCL24), Mipla), any C
chemokine (e.g., XCL1, XCL2), any CX3C chemokine (e.g., CX3CL1)), tumor
necrosis
factor (e.g., TNF-(x, TNF-pi)), negative regulators (e.g., PD-1, IL-T) and /
or any of the
cellular components (e.g., kinases, lipases, nucleases, transcription-related
factors (e.g.,
IRF-1, IRF-7, STAT-5, NFKB, STAT3, STAT1, IRF-10), and / or cell surface
markers
suppressed or induced by such immunomodulators) involved in the expression of
such
immunomodulators. The presence, absence or altered expression may be detected
within
cells of interest or near those cells (e.g., within a cell culture
supernatant, nearby cell or
tissue in vitro or in vivo, and / or in blood or plasma). Administration of
the immunogen
may induce (e.g., stimulate a de novo or previously undetected response),
enhance and /
or suppress an existing response against the immunogen by, for example,
causing an
increased antibody response (e.g., amount of antibody, increased affinity /
avidity) or an
increased cellular response (e.g., increased number of activated T cells,
increased affinity
/ avidity of T cell receptors). In certain embodiments, the immune response
may be
13
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protective, meaning that the immune response may be capable of preventing
initiation or
continued infection of or growth within a host and / or by eliminating an
agent (e.g., a
causative agent, such as HIV) from the host.
The formulations described herein may include one or more immunogen(s) from a
single source or multiple sources. For instance, immunogens may also be
derived from
or direct an immune response against one or more viruses (e.g., viral target
antigen(s))
including, for example, a dsDNA virus (e.g. adenovirus, herpesvirus, epstein-
barr virus,
herpes simplex type 1, herpes simplex type 2, human herpes virus simplex type
8, human
cytomegalovirus, varicella-zoster virus, poxvirus); ssDNA virus (e.g.,
parvovirus,
papillomavirus (e.g., El, E2, E3, E4, E5, E6, E7, E8, BPV 1, BPV2, BPV3, BPV4,
BPV5
and BPV6 (In Papillomavirus and Human Cancer, edited by H. Pfister (CRC Press,
Inc.
1990); Lancaster et al., Cancer Metast. Rev. pp. 6653-6664 (1987); Pfister, et
al. Adv.
Cancer Res 48, 113-147 (1987)); dsRNA viruses (e.g., reovirus); (4)ssRNA
viruses (e.g.,
picornavirus, coxsackie virus, hepatitis A virus, poliovirus, togavirus,
rubella virus,
flavivirus, hepatitis C virus, yellow fever virus, dengue virus, west Nile
virus); (-)ssRNA
viruses (e.g., orthomyxovirus, influenza virus, rhabdovirus, paramyxovirus,
measles
virus, mumps virus, parainfluenza virus, respiratory syncytial virus,
rhabdovirus, rabies
virus); ssRNA-RT viruses (e.g. retrovirus, human immunodeficiency virus
(HIV)); and,
dsDNA-RT viruses (e.g. hepadnavirus, hepatitis B). Immunogens may also be
derived
from other viruses not listed above but available to one of skill in the art.
With respect to HIV, immunogens may be selected from any HIV isolate. As is
well-known in the art, HIV isolates are now classified into discrete genetic
subtypes.
HIV-1 is known to comprise at least ten subtypes (A, B, C, D, E, F, G, H, J
and K). HIV-
2 is known to include at least five subtypes (A, B, C, D, and E). Subtype B
has been
associated with the HIV epidemic in homosexual men and intravenous drug users
worldwide. Most HIV-1 immunogens, laboratory adapted isolates, reagents and
mapped
epitopes belong to subtype B. In sub-Saharan Africa, India and China, areas
where the
incidence of new HIV infections is high, HIV-1 subtype B accounts for only a
small
minority of infections, and subtype HIV-1 C appears to be the most common
infecting
subtype. Thus, in certain embodiments, it may be preferable to select
immunogens from
HIV-1 subtypes B and / or C. It may be desirable to include immunogens from
multiple
14
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HIV subtypes (e.g., HIV-1 subtypes B and C, HIV-2 subtypes A and B, or a
combination
of HIV-1 and HIV-2 subtypes) in a single immunological formulation. Suitable
HIV
immunogens include ENV, GAG, POL, NEF, as well as variants, derivatives, and
fusion
proteins thereof, for example. Any of these may be encoded by a polynucleotide
within a
recombinant vector, and / or used in combination with a recombinant vector as
part of an
immunization strategy.
Immunogens may also be derived from or direct an immune response against one
or more bacterial species (spp.) (e.g., bacterial target antigen(s))
including, for example,
Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella
pertussis),
Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella
abortus, Brucella
canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g.,
Campylobacter
jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci,
Chlamydia
trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium
difficile,
Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g.,
Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis,
enterococcus faecum), Escherichia spp. (e.g., Escherichia coli), Francisella
spp. (e.g.,
Francisella tularensis), Haemophilus spp. (e.g., Haemophilus influenza),
Helicobacter
spp. (e.g., Helicobacter pylori), Legionella spp. (e.g., Legionella
pneumophila),
Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria
monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium
tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp.
(e.g.,
Neisseria gonorrhea, Neisseria meningitidis), Pseudomonas spp. (e.g.,
Pseudomonas
aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp.
(e.g., Salmonella
typhi, Salmonella typhinurium), Shigella spp. (e.g., Shigella sonnei),
Staphylococcus spp.
(e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus
saprophyticus,
coagulase negative staphylococcus (e.g., U.S. Pat. No. 7,473,762)),
Streptococcus spp.
(e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus
pyrogenes),
Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio
cholerae), and
Yersinia spp. (Yersinia pestis). Exemplary antigens may include, for example,
phtE (also
"protein E"), PcpA ("protein A"), LytB ("protein B"), and PhtD ("protein D")
(see, e.g.,
Examples 2 and 4 herein). Immunogens may also be derived from or direct the
immune
CA 02697804 2010-03-17
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response against other bacterial species not listed above but available to one
of skill in the
art.
Immunogens may also be derived from or direct an immune response against one
one or more fungal species (spp.) may be detected such as, for example,
Actinomyces
spp. (e.g., A. israelii, A. bovis, A. naeslundii), Allescheria spp. (e.g., A.
boydii),
Aspergillus spp. (e.g., A. fumigatus, A. nidulans), Blastomyces spp. (e.g., B.
dermatidis),
Candida spp. (e.g., C. albicans), Cladosporium spp. (e.g., C. carrionii),
Coccidioides spp.
(e.g., C. immitis), Cryptococcus spp. (e.g., C. neoformans), Fonsecaea spp.
(e.g., F.
pedrosoi, F. compacta, F. dermatidis), Histoplasma spp. (e.g., H. capsulatum),
Nocardia
spp. (e.g., N. asteroids, N. brasiliensis), Keratinomyces spp. (e.g., K.
ajelloi), Madurella
spp. (e.g., M. grisea, M. mycetomi), Microsporum spp. (e.g., M. adnouini, M.
gypseum,
M. canis), Mucor spp. (e.g., M. corymbifer, Absidia corymbifera),
Paracoccidioides spp.
(e.g., P. brasiliensis), Phialosphora spp. (e.g., P. jeansilmei, P.
verrucosa), Rhizopus spp.
(e.g., R. oryzae, R. arrhizus, R. nigricans), Sporotrichum spp. (e.g., S.
Schenkii), and
Trichophyton spp. (e.g., T. mentagrophytes, T. rubrum). Immunogens may also be
derived from other fungal species not listed above as would be understood by
one of skill
in the art.
Immunogens may also be derived from or direct an immune response against one
or more parasitic organisms (spp.) (e.g., parasite target antigen(s))
including, for
example, Ancylostoma spp. (e.g., A. duodenale), Anisakis spp., Ascaris
lumbricoides,
Balantidium coli, Cestoda spp., Cimicidae spp., Clonorchis sinensis,
Dicrocoelium
dendriticum, Dicrocoelium hospes, Diphyllobothrium latum, Dracunculus spp.,
Echinococcus spp. (e.g., E. granulosus, E. multilocularis), Entamoeba
histolytica,
Enterobius vermicularis, Fasciola spp. (e.g., F. hepatica, F. magna, F.
gigantica, F.
jacksoni), Fasciolopsis buski, Giardia spp. (Giardia lamblia), Gnathostoma
spp.,
Hymenolepis spp. (e.g., H. nana, H. diminuta), Leishmania spp., Loa loa,
Metorchis spp.
(M. conjunctus, M. albidus), Necator americanus, Oestroidea spp. (e.g.,
botfly),
Onchocercidae spp., Opisthorchis spp. (e.g., O. viverrini, O. felineus, O.
guayaquilensis,
and O. noverca), Plasmodium spp. (e.g., P. falciparum), Protofasciola robusta,
Parafasciolopsis fasciomorphae, Paragonimus westermani, Schistosoma spp.
(e.g., S.
mansoni, S. japonicum, S. mekongi, S. haematobium), Spirometra
erinaceieuropaei,
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Strongyloides stercoralis, Taenia spp. (e.g., T. saginata, T. solium),
Toxocara spp. (e.g.,
T. canis, T. cati), Toxoplasma spp. (e.g., T. gondii), Trichobilharzia
regenti, Trichinella
spiralis, Trichuris trichiura, Trombiculidae spp., Trypanosoma spp., Tunga
penetrans, and
/ or Wuchereria bancrofti. Immunogens may also be derived from or direct the
immune
response against other parasitic organisms not listed above but available to
one of skill in
the art.
Immunogens may be derived from or direct the immune response against tumor
target antigens (e.g., tumor target antigens). The term tumor target antigen
(TA) may
include both tumor-associated antigens (TAAs) and tumor-specific antigens
(TSAs),
where a cancerous cell is the source of the antigen. A TA may be an antigen
that is
expressed on the surface of a tumor cell in higher amounts than is observed on
normal
cells or an antigen that is expressed on normal cells during fetal
development. A TSA is
typically an antigen that is unique to tumor cells and is not expressed on
normal cells.
TAs are typically classified into five categories according to their
expression pattern,
function, or genetic origin: cancer-testis (CT) antigens (e.g., MAGE, NY-ESO-
1);
melanocyte differentiation antigens (e.g., Melan A/MART-1, tyrosinase, gp100);
mutational antigens (e.g., MUM-1, p53, CDK-4); overexpressed `self antigens
(e.g.,
HER-2/neu, p53); and, viral antigens (e.g., HPV, EBV). Suitable TAs include,
for
example, gplOO (Cox et al., Science, 264:716-719 (1994)), MART-1/Melan A
(Kawakami et al., J. Exp. Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et
al., J. Exp.
Med., 186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Immunol.,
24:759-764
(1994)), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom
et al., J. Immunol., 130:1467-1472 (1983)), MAGE family antigens (e.g., MAGE-
1,
2,3,4,6, and 12; Van der Bruggen et al., Science, 254:1643-1647 (1991); U.S.
Pat. Nos.
6,235,525), BAGE family antigens (Boel et at., Immunity, 2:167-175 (1995)),
GAGE
family antigens (e.g., GAGE-1,2; Van den Eynde et al., J. Exp. Med., 182:689-
698
(1995); U.S. Pat. No. 6,013,765), RAGE family antigens (e.g., RAGE-1; Gaugler
et al.,
Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526), N-
acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med., 183:1173-1183
(1996)),
p15 (Robbins et al., J. lmmunol. 154:5944-5950 (1995)), 13-catenin (Robbins et
al., J.
Exp. Med., 183:1185-1192 (1996)), MUM-1 (Coulie et al., Proc. Natl. Acad. Sci.
USA,
17
CA 02697804 2010-03-17
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92:7976-7980 (1995)), cyclin dependent kinase-4 (CDK4) (Wolfel et al.,
Science,
269:1281-1284 (1995)), p21-ras (Fossum et at., Int. J. Cancer, 56:40-45
(1994)), BCR-abl
(Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald et al., Proc.
Natl. Acad. Sci.
USA, 92:11993-11997 (1995)), p185 HER2/neu (erb-B1; Fisk et al., J. Exp. Med.,
181:2109-2117 (1995)), epidermal growth factor receptor (EGFR) (Harris et al.,
Breast
Cancer Res. Treat, 29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et
al., J.
Natl. Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087;
5,571,710;
6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and, EP 784483);
carcinoma-
associated mutated mucins (e.g., MUC-1 gene products; Jerome et al., J.
Immunol.,
151:1654-1662 (1993)); EBNA gene products of EBV (e.g., EBNA-1; Rickinson et
al.,
Cancer Surveys, 13:53-80 (1992)); E7, E6 proteins of human papillomavirus
(Ressing et
al., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA; Xue et
al., The
Prostate, 30:73-78 (1997)); prostate specific membrane antigen (PSMA; Israeli,
et al.,
Cancer Res., 54:1807-1811 (1994)); idiotypic epitopes or antigens, for
example,
immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al., J.
Immunol.,
153:4775-4787 (1994)); KSA (U.S. Patent No. 5,348,887), kinesin 2 (Dietz, et
al.
Biochem Biophys Res Commun 2000 Sep 7;275(3):731-8), HIP-55, TGF(3-1 anti-
apoptotic factor (Toomey, et al. Br J Biomed Sci 2001;58(3):177-83), tumor
protein D52
(Bryne J.A., et al., Genomics, 35:523-532 (1996)), H1FT, NY-BR-1 (WO
01/47959),
NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87 and NY-BR-96 (Scanlan, M. Serologic
and Bioinformatic Approaches to the Identification of Human Tumor Antigens, in
Cancer
Vaccines 2000, Cancer Research Institute, New York, NY), and / or pancreatic
cancer
antigens (e.g., SEQ ID NOS: 1-288 of U.S. Pat. No. 7,473,531). Immunogens may
also
be derived from or direct the immune response against include TAs not listed
above but
available to one of skill in the art.
Vaccines suitable for preparation using the systems described herein are
typically
"multivalent". A multivalent vaccine is an antigenic preparation including
more than one
infectious agent or several different antigenic determinants of a single
agent. For
example, described herein are multivalent vaccines containing at least two to
five
different recombinant proteins formulated as a combination vaccine. The system
described herein is suitable for the development of biopharmaceutical
compositions that
18
CA 02697804 2010-03-17
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may be used anywhere from concept through Phase ' III clinical testing. For
instance,
Phase I/II typically requires less than 200 doses for a trial, while a two to
five liter (2-5 L)
final formulation bulk size is needed.
Active agents may also be antibodies. The term "antibody" or "antibodies"
includes whole or fragmented antibodies in unpurified or partially purified
form (i.e.,
hybridoma supernatant, ascites, polyclonal antisera) or in purified form. A
"purified"
antibody is one that is separated from at least about 50% of the proteins with
which it is
initially found (i.e., as part of a hybridoma supernatant or ascites
preparation).
Preferably, a purified antibody is separated from at least about 60 %, 75 %,
90 %, or 95 %
of the proteins with which it is initially found. Suitable derivatives may
include
fragments (i.e., Fab, Fab2 or single chain antibodies (Fv for example)), as
are known in
the art. The antibodies may be of any suitable origin or form including, for
example,
murine (i.e., produced by murine hybridoma cells), or expressed as humanized
antibodies, chimeric antibodies, human antibodies, and the like. Methods of
preparing
and utilizing various types of antibodies are well-known to those of skill in
the art and
would be suitable in practicing the present invention (see, for example,
Harlow, et al.
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Harlow,
et al.
Using Antibodies: A Laboratory Manual, Portable Protocol No. 1, 1998; Kohler
and
Milstein, Nature, 256:495 (1975)); Jones et al. Nature, 321:522-525 (1986);
Riechmann
et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol., 2:593-596
(1992);
Verhoeyen et al. (Science, 239:1534-1536 (1988); Hoogenboom et al., J. Mol.
Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole et al.,
Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.
Immunol.,
147(1):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg
et al.,
Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et
al., Nature
Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826
(1996);
Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S.
Pat. Nos.
4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and,
5,661,016). In
certain applications, the antibodies may be contained within hybridoma
supernatant or
ascites and utilized either directly as such or following concentration using
standard
techniques. In other applications, the antibodies may be further purified
using, for
19
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example, salt fractionation and ion exchange chromatography, or affinity
chromatography using Protein A, Protein G, Protein A/G, and / or Protein L
ligands
covalently coupled to a solid support such as agarose beads, or combinations
of these
techniques. The antibodies may be stored in any suitable format, including as
a frozen
preparation (i.e., -20 C or -70 C), in lyophilized form, or under normal
refrigeration
conditions (i.e., 4 C). When stored in liquid form, it is preferred that a
suitable buffer
such as Tris-buffered saline (TBS) or phosphate buffered saline (PBS) is
utilized. The
antibodies described herein may be prepared as injectable preparations, such
as in
suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable
vehicles
and solvents that may be utilized include water, Ringer's solution, and
isotonic sodium
chloride solution, TBS and PBS, among others. It is preferred that the
antibodies be
suitable for use in vivo.
Suitable hormones include but are not limted to antidiuretic hormone,
proopiomelanocortin, luteinizing hormone, follicle stimulating hormone,
adrenocorticotrophic hormone, growth hormone, prolactin, melanocyte
stimulating
hormone, thyroid stimulating hormone, insulin, triiodothyronine, thyroxine,
cortisol,
dehydroepiandrostendione, an estrogen (e.g., estradiol, estrone, estriol),
progesterone,
testosterone, dihydrotestosterone, inhibin, progesterone, and estriol, for
example.
Suitable, exemplary growth factors include but are not limited to bone
morphogenic
proteins (BMPs), epidermal growth factor (EGF), erythropoietin (EPO),
fibroblast growth
factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-
macrophage
colony stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9),
hepatocyte
growth factor (HGF), insulin-like growth factor (IGF), myostatin (GDF-8),
neurotrophins
(e.g., nerve growth factor (NGF)), platelet-derived growth factor (PDGF),
thrombopoietin
(TPO), transforming growth factor alpha (TGF-a), transforming growth factor
beta
(TGF-0), and vascular endothelial growth factor (VEGF), among others.
As described above, in certain embodiments, one or more additional components
may also be added to form a final formulation. In some embodiments, the active
agent
may be an antigen and / or the one or more additional components may be one or
more
adjuvants. An immunogen may also be administered in combination with one or
more
adjuvants to boost the immune response. Adjuvants may also be included to
stimulate or
CA 02697804 2010-03-17
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enhance the immune response. Non-limiting examples of suitable adjuvants
include
those of the gel-type (e.g., aluminum hydroxide/phosphate ("alum adjuvants"),
calcium
phosphate), of microbial origin (muramyl dipeptide (MDP)), bacterial exotoxins
(cholera
toxin (CT), native cholera toxin subunit B (CTB), E. coli labile toxin (LT),
pertussis toxin
(PT), CpG oligonucleotides, BCG sequences, tetanus toxoid, monophosphoryl
lipid A
(MPL) of, for example, E. coli, Salmonella minnesota, Salmonella typhimurium,
or
Shigella exseri), particulate adjuvants (biodegradable, polymer microspheres),
immunostimulatory complexes (ISCOMs)), oil-emulsion and surfactant-based
adjuvants
(Freund's incomplete adjuvant (FIA), microfluidized emulsions (MF59, SAF),
saponins
(QS-21)), synthetic (muramyl peptide derivatives (murabutide, threony-MDP),
nonionic
block copolymers (L121), polyphosphazene (PCCP), synthetic polynucleotides
(poly
A :U, poly I:Q, thalidomide derivatives (CC-4407/ACTIMID)), RH3-ligand, or
polylactide glycolide (PLGA) microspheres, among others. Fragments, homologs,
derivatives, and fusions to any of these toxins are also suitable, provided
that they retain
adjuvant activity. Suitable mutants or variants of adjuvants are described,
e.g., in WO
95/17211 (Arg-7- Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO
95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that can
be
used in the methods and compositions of the invention include, e. g., Ser-63-
Lys, Ala-69-
Gly,Glu-110-Asp, and Glu-112-Asp mutants. Other suitable adjuvants are also
well-
known in the art.
As an example, metallic salt adjuvants such alum adjuvants are well-known in
the
art as providing a safe excipient with adjuvant activity. The mechanism of
action of these
adjuvants are thought to include the formation of an antigen depot such that
antigen may
stay at the site of injection for up to 3 weeks after administration, and also
the formation
of antigen/metallic salt complexes which are more easily taken up by antigen
presenting
cells. In addition to aluminium, other metallic salts have been used to adsorb
antigens,
including salts of zinc, calcium, cerium, chromium, iron, and berilium. The
hydroxide
and phosphate salts of aluminium are the most common. Formulations or
compositions
containing aluminium salts, antigen, and an additional immunostimulant are
known in the
art. An example of an immunostimulant is 3-de-O-acylated monophosphoryl lipid
A (3D-
MPL).
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One or more cytokines and / or chemokines may also be suitable adjuvants
(Parmiani, et al. Immunol Lett 2000 Sep 15; 74(1): 41-4; Berzofsky, et at.
Nature
Immunol. 1: 209-219). Suitable cytokines include, for example, interleukin-2
(IL-2)
(Rosenberg, et at. Nature Med. 4: 321-327 (1998)), IL-4, IL-7, IL-12 (reviewed
by
Pardoll, 1992; Harries, et al. J. Gene Med. 2000 Jul-Aug;2(4):243-9; Rao, et
al. J.
Immunol. 156: 3357-3365 (1996)), IL-15 (Xin, et at. Vaccine, 17:858-866,
1999), IL-16
(Cruikshank, et al. J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. Cancer Res.
Clin. Oncol.
2001. 127(12): 718-726), GM-CSF (CSF (Disis, et at. Blood, 88: 202-210
(1996)),
tumor necrosis factor-alpha (TNF-a), or interferon-gamma (INF-y). Chemokines
may
also be utilized. For example, fusion proteins comprising CXCL10 (IP-10) and
CCL7
(MCP-3) fused to a tumor self-antigen have been shown to induce anti-tumor
immunity
(Biragyn, et al. Nature Biotech. 1999, 17: 253-258). The chemokines CCL3 (MIP-
la)
and CCL5 (RANTES) (Boyer, et at. Vaccine, 1999, 17 (Supp. 2): S53-S64) may
also be
of use in practicing the present invention. Other suitable cytokines and
chemokines are
known in the art.
Formulations produced as described herein may be prepared as pharmaceutical
compositions. The pharmaceutical composition may be administered orally,
parentally,
by inhalation spray, rectally, intranodally, or topically in dosage unit
formulations
containing conventional pharmaceutically acceptable carriers, adjuvants, and
vehicles.
The term "pharmaceutically acceptable carrier" or "physiologically acceptable
carrier" as
used herein refers to one or more formulation materials suitable for
accomplishing or
enhancing the delivery of a nucleic acid, polypeptide, or peptide as a
pharmaceutical
composition. A "pharmaceutical composition" may be a composition comprising a
therapeutically effective amount of an active agent contained within a
formulation. The
terms "effective amount" and "therapeutically effective amount" each refer to
the amount
of active agent required to observe the desired therapeutic effect (e.g.,
induce or enhance
and immune response).
Injectable preparations, such as sterile injectable aqueous or oleaginous
suspensions, may be formulated according to known methods using suitable
dispersing or
wetting agents and suspending agents. The injectable preparation may also be a
sterile
injectable solution or suspension in a non-toxic parenterally acceptable
diluent or solvent.
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Suitable vehicles and solvents that may be employed are water, Ringer's
solution, and
isotonic sodium chloride solution, among others. In addition, sterile, fixed
oils are
conventionally employed as a solvent or suspending medium. For this purpose,
any
bland fixed oil may be employed, including synthetic mono- or diglycerides. In
addition,
fatty acids such as oleic acid find use in the preparation of injectables.
Pharmaceutical compositions may take any of several forms and may be
administered by any of several routes. The compositions may be administered
via a
parenteral route (intradermal, intramuscular or subcutaneous) to induce an
immune
response in the host. Alternatively, the composition may be administered
directly into a
lymph node (intranodal) or tumor mass (e.g., intratumoral administration).
Preferred
embodiments of administratable compositions include, for example, one or more
active
agents in liquid preparations such as suspensions, syrups, or elixirs.
Preferred injectable
preparations include, for example, nucleic acids or polypeptides suitable for
parental,
subcutaneous, intradermal, intramuscular or intravenous administration such as
sterile
suspensions or emulsions. For example, active agents may be prepared in
admixture with
a suitable carrier, diluent, or excipient such as sterile water, physiological
saline, glucose
or the like. The composition may also be provided in lyophilized form for
reconstituting,
for instance, in isotonic aqueous, saline buffer. In addition, the
compositions can be co-
administered or sequentially administered with one another, other antiviral
compounds,
other anti-cancer compounds and/or compounds that reduce or alleviate ill
effects of such
agents.
As previously mentioned, while the compositions described herein may be
administered as the sole active pharmaceutical agent, they can also be used in
combination with one or more other compositions or agents (e.g., other
immunogens, co-
stimulatory molecules, adjuvants). When administered as a combination, the
individual
components can be formulated as separate compositions administered at the same
time or
different times, or the components can be combined as a single composition. In
one
embodiment, a method of administering to a host a first form of an immunogen
and
subsequently administering a second form of the immunogen, wherein the first
and
second forms are different, and wherein administration of the first form prior
to
administration of the second form enhances the immune response resulting from
23
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administration of the second form relative to administration of the second
form alone, is
provided. Also provided are compositions for administration to the host. For
example, a
two-part immunological composition where the first part of the composition
comprises a
first form of an immunogen and the second part comprises a second form of the
immunogen, wherein the first and second parts are administered separately from
one
another such that administration of the first form enhances the immune
response against
the second form relative to administration of the second form alone, is
provided. The
immunogens, which may be the same or different, are preferably derived from
the
infectious agent or other source of immunogens. The multiple immunogens may be
administered together or separately, as a single or multiple compositions, or
in single or
multiple recombinant vectors.
A kit is also provided which may include a system comprising a buffer
reservoir,
multiple reservoirs of active agents, each reservoir containing a different
active agent or
combination of active agents, one or more pumps, one or more sterilizing
filters, multiple
single-use, pre-sterilized bags, each bag containing a formulation of an
active agent or
combination of active agents corresponding to those in the reservoirs, a
station for mixing
the formulations contained within the bags with one another, which optionally
contain
one or more additional components (e.g., an adjuvant) to form a final
formulation. The
kit may also include some or all of these components such as, for example, one
or more
buffer reservoirs, one or more reservoirs of active agents, one or more
sterilizing filters,
one or more bags containing a formulation of active agent and / or one or more
additional
components (e.g., adjuvant). These components may be adapted for use in a
system
comprising one or more pumps. Additionally, the kit can include instructions
for using
these components to prepare the formulations described herein.
Certain embodiments are further described in the following examples. These
embodiments are provided as examples only and are not intended to limit the
scope of
the claims in any way.
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EXAMPLES
Example 1
Materials and Methods
Equipment used in a sterile, closed, disposable system cannot be intrusive,
meaning, no part of the equipment can come in direct contact with the product,
unless
this part is also sterile, and such that it maintains the sterility and closed
system of the
overall assembly. Also due to small-scale processing, each piece of equipment
and device
is small and portable so that the system can be transferred easily from lab
bench scale to a
.cleanroom without doubling capital.
The Wave 20/50EH Electric WaveMixer with Touchpanel (GE Healthcare Life
Sciences) is an electrical rocker where bags are placed in a SS holder that
fits on a base
and unit provides mixing with heater and temperature control for thawing,
warming and
mixing applications. The Wave concept of non-invasive mixing provides low
fluid
velocity to reduce shear forces and protect products from damage and foaming.
Agitation
is achieved using gravity to accelerate the fluid contained in the bag. The
wave sweeps up
solids and disperses them into the liquid. Direction reversals cause a
reciprocating chaotic
motion (Source: Singh, 2000). This unit is used for mixing of bulk
ingredients, in-process
and final formulations in bags.
The BLH/Vishay Kis 3 Shear Beam Load Cell (Vishay BLH) with support post
and bracketing assembly was used to weigh suspended bags during the dispensing
of
formulation ingredients. The load cell works similarly to a scale, however, it
measures
strain based on shear and is more accurate and precise. There is limited
interference from
nearby assemblies as the bags are suspended and tubing secured using weighted
tubing
holders. Bags were primed, tared and weighed using the device with a
microprocessor-
based control and panel readout. The accuracy of these units is 0.02%, and
there are no
effects of reading by thermal or vibration interference, and the device has
moveable load
points. The device also withstands both high lateral forces and have a wide
temperature
range of -40 to +80 C.
The Sartochek Filter Integrity Tester unit (Sartorius Stedim Biotech S.A.) is
an
automatic standard, microprocessor-controlled filter integrity tester to test
the integrity of
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vent and liquid membrane filters. It is used for its bubble point testing to
test integrity of
the filters from the multivalent disposable formulation system.
A peristaltic pump was used to non-invasively and gently pump and dispense
liquids from one container or system to another. This provides more control
for fluid
movement in the disposable formulation system.
The Wave Biotech Hot Lips Tube Sealer (Wave Europe Pvt. Ltd) was used to seal
the outside of the tubing while the inside remains sterile preventing leakage
or contact
with foreign materials and equipment. It can be used with liquid-filled
thermoplastic
tubing such as C-Flex.
The Disposable Bag Assemblies (TC-TECH) (Thermo Fisher Scientific)
assemblies consisting of bags, tubing, connectors and filters are custom-
designed
specifically for the purposes of the multivalent formulation. They are
designed by the
end-user and bag manufacturer (formerly Sartorius/Stericon, now Thermofisher),
assembled, sealed in bags and then gamma irradiated by a validated process.
Bags: TC-TECH/Thermofisher, film AF-793 with a ULDPE main product contact
layer. Bags used in the system range from 60mL to 5L. The 1L and 5L bags
contain a 2 x
3/8" Teflon-coated stir bar (component numbers CX22782S and SV20887.01)
intended
for mixing on a stir plate.
C-Flex Tubing: Opaque TPE tubing is heat sealable and weldable. Low protein
binding minimizes potential for active ingredient loss. Tubing is fully
characterized in
accordance with USP 24 guidelines. Formulation 072, Shore A, 60. Formulation
050,
Shore A, 50.
Sartorius Sartopore 2 filters (Sartorius Stedim): These filters are gamma
irradiated in a full assembly provided by Thermofisher prior to use. The
filters consist of
a 0.45 gm asymmetric polyethersulphone (PES) filter followed by a 0.2 m
asymmetric
PES end filter, and exhibit broad chemical compatibility of pH 1 through 14.
The MGA Technologies Tube Welder is a sterile, connecting device that was
developed by MGA Technologies to improve sterility assurance during aseptic
connections between pieces of C-Flex tubing. The device operates by using a
heated
Teflon blade (215 C) that cuts tubing. While hot and in contact with the blade
the tubing
ends to be connected are aligned and pressed together. Tubing can be dry or
moist (but
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not liquid-filled) for the operation. As the tubing cools a sterile weld is
formed and
during the process the internal bore of the tubing is never exposed to the
external
atmosphere. The connection is performed without open aseptic manipulation.
The following is a list of calibration and validation required for equipment
used
for the formulation process (Table 1).
Table 1
Disposables Equipment Calibration and Validation Requirements
Equipment Calibration Validation (required for GMP)
Peristaltic pump NO NO
Tube welder YES IO ,
Tube sealer YES IOQ, PQ through broth
Load cells YES NO
Magnetic stirrer NO NO
Filter Integrity YES IOQ
During this process, the formulator must obtain formulation ingredients in
closed
containers with weldable tubing, with sterility and/or bioburden, specific
gravity and
concentration test records (as applicable); ensure all equipment is fully
operational with
maintenance/use logbooks in place, calibrated, validated (where required) and
setup at
the desired area for formulation; assemble the configuration (e.g., tube
welding one bag
to another); relocate the mobile equipment during different parts of the
formulation; and,
filter integrity testing, pre- and post-filtration; observe the load cell
control panels during
pump dispensing to ensure the dispensing weight meets calculated target;
observe the
lines for air bubbles and ingredients at certain times in the process; clamp
the lines with
haemostats; and, execute and populate the batch-specific procedural documents.
Calculations for a multivalent formulation can be complicated, especially when
an
intermediate formulation and several ingredients are required (e.g.
excipients). It is
convenient to setup the calculations in a spreadsheet that has entry cells for
"known",
variables (shown in the bold squares) and with formulas for calculations for
outputs.
Formulation volumes are back-calculated based on the number of filled vials
required for
the study. In some cases, there is only so much material (e.g. protein) to
work with, so
this can be a limiting factor. Volumes to dispense are based on weight by way
of the
known specific gravity of the ingredients.
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As described herein, studies have been performed to develop the formulation
process in the disposables system, testing antigen concentration and aluminum
content as
major outcomes. One study compared two different processing scenarios for a
quadravalent formulation. Another study optimized production of bivalent and
trivalent
formulations.
Example 2
Study CA-08-162A
The single-use assemblies used in this Example consisted of two (2) and three
(3)
D bags connected to a manifold of tubing, connectors, and filters. These were
custom-
made by the bag manufacturer, assembled, sealed into bags, and gamma-
irradiated using
a validated sterilization method. Selected primarily for their inert
compatibility
properties, gamma-irradiation stability, quality testing, biological safety
testing, and low
leachables/extractables profile (Cardona and Allen, 2006), the film and tubing
remained
constant throughout these experiments. The bags, tubing and filters were
supported by
stands and holding apparatuses assuring proper alignment and dispensing
control for the
connections.
The process was designed for a vaccine formulation comprising proteins,
adjuvants and excipients in the final product. The stages encompassed include
filtration,
intermediate formulation, final formulation, and blending at the bulk product
stage (Figs.
1-4).
A number of filtration studies were carried out to demonstrate that process
outputs
fall within expected error ranges or satisfy pre-determined criteria for
successful
multivalent filtration and formulation. All experimental processes were
performed at
ambient temperature. On the recommendation of leading filter manufacturers for
filtration of proteins, each of Filter M, Filter E and Filter S were selected
based on four
critical specifications: 1) an appropriate surface area for the volumes
required; 2)
membrane types and construction suitable for sterile filtration of recombinant
proteins
(up to 100 000 Daltons) and buffers; 3) filter membrane materials are designed
for low
binding of proteins; 4) filters are hydrophilic, wettable without use of
wetting agent and
can be gamma-irradiated (Cardona and Inseal, 2006). Filter M has a
polyvinylidene
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fluoride (PVDF) membrane, Filter E has both PVDF and polyethersulphone (PES)
membranes, and filter S membrane is PES (Table 2).
Table 2
Sterile Filter Technical Information
0.22 m
PVDF Polycarbonate Stacked disk 100 cm2
filter
0.45 gm+
0.2 gm
PES Polypropylene Pleated, 150 cm2
asymmetric
capsule
0.45 m +
PVDF + 0.22 gm 200+
E PES Polypropylene Pleated 220cm2
capsule
Physical studies compared the rate of filtration and pressure change of the
three
sterilizing grade filters (E, M, and S) after filtration in one embodiment of
the present
invention of up to five antigens (proteins E (antigen phtE), A (antigen PcpA),
B (antigen
LytB), and D (antigen PhtD)) consecutively and in random order. These anigens
can be
isolated from the native organism or recombinantly produced. In this
embodiment these
antigens were recombinantly produced from cloned genes from a Streptococus
pneumonia bacterium. Constant pump speed at infeed was applied. An increase in
pressure at the filter could indicate pore clogging. This effect is likely
caused by
aggregation of the proteins at the filter membrane (Sharma et al., 2008).
Comparing the
filtration rate of multiple proteins through the smaller disc version of the
membrane did
not have the same results as the capsule or stacked disk system. For this
reason, and to
verify the actual filtration assembly system, further experiments were done
with the
scalable dual in-line capsule/stacked disk filters and filter testing assembly
to represent
actual filter size, geometry, type of symmetry, volumes, and setup used.
Measuring the
effluent should be tested to ensure minimal protein loss, (Cordona and Inseal,
2006).
Lower flushing volumes reduce waste and time of processing while still
maintaining a
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high quality of filtrate. A second study was performed to measure the volume
of buffer
necessary to flush the filters to prevent cross-contamination of the protein
intermediates
prior to final blending. Finally, with the best filtration system selected,
percentage
protein loss was tested.
The three filters did not clog during antigen filtration and there was no
pressure
increase, therefore all three filters can be used for the filtration of the
antigens tested.
There was no direct correlation between order of the proteins added and
filtration rate.
However, based on the design, for a protein that requires different
ingredients (e.g.,
excipients) in the intermediates, it should be processed and filtered last.
Filter S had the
highest filtration rate at 43-50 mL/min, followed by Filter E at 41-50 mL/min,
and then
Filter M at 32-48 mL/min. To properly flush each antigen between filtrations
for
intermediate formulation, Filter S required 150-200 mL, Filter M required 200
mL and
Filter E required >300 mL of buffer. Filter E had the largest capsule holding
volume.
Filter S was selected as the best choice as it met all criteria, had a higher
filtration rate,
and used the least amount of flushing volume. Satisfactory results were
obtained using
the dual Filter M assembly, the dual Filter S assembly, and a dual Filter E
assembly. For
each filter, the order of proteins was selected randomly with buffer flushing
between each
protein addition at constant pump speed.
Filter S was then tested for protein loss during filtration of a bivalent
formulation;
however, no determinable loss of the individual proteins occurred, as Protein
A was
below targeted concentration by an average of 3.9% and Protein D was above
targeted
concentration by an average of 9.4% after dual filtration. Target
concentration range of
final product of 30% per protein (inclusive of assay variability) was
therefore met.
In this small scale system, ingredient addition is based on product specific
gravity,
desired volumes by weight, and zeroing of bag weight in-line prior to
addition. Small
bags (1L) in series, such as those containing intermediates, are prone to
moving around
on scales or balances, leading to inaccuracies when attempting to measure
weight in bags.
For this system, load cells supported by a post and bracketing assembly were
designed to
weigh suspended bags during addition. They were selected for their ability to
withstand
measurement disturbances from side loads (bag swaying) and they have moveable
load
points, making it convenient to hang bags of different configurations. In
addition,
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according to the manufacturer, the load cells have high individual accuracy
with a
combined error of 0.02% and repeatability of 0.01%, and designed to discount
measurements due to thermal or vibration interference. Bags were primed,
tared, and
weighed using the device with a microprocessor-based control with display.
Readings from the load cells once ingredients were pumped into the hanging
bags
had an average percentage difference of 0.15% (n 35, practical minimum and
maximum weights applied) compared with target weight, largely due to human
error.
Aluminum content samples were taken after intermediate individual adjuvanted
proteins
and final multivalent formulations with use of hanging load cells. Results
were well
within acceptable final product limits of 0.28 0.1 mg Al / 0.5 mL.
Example 3
Study CA-08-010
The purpose of this study was to formulate a multivalent product successfully
and
accurately. The study tested two different scenarios: the "old" process
(Figure 5), which
was the same process as the single-valent formulation such that all
ingredients are added
at one time, versus a "new" process (Figures 6 and 7) where intermediates made
of stock
individual adjuvanted antigen formulations are mixed to allow for binding,
then blended
in a final step.
Tables 3 and 4 summarize the testing matrix for CA-08-010. ID "A" was used as
a control since there was no adjuvant in this formulation. PBS was no longer
the buffer of
choice, however, an assay had already been developed with this buffer and some
of the
antigens. For this study final formulation protein concentrations by HPLC,
Aluminum
content analysis by ICP and particle size by Mastersizer were compared for
Scenarios 1
and 2 (IDs "C" and `B"). Aluminum hydroxide (AIOOH) was the adjuvant of
choice,
however, as the analytical testing lab was still developing the HPLC testing
method for
A1OOH bound antigens, Scenario 1 was also performed with the previous adjuvant
and
buffer used, Aluminum phosphate (A1PO4) and PBS as represented by ID "D". This
way,
if the results for ID "C" were skewed or offset, it could be confirmed by ID
"D" if it was
due to the process or the HPLC assay. Chromatographs were to show any
detectable
cross-contamination of antigens in the single-valent intermediates.
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The antigens used (Proteins A, B, D and C) were prepared at a concentration of
200 g/mL per intermediate bag for Scenario 1 and final formulated
concentrations of 20
gg/mL/protein for all scenarios. The disposable bags used for all scenarios
were TC-
TECH using C-Flex tubing and Sartopore 2 filters.
Table 3
Testing Matrix for CA-08-010
ID A B C D
Formulation Scenario 2 Scenario 2 Scenario 1 Scenario 1
process
fixing Parameters Wave Mixer Wave Mixer Wave Mixer Wave Mixer
Adjuvant nadjuvanted 1OOH, target IOOH, target lPO4, target
1.25 m /mL 1.25 mg/ml, m /mL
mM Sodium 10 mM Sodium
Phosphate pH 7.2 10 mM Tris-HCL 10 mM Tris-HCL Phosphate pH 7.2
Buffer with 150 mM Buffer pH 7.4 150 Buffer pH 7.4 150 with 150 mM
Sodium Chloride M NaCI (TBS) M NaCI (TBS) Sodium Chloride
PBS) PBS)
Concentration Bulk /A /A 00 pg/mL (-6x) 00 pg/mL (-6x)
ncen
(Total protein)
Final Formulated 80 g/mL 80 g/mL 80 g/mL 0 gg/mL
Concentration
Each Antigen 0 g/mL= protein 0 g/mL= protein 0 g/mL= protein 0 mL= p
rotein
Concentration
/A /A la, 2a, 3a, 4a after la, 2a, 3a, 4a after
ample points .5h mixing .5h mixing
inal formulation inal formulation Final formulation Final formulation
after 30min mixing after 30min mixing after 30min mixin after 30min mix,-
inal formulation:
0 gg/mL=protein Intermediates: Intermediates:
80 gg/mL total inal formulation: 00 g/mL=protein 00 g/mL=protein
resting Outcome Suitable fluid path 0 g/mL=protein inal formulation: Final
formulation:
reduced pressure 80 g/mL total 0 g/mL=protein 0 g/mL=protein
Puild-up), 80 g/mL total 0 gg/mL total
x erience
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Table 4
Observations and Actions from CA-08-010
Observation Action
Both processes worked well No action necessary.
Many steps for both, time consuming for Time consuming due to tube welding
custom
setup assemblies onsite. Once process becomes finalized,
the bag assembly supplier (e.g. Thermofisher) will
provide a gamma sterilized pre-made assembly to
minimize setup time.
Aggregation (observed as white flakes) ID "B" was performed before ID "C", and
it was
occurred with Scenario 2, ID "B" before believed that possibly the aggregation
was due to the
mixing order of component addition: proteins, buffer,
adjuvant. Concerned this may be observed in "C", the
order was changed for ID "C" to adjuvant, protein,
buffer for the intermediate formulation and no
aggregation was observed
Sedimentation of adjuvant during mixing Additional mixing studies required to
test different
using the Wave Mixer mixing technologies and parameter optimization of
Wave Mixer.
"Flashing" from the tube welder Flashing occurs when a weld is made
unsuccessfully
occurred between the two pieces of tubing where the contents
of the tubing remain integral, however, the fusion
does not leave a sufficient opening for fluid to flow
through the inner welded diameter of the tubing.
Believed to have occurred by using wet tubing or not
"popping" the tubing immediately after a tube weld.
In order to quantify the dispensing accuracy of the ingredients being added to
the
formulation, HPLC measured the protein concentration in the intermediates and
final
formulations. For the intermediate concentrations, A1OOH and AIPO4 adjuvanted
protein
formulations were within a range of 30% except for Protein A intermediate
which read
low for both adjuvanted formulations due to issues with reference standard.
New
desorption methods are shown.
In these intermediates of the individual stock antigen formulations, it is
also
important to ensure there is minimal or no residual protein (cross-
contamination) from
the other antigens during the process that may have been carried into the bags
during
formulations. Based on the four antigen intermediate formulations tested,
there were no
measurable residuals, thus confirming purity of single-antigen intermediates.
For the
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A1OOH adjuvanted formulations, it is more difficult to conclude due to
desorption issues
that resulted in lower concentrations of the antigens as well as shoulders
present in the
peaks, even for the unadjuvanted formulations (not shown).
The chromatograms of the final formulations prepared as in Scenario 1 displays
peaks of the individual antigens present for the formulation with A1PO4
adjuvantation.
For the A1OOH adjuvanted formulations, it is more difficult to conclude due to
desorption issues that resulted in lower concentrations of the antigens as
well as
shoulders present in the peaks, even for the unadjuvanted formulations (not
shown).
Using crude testing methods and first-time processing scenarios (1 -
preadsorbed
antigens, 2 - antigens adsorbed after blending), A1OOH and A1PO4 adjuvanted
protein
final formulations and the "old" desorption method, all samples for both A1OOH
and
A1PO4 adjuvanted formulations and Scenarios 1 and 2 were within a 30% range
of final
formulation per antigen. Using the new desoprtion method, however, the Protein
D
samples were recalculated against a new standard curve showing much higher
values
were obtained. Protein A values are offset due to reference standard
discrepancies.
Adjuvant concentration was measured by aluminum content using Inductively
Coupled Plasma Atomic Emission Spectrometry as a measure of bulk product
homogeneity of suspension. Adjuvant concentrations of intermediate stock and
final
formulations in bags were within the target ranges ( 0.1 mg Al/0.5 mL) for
both A1OOH
and A1PO4.
Each intermediate and final formulation tested by Mastersizer showed
consistent
distribution of particle sizes at 50% distribution and lower for both A1OOH
and A1PO4
adjuvanted formulations. The A1PO4 readings are well within the expected
values for the
control (5-12 um).
Several studies were performed for mixing optimization of the stock antigen,
intermediate and final formulations in disposable bags. Major observations for
these
studies included observation of overall mixing efficiency, presence of
unwanted visible
aggregation, homogeneity, foaming, dead pockets in the bags, and spattering
(when there
is accumulation of adjuvant on the inner top of the bag). Four different
mixing systems
were attempted in these studies. Mixing optimization studies were performed by
visual
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observation of 1L bags containing adjuvanted intermediate and 5L bags with
adjuvant
solutions mixed using a Wave Mixer, Recirculation Line, Rotating Drum, and
Stir plate.
The Wave Mixer is designed for mixing liquids in disposable bags up to 20 kg
using Wave motion technology. For this reason, and because the current system
using a
stir bar and Stir Plate which are difficult to setup and control, it was
tested and optimized
more than any of the other systems. A recirculation line was also tested where
two tubing
lines coming from the bag were welded and looped through a peristaltic pump to
keep the
line in circulation. A rotating drum (used primarily for rotating syringes)
was tested by
affixing a 1L bag to it using cable ties.
Example 4
Study CA-07-120
To optimize parameters of the WAVE mixer instrument and to compare mixing
effectiveness of the WAVE mixer with the stir plate using A1PO4 adjuvanted
products.
The formulation of 3750m1 of 3mg/ml AIPO4+20ug/ml Protein D + PBS in a 5L
TCTECH bag was tested. Wave Mixer operating parameters for 5L bag at 40 rpm, 6
for
30min reduces foaming and pooling over other settings as can be seen in Table
5.
Table 5
Preliminary Optimal Wave Mixer Settings
CA-07-120 Low -6 rpm. Med-25rpm High-40 rpm
High-12 Dead pockets some Dead pockets some Speed excessive
foaming large air foaming large air Excessive
bubbles bubbles foaming
Bag moves
around
Med-6 Minimal mixing Incomplete mixing Minimal adjuvant
pooling at bottom
layer
Air bubbles
Low-2 No mixing Incomplete mixing Incomplete
mixing
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Example 5
Study CA-08-044
To perform visual observations of adjuvanted product mixed using the WAVE
mixer instrument. Parameters tested: 10 at 20 rpm for 30 min or 6 at 40 rpm
for 30min.
Formulations: 20ug/ml Protein B +PBS in ALOOH in 5L TC-TECH bags at 500
mL and 3000 mL capacity; 20ug/ml Protein D +PBS in ALOOH in 4 x 1L TCTECH
bags at 200 mL and 750 mL capacity.
Outcomes: Optimal settings with 5L bag were at 10 at 20 rpm for 30 min or 6
at
40 rpm for 30min. Settling occurs at both settings for 4xlL bags. Alum
settling occurs
more at maximum volumes then at minimum volumes
Example 6
Study CA-08-050
Objective: To perform visual observation of concentrated stock A1OOH adjuvant
and adjuvanted intermediate bulk formulations mixed using the WAVE mixer,
recirculation line, rotating drum, and stir plate. Blue dextran was used to
bind to the
A1OOH adjuvant for phase separation to identify sedimentation.
Formulations: 1) 5L TCTECH bag at 200 mL and 750 mL capacities: 24.30
mg/m1 ALOOH w/0.01 % Blue dextran; 2) 1L TCTECH bag at 200 mL and 750 mL
capacities: 1.25 mg/ml Al0OH w/200ug/ml Protein A w/0.005 % Blue dextran in
TBS.
Mixing time: up to 30 minutes
Assays: 1. Visual inspection 2. Aluminum content analysis
Outcome: Wave Mixer; operating parameters were at 40 rpm, 6 for both 5L and
1L bags.
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Table 6
Wave Mixer Mixing Efficiencies in IL and 5L bags
Good No Homogene Minimal No Dead Spattering
Mixing (low Aggregation ous Foaming Pockets
shear) (uniphase,
no
settling)
5L Bag
high
volume
5L Bag
low
volume
with
clamp
1L Bag
high
volume
1L Bag
low
volume
Recommendations resulting from these studies: 1) Tap 5L bag occasionally to
break
spattering; 2) Use clamp when 5L bag is at low volumes to reduce dead pockets.
Recirculation Line: operating parameters at fastest pump speed (10)
Table 7
Recirculation Line Mixing Efficiencies in 5L bags
Good No Homogeneous Minimal No Dead Spattering
Mixing (low Aggregation (uniphase, no Foaming Pockets
shear) settling)
5L Bag slight
high
volume
5L Bag
low
volume
with
clamp
37
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Recommendations from these studies: 1) 5L bags must be suspended slightly
angled
from the vertical hanging position to avoid tubing from folding and affecting
flow rate; 2)
pump should be tested at slower speeds for optimization; 3) 1L bags not tested
as each
intermediate bag would require a recirculation line and this would take up a
significant
amount of processing area, time and setup. It would also be difficult to
control; 4) use
clamp when 5L bag to reduce dead pockets.
Rotating Drum
Refer to Table 8; operating parameters at highest RPM on unit.
Table 8
Rotating Drum Mixing Efficiencies in IL bags
Good No Homogeneous Minimal No Dead Spattering
Mixing Aggregation (uniphase, no Foaming Pockets
(low settling)
shear
1L Bag high
volume
1L Bag low
volume
Recommendations from these studies: 1) time consuming and difficult to setup
due to
cable tying and wheel configuration; 2) drum should be tested at slower speeds
for
optimization; and, 3) significant foaming when bags at high volume.
Stir Plate
Refer to Table 9; operating parameters at 400 RPM
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Table 9
Stir Plate Mixing Efficiencies in IL bags and 2L and 5L Bottles
Good No Homogeneous Minimal No Dead Spattering
Mixing Aggregation (uniphase, no Foaming Pockets
(low settling)
shear)
1L Bag high
volume
1L Bag low
volume
5L Bottle
(control)
2L Bottle
(intermediate
formulation
control)
Recommendations from these studies: As foaming was observed at low volumes,
test at
lower speeds and low volumes to reduce foaming.
The aluminum content results in all mixing systems tested for both 5L and 1L
bags were within 90% of the control (stir plate using glass bottle with stir
bar). This is
well within the aluminum content release criteria for final product which is
0.1 mg All
0.5 mL. Therefore aluminum content results were consistent with all mixing
systems
showing good homogeneity in bags.
From these results, it was determined that the rotating drum created too much
foaming (even without a surfactant) and the recirculation line was difficult
to setup to
ensure all parts of the bag were recirculating efficiently. The stir plate and
Wave Mixer
were successful though the speed on the stir plate required optimization as
400rpm
created too much foaming at lower volumes.
Example 7
Study CA-08-065
To further optimize parameters of the WAVE mixer instrument by mixing
adjuvanted intermediate in IL bag with a worst case formulation. According to
CA-07-
120 and CA-08-044 study, most optimization settings of the WAVE mixer
instrument
39
CA 02697804 2010-03-17
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were performed for low concentrated formulations and without Tween. For mixing
of an
adjuvant intermediate in IL bag, a worst case formulation was defined for an
adjuvanted
intermediate with Tween 80, higher protein concentration and aluminum
hydroxide. This
study will determine optimal WAVE mixer parameters for said worst-case
adjuvanted
intermediate in 1L bag at high and low volumes.
Formulation: Adjuvanted intermediate (1.25 mg/mL AIOOH, 400 g/ml protein,
0.05 %Tween 80 in TBS) in 1L bag. Order added: Adjuvant, protein, Tween 80,
and TBS.
Table 10 describes the results from the angles and speed of the Wave Mixer
tested.
Table 10
Wave Mixer Pitch and Speed For 1L Bags with ALOOH, 0.05% Tween 80, and
ProteinA Protein in TBS
Speed
Pitch Low -10r pm Med - 20r pm Med - 30 rpm High - 40 rpm
High-12 Not efficient Not efficient Some foaming High foaming
mixing mixing No adjuvant
settling
occurred.
No dead
pockets.
Mix completely.
Med-10 Not efficient High foaming
mixing
Low-6 Not efficient
mixing
Optimal mixing settings for a 1 L bag with worst-case intermediate formulation
was at 30
rpm with a 12 tilt.
Example 8
Study CA-08-064
This study was performed to evaluate the effect of mixing processes (stir bar
and
wave mixer) of adjuvanted and non-adjuvanted products, perform visual
observation, and
characterize aggregation & foaming of Tween 80 using these mixing processes.
Protein
C antigen required addition of a surfactant such as Tween 80 in order to
reduce the
CA 02697804 2010-03-17
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potential for aggregation, however, Tween 80 has a tendency to increasing
foaming
during mixing (stirring and/or shaking). This study represents the worst case
situation
with an antigen prone to aggregation and the presence of the foaming
surfactant added to
the intermediate and final formulations to evaluate whether the optimized
mixing process
from previous studies is also applicable.
After reviewing information provided by Wave Biotech (Source: Singh, 2000), it
was confirmed that sedimentation had occurred in CA-08-010 in the 1L bags due
to
pooling at the bottom center of bag where the tilt angle for these smaller
bags was not
enough to create a sufficient velocity for movement of the adjuvant
particulates to travel
any great distance (e.g. caught in momentum of the wave). For this reason,
different
parameters on the Wave Mixer were tested for the 1L bags as the bag
configuration and
geometry is different for the longer, larger 5L bags though the same settings
had been
used in the past.
Formulation: Alhydrogel: 24.35 2.43 mg/ml ALOOH,.
Tris Buffered Saline (TBS):
protein antigen: 1) Protein A , 796.6ug/ml; 2) Protein D(1244.2ug/ml); 3)
Protein C ,
381.72ug/ml.
2% TWEEN80 in TBS: Lot#10002884-199-EX
Assays: 1. Visual inspection; 2. Aluminum content analysis
Outcomes: Table 11 describes the mixing efficiencies with Wave Maxer and Stir
Plate
for 1L and 5L bags at the various settings tested.
Table 11
Mixing Efficiency of JL and 5L bags with Wave Mixer and Stir Plate at Various
Settings
Good No Homogeneous Minimal No Spattering
Mixing Aggregation (uniphase, no Foaming Dead
(low settling) Pockets
shear)
Intermediate formulation unadjuvanted: 200u /ml Protein C w/0.05 % Tween 80 in
TBS
Wave Mixer-
30rpm/12 ,
750 ml in 1 L bag
Wave Mixer-
41
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30rpm/ 12 ,
200 ml in 1 L bag
Stir Bar-200rpm Not
750 ml in 1 L bag efficient
mixing
Stir Bar-300rpm
750 ml in 1 L bag
Stir Bar-400rpm
750 ml in 1 L bag
Stir Bar-200rpm Not
200 ml in 1 L bag efficient
mixing
Stir Bar-300rpm
200 ml in 1 L bag Stir Bar-400rpm
200 ml in 1 L bag
Intermediate formulation adjuvanted: 200ug/ml Protein C w/0.05 % Tween 80 in
TBS, 1.25mg/ml
ALOOH)
Wave Mixer-
3Orpm/12 ,
750 ml in 1 L bag
Wave Mixer-
30rpm/12 ,
200 ml in 1 L bag
Stir Bar-200rpm Not
750 ml in 1 L bag efficient
mixing
Stir Bar-300rpm Less
750 ml in 1 L bag foam
Stir Bar-400rpm More
750 ml in 1 L bag foam
Stir Bar-200rpm Not
200 ml in 1 L bag efficient
mixing
Stir Bar-300rpm .4 -7 Use bag
200 ml in 1 L bag clam
Stir Bar-400rpm
200 ml in 1 L bag
Final formulation adjuvanted: 1.25mg/ml ALOOH, 100ug/ml Protein A & Protein D,
50ug/ml
Protein C, w/0.05 % T80 in TBS)
Wave Mixer
40rpm/6
3750 ml in 5 L bag
Stir Bar-400rpm
3750 ml in 5 L bag
Wave Mixer-
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20rpm/ 10
3750 ml in 5 L bag
Wave Mixer-
30rpm/10
3750 ml in 5 L bag
Stir Bar-500rpm
3750 ml in 5 L bag
Aluminum Content Analysis: The aluminum content results in all mixing systems
tested
for both intermediate 1L bags and final formulations in 5L bags were within
90% of the
control (stir plate using glass bottle with stir bar). This is well within the
aluminum
content release criteria for final product which is 0.1 mg Al/ 0.5 mL.
Therefore
aluminum content results were consistent with all mixing systems showing good
homogeneity in bags.
The overall summary for mixing studies is shown below for the formulations
tested. The best parameters can be seen in the tables above in this section of
the report.
Mixing efficiency, homogeneity, foaming, dead pockets and spattering is
dependent on: 1)
the mixing system selected; 2) mixing system parameters (e.g. rpm, tilt
angle); 3) bag size
and shape; 4) formulation ingredients and concentrations; and, 5)
configuration of the bag
as it is placed on/in the mixing system (e.g. vertical, bag clamp). For future
formulations
(those tested) with the 5L bag, the Wave Mixer will be used as the preferred
mixing system
as this provided the best results. For intermediate formulations with the 1L
bag, either the
Wave mixer or the stir plate should be used as the rotating drum is difficult
to setup and
caused foaming without testing with Tween 80 in the formulation. However,
should
mixing while blending of the intermediates be required, only the stir plates
should be used
due to processing setup limitations. Table 12 summarizes the mixing efficiency
results
from studies CA-07-120, CA-08-044, CA-08-050, CA-08-064, CA-08-065 for various
systems tested with single-use disposable bags. The Wave Mixer produced best
results for
both 1L intermediate and 5L final formulation bags.
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CA 02697804 2010-03-17
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Table 12
No Visible Homogenous Minimal No Dead No
Aggregation (uniphase, Foaming Pockets Spattering
no setting)
Containers on Stir Plates
1L
intermediate
bag
5L bag 500 m OK
5L bottle
(control)
2L
intermediate
bottle
(control)
Recirculating Line
5L bag
Rotating Drum
1L
intermediate
bag
Wave Mixer
5L bag
1L
intermediate
bag
Example 9
Filtration Studies
Filtration studies have been performed with the multivalent antigens. Three
filters
have been tested and compared to determine which one is the most suitable for
the
filtration of the five test proteins: Millipak 20 from Millipore, Sartopore 2
from Sartorius
and EBV from Pall. Up to five antigens were filtered through the same filter
and buffer
was flushed through the filter between each protein filtration to remove the
residual
proteins from the filter for intermediate bulk stock antigens. The different
parameters
that are analyzed during the process were the pressure in the filter, the flow
rate and the
amount of protein in the wash buffer. The data obtained enabled us to
determine the best
order of filtration, the volume of buffer necessary to remove the proteins
from the filter,
44
CA 02697804 2010-03-17
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and the decay ratio of the proteins. Further analysis enabled to determine the
loss of
protein in the filters during the filtration.
The objective of this study was to compare three filters regarding the
filtration of
the five test antigens, and to determine which filter(s) is (are) most
suitable for the
filtration of the test antigens, based on the pressure in the filter during
the filtration, the
decay ratio, the protein loss and the volume of buffer necessary to flush the
filters. An
increase of pressure in the filter could indicate a plugging of the pores and
thus
aggregation of the proteins at the filter face or interaction between the
protein and the
filter (Sharma et al., 2008).
The study also determined the optimal order of filtration for each filter and
confirm if there was a detectable amount of protein lost in the filter. The
scope of this
study included the filtration of five antigens: Protein A, Protein D, Protein
E, Protein
Band Protein C. TBS-Tween 80 (2%). Small scale studies were first performed to
determine the order of filtration for the antigens. The antigens were then
sterile-filtered
at full scale through three different filters: Millipak 20 (Millipore, part #
MPGL02GH2),
EBV (Pall, part # 1EBV7PH4) and Sartopore 2 (Sartorius, part # 5441307H4G).
Millipak 20, EBV and Sartopore 2 have been recommended by the suppliers for
multi-
antigen filtration, based on two critical specifications: 1) the surface
filtrations were
adapted for the volume of protein solutions and buffer to be filtered; and, 2)
the PVDF or
PES membranes and the geometries are suitable for the filtration of
recombinant proteins
which can be up to 100 000 Daltons in size. Direct comparison filtration
studies and
bacterial retention studies were performed. Millipak 20 disposable filter
units are stacked
disc filters designed for the removal of particles and microorganisms from
liquids and
gases.
Table 13
Millipak 20 Specifications
Support Material Polycarbonate
Configuration Stack Disk
Vent Cap Material PVDF
Filter Brand Name Durapore
Bubble Point at 23 C ?3450 mbar (50 psig) air with water
CA 02697804 2010-03-17
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Max Inlet Pressure 5.2 bar at 25 C
Process Volume 10 L
Connections, Inlet/Outlet 6 mm (1/4 in.) Hose Barb with bell
Filter pore size 0.22 gm
Max Differential Pressure 4.1 bar @ 25 C; 1.7 bar @ 80 C; 0.35 @
123 C
Capsule Type Liquid
Filtration Area 100 cm
Filter Material Hydrophilic PVDF
Flow rate 1.5 L/min @ 1.75 bar P
Sartopore 2-y- capsules (Sartorius Stedim Biotech; Table 14) are 0.2 m rated
sterilizing grade filter capsules designed for connection to flexible-bag-
container-systems
prior to sterilization by y-irradiation.
Table 14
Sartopore 2 Specifications
Support Material Polypropylene
Vent Cap Material Polyethersulfone
Filter pore size 0.45 + 0.22 m
Max Differential Pressure 4 bar at 20 C; 2 bar at 80 C
Capsule Type Liquid
Filtration Area 150 cm
Filter Material Polyethersulfone, assymetric
Pall's Mini Kleenpak sterilizing capsule filters are compact pharmaceutical-
grade capsule
filters featuring low hold-up volumes.
46
CA 02697804 2010-03-17
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Table 15
EBV Specifications
Support Material Polypropylene
Max Operating Pressure 4.1 bar at 38 C; 2.1 bar at 80 C
Filter pore size 0.45 gm + 0.22 m
Capsule Type Liquid
Filtration Area 200 cm +220 cm
Filter Material PVDF +PES
Flow rate 322 mL/min/100 mbar
A double filtration was performed to satisfy GQD recommendations of having a
"redundant" sterilizing grade filter (GQ_000795). All the antigens were
filtered through
the same filter and collected separately after filtration. Between each
protein filtration,
the filters were flushed with buffer in order to remove the protein from the
filters. A
pressure gauge and the measurement of the flow rate were used to determine if
there is
any increase of pressure during the filtration, e.g. if the filter starts to
clog. The assembly
is illustrated in Figure 8. The wash fractions, after each protein filtration,
were analyzed
by BCA to determine the volume of buffer necessary to remove the proteins from
the
filters.
The reagents used for this study are:
^ 3 x 5 L of TBS buffer (iris 10 mM with NaCI 150 mM), pH 7.4. lot #
C12431
^ 700 mL of Protein A protein solution, 979 pg/mL,
^ 500 mL of Protein D protein solution, 1311 g/mL,
^ 200 mL of Protein D protein solution, 1143 pg/mL,
^ 700 mL of Protein E protein solution, 848 pg/mL,
^ 700 mL of Protein B protein solution, 1038 g/mL,
^ 700 mL of Protein C protein solution, 491 g/mL,
^ Tween 80, batch # 0001766,
The materials used for this study included:
47
CA 02697804 2010-03-17
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^ 15x sterile plastic bottles (250 mL) for the filtered proteins
^ 60x sterile plastic bottles (60 mL) for the flushing buffer
^ 6x 250 mL sterile graduated cylinders
^ lx 500 mL container to collect waste
^ lx Pressure gauge
^ 3x sterile C-flex tubing to be connected to the pressure gauge
^ 6x C-flex Y tubing
^ Balance scale
^ Stopwatch
^ 7x tubing clamps
^ Several sterile powder-free surgical gloves, polyester or disposable
Tyvek lab coat, safety glasses
^ 2x Millipak 20 filter, part # MPGL02GH2
^ 2x Sartopore 2 filter, part # 5441307H4G
^ 2x EBV filter, part # KA02EBVP2S
BCA assays were performed on the wash buffer samples, as per instructions..
The protein solutions, the buffer fractions and the TBS-Tween 80 were
collected after
filtration to be analyzed by BCA. A sample of the proteins solutions and TBS-
Tween 80
solution before filtration was collected. The samples were stored at 2-8 C.
The equipments used for this study included:
^ Biocontainment hood: equipment BCC 1066, functional location
B93BOO00009, tech ID B67BCC005, environmental H.V.A.C.
performance certification date 22feb2008
^ Peristaltic pump: Easyload Masterflex LIS standard drive, model
7518-00
^ Tube welder: ID #TUW 1010, bld93 room 121, certified on 04feb2008
Testing with the Milliex (Millipak) disc filter showed that filtration
throughout
capacity of the antigen component was susceptible to its position in the
filtration
sequence. The five antigens and the TBS-Tween 2% were filtered through the
same
filters.
48
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The volume filtered through the Millipak 20 filter as a function of time for
each
antigen was acceptable. The pressure applied by the peristaltic pump is
constant during
the filtration of the five antigens. The flow rate is constant during the
filtration for all the
proteins. There is no increase of pressure. The amount of protein in the wash
fractions,
for each antigen, was acceptable.
The volume of buffer necessary to remove the protein from the filters down to
a
level lower than the limit of detection is > 200 mL for Protein A, Protein D,
Protein E and
Protein B. The less concentrated sample in the calibrating curve is 20 g/ml,,
so the'
amount of protein in a sample is considered insignificant when the
concentration is below
20 gg/mL. Due to the presence of Tween 80 in Protein C wash fractions, and
because of
the interference of the Tween 80 in the BCA assay, HPLC analyses was performed
on
Protein C wash fractions.
The volume of protein solutions filtered through the Sartopore 2 filter as a
function of time was acceptable. The flow rate is constant during the
filtration of all the
proteins. There is no increase of pressure. The amount of protein in the wash
fractions,
for each antigen, was acceptable. The volume of buffer necessary to remove the
protein
from the filters down to a level lower than the limit of detection is > 150 mL
for
ProteinA, Protein D and ProteinE, and > 200 mL for ProteinB. Due to the
presence of
Tween 80 in Protein C wash fractions, and because of the interference of the
Tween 80 in
the BCA assay, HPLC analyses was performed on Protein C wash fractions. The
volume of protein solutions filtered as a function of time was acceptable. The
flow rate is
constant during the filtration of all the antigens. The pressure does not
increase. The
amount of protein in the wash fractions, for each antigen, was acceptable. The
volume of
buffer necessary to remove protein from the filters down to a level lower than
the limit of
detection is > 300 mL for ProteinA, ProteinD, Protein E and ProteinB.
The following experiment was performed to determine the protein loss in the
Sartopore 2 filter during the filtration. Protein Aand Protein D were filtered
through two
Sartopore 2 filters and diluted with TBS to a target concentration of 100
g/mL. Three
runs have been performed for each protein dilution. The diluted samples were
then
analyzed by BCA to determine the concentration (Table 16).
49
CA 02697804 2010-03-17
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Table 16
Sartopore 2 - Protein Assay Loss
Concentration of Protein Concentration of Protein D
Run # A ( g/mL) (tg/mL)
Starting material 816.76 16.32 894.64 f 48.07
1 99.64 3.78 102.33 f 7.22
2 92.56 2.68 120.56 f 2.02
3 96.32 3.37 105.28 4.56
Average 96.14 4.23 109.39 f 9.5
For Protein A, the concentration obtained for the three runs were below the
targeted concentration by an average of 3.9 %. For Protein D, the
concentration obtained
for the three runs were above the targeted concentration by an average of
9.4%.
Considering the three runs for each protein all together, the targeted
concentration of 100
gg/mL is within the interval given by the standard deviation. Thus there is no
significant
loss of protein in the filter during the filtration of Protein A and Protein D
through the
Sartopore 2 filter.
As observed in these experiments:
^ The three filters did not clog during the antigens filtration, so all three
filters can be used for the filtration of the antigens.
= There is no increase of pressure in the filters during the filtration of the
five antigens, for the 3 filters tested. Thus the order of filtration is not
relevant. The only limitation is that Protein C should be filtered last, if
Tween 80 will be used in the process. Some proteins such that have
been reported as being stickier than other proteins, should be filtered
last.
^ Sartopore 2 requires 150-200 mL of buffer, Millipak 20 requires 200
mL and EBV requires 300 mL. Thus Sartopore 2 would be the best
choice regarding the wash volumes.
^ There is no loss of protein during the filtration of Protein A and
Protein D through the Sartopore 2 filters.
CA 02697804 2010-03-17
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Based on these results, the optimal primary filter of choice to use in the
multivalent formulation is the 2 x Sartopore 2 filters in series because it
limits the loss of
protein during the filtration and the volume of flushing buffer. An exemplary,
suitable
alternative is the Millipak 20 filter due to a limited volume of flushing
buffer required,
however studies would be required to confirm loss of any protein during
filtration.
Example 10
Study CA-08-077
The following study uses parameters optimized in the sections to follow: 1)
mixing parameters: Wave Mixer for 5L final formulations and stir plate for 1L
intermediate bags; 2) filtration: 2 x 0.2 um Sartopore 2 filters in series
with 150 mL
buffer flushing volumes; 3) Process Scenario 1 (Figures 9 and 101: preadsorbed
intermediates and final blending. Formulation ingredients include: Protein D,
Protein A,
Protein C and A1OOH Adjuvant (for adjuvanted formulations), TBS, and Tween 80
(for
Protein C antigen). The objective of this study was to formulate a multivalent
product
successfully and accurately and to optimize mixing time of multivalent
products, and to
ensure homogeneous product throughout Beginning, Middle, and End sampling of
final
container, and a suitable seal using flip-off caps. Bivalent and trivalent
formulations
were made with adjuvant. The trivalent was also formulated in unadjuvanted
form.
Formulation ingredients included: 1) Protein D Protein - Purified
concentration values
based on HPLC assay; 2) Protein A Protein - Purified concentration values
based on
HPLC assay; 3) Protein C Protein - Purified concentration values based on HPLC
assay;
4) 5 L TC-TECH bag with pooled A1OOH at 23.34 mg/mL (concentration may vary
slightly (20.01-24.45 mg/mL) depending on CofA); 5) 5 L 110 mM Tris-HCL Buffer
pH
7.4 150 mM NaCl; and, 6) Tween 80, Plant origin, EP grade. Table 17 describes
the
study formulation matrix.
Table 17
CA-08-077 Sampling Matrix
ID CA-08-077-A CA-08-077-B CA-08-077-C
Formulation Scenario 1 Scenario 1 Scenario 2
process Trivalent (adj) Bivalent ad') Trivalent unad'
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Wave Mixer settings for Wave Mixer settings for
5L final formulation: 20 5L final formulation: 20 Wave Mixer
Mixing rpm, 100 rpm, 100 settings for 5L:
Parameters Stir Plate settings for 1L Stir Plate settings for 1L 20 rpm, 10
during final formulation: during final formulation:
400 m 400 m
Adjuvant A1001-1, target 1.25 A100H, target 1.25 N/A
mg/mL mg/mL
Buffer TBS, pH 7.4, with 0.05 % TBS, pH 7.4,
TBS, pH 7.4 with 0.05 %
Tween 80 Tween 80
Intermediate Bulk 400 g/mL N/A
Concentration
Each Antigen 20 g/mL-protein 20 20
Concentration g/mL-protein g/mL=protein
Intermediate bags la, 2a, 3a after 30 min, lh N/A
and 3.5h mixin
Sample points Final
Final formulation after 30 Final formulation after formulation after
min, lh and 3.5h mixing 30min mixing
30min mixing
Intermediates: Final
400 g/mL=protein
Final formulation: formulation:
Testing Outcome Final formulation: 20 g/mL-protein 20 g / mL
20 g/mL-protein 40 g/mL total protein
60 /mL total 60 g / mL total
Assays: Samples were be tested for total protein concentration by: 1) RP-HPLC -
total
protein assay + individual proteins; SDS-PAGE and % Adsorption; 2) HPLC -
total
protein assay +individual proteins (PD CA); 3) Aluminum Content (Bodycote);
and, 4)
Stability Testing. The desired target accuracy was 15% (inner target), and
the desired
Release Testing accuracy (outer target) was X30%. The Intermediate Formulation
was
to be > 400 g/mL, and the Final Formulation > 20 g/mL/protein. The target
aluminum
content in ALOOH was 0.28 0.10 mg Al/0.5 mL.
Formulation steps of the intermediates and final formulation for the
adjuvanted
trivalent are shown below as this process is most complex as compared with
unadjuvanted. The bivalent adjuvanted final formulation was made using the
intermediates of Protein A and Protein D from the trivalent and the only
difference is Part
B of the procedure where only two intermediates were used.
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CA-08-077-A - Scenario 1, Trivalent (adj)
1) Set up formulation bag manifold system and formulate to 3750 mL (75 % of
5L bag) and according to BPR 300-FF-04 where possible (Figure 11,
representing sampling locations). Perform only if pre-filter integrity testing
is
required.
2) Calibrate load cells.
3) Scenario 1. Part A (Note: filtration is performed using 2 in-line 0.2 um
Sartopore 2 filters):
a. Make all appropriate weld connections.
b. Prime proteins just downstream of each respective "T" junction of
manifold. Ensure minimal air bubbles in line.
c. Prime Tween 80 in TBS to respective "T" junction of manifold. Ensure
minimal air bubbles in line.
d. Prime main line with diluent to priming start mark of bioburden bag and
pull -10 mL bioburden from diluent to bioburden bag.
e. Flush main line to Waste 2 with approximately 200 g diluent.
f. Pre-FIT (not performed for study). Allow diluent to be pushed into Waste
bag 1 during FIT.
g. Prime diluent to each intermediate bag "fill start" mark.
h. Flush main line to Waste 1 with 200 g adjuvant.
i. Add required amount of adjuvant to each individual intermediate bag.
j . Flush main line with 200 g buffer to waste bag 2.
k. Start with concentrated, purified protein container (Container 1) closest
to
filter assembly and add required amount of protein to respective
intermediate bag starting with Bag la (closest to filter assembly).
1. Pull 10 mL of protein into bioburden sampling bag.
in. Add required amount of buffer to intermediate bag (must be >150mL or
additional flushing will be required up to 150 mL).
n. Repeat steps k to in for second protein Container 2/Bag 2a and so on. For
Protein C protein requiring Tween 80 perform the following steps:
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i. Repeat step k and 1 for ProteinC
ii. Add required amount of Tween 80.
iii. Pull 10 mL Tween 80 into bioburden sampling bag.
iv. Add required amount of diluent.
o. Seal adjuvant container and line just between waste bag 1 and bag la.
p. Perform Post FIT.
4) Sampling
a. Stir using Wave Mixer for 30 min, l hour and 3.5 hours, sample up to
50 mL using sample bags at each time point for each intermediate.
Sampling must be done when intermediates are in suspension. Ensure
sampling line is flushed into a waste container (last 50 mL bag) prior
to each in-series sample time point.
b. Sampling will be done in gamma irradiated sampling bags. From these
bags, they will be loaded aseptically into standard 3 mL serum vials at
sampling 3x each under a GLP hood (except for container integrity) at
a volume of approximately 2.5 mL/vial.
c. At minimum, aluminum content and protein concentration will be
tested for each sample point.
5) Storage: After mixing, store intermediates at 2-8C until required for Part
B.
Record in/out storage time/date.
6) Scenario 1, Part B
a. Make all appropriate weld connections except adjuvant can be welded
at time of adjuvant addition.
b. Mix intermediate bags for 30 min each on stir plates at 400 rpm prior
to drawing from each of them.
c. Prime main line with 50 mL of la intermediate bag to waste.
d. Prime main line with 50 mL of 2a intermediate bag to waste.
e. Prime main line with 50 mL of 3a intermediate bag to waste.
f. Flush main line with -200 mL diluent to Waste. Prime to "fill start"
mark of 5L final formulation bag.
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g. Tare 5L bag, then add each protein from intermediate bags to
formulation container (1:1:1). Start with closest intermediate bag to
formulation in order from right to left (3a - >2a - >I a).
h. Without flushing, tare and add required amount of diluent and seal
connections on the main line.
i. Mix adjuvant in bag for at least 30 minutes on the Wave Mixer. Weld
the line to a clean line on the formulation bag.
j. Prime line with adjuvant to fill start mark. Tare and top-up formulation
bag with adjuvant.
k. Seal lines.
1. Seal connections and stir using Wave mixer at 20 rpm, 10 degrees for
30 min.
7) Sampling
a. Sample up to 4x50 mL using sample bags. Sampling must be done
when formulation bulk is in suspension. Ensure sampling line is
flushed into a waste container (last 50 mL bag) prior to each in-series
sample time point.
b. Sampling will be done in gamma irradiated sampling bags. From these
bags, they will be loaded aseptically into standard 3 mL serum vials at
sampling 3x each under a GLP hood (except for container integrity) at
a volume of approximately 2.5 mL/vial.
c. At minimum, aluminum content and protein concentration will be
tested for each sample point.
HPLC was performed at two laboratories, and there were differences in the
results
obtained from these labs because the assays and standards are neither
identical nor
validated (e.g. in the case of A1OOH adjuvanted proteins and Protein C protein
formulations). Desorption issues surround the Protein C formulations so these
results
have been omitted. For the intermediate formulations, HPLC resulted in values
for both
intermediate formulations were within a 30% range. For Protein D, the
results for the
400 gg/mL target values vary up to 20% (average 322 g/mL) while the
Formulations
lab results vary by up to 7% (average 427 pg/mL). For Protein A, the results
for the
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400 pg/mL target values vary by 4% (average 387 pg/mL) while the
Formulations lab
results vary by up to 5% (average 418 gg/mL).
Based on the HPLC results, values for final formulations were within a
criteria
range of 30% for the Bivalent formulation. The lab results for the 20
gg/mL/protein
target values vary by 15%. Results for these samples were: Protein A= 19
gg/mL and
Protein D = 23 g/mL. The results for the 20 gg/mL/protein target values vary
by up to
21 %. Results for these samples were: Protein A= 18 g/mL and Protein D= 24
gg/mL.
From these results, it can be concluded that the Bivalent final formulation
process was
successful based on accuracy as it was within a final formulation criteria off
30%.
For GMP clinical lot implementation, a risk-based approach may be followed,
taking the following aspects of a multivalent formulation process based on
quality, purity,
operator safety, product identity and sterility into consideration:
= Omitting vent filter integrity testing of in-process filters (e.g.
intermediate
containers, waste). Currently, a very time-consuming step that may not be
value-added.
= Operator error due to complex process, lack of training
= Percent (%) error and variation of protein assay methodolgy (HPLC, BCA)
varies from site to site and makes it difficult to confirm in-process and
final
concentration of individual antigens
= Pressure build-up in system may be a safety hazard or lead to back-flushing
= Unexpected aggregation of intermediates or final formulated bulk
= Robustness and repeatability of process
= Flashing from tube welding and causing re-welding of wetted tubing lines
= Wave mixer safety hazards (e.g. pinching of fingers)
Example 11
Broth Formulation Process
A broth formulation process was designed using the disposable bag assembly (or
glass bottles as a backup) as a worst case to validate the multivalent
formulation that
encompasses the following: single-antigen formulation, multi-antigen
formulation,
intermediate formulations, sampling, dilutions, filtration formulations with
treated
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adjuvanted, untreated adjuvanted. and unadjuvanted. The worst case includes
the
maximum number of tube welds and seals compared to actual processes used. In
addition
it will include sterile connectors (e.g. Pall Kleenpak(V). A diagram of an
exemplary
configuration is shown in Figure 12. Tryptic Soy Broth (TSB) is passed through
each
line, challenging the assembly lines representing product ingredients and
bags.
Conclusions Derived from Examples 1-11
To summarize, a suitable disposables formulation process has been designed for
a
multivalent final bulk product with the following conclusions:
= Intermediate and final bulk formulation adjuvant homogeneity is within 0.1
mg Al/0.5 mL.
= Each individual antigen is preadsorbed with A1OOH successfully as a stock
intermediate supply within desirable protein concentrations ( 30% or better).
= Performance of a pre-adsorbed intermediate step before final blending is as
or
more accurate as filtering, blending then adjuvanting purified antigens in one
step.
= Particle size distribution of adjuvanted formulations is within expected
ranges.
= Wave Mixer is most efficient mixing for intermediate and final bulk product
during formulation, however, mixing will be performed with a stir plate for
mixing intermediates during final formulation so that mixing and dispensing
of the 1L bags can occur simultaneously.
= Using a 2 x Sartopore 2 filtration as primary filter assembly of choice due
to
reduced flushing volumes (2 x Millipak 20s and 2 x EBV filters as
alternatives) and confirmation of overall performance.
= Flushing filtration and disposables line assembly with at least 150 mL
buffer
between antigen addition to intermediates is required.
= No quantifiable amounts of process-induced residual proteins were detected
in
the single-antigen intermediate formulation using an HPLC indicating assay
= No specific protein order is required for filtration using the Sartopore 2
filters
at full scale based on performance studies (none required for Sartopore 2
filters)
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Example 12
Studies were performed in order to optimize the formulation and filling
processes
for an example Trivalent composition of proteins from Streptococcus pneumoniae
(PhtD
+PcpA +P1yD1) products prior to manufacturing of the toxicological lots. The
goal of
these studies was to determine the most efficient parameters during the
formulation and
the filling to ensure the final product is sterile and the concentrations are
within
acceptable ranges. The TBS buffer was formulated at pH 7.4, with 50 mM Tris
and 150
mM NaCl. The antigens were PcpA, PhtD and PlyD 1. PcpA and PhtD are in
solution in
the TBS buffer. PlyDi purified bulk antigen is supplied in solution in TBS
buffer with
residual Tween 80 (0.05 %). The phosphate-treated hydroxide (PTH) aluminum
adjuvant
(A1PO4) contained 5.6 mg Al/mL and 2 mM NaPO4, in solution in the TBS buffer.
The
process was performed in a closed, disposable assembly. The assembly was
considered
closed downstream of the final filter. The assembly is pre-sterilized as
provided by the
manufacturer.
Optimization and toxicity lot testing was carried out using the systems
described
in Figure 3 (A1PO4-adjuvanted formulation) and Figure 4 (unadjuvanted
formulation).
Kleenpak sterile connectors were used in-process where possible (e.g., for
buffer
addition, see diamond-shaped connectors in Figs. 3 and 4). As shown in Fig. 3,
the
adjuvant source was relocated from near the final formulated bulk bag to
before the
intermediate bags to allow for unidirectional pumping. Adjuvant concentrations
were
also increased from 0.56 mg Al/mL in the intermediates to eliminate addition
of adjuvant
to the final bulk formulation to compensate for dilution (Fig. 3). The waste
bag was also
positioned at the end of the process line in order for correct fluid
displacement and
ingredient addition (Fig. 3). A Pendotech pre-sterile in-line pressure sensor
(supplied by
Pall) to measure the pressure pre-filtration for indication of clogging and/or
line blockage
(Figs. 3 and 4). The bioburden bag from before the redundant (first filter) to
between the
first and second filter as this is more compliant with the regulatory
guidelines as the
sample is pulled just prior to the final sterilizing filter (Figs. 3 and 4).
The load cell
apparatus (Fig. 3) was utilized as "stand-alone" system that was not affixed
to the
formulation table to prevent unwanted vibrations or interference, provide
improved
stability, and a single control panel was used, instead of one control panel
for each cell.
58
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Regarding the filterability studies, any of the following five filters are
suitable for
the sterile filtration of a trivalent or monovalent PhtD, PcpA, or P1yD1
product: Sartorius
Sartopore 2, Pall EDF, Pall EBV, Pall EKV, Millipore Millipak 20 (Table 18).
Sartopore 2 was selected as the filter of choice. The Sartopore 2 filters
challenged with
B. diminuta in solution in the Trivalent product were able to retain 100% of
the bacteria.
Thus this filter can be safely used for the sterile filtration of the
trivalent product. The
minimum bubble point was previously tested in an MTECH report (C#010578) with
the
same buffer flushing solution (TBS) as the wetting agent and is 30. The
maximum bubble
point was recommended as 55 PSI by MTECH, however, at times this can be
exceeded
due to the surface tension of the buffer on the filter. A maximum parameter of
> 55PSI is
now indicated in the batch production records for the trivalent and related
products.
Table 18
Filters SHF SHC Milli ak20 Sartopore2 EBV EKV EDF
Number of 1 2 1 2 2 2 2
layers
First layer PES PES PVDF PES PES PES PES
material
First layer 0.22 0.5 pm 0.22 pm 0.45 pm 0.45 0.65 pm 0.45
pore size pm pm pm
Second - PES - PES PES PES PVDF
layer
material
Second - 0.22 - 0.22 pm 0.22 0.22 pm 0.22
layer pore pm pm pm
size
The mixing studies showed that the best parameters to maintain a trivalent
adjuvanted product homogeneous are 350 rpm, with proper purging for bottom bag
samples, for at least 30 minutes for an adjuvanted product formulated in a 3L-
3D bag. A
clear plastic bag holder (Fig. 15; other materials may also be used, such as
stainless steel)
with a hole at the bottom was used to prevent the bag from moving on the
surface of the
magnetic stirrer and so that visibility of settling and volume levels is
possible. The
aggregation studies indicated that a magnetic stirrer induces more aggregation
than a
WaveMixer on a trivalent unadjuvanted product. The particulates are
approximately 10
times bigger. However, the aggregated particulates are not visible and do not
induce any
59
CA 02697804 2010-03-17
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variation in the protein concentration. Mixing conditions of unadjuvanted
product on the
WaveMixer are 20 rpm, 10 for a minimum of 10 minutes.
Leachables studies were performed on the TC-Tech bags with several ingredients
including stock Aluminum Hydroxide Adjuvant, Phosphate Treated Hydroxide
Adjuvant,
Tween-containing product and representative final product using the system
shown in
Fig. 16. Bags containing 0.05 % Tween 80 showed an unidentified peak that may
have
been Tween related or a leachable from the bag due to the presence of Tween.
Bags,
representing final product with residual Tween 80, containing 0.025 % Tween 80
in TBS
and Phosphate Treated Hydroxide, showed no peaks up to 1 month. This was
consistent
with findings from an MTECH study done on the bags using a higher
concentration of
Tween (0.5%, 5000 ppm, C012431). After 6 months, for the stock phosphate
treated
hydroxide or final product without Tween 80 residual, one unidentified peak
was
detected by HPLC/UV. All other results showed no noncarcinogenic, non-toxic
leachables above reportable values.
The optimization runs showed that the mixing and formulation parameters were
suitable to maintain the proteins and aluminum concentrations stable
throughout the
filling process. Some investigations were performed to determine low protein
and
aluminum concentrations detected in some lots were related to the assay
methodology or
change of columns. The product appearance, based on visual inspection
performed during
the optimization and toxicity lot runs is described as clear, colorless
solution for
unadjuvanted Trivalent product, and a white, cloudy suspension for adjuvanted
Trivalent
product. Five optimization runs with Trivalent products were conducted. The
optimization runs were performed with a Trivalent unadjuvanted product and the
Trivalent adjuvanted products. Two optimization runs at full scale with
adjuvanted, high
dose (100 gg/mL/protein, PhtD +PcpA + PlyD 1) mixed in formulated bulk in a 3D
3L
bag for final blending (min 30 minutes) and filled (minimum 30 minutes) at 350
rpm on a
stir plate. Two optimization runs were also performed using unadjuvanted, high
dose
(100 g/mL/protein, PhtD +PcpA + P1yD 1) mixed in formulated bulk in 5L a bag
for
final blending (min 10 minutes) using 20 rpm, 10 on a WaveMixer. Optimization
runs
for adjuvanted, low dose (20 g/mL/protein, PhtD + PcpA + P1yD1) mixed as a
formulated bulk in 3D 3L bag for final blending (minimum 30 minutes) and
filled
CA 02697804 2010-03-17
APL-09-02-CA
(minimum 30 minutes) using 350 rpm on stir plate. The formulated bulk and
filled vials
were analyzed for protein content and, where applicable, aluminum and
phosphorous
content. Samples were analyzed for protein content by HPLC, with an expected
target
range for high dose is 70-130 g/mL and for low dose is 14-26 .tg/mL. Samples
were
analyzed for aluminum and phosphorous content by with an expected target range
for
aluminum content was 0.56 0.2 mg Al/mL. Samples were also checked for visual
inspection to define an appropriate description for product appearance for
both
adjuvanted and unadjuvanted trivalent products. The high dose formulations are
representative of high, medium and low dose. The samples were analyzed for
protein
content by HPLC, where applicable though it was found that these samples were
difficult
to pull as there was insufficient representative material in the intermediates
that remained
after final blending. Samples were also taken during filling at the beginning,
middle and
end to be analyzed for protein content to ensure the homogeneity of the
formulated bulk.
Samples were also taken from the beginning and the end of the filling process
to be
analyzed for aluminum content and phosphorous content to ensure homogeneity.
The protein concentrations for the unadjuvanted trivalent high dose
formulations
were acceptable, although the concentration for PhtD was low in one instance
PlyD i was
deemed as not a reportable value. For these samples, the protein
concentrations were
consistent throughout the beginning, middle and end filling samples tested
indicating no
adverse trending during filling of unadjuvanted product.
The protein concentrations for the adjuvanted trivalent high dose formulations
were acceptable. The aluminum content for these samples were acceptable for
beginning
and end samples; phosphorous was reported at about 2.2 - 2.3 mM (trending
only; one
sample was reported as low for unknown reasons). For these samples, the
protein
concentrations were consistent throughout the beginning, middle and end
filling samples
tested indicating no adverse trending during filling of unadjuvanted product.
The protein
and adjuvant concentrations were consistent throughout the filling process
indicating
product homogeneity is maintained during mixing and filling.
Table 19 shows the results obtained for the different optimization and
toxicity
runs for protein content, aluminum content and phosphorous content.
61
CA 02697804 2010-03-17
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CA 02697804 2010-03-17
APL-09-02-CA
All references cited and / or listed herein are hereby incorporated into this
disclosure in their entirety. While certain embodiments have been described in
terms of
the preferred embodiments, it is understood that variations and modifications
will occur
to those skilled in the art. Therefore, it is intended that the appended
claims cover all
such equivalent variations that come within the scope of the following claims.
63
CA 02697804 2010-03-17
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REFERENCES
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Yield Biotech
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Roberson, et al. Engineering Fluid Mechanics, Sixth Edition, Wiley, 1997
Motzkau, et al. The .Importance of Vendor Validation Services: Experience and
Economics, BioProcess International, September 2005.
Sharma, et al. Filter Clogging Issues in Sterile Filtration, Biopharm
International, April
2008
Baumfalk, et al. Integrity Testing in the Pharmaceutical Process Environment,
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Priebe, et at. Choosing and Scaling Up Right Filter Combo, Bioprocessing
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Genetic Engineering News, March 1, 2006
Luckiewicz, E. Elements of Applied Process Engineering Course Notes, Center
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Professional Advancement, New Brunswick, NJ, March 2004
Doran, P. Bioprocess Engineering Principles, Academic Press, 1995
Cardona, M. Considerations for Buffer Filtration, Contamination Control,
June/July 2005
EMEA, Manufacture of the Finished Dosage Form (Directive 81/852/EEC as
amended),
December 1995
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CA 02697804 2010-03-17
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US Food and Drug Administration. Sterile Drug Products Produced By Aseptic
Processing, Current Good Manufacturing Practices (Guidance for Industry),
September
2004
ICH Harmonised Tripartite Guideline, Quality Risk Management Q9, Current Step
4
version, November 2005
Phillips, C. It's Not Whether but Rather What and How to Implement, Bioprocess
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Singh, V. BioProcess Tutorial, Non-invasive mixing in bags, Feb 2000
Sharma, et al. Filter clogging issues in sterile filtration, Biopharm
Internation, April 2008
CA 02697804 2010-03-17
PCL XL error
Subsystem: KERNEL
Error: IL LegaLOperatorSequence
Operator: Readlmage
Position: 14371
