Note: Descriptions are shown in the official language in which they were submitted.
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Apparatus and Method for Producing Porous Polymer Particles
Field of the Invention:
The present invention relates to an apparatus and
process for the formation of porous polymer particles for use
in chromatography techniques.
Background:
The capacity of certain porous support particles to
cause selective retardation based on either size or shape is
well known. Such particles are used in chromatographic
separation techniques, for example gel filtration, to separate
biological macromolecules, e.g. proteins, DNA, RNA
polysaccharides and the like. The sieving particles are
characterized by the presence of a microporous structure that
exerts a selective action on the migrating solute
macromolecules, restricting passage of larger particles more
than that of the smaller particles. Thus, the utility of
sieving lies in the capacity of the particles to distinguish
between molecules of different sizes and shapes.
Affinity chromatography is a chromatographic method
used for the isolation of proteins and other biological
compounds. This technique is performed using an affinity
ligand attached to a support particle and the resulting
adsorbent packed into a chromatography column. The target
protein is captured from solution by selective binding to the
immobilized ligand. The bound protein may be washed to remove
unwanted contaminants and subsequently eluted in a highly
purified form.
e.v
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Good separation using chromatography techniques depends
on the size of particles, the size distribution of particles
and the porosity of the particles. The beads, once packed into
a column, should be of a high strength in order to support the
liquid flow rates observed during purification and column
regeneration. The effect of polymer concentration and other
preparation parameters on agarose particle porosity and
strength are presented in S. Hjerten and K.O. Eriksson,
Analytical Biochemistry, 137, 313-317 (1984). Additional
fundamental information is presented in Studie"s on Structure
and Properties of Agarose, A. S. Medin, pH.D. Thesis, Uppsala,
1995. The description of chemical additives that help to
improve the agarose particle porosity are found in M. Letherby
and D.A. Young, J. Chem. Soc., Faraday Trans. 1, 77, 1953-1966
(1981) and in M. Tako and S. Nakamura, Carbohydrate Research,
180, 277-284 (1988).
Many particle formation methods and apparatus have
been developed using centrifugal action to divide a liquid
or into droplets or particles. Rotary atomizer machines in
general are discussed in the text Spray Drying Handbook, K.
Masters, Fifth edition, Longman Scientific & Technical,
Longman Group UK Limited. Other relevant references related
to atomization are Atomization and Sprays, A. Lefebvre,
Hemisphere Publications, 1989 and Liquid Atomization, L.
Bayvel and Z. Orzechowski, Taylor and Francis, 1993. A
fundamental theory used in the present invention is known
as "spray congealing", based on spray drying principles
with the exception that solidification is the objective
instead of drying. Traditional emulsion based methods for
agarose bead preparation are described in, for
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example, Studies on Structure and Properties of Agarose, A. S.
Medin, pH.D. Thesis, Uppsala, 1995 and in "The Preparation of
Agarose Spheres for Chromatography of Molecules and
Particles", Biochimica et Biophysica Acta, 79, 393-398 (1964).
The particle size distribution produced by known
apparatus and methods require further sorting steps or
procedures in order to select particles of uniform size
required for chromatography. The additional sorting steps
introduce further costs that could be avoided if the factors
determining size distribution of the particles and operating
variables are closely controlled. Without additional sorting
steps, the products manufactured by conventional rotary
atomization or emulsion techniques cannot be used in
applications where the size distribution of the particles must
be very narrow. For example, when using particles in blood
purification applications, small particles must be avoided as
small particles could be caught by the carrier fluid and would
result in contamination of the purified material. Of course a
narrow particle size distribution improves performances of
particles in many applications, including chromatographic
applications.
Operating variables that influence droplet size
produced from atomizer wheels and hence particle size include
speed of rotation, wheel diameter, wheel design, feed rate,
viscosity of feed and air, density of feed and air and surface
tension of feed.
The atmosphere within which a particle passes is
important in order to avoid reduction of pore size. In
particular, humidity and temperature control avoids particle
desiccation during polymerization and gelling stages.
Particle desiccation reduces pore size. It is desirable to
8 .r.~ ~= s =--r[~ a.""'^ (.'y ("r"''" r
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have a machine and process to produce particles using
centrifugal action in such a manner as that the particles have
a narrow particle size distribution with both high porosity
and flow.
Lengthy consideration of prior art devices and
processes has identified a number of factors that may be
responsible for the wider size distribution of particles.
Such factors include interruptions on the wheel surface that
may impede radial acceleration of the particle solution and
adhesion to the surface of the wheel; lack of adequate
temperature control on the atomizer wheel that may result in
changes in feed viscosity and particle structure; and
uncontrolled airflow patterns at the perimeter of the atomizer
wheel that may result in particle twinning due to collisions
between particles prior to gelation and in undesired drying of
the particles due to a modification in their path down from
the wheel to the collecting liquid.
Summary of the Invention:
Applicants have recognized that control of humidity
and temperature within specific parameters in the immediate
area of the atomizer wheel will yield particles of a narrower
size distribution than previously possible with both good
porosity and rigidity.
Specifically, It has been discovered that the air
flow rate, temperature and humidity may be controlled in the
immediate area of the atomizer wheel with sufficient accuracy
to produce particles of a narrow size distribution. Control
of temperature and humidity is achieved by the combination of
temperature and humidity control means and an enclosure
*'rc *~ n ~ `rI ".. ~~ Me, e t..!s ~''wt % Fly I7 2 rF m T,.; ~'
f~ ~'] ~" Ah / 1 . '" G3
~--r,
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comprising an aperture, thus partially enclosing the atomizer
machine.
The apparatus and method of the present invention
5 produce particles having improved properties including very
good bead shape and a narrower size distribution than possible
with conventional production apparatus and methods. The
apparatus and method are particularly well suited for the
production of agarose beads for use in chromatography.
According to a first broad aspect, the invention
provides an atomizer machine for the production of porous
polymer particles, comprising:
a) an atomizer wheel having an edge, wherein the wheel is
rotatable about an axis;
b) a distributor for depositing polymer in fluid state to the
wheel;
c) a catch tray disposed under the atomizer wheel to collect
the polymer particles formed as a result of ejection of the
polymer from the edge as the atomizer wheel rotates;
d) an enclosure, enclosing the atomizer wheel, the
distributor and the catch tray, the enclosure defining a
partition between an interior environment of the atomizer
machine and an exterior environment of the atomizer machine;
e) an aperture on the enclosure allowing a gaseous exchange
between the interior environment of the atomizer machine and
the exterior environment of the atomizer machine.
In an embodiment, the above-mentioned aperture is of
variable size.
In an embodiment, the above-mentioned enclosure
includes a peripheral wall surrounding the atomizer wheel, the
a-,a 1 _ i'=:.c;, .,_. ~ t~ li .~L1 ~~a~ ~~ i~ u ~i~ ~~'.~u ~
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distributor and the catch tray and a roof portion covering the
peripheral wall.
In an embodiment, the above-mentioned peripheral
wall is generally circular.
In an embodiment, the above-mentioned aperture
extends circumferentially along the peripheral wall.
In an embodiment, the above-mentioned peripheral
wall includes an upper portion and a lower portion, the
aperture being defined between the upper portion and between
the lower portion.
In an embodiment, the above-mentioned atomizer
machine further comprises an actuator to displace the upper
portion and the lower portion with relation to one another to
vary the size of the aperture.
In an embodiment, the above-mentioned actuator is
operative to displace the upper portion along the axis to vary
the size of the aperture.
In an embodiment, the above-mentioned atomizer
machine includes a temperature control unit to regulate a
level of temperature in the interior environment.
In an embodiment, the above-mentioned atomizer
machine includes a humidity control unit to regulate a level
of humidity in the interior environment.
In an embodiment, the above-mentioned temperature
control unit comprises at least one of: a unit for controlling
a size of the aperture, a unit for controlling a level of
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temperature of the distributor and wheel, a unit for
controlling a level of temperature and a flow rate of water in
the catch tray, at least one valve providing at least one
respective vapor stream at a periphery of the atomizer wheel,
over the wheel and in the enclosure, and at least one steam
trap for de-misting air in the interior environment and
preventing water droplets from falling on the atomizer wheel.
In an embodiment, the above-mentioned humidity
control unit comprises at least one of: a unit for controlling
a size of the aperture, a unit for controlling a level of
temperature of the distributor and wheel, a unit for
controlling a level of temperature and a flow rate of water in
the catch tray, at least one valve providing at least one
respective vapor stream at a periphery of the atomizer wheel,
over the wheel and in the enclosure, and at least one steam
trap for de-misting the air in the interior environment and
preventing water droplets from falling on the atomizer wheel.
In an embodiment, the above-mentioned atomizer
machine further comprises a monitor capable of indicating a
level of temperature in the interior environment.
In an embodiment, the above-mentioned atomizer
machine further comprises a monitor capable of indicating a
level of humidity in the interior environment.
In an embodiment, the above-mentioned atomizer
machine further comprises a trajectory control means to
control a trajectory of the particles from a periphery of the
atomizer wheel to the catch tray.
In an embodiment, the above-mentioned trajectory
control means comprises a unit for controlling a size of the
... f' , i= +' .~ + ..... ~'`
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aperture, disposing steam valves at the periphery of the
atomizer wheel, over the atomizer wheel and directly into the
enclosure, and controlling airflow patterns at the periphery
of the atomizer wheel.
In an embodiment, the above-mentioned atomizer
machine further comprises a reactor for producing the polymer
and at least one temperature controlled conduit for feeding
the polymer to the distributor.
In an embodiment, the above-mentioned at least one
conduit consists of a double jacket tube defining an inner
passage for feeding the polymer to the distributor and an
outer envelope surrounding the inner passage, through which
outer envelope a temperature liquid is flowed to control a
level of temperature of the polymer.
In an embodiment, the above-mentioned distributor
rotates in the same direction as the atomizer wheel.
In an embodiment, the above-mentioned distributor
-comprises a plurality of holes.
In an embodiment, the above-mentioned plurality of
holes are disposed in a circle.
In an embodiment, the above-mentioned distributor
has 24 holes.
In an embodiment, the above-mentioned atomizer wheel
has a flat surface.
In an embodiment, the above-mentioned atomizer
machine further comprises a shaft for receiving the atomizer
a ^iav;.a
Zl ':o
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wheel, the shaft being conical and tapered so as to reduce
vibrations during rotation of the atomizer wheel.
In an embodiment, the above-mentioned atomizer
machine further comprises a shaft for receiving the atomizer
wheel, the shaft having a threaded section for securing the
atomizer wheel to the shaft.
In an embodiment, the above-mentioned atomizer
machine further comprises a sorting bin for receiving and
sorting the particles from the catch tray.
In an embodiment, the above-mentioned atomizer wheel
has a perimeter, the perimeter having radially projecting
teeth.
In an embodiment, the above-mentioned atomizer
machine further comprises at least one baffle disposed within
the enclosure for regulating air fl'ow within the internal
environment.
In an embodiment, the above-mentioned at least one
baffle is a plurality of baffles.
In an embodiment, the above-mentioned plurality of
baffles is 4 baffles.
According to a second broad aspect, the invention
provides a method for producing polymer particles, the method
comprising:
a) providing an atomizer wheel, distributor and a catch tray
enclosed by an enclosure defining a partition between an
interior and an exterior environment and having an aperture
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for allowing gaseous exchange between the interior and the
exterior environment; and
b) allowing gaseous exchange through the aperture thereby to
regulate at least one condition of temperature, humidity or
5 air flow within the interior environment.
In an embodiment, the above-mentioned method further
comprises varying a size of the aperture to vary a rate of
gaseous exchange.
In a further embodiment, the above mentioned
enclosure comprises a dome. The dome partially enclosing the
atomizer machine at once creates an open-system and creates a
zone surrounding the machine. The open system is necessary to
obtain an air flow current from within the zone to the
exterior of the zone. The air flow current contributes to the
control of temperature and humidity by preventing build-up of
heat within the immediate vicinity of the wheel as a result of
rapid rotation of the wheel. The creation of a zone
surrounding the machine is necessary to define an area within
which a desired temperature and humidity profile may be
maintained. It has been found that accurate control of the
factors that determine the temperature and humidity
surrounding the machine is not possible in absence of a
structure that defines a zone within which the temperature and
humidity control means operate to maintain the desired
temperature and humidity profile. Advantageously, the dome is
adjustable, thus providing adjustment of the aperture size, to
compensate for variations in the factors that affect the
temperature and humidity in the immediate vicinity of the gel
and particles.
Temperature, humidity and turbulence of the air
surrounding the apparatus and inside the apparatus affect the
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v' l
0
r$_'
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properties of beads: porosity, flow, average particle size,
particle size distribution, bead shape and non-specific
binding.
In a further embodiment, the invention further
provides an atomizer machine for the production of porous
polymer particles having a narrow size distribution
comprising:
a) an atomizer wheel rotating about an axis;
b) a distributor for providing a uniform thin layer of a
gelatinous polymer on the wheel;
c) a shaft connecting the wheel to a rotor;
d) a catch tray disposed under the wheel to collect the
particles;
e) a dome partially enclosing the atomizer wheel and catch tray
so as to maintain an open system and defining a zone
surrounding the wheel and catch tray;
f) a means for temperature and humidity control for creating
and maintaining a temperature and humidity gradient within
the zone;
wherein the gelatinous polymer deposited on the rotating wheel
moves to the periphery of the wheel under action of
centrifugal force, the film being broken into free flying
particles at the edge of the wheel.
These and other aspects of the invention shall
become apparent to those of ordinary skill in the art upon
consideration of the following description of specific
embodiments in conjunction with the accompanying drawings.
Brief Description of the Drawings:
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l 1 = . .. ~ . r
arsai} ~"~ai t..pl m~~,d C~ d~ Wax'=' ~t t~a G.,r=~ r3 1. l~s.~ an t.
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In the drawings:
Figure 1 illustrates an apparatus for producing porous
particles according to an embodiment of the invention;
Figure 2 illustrates the central column of the apparatus of
Figure 1 showing bottom and top steam diffusers and shaft-
wheel-distributor assembly;
Figure 3 illustrates the shaft-wheel-distributor assembly of
Figure 2;
Figure 4 is a bottom view of the bottom steam diffuser of
Figure 2 that distributes steam to the edge of the wheel;
Figure 5 is a side view of the bottom steam diffuser of Figure
2;
Figure 6 is a top view of the top steam diffuser of Figure 2
and a partial side view of the top steam diffuser of Figure 2;
and
Figure 7 is a bottom view of the top steam diffuser of Figure
2 and a partial side view of the top steam diffuser of Figure
2.
Detailed Description of the Invention:
One embodiment of the liquid atomization apparatus
of the invention is illustrated in Figure 1. A solution
comprising a polymer is prepared in reactor (1). Solid
particles are formed from the solution in a beader (2). A
heated tube (12) connects the reactor (1) to the beader (2).
~ ~l c EJ E ~ il ~~ c at ll l l'~a' ~
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The apparatus and method of the invention will be described in
conjunction with the production of agarose beads. However,
the apparatus may be used to produce particles of any other
polymer.
The polymer is first slowly poured into a solvent,
in an embodiment, at room temperature, under vigorous stirring
in a sealed stainless steel reactor (1), yielding a mixture.
Suitable solvents include, but are not limited to, water,
aqueous salt solutions, and organic solvents. The mixture is
heated up to over 90 C to allow a complete dissolution of the
agarose, forming a solution. The solution is quickly cooled
down to an intermediate temperature between dissolution and
gelling temperatures, where a special additive can be added to
the solution in order to improve bead porosity. This additive
or, chemical can be any chemical that helps obtain improved
porosity, such as a salt (for example, ammonium sulfate) or a
surfactant, in an embodiment, ammonium sulfate. Then, the
solution is slowly cooled down to the process temperature,
close enough to the gelling temperature, at a rate, for
example, of up to about 0.5 C/min, in an embodiment, not more
than 0.1 C/min.
Once the gel has reached its process temperature, it
is pumped through a heated tube (12) maintained at the gel
process temperature, from the reactor (1) to a nozzle (42),
using a gear pump (11) also maintained at the gel process
temperature by means of a pump head heater (not illustrated).
The gel is supplied to a distributor (40) by the nozzle (42),
and evenly distributed on the atomization wheel (39) by means
of a distributor (40). A thin uniform layer is formed by both
centrifugal force and the use of the distributor (40), and
split by teeth (43) into filaments, which are broken in
uniform sized spheres by the air flowing at the atomization
~S ,õ [7 f^r7 ~`-r m""'~ .ve ~; ~~ =-i sir~^~i ;F1 `.~ {"'P T,~"'e .~"; ,,.
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wheel (39) edge. The beads travel through the surrounding air
in the dome (13), where relative humidity and temperature are
accurately controlled (from hot and humid at the atomization
wheel (39) edge to less hot and less humid air at the catch
tray (14) level) before they fall into the catch tray (14).
The temperature and humidity profiles in the dome (13),
between the atomization wheel (39) and surface of the catch
tray (45), are accurately controlled in order to make sure
that the bead formed turns into solid phase prior to reaching
the catch tray surface. A liquid, for example water, is
continuously recirculated at a flow rate in a closed loop from
the catch tray (14) to a siever (20) and back to the catch
tray via a recirculation pump (23). The flow rate is adjusted
in such way that the surface of the catch tray (45) is always
covered with a thin continuous layer of liquid. A heat
exchanger (21) is installed in the inlet reservoir (22) of the
recirculation pump (23) to control the catch tray (14)
temperature. The beads can be collected at the outlet of the
siever machine (20) in a sealed bucket (24) for packaging.
The beader (2) thus contains a dome (13) and a catch
tray (14). The dome (13) is not attached to the catch tray
(14), leaving the beader (2) open for air exchanges with the
production room. The dome skirt (15) controls the gap between
the dome (13) and the catch tray (14), which is responsible
for the fresh air inlet into the process.. Therefore the dome
(13) defines a zone surrounding the apparatus and partially
encloses the atomizer wheel in an open system. In certain
embodiments, the temperature and humidity of the ambient air
in the production room should be accurately controlled between
20-23 C and 25-75% humidity, respectively, in order to get an
adequate temperature and humidity profile in the dome (13).
Deviations from these recommended adjustments could be
compensated by variations in other process parameters such as
. IN
~:u ~ i., J ~! rs ~ t_a c. t 7 i~ 6
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the gap between the dome (13) and the catch tray (14) as an
example.
The catch tray (14), which has a slope from the
5 center to the edge, collects the beads off the atomization
wheel in a liquid that is in continuous recirculation. In
order to allow the dome to move up and down for maintenance,
cleaning and atomization wheel (39) installation, a system of,
for example, at least one rod with an air cylinder, for
10 example, three rods (18) with three air cylinders (19), may be
used. A rigid structure (44) stabilizes the dome and avoids
any instability that could result in vibration or movements of
the dome (13).
15 Two columns are included in the beader: a top column
(4), which is attached to the dome (13), and a bottom column
(3), which is attached to the catch tray (14) These two
columns are clearly illustrated in drawings 2 to 7. The
bottom column (3) holds the atomization wheel and helps in the
control of temperature and humidity profiles in the dome (39),
while the top column (4) controls the environment over the
atomization wheel and helps in the control of the temperature
and humidity in the dome (13). For maximum advantage, these
two columns should be centered at all times. Both the rods
(18) and the rigid structure (44) of the dome (13) guarantee
centering of the two columns (3) and (4). A main steam supply
(5) is split in two steam lines (6) and (7), that are required
for the control of both temperature and humidity in the dome
(13).
A collecting liquid (water in the illustrated
example) is distributed from the inlet reservoir (22) to the
catch tray (14) through a splitter (16) which can be located
at the center of the catch tray (14) The liquid forms a
if 7tL; , (
~u ~ Yõ iL.7 ~h.9
V"is~Z
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uniform and evenly distributed thin film on the catch tray
surface (45) and ends in tubes (17) that are connected to the
siever (20). For maximum advantage, the catch tray surface
(45) should be continuously covered by a thin layer of liquid
in order to prevent drying of the beads as they fall on the
catch tray (14). The liquid flow rate in the catch tray (14)
and its temperature affect the control of humidity and
temperature in the dome (13).
The center part of the dome is illustrated in figure
2. A flat atomization wheel (39) that, in certain
embodiments, can have radially projecting teeth (43) at an
edge thereof, is covered by a distributor (40), which is
centered with the atomization wheel (39) and, in certain
embodiments, rotates at the same speed as the atomization
wheel. A plate (58) is screwed to the tapered shaft (29),
keeping the atomization wheel-distributor assembly in place.
The distributor (40) is the recipient for the gel that comes
out from a nozzle (42) . The gel falls on a distributor lip
(67), which is filled with holes (66), allowing the gel to be
evenly distributed at the bottom of the distributor (40).
These holes should occupy almost all the distributor lip (67)
surface andbe spaced in such a way that sufficient strength
of the distributor (40) is maintained. An inside cylinder
(60) of the distributor (40) is longer than an outside
cylinder (59) giving a constant and reproducible gap between
the atomization wheel (39) and the distributor (40) This
design avoids the use of spacers, which would unbalance the
atomization wheel-distributor assembly and increase particle
size distribution. In mounting high speed rotary bodies it is
advantageous that the rotating mass be balanced. A suitable
mounting arrangement for securely positioning the wheel on the
rotatable shaft is to use a tapered shaft (29) in order to
make atomization wheel-shaft alignment easy and reproducible
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and to maintain balancing. The inside of the atomization
wheel (39) is machined with the same slope as the tapered
section (61) of the tapered shaft (29). In an embodiment, the
atomization wheel (39) does not touch the bottom of the
tapered section (61) but is supported by the tapered section
(61) itself. This has been designed to avoid any screwed part
that would make the alignment-difficult to reproduce. For
maximum advantage, the tapered shaft-atomization wheel-
distributor assembly should be balanced, advantageously, at
all speeds within the rotation speed range utilized, to,
eliminate vibration.
The atomization wheel (39) stands over a bottom
steam diffuser (31), which helps the regulation of temperature
and humidity in the dome (13) and in the area close to the
atomization wheel (39) . The bottom steam diffuser (31) is
connected to a bottom steam line (7) where a steam trap (48)
removes any steam condensate located in the bottom steam line
(7). A needle valve (65), located as close as possible to the
steam trap (48), accurately controls the steam flow rate to
the bottom steam diffuser (31). Steam is distributed into the
dome (13) via slots (50) located on the side of the bottom
steam diffuser (31). A bottom plate (46) and a top plate (38)
are part of the bottom steam diffuser (31) and can be affixed
to it using screws (49), for example. A drain (47) allows the
evacuation of any condensation that could occur in the bottom
steam diffuser (31) and avoids water accumulation that would
result in steam bubbling and result in a change in humidity
and temperature conditions in the dome (13). An annular plate
(30) having holes, for example, very small holes, covers the
side of the bottom steam diffuser (31). The small holes of
the annular plate (30) cover a limited sector of the annular
plate (30) . For example, the sector may be defined by the
first 60 starting at the bottom of the annular plate (30), in
{'rs ~ r7 ~1 ry.?J i. p ,,~n- i /'7' Fl n 1~"a n a^
'a
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order to guide the steam in the dome (13) and not under the
atomization wheel (39) or at the atomization wheel (39) edge,
close to the teeth (43). The bottom column (3) also holds a
motor (not illustrated) that controls atomization wheel (39)
RPMs (revolutions per minute).
The top steam diffuser (26) is connected to the top
column (4) using flange (28), spacer (25) and connection (27).
The spacer (25) and connection (27) avoid any chimney effect
in the top column (4) that could result from the high spinning
rate of the atomization wheel and thus affect the temperature
and humidity conditions in the dome (13) and in the area close
to and above the atomization wheel (39). The steam in the top
steam line (6) goes through a demister (69) where most water
drops resulting from steam condensation are removed. A steam
trap (68) completes condensate removal from the top steam line
(6). The top steam line (6) is then split into three steam
lines (8), (9) and (10), where needle valves (62), (63) and
(64) respectively, accurately control the steam flow rate in
the areas of the top steam diffuser (26) . The first steam
line (10) is split into a group of holes (55) located
immediately above the distributor (40), and keeps the air
above the distributor (40) fully saturated in order to prevent
the liquid sprayed from drying under the effect of the fast
air flow rate generated by pumping caused by the rotation of
the atomization wheel (39). The second steam line (9) is
split into a second group of holes (54) forming a circle
located outside the distributor (40) but still above the
atomization wheel (39) . This second steam line (9) is also
required to avoid drying of the liquid on the atomization
wheel (39) but also to maintain the required temperature
profile above the atomization wheel (39). A ring (57)
restricts exchanges between the dome (13) and the area above
the atomization wheel (39) and helps to control temperature
~"'~ {`~ l' Vi"'3 ~'u ~1 s, ya r.-^. n n._sm-+= 7m :r-b Sm S"r F"+^ ~^= ,.~u
~ ,~ ~
E, b~ l';i Ll
CA 02417285 2003-01-27
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and humidity conditions above the atomization wheel (39). The
third steam line (8) supplies steam to a group of holes (53)
located above the atomization wheel (39) but outside the ring
(57), directing steam in the dome (13), close to the
atomization wheel (39) edge.
The combination of appropriate adjustments to the
following process parameters combined with the presence of a
demister (69), steam traps (48) and (68), bottom steam
diffuser (31) and top steam diffuser (26) controls the
temperature and humidity profiles in the dome (.13), in the
area above the atomization wheel and at the atomization wheel
edge: distance between the dome (13) and the catch tray (14),
steam pressure, temperature and flow rate of the liquid in the
catch tray, humidity and temperature of the air surrounding
the apparatus (production room), needle valves (62), (63),
(64), (65) adjustments, distance between the atomization wheel
(39) and the ring (57) of the top steam diffuser (51),
atomization wheel (39) spinning rate, distance between the
atomization wheel (39) and the surface of the catch tray (45).
These parameters control temperature and humidity profiles and
are adjusted according to the product manufactured and desired
properties.
Although various embodiments of the invention are
disclosed herein, many adaptations and modifications may be
made within the scope of the invention in accordance with the
common general knowledge of those skilled in this art. Such
modifications include the substitution of known equivalents
for any aspect of the invention in order to achieve
substantially the same result in substantially the same way.
Numeric ranges are inclusive of the numbers defining the
range. In the claims, the word "comprising" is used as an
open-ended term, substantially equivalent to the phrase
CA 02417285 2003-01-27
WO 02/12374 PCT/CA01/01126
"including, but not limited to". The following examples are
illustrative of various aspects of the invention, and do not
limit the broad aspects of the invention as disclosed herein.
5 Examples
Particles produced by the apparatus and method of
the present invention have a very narrow size distribution as
illustrated by the following examples describing agarose bead
10 manufacture. The polymer preparation steps and temperatures
and most process parameters are specific to agarose
preparation and could differ depending on the polymer used for
particle formation.
15 Example 1: Preparation of 4~ 100 ,um agarose beads
300 g of agarose was slowly poured in 4.25L of
purified water under vigorous mixing. This solution was
heated up to 97-99 C for 30 minutes and cooled down to 70 C.
20 A heating/cooling fluid was used in the jacket of the reactor
to control the temperature accurately. 750 mL of a 0.75M
ammonium sulfate solution, maintained at 70 C, was added very
slowly and under vigorous stirring to the previous agarose
solution in order to prevent local salting out, which would
result in the formation of lumps. The final solution was
cooled to 56-57 C at a rate not more than 0.1 C/min.
In the meantime, the beader was started for
stabilization. Atomization wheel-column centering was
checked, and the distance between the atomization wheel and
the top column was adjusted to 15 mm. The dome opening
(distance between the dome and the catch tray) was adjusted to
7 cm. The atomization wheel speed was adjusted to 4900-5100
RPM , needle valves were all adjusted at 7 and steam pressure
p~`,;'" RJ f7 I^"'y ~ ';^" F - n In' . "" ~' ~,.~ "~ =~-_m ~''~
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21
was set at 5psig at the boiler outlet. These steam
adjustments allowed the control of dome temperature at 36-39 C
close to the edge of the atomization wheel and were adequate
for the product manufactured and the size of the dome.
Approximately 60L/minute of purified water maintained at 16-
19 C were recirculated in the catch tray to ensure that the
catch tray surface is continuously covered with a thin film of
water. This water flow rate is also appropriated for the
control of temperature and humidity in the dome. Resulting
stabilized beader temperatures were the following:
Atomization wheel temperature: 56-60 C
Catch tray temperature: 16-19 C
Dome temperature close to the atomization wheel edge: 37-39 C
Temperature at area above atomization wheel: 71-73 C
Once the beader was stabilized and the gel at the
right temperature, the gear pump was turned on, feeding 1.6L
of gel/hr to the atomization wheel. The following properties
were recorded:
~ Gn h ~t';:.~~V}~
'771
t L~
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22
Properties / Lot Specifica- Lot Lot Lot
tions 000612381 001016434 001017435
Porosity
Thyroglobulin 0.35 - 0.53 0.44 0.48 0.48
Apoferritin 0.50 - 0.76 0.60 0.63 0.64
(3-1-\mylase 0.54 - 0.80 0.64 0.67 0.68
Alcohol 0.58 - 0.86 0.69 0.72 0.72
Dehydrogenase
Albumin 0.61 - 0.91 0.72 0.74 0.76
(Bovine Serum)
Carbonic 0.68 - 1.00 0.85 0.85 0.87
Anhydrase
Pressure vs Flow > 20cm/hr 35 22 22
Particle Size
Analysis
Average N/A 106 103 101
Size ( m)
% Between N/A 98% 97.4% 90%
76-140 microns
before sieving
% Between Greater than 99.7% 99% 99%
76-140 microns 95%
after sieving
Non-Specific Less than 8 3.1 2.5 2.2
Binding g cyt. /ml
gel
Microscopy Less than 3% 0.89% 0.7% 0.9%
broken,
fused,
damaged
beads
Process reproducibility has been demonstrated and is clearly
documented. The particle size distribution before sieving is
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23
very narrow, a lot more than any equivalent product available
on the market at the moment. The distribution can be
significantly improved by sieving, without significantly
reducing the global yield. As an example, a 5L batch prepared
as above gave reproducibly 6.5 to 6.8L of beads.
Example 2: Preparation of 4-2r 100 ,um agarose beads using
apparatus further comprising baffles
A plurality of 3 inch or 6 inch baffles were
inserted into the dome in order to get a more homogeneous
temperature profile in the dome. Four baffles were equally
distributed on the inside of the dome, vertically, to inhibit
the effect of room conditions in the dome. With the baffles
in place, the dome is less sensitive to ambient conditions,
and dome conditions are more easily reproduced. In addition,
a wider range of particle size can be produced in the same
apparatus when the baffles are used. The increase in disk RPM
required to produce smaller particles affects the air pattern
and hydrodynamics in the dome. The presence of baffles makes
the temperature profiles less dependant on disk RPM. Also,
the production of large particles (above 200 microns) resulted
in projection of particles on the walls of the dome, due to
the inertia of the particles produced. The presence of
baffles affects the air pattern in such a way that the
particles formed fall closer to the bottom column, making
possible the manufacture of large particles without changing
the design of the equipment.
4% 100 microns agarose beads have been manufactured
using conditions in the example above to demonstrate that the
presence of baffles do not affect particle properties. Lot
001103443 was manufactured using 6" baffles, while lot
W? '!''.
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24
001113447 was manufactured using 3" baffles. Lot 000612381 is
reproduced in the table for comparison purposes.
Properties / Lot Specifica- Lot Lot Lot
tions 000612381 001113447 001103443
Porosity
Thyroglobulin 0.35 - 0.53 0.44 0.45 0.43
Apoferritin 0.50 - 0.76 0.60 N/A N/A
(3-Amylase 0.54 - 0.80 0.64 N/A N/A
Alcohol 0.58 - 0.86 0.69 N/A N/A
Dehydrogenase
Albumin 0.61 - 0.91 0.72 N/A N/A
(Bovine Serum)
Carbonic 0.68 0.85 N/A N/A
Anhydrase
Pressure vs Flow > 20cm/hr 35 28 24
Particle Size
Analysis
Average N/A 106 101 100
Size ( m)
% Between N/A 98% 97.0% 97.2%
76-140 microns
before sieving
% Between Greater than 99.7% 98.9% 98.6
76-140 microns 95%
after sieving
, `:. S f ~I u.~a -^3
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WO 02/12374 PCT/CA01/01126
Non-Specific Less than 8 3.1 N/A N/A
Binding g cyt. /ml
gel
Microscopy Less than 30 0.890 1.90 1.50
broken,
fused,
damaged
beads
Example 3: Preparati.on of 5~ 200,um agarose beads
A procedure similar to the one described for the
5 preparation of 40 100 microns agarose beads has been applied
for the manufacturing of 200 microns agarose beads at the 2L
scale. The differences are set forth in the present
description.
10 110 g of agarose was slowly poured in 1.7L of
purified water under vigorous mi.xing. This solution was
heated up to 97-99 C for 30 minutes and cooled down to 70 C.
300 mL of a 0.75M ammonium sulfate solution, maintained at
70 C, was added very slowly and under vigorous stirring to the
15 previous agarose solution. The final solution was cooled to
55-57 C at a rate not more than 0.1 C/min.
In the meantime, the beader was started for
stabilization. The distance between the atomization wheel and
20 the top column was adjusted to 15 mm. The dome opening was
adjusted to 4 cm. The atomization wheel speed was adjusted to
about 2000 RPM, needle valves were all adjusted at 3 and steam
pressure was set at 5psig at the boiler outlet. These steam
adjustments allowed the control of dome temperature at 47-50 C
25 close to the edge of the atomization wheel and were adequate
for the product manufactured and the size of the dome.
r"~ ~" ~'I { -T ^"3 e^rsn m ,{ '~ ^~ ~:e~ !'^~ fl n G"-~ ~~-~7c.f~~,o
F I~.I .- 5 Y. nn ~ :-'li ~~'I ,~ I~
A1.1'.U,~ ~.r.~ lltL~ '~e au1 t~ Ll ~~ ``~l'$~ h~ {~~-,u',J ~y~ J,~~r'~ ~JNi
L.~~.U
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26
Approximately 60L/minute of purified water maintained at 14-
16 C was recirculated in the catch tray. Resulting stabilized
beader temperatures were the following:
Atomization wheel temperature: Not available due to the dome
opening
Catch tray temperature: 14-16 C
Dome temperature close to the atomization wheel edge: 47-50 C
Temperature at area above atomization wheel: 78-80 C
Once the beader was stabilized and the gel at the
right temperature, the gear pump was turned on, feeding about
2.8L of gel/hr to the atomization wheel. The following
properties were recorded:
Properties / Lot Specifica- Lot Lot
tions 001116450 001123453
Porosity
Thyroglobulin N/A 0.02 0.03
Apoferritin N/A 0.46 0.11
(3-Amylase N/A 0.63 0.64
Alcohol N/A 0.70 0.73
Dehydrogenase
Albumin N/A 0.71 0.74
(Bovine Serum)
Carbonic N/A 0.85 0.86
Anhydrase
Pressure vs Flow N/A 114 106
Particle Size
Analysis
Average N/A 200 199
Size ( m)
Gsy
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% Between N/A N/A N/A
150-300 microns
before sieving
% Between N/A 96% 96%
150-300 microns
after sieving
Non-Specific N/A N/A N/A
Binding
Microscopy N/A 2.5 3.9
Example 4: Preparation of 4-t 125,um agarose beads
The procedure described for the production of 4% 100
microns agarose beads has been applied for the production of
4% 125 microns agarose beads at the 3L scale. The differences
are set forth in the present description.
180 g of agarose was slowly poured in 2.55L of
purified water under vigorous mixing. This solution was
heated up to 97-99 C for 30 minutes and cooled down to 70 C.
450 mL of a 0.75M ammonium sulfate solution, maintained at
70 C, was added very slowly and under vigorous stirring to the
previous agarose solution. The final solution was cooled to
55-57 C at a rate not more than 0.1 C/min.
In the meantime, the beader was started for
stabilization. The distance between the atomization wheel and
the top column was adjusted to 15 mm. The dome opening was
adjusted to 7 cm. The atomization wheel speed was adjusted to
3700-3800 RPM, needle valves were all adjusted at 4-5 and
steam pressure was set at 5psig at the boiler outlet. These
steam adjustments allowed the control of dome temperature at
35-37 C close to the edge of the atomization wheel and were
er, `~ .uuc1 E~' +U:~~ ~~~+ ~~/ e~x.~ ~Lf u' L 1z-?. .no
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28
adequate for the product manufactured and the size of the
dome. Approximately 60L/minute of purified water maintained
at 14-16 C were recirculated in the catch tray. Resulting
stabilized beader temperatures were the following:
Atomization wheel temperature: 56-58 C
Catch tray temperature: 14-16 C
Dome temperature close to the atomization wheel edge: 35-37 C
Temperature at area above atomization wheel: 71-75 C
Once the beader was stabilized and the gel at the
right temperature, the gear pump was turned on, feeding about
1.9L of gel/hr to the atomization wheel. The following
properties were recorded:
Properties / Lot Specifica- Lot Lot Lot
tions 010118464 010124465 010125466
Porosity
Thyroglobulin 0.35 - 0.53 0.45 0.47 0.48
Apoferritin 0.50 - 0.76 N/A N/A N/A
(3-Amylase 0.54 - 0.80 N/A N/A N/A
Alcohol 0.58 - 0.86 N/A N/A N/A
Dehydrogenase
Albumin 0.61 - 0.91 N/A N/A N/A
(Bovine Serum)
Carbonic 0.68 - 1.00 N/A N/A N/A
Anhydrase
Pressure vs Flow > 30cm/hr 44 53 45
Particle Size
Analysis
Average N/A 124 120 123
Size (pm)
~ .+a= ~ r=-r-, ~ ~ r-=n ~p,~ ~-,~ q~ ~-a m-3 c~
. . :a i =., a ~
<~,~~ u t~ii v~,2a
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29
% Between N/A N/A N/A N/A
95-165 microns
before sieving
% Between Greater than 99.5% 98.5% 99.5%
95-165 microns 95%
after sieving
Non-Specific Less than 8 N/A N/A N/A
Binding g cyt. /ml
gel
Microscopy Less than 3% 1.3 1.3 0.6
broken,
fused,
damaged
beads
Example 5: Preparation of 4-b 60,um agarose beads
The procedure described for the production of 4% 100
microns agarose beads has been applied for the production of
4% 60 microns agarose beads at the 2L scale. The differences
are set forth in the present description.
120 g of agarose was slowly poured in 1.7L of
purified water under vigorous mixing. This solution was
heated up to 97-99 C for 30 minutes and cooled down to 70 C.
300 mL of a 0.75M ammonium sulfate solution, maintained at
70 C, was added very slowly and under vigorous stirring to the
previous agarose solution. The final solution was cooled to
56-58 C at a rate not more than 0.1 C/min.
In the meantime, the beader was started for
stabilization. The distance between the atomization wheel and
the top column was adjusted to 15 mm. The dome opening was
adjusted to 7 cm. The atomization wheel speed was adjusted to
rr, ,,,
GI ~7nu ~X ~ IJ t, v tr-=+
eun ''wL +':.i r,r~ ~] IJ ~l J
CrC~
CA 02417285 2003-01-27
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7200 RPM , needle valves were all adjusted at 7 and steam
pressure was set at 5psig at the boiler outlet. Those steam
adjustments allowed the control of dome temperature at 33-35 C
close to the edge of the atomization wheel and were adequate
5 for the product manufactured and the size of the dome.
Approximately 60L/minute of purified water maintained at 16-
19 C were recirculated in the catch tray. Resulting
stabilized beader temperatures were the following:
10 Atomization wheel temperature: 56-58 C
Catch tray temperature: 16-19 C
Dome temperature close to the atomization wheel edge: 33-35 C
Temperature at area above atomization wheel: 68-70 C
15 Once the beader was stabilized and the gel at the
right temperature, the gear pump was turned on, feeding about
0.6L of gel/hr to the atomization wheel. The following
properties were recorded:
Properties / Lot Specifica- Lot Lot Lot
tions 001018436 001019437 001108446
Porosity
Thyroglobulin >0.20 0.56 0.47 0.45
Apoferritin N/A N/A N/A N/A
(3-Amylase N/A N/A N/A N/A
Alcohol N/A N/A N/A N/A
Dehydrogenase
Albumin N/A N/A N/A N/A
(Bovine Serum)
Carbonic N/A N/A N/A N/A
Anhydrase
' G'uy,s7 .,aaN;~ Li i q,~' W~S d;J ~Fas m~w~ 6sC"(~ Ci 9'~w LHw
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31
Pressure vs Flow > 5 cm/hr 10 7 7
Particle Size
Analysis
Average N/A 59 61 59
Size (pm)
% Between N/A 92.3% 90.5% 86.7%
30-95 microns
before sieving
% Between N/A 91.3% 90.5% 90.5%
30-95 microns after
sieving
Non-Specific N/A N/A N/A N/A
Binding
Microscopy Less than 3% 0% 1.3% 0.8%
broken,
fused,
damaged
beads
Again lot 001108446 was manufactured using the 6" baffles as
described in the example above and compared to the standard
material to demonstrate that the presence of baffles do not
affect the particle properties.
Example 6: Preparation of 6~ 100,um agarose beads
The procedure described for the production of 4% 100
microns agarose beads has been applied for the production of
6% 100 microns agarose beads at the 5L scale. The differences
are set forth in the present description.
380 g of agarose was slowly poured in 4.25L of
purified water under vigorous mixing. This solution was
heated up to 97-99 C for 30 minutes and cooled down to 70 C.
( ~ ~~ i. ~ ; . o y x
ez!, ~w~ ~~r a
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32
750 mL of a 0.75M ammonium sulfate solution, maintained at
70 C, was added very slowly and under.vigorous stirring to the
previous agarose solution. The final solution was cooled to
59-61 C at a rate not more than 0.1 C/min.
In the meantime, the beader was started for
stabilization. The distance between the atomization wheel and
the top column was adjusted to 15 mm. The dome opening was
adjusted to 7 cm. The atomization wheel speed was adjusted to
4900-5100 RPM , needle valves were all adjusted at 7-9 and
steam pressure was set at 5psig at the boiler outlet. Those
steam adjustments allowed the control of dome temperature at
37-39 C close to the edge of the atomization wheel and were
adequate for the product manufactured and the size of the
dome. Approximately 60L/minute of purified water maintained
at 16-20 C were recirculated in the catch tray. Resulting
stabilized beader temperatures were the following:
Atomization wheel temperature: 59-63
Catch tray temperature: 16-20 C
Dome temperature close to the atomization wheel edge: 37-39 C
Tempetature at area above atomization wheel: 71-74 C
Once the beader was stabilized and the gel at the
right temperature, the gear pump was turned on, feeding about
1.7L of gel/hr to the atomization wheel. The following
properties were recorded:
Properties / Lot Specifica- Lot Lot Lot
tions B28902 B32904 001026439
Porosity
Thyroglobulin 0.25-0.44 0.28 0.31 0.27
Apoferritin 0.39-0.59 0.44 0.47 N/A
! r ~ ~ ~~ r
4ta:-~) 4,.,,1.; =<cr b U G.
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33
(3-Amylase 0.48-0.72 0.50 0.52 N/A
Alcohol 0.49-0.73 0.56 0.57 N/A
Dehydrogenase
Albumin 0.52-0.78 0.59 0.62 N/A
(Bovine Serum)
Carbonic 0.66-0.98 0.73 0.77 N/A
Anhydrase
Pressure vs Flow >45 cm/hr 60 64 52
Particle Size
Analysis
Average N/A 101 101 102
Size (p.m)
% Between N/A 80.3 80.1 68%
76-140 microns
before sieving
% Between >95% 99% 99% 99.4%
76-140 microns
after sieving
Non-Specific Less than 8 1.7 2.9 N/A
Binding g cyt. /ml
gel
Microscopy Less than 3% 0% 0.3% 1.6
broken,
fused,
damaged
beads
Batch 001026439 was produced using a higher pump flow rate.
According to the theory, the pump flow rate could be
significantly increased without affecting the product quality.
This has been confirmed with lot 001026439, where the pump was
increased to its limit and delivering about 3.5L/hr of gel on
the atomization wheel without affecting the properties. Only
the particle size distribution before sieving was slightly
broader when the pump flow rate is increased, resulting in a
t RY f~.a
~i z 7 ~
~j `J' l; V,c~u ~~ Lf W' q~lY~ ,.,,._n W~'.~ ~, 1 u 41 uu3 !~ =9 ~
CA 02417285 2003-01-27
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34
lower product yield. Therefore the gel flow rate fed to the
atomization disk is not limited to the examples above, higher
and lower feed rates can result in the same product.
In an embodiment, the atomizer machine may further
comprise at least one baffle, in a further embodiment, a
plurality of baffles, in a further embodiment, 4 baffles,
which is/are disposed within the enclosure and can
affect/regulate the air pattern in the interior environment.
The particles produced by the apparatus and process
of present invention may be used in all the chromatographic
and electrophoretic methodologies for industrial purification
purposes including affinity chromatography, gel filtration,
ion-exchange chromatography, as support for grafting different
types of ligands; and coating rigid spheres of glass or
plastic for types of chromatographic applications.
e,,~'i
