U.S. patent application number 14/386085 was filed with the patent office on 2015-02-12 for micro flow filtration system and integrated microfluidic element.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Adelbert Grossmann, Nadine Losleben, Sascha Lutz, Norbert Oranth.
Application Number | 20150041375 14/386085 |
Document ID | / |
Family ID | 47989002 |
Filed Date | 2015-02-12 |
United States Patent
Application |
20150041375 |
Kind Code |
A1 |
Oranth; Norbert ; et
al. |
February 12, 2015 |
MICRO FLOW FILTRATION SYSTEM AND INTEGRATED MICROFLUIDIC
ELEMENT
Abstract
A micro fluid filtration system (100) preferably for increasing
the concentration of components contained in a fluid sample has a
fluid circuitry (1). The fluid circuitry (1) comprises the
following elements: A tangential flow filtration element (7)
capable for separating the fluid sample into a retentate stream and
a permeate stream upon passage of the fluid, an element for pumping
(3) for creating and driving a fluid flow through the fluid
circuitry (1) and at least one element for obtaining information
about the properties of the fluid sample within the circuitry. The
circuitry further comprises a plurality of conduits (24) connecting
the elements of the fluid circuitry (1) through which a fluid
stream of the fluid sample is conducted. The circuitry (1) has a
minimal working volume of at most 5 ml, which is the minimal fluid
volume retained in the elements and the conduits (24) of the
circuitry (1) such that the fluid can be recirculated in the
circuitry (1) without pumping air through the circuitry (1). An
integrated microfluidic element (20) of the circuitry (1) contains
the functionality of at least two elements of the group of elements
of the circuitry (1).
Inventors: |
Oranth; Norbert;
(Voerstetten, DE) ; Losleben; Nadine; (Mannheim,
DE) ; Lutz; Sascha; (Neustadt, DE) ;
Grossmann; Adelbert; (Eglfing, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
47989002 |
Appl. No.: |
14/386085 |
Filed: |
March 25, 2013 |
PCT Filed: |
March 25, 2013 |
PCT NO: |
PCT/EP2013/056294 |
371 Date: |
September 18, 2014 |
Current U.S.
Class: |
210/90 ;
210/85 |
Current CPC
Class: |
B01L 2400/0487 20130101;
F16B 33/006 20130101; B01L 2300/0816 20130101; B01D 2311/14
20130101; B29C 70/72 20130101; F16B 33/004 20130101; B01D 63/088
20130101; B01L 3/502753 20130101; B29K 2105/08 20130101; B29L
2031/727 20130101; F16B 35/00 20130101; G01N 2001/4088 20130101;
B01D 61/147 20130101; B01L 2300/088 20130101; B01L 3/502715
20130101; B01L 2300/0864 20130101; Y10T 403/473 20150115; B01D
2311/25 20130101; B01D 2315/10 20130101; B01D 63/005 20130101; B01L
2200/146 20130101; B01L 2300/0681 20130101; B01D 61/22 20130101;
Y10T 403/56 20150115; B01D 63/087 20130101; B01L 2300/0627
20130101; B01L 2400/0478 20130101; B01L 2200/0647 20130101; B29C
45/14 20130101; B01D 2313/90 20130101; B01D 2311/246 20130101 |
Class at
Publication: |
210/90 ;
210/85 |
International
Class: |
B01D 63/00 20060101
B01D063/00; B01D 61/22 20060101 B01D061/22; B01D 61/14 20060101
B01D061/14 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 29, 2012 |
EP |
12162011.6 |
Claims
1. A microfluidic flow filtration system having a fluid circuitry
(1), the fluid circuitry (1) comprising the following elements: a
tangential flow filtration element (7) having a feed inlet (12), a
retentate outlet (13), a permeate outlet (14) and a membrane (15)
capable for separating the fluid sample into a retentate stream and
a permeate stream upon passage of the fluid sample into the
tangential flow filtration element (7) through the feed inlet (12),
an element for pumping for creating and driving a fluid flow of the
fluid sample through the fluid circuitry (1) and the tangential
flow filtration element (7), at least two elements for obtaining
information about the properties of the fluid sample within the
fluid circuitry (1); and a plurality of conduits (24) connecting
the elements to the fluid circuitry (1) through which a fluid
stream of the fluid sample is conducted; wherein a minimal working
volume of the fluid circuitry (1) being defined by the minimal
fluid volume retained in the elements and the conduits (24), such
that the fluid can be recirculated in the fluid circuitry (1)
without pumping air through the fluid circuitry (1), the minimal
working volume of the fluid circuitry (1) is at most 5 ml, the
functionality of at least two elements of the group of elements of
the circuitry (1) is integrated in one integrated microfluidic
element (20), the one integrated microfluidic element (20) defines
a volume element having a volume which is at most one fourth of the
minimal working volume of the fluid circuitry (1).
2. The microfluidic flow filtration system according to claim 1,
characterized in that the integrated microfluidic element (20)
integrates the functionality of at least two elements for obtaining
information about the properties of the fluid sample, wherein
preferably one element for obtaining information about the
properties is an optical measuring element for determining the
concentration of the components contained in the fluid sample and
one element for obtaining information about the properties is a
measuring element for determining the viscosity of the fluid
sample, and further preferably the optical measuring element for
determining the concentration is a cuvette (6) and/or the measuring
element for determining the viscosity is at least one element for
determining pressure preferably in combination with a capillary
channel element.
3. The microfluidic flow filtration system according to claim 1,
characterized in that the integrated microfluidic element (20)
provides the functionality of a cuvette (6) or a capillary channel
element for determining the concentration, preferably in form of a
transparent capillary (28), and of at least one pressure sensor
(26, 27), preferably of at least two pressure sensors (26, 27), for
determining the viscosity.
4. The microfluidic flow filtration system according to claim 1,
characterized in that the integrated microfluidic element (20)
provides the functionality of at least one element for determining
pressure, preferably a pressure sensor, and the tangential flow
filtration element (7).
5. The microfluidic flow filtration system according to claim 1,
characterized in that the integrated microfluidic element (20)
having a housing and the tangential flow filtration element (7)
having a TFF-housing which is part of the housing of the
microfluidic element (20), wherein preferably the membrane of the
tangential flow filtration element (7) is disposable.
6. The microfluidic flow filtration system according to claim 1,
characterized in that the circuitry (1) further comprises a
reservoir element (2) suitable for containing a fluid, the
reservoir element (2) being integrated in the fluid circuitry (1)
has at least a reservoir inlet and reservoir outlet both in
connection to the circuitry (1), which is a reservoir or a tank
containing the fluid sample, wherein the reservoir element (2) is
also a member of the group of elements of the circuitry (1).
7. The microfluidic flow filtration system according to claim 1,
characterized in that the volume of the reservoir (2) is at most 10
ml, preferably at most 1 ml, particularly preferably at most 0.7 ml
and also particularly preferably at most 0.5 ml.
8. The microfluidic flow filtration system according to claim 1,
characterized in that the circuitry (1) further comprises at least
a valve element (9); and/or a hollow fiber element; and/or a
regulator element (8) for regulating the flow of the fluid through
the fluid circuitry, which is preferably is a pump, and/or a
pressure regulation element for regulating the pressure of the
fluid in the fluid circuitry, which is preferably a valve, and/or
an element for determining pressure data which is one or more
pressure sensors (4, 5, 39); and/or an optical detection element
(6), wherein these elements are also members of the group of
elements of the circuitry (1).
9. The microfluidic flow filtration system according to claim 1,
characterized in that the minimal working volume of the fluid
circuitry (1) is at most 1 ml, preferably at most 500 .mu.l,
further preferably at most 200 .mu.l and particularly preferably at
most 100 .mu.l.
10. The microfluidic flow filtration system according to claim 1,
characterized in that the conduits (24) have an internal diameter
of at most 1.5 mm, preferably of at most 1 mm, particularly
preferably of at most 0.7 mm or also particularly preferably of at
most 0.1 mm wherein a conduit (24) is preferably a channel, or a
passage in an element of the circuitry, or a pipe, or a tubing.
11. An integrated microfluidic element (20) for a microfluidic flow
filtration system with a fluid circuitry (1) having a minimal
working volume of at most 5 ml, preferably for a system (100)
according to any one of the preceding claims, characterized in that
the integrated microfluidic element (20) defines a volume element
having a volume which is at most one fourth of the minimal working
volume of the fluid circuitry of the micro flow filtration system,
and provides the functionality of at least two elements of the
group of elements of the fluid circuitry (1), wherein the group of
elements comprises a tangential flow filtration element (7), an
element for pumping suitable for creating and driving a fluid flow
and at least two elements for obtaining information about the
properties of the fluid sample within the circuitry.
12. The integrated microfluidic element according to the preceding
claim 11, characterized in that the element for obtaining
information about the properties of the fluid sample within the
circuitry is an optical measuring element for obtaining information
about the concentration of the components contained in the fluid
sample and/or an element for determining the physical properties of
the fluid sample, preferably for determining the viscosity of the
fluid sample.
13. The integrated microfluidic element according to claim 11,
characterized in that the optical measurement element is a
transparent cuvette (6) and the two elements for obtaining
information about the properties of the fluid sample which are
preferably pressure sensors (4, 5, 26, 27).
14. The integrated microfluidic element according to claim 11,
characterized in that it comprises the functionality of the
tangential flow filtration element (7) and of two elements for
obtaining information about the properties of the fluid sample,
which are preferably pressure sensors (4, 5, 26, 27), and
preferably but not mandatory in addition the functionality of the
pressure regulator element (8).
15. The integrated microfluidic element according to claim 11,
characterized in that it comprises the functionality of a capillary
(28) or capillary channel and of two elements for obtaining
information about the properties of the fluid sample, which are
preferably pressure sensors (4, 5, 26, 27).
Description
[0001] The invention relates to a micro flow filtration system
preferably for increasing the concentration of a component
contained in a small volume fluid sample. The system comprises a
micro tangential flow filtration element having a semipermeable
membrane capable for separating the fluid sample into a retentate
stream and a permeate stream upon passage of the fluid sample. The
fluid circuitry further comprises a pumping element, at least one
element for obtaining information about the properties of the fluid
sample within the circuitry (e.g. two or more pressure sensors for
obtaining information about the viscosity of the sample or the
determination of the transmembrane pressure or an optical cuvette
for obtaining information about the concentration of components of
the fluid sample), a plurality of conduits connecting the elements
to the circuitry and optionally a flow regulator.
[0002] Flow filtration systems for filtrating a fluid sample using
a semipermeable membrane for purposes of purification or
concentration of components contained in the fluid sample are well
known in the state of the art. These systems are used to remove
particulate or molecular contaminants in the case of a purification
or are used to increase the concentration of a component in a fluid
for example for laboratory analysis. Such systems can also be
applied to exchange the solvent containing a molecule or
particulate of interest by diafiltration. The membrane of such
filtration systems can be located in a normal orientation to the
flow direction of the fluid sample in a way that the membrane
covers the complete diameter of the flow channel which is
characterized as a normal flow filtration or dead-end filtration,
or the membrane surface can be located essentially parallel to the
flow of the liquid sample which is known as tangential flow
filtration system (TFF-system).
[0003] Tangential flow filtration systems have the advantage that
due to the direction of the flow of the sample which is essentially
parallel to the membrane surface an automatically sweeping and
cleansing takes place so that often higher fluxes and higher
throughputs can be attained with such systems in relation to
corresponding normal flow filtration systems. Further, a large
fraction of sample flows continuously over the membrane surface so
that a clogging, fouling, or a concentration polarization is
discouraged in such systems. With respect to these and other
advantages tangential flow filtration systems (TFF-system) are
often used in biotechnological and drug manufacturing
processes.
[0004] During the passage of the fluid through the tangential flow
filtration element having a semipermeable membrane the components
of the solution that are smaller than the pore size of the membrane
flow through the membrane as permeate stream while larger
components are retained in a retentate stream. The retentate stream
is recirculated in the flow circuitry and is pumped across the
membrane again in a continuous fashion. Such TFF-systems are used
to significantly reduce the volume of the sample solution as a
permeate stream is withdrawn from the system. So, the sample
solution becomes concentrated when the system is driven in a
concentration mode.
[0005] In other applications, the separation of two or more
components in the solution such as a buffer has to be performed.
Therefore, an exchange buffer solution (diafiltrate solution)
typically but not obligatory not containing the component to be
separated is added to the system so that one component is withdrawn
as permeate stream and exchanged by another component, i.e. by the
exchange buffer solution, so that in the end, for example, one
buffer is exchanged by another buffer. A diafiltration mode and a
concentration mode can be performed in the same system using
special control strategies.
[0006] The document WO 2006/026253 A2 describes a tangential flow
filtration (TFF) process development device to transfer the results
of a lab-scale TFF-system to an industrial-production-scale-system.
The information and data relevant for "scaling up" to industrial
scale processes are collected automatically. Therefore, a fully
automated TFF-system is provided capable of concentrating 0.5 l to
5 l batches of a reservoir to a minimum volume of approximately 20
ml or less than 20 ml. So, the minimal recirculation volume (also
called minimal working volume) of the system is approximately 20 ml
(or a little less). To achieve such minimal recirculation volume a
special tank for storing the fluid is used.
[0007] The tank for housing the reservoir has a mixing zone located
at a downstream end of the reservoir. The inlet and outlet of the
tank are positioned in the mixing zone to enable a resulting volume
for the concentrate of about 20 ml. Although the reservoir's inlet
and outlet is positioned at the bottom of the tank, the minimal
value of the minimal recirculation volume is restricted to
approximately 20 ml or a little less due to the relatively large
scale of the tank with a maximum volume of 0.5 l to 2 l or 5 l. The
minimum recirculation volume is in the range of 20 ml and may be
reduced to approximately 15 ml or 10 ml with such a system but not
to a lower value. So, the proposed system can not be used or
optimized to receive an end volume of the concentrated solution of
less than 10 ml at the end of the concentration process.
[0008] Tangential flow filtration systems are often used in
production processes of substances useful for biotechnological,
chemical, therapeutical or diagnostic applications to increase the
concentration of these desired substances. Because the start
materials and solutions used in these production processes are
often expensive and rare, in advance to the industrial scale
process a lab-scale filtration process is installed using only
small amounts and volumes of the solution and the samples. This is
especially important if the components are proteins or the like
which are sensitive of damages due to shear forces and other forces
existing in large-scale devices. For example biotechnologically
produced proteins which have to be concentrated in a solution are
extremely costly and on the other side extremely sensitive of
damages due to the forces which arises in industrial scale process
devices. Therefore, the risk to concentrate such proteins in a
solution in industrial scale devices is high. Further, these
proteins are often not available in a larger amount.
[0009] So, there is a strong demand in the industrial process
development for a flow filtration device being able to concentrate
a component of a solution preferably by the factor of more than 10
and using a start volume that is at most 20 ml.
[0010] It is an object of the invention to provide an optimized
TFF-system being able to handle a start volume of a solution of at
most 20 ml, preferably at most 10 ml, and to concentrate the
components of the solution. The resulting volume of the
concentrated solution at the end of the concentration process
should preferably be at most 2 ml, particularly preferably at most
1 ml, particularly preferably at most 100 .mu.l. The filtration
process should be fast and economical, repeatable and accurate.
[0011] The problem is solved with a microfluidic flow filtration
system for increasing the concentration of components contained in
a fluid sample with the features according to claim 1. The object
is also solved with an integrated microfluidic element according to
claim 11.
[0012] The micro flow filtration system according to the invention
for increasing the concentration of components contained in a fluid
sample has a fluid circuitry in form of a loop in which the fluid
sample or solution is recirculated. The fluid circuitry comprises a
tangential flow filtration element (TFF-element), an element for
pumping which is suitable for increasing and driving a fluid flow
of the fluid sample through the fluid circuitry and through the
tangential flow filtration element, at least two elements for
obtaining information about the properties of the fluid sample
within the circuitry, and a plurality of conduits connecting the
elements to the fluid circuitry through which the fluid stream of
the fluid sample is conducted. Optionally, the fluid circuitry
comprises also a regulator element for regulation the pressure
and/or the flow in the circuitry.
[0013] The fluid circuitry has a minimal working volume which is
also called minimal recirculation volume. This volume is the amount
of fluid which has to be retained in the fluid circuitry such that
the fluid can be recirculated in the circuitry without pumping air
through the circuitry. In other words, the elements of the fluid
circuitry and the conduits connecting these elements have to be
filled with a fluid in such a manner that a continuous fluid flow
in the circuitry can be arranged. The minimal working volume
depends on the elements used in the circuitry. For example, if
tubings for transporting the fluid are installed in the circuitry,
these tubings have also a non-negligible contribution to the
minimal working volume. The minimal working volume of the circuitry
of the micro fluid filtration system according to the invention is
at most 5 ml. Preferably, the minimal working volume of the
circuitry is even smaller, e.g. preferably at most 2 ml, further
preferably at most 1 ml, particularly preferably at most 100
.mu.l.
[0014] According to the invention the fluid circuitry contains also
an integrated microfluidic element in which the functionality of at
least two elements of the group of elements of the circuitry is
integrated. So, the integrated microfluidic element is an element
or a component of the circuitry in which the function of at least
two of the above mentioned elements of the circuitry is
implemented. So, instead of the two separate elements, which are
substituted, only one integrated microfluidic element is arranged
in the circuitry. Preferably, the integrated microfluidic element
is arranged in the retentate circuitry of the fluid circuitry and
not in the permeate circuitry. Especially in a concentration mode
such positioning of the microfluidic element is advantageous
because the functionality of two elements of the circuitry is
provided in only one element and the working volume of the system
is decreased or reduced.
[0015] The integrated microfluidic element defines a volume element
having a volume. So, a discrete volume can be assigned to the
integrated microfluidic element. The volume of the element is not
larger than one fourth of the minimal working volume of the fluid
circuitry. In a preferred embodiment this sub-volume of the
integrated microfluidic element is at most 20% of the working
volume of the fluid circuitry, preferably at most 15% and further
preferably at most 10%. The smaller the sub-volume of the
integrated microfluidic element is, the smaller is the influence to
the total working volume of the system which means to the total
working volume of the fluid circuitry. In a preferred embodiment
the volume of the integrated microfluidic element (sub-volume) is
at least 500 .mu.l, preferably at most 200 .mu.l.
[0016] This has the advantage that the integrated microfluidic
element can be of a smaller size than the two substituted elements.
The integrated microfluidic element has a volume that is smaller
than the volume of the two substituted elements together with the
conduits connecting these elements. So, the contribution of the
integrated microfluidic element to the minimal working volume is
sufficiently smaller than the contribution of two separate elements
of the fluidic circuitry and the conduit connecting these elements.
Especially the fact that the connecting conduit and the substituted
elements have to be connected together increases the contribution
to the minimal working volume because also the fittings and tubings
or fluidic channels to connect the conduit to the elements play a
non-negligible role.
[0017] So, using an integrated microfluidic element allows
combining the functionality of two elements in one volume element.
This has a positive influence to the minimal working volume which
is reduced. Even very small amounts of fluid (less than 15 ml,
preferably less than 5 ml) can be processed in the flow filtration
system wherein in a concentration mode the concentration of the
fluid can be raised by the factor of 100 and more.
[0018] In a preferred embodiment of the microfluidic filtration
system the circuitry further comprises a reservoir element suitable
for containing a fluid wherein the reservoir element is integrated
into the fluid circuitry loop. The reservoir element has a
reservoir inlet and a reservoir outlet both in connection to the
circuitry. Preferably the inlet and the outlet of the reservoir are
arranged at the bottom of the reservoir which can also be a tank or
the like. This assures that the contribution of the reservoir to
the minimal working volume of the circuitry is relatively
small.
[0019] Preferably the volume of the reservoir is at most 20 ml, in
particular at most 10 ml. During the invention it was recognized
that the size of the reservoir can be further reduced to a maximum
volume of the reservoir of 5 ml. In a preferred embodiment the
reservoir has a volume of at most 2 ml, very preferably of at most
1 ml. If very small solution volumes have to be processed the
volume of the reservoir can be further reduced to a volume of at
most 0.7 ml or preferably of at most 0.5 ml. Especially if fluid
batches of approximately 0.5 ml or less have to be processed in the
microfluidic filtration system, the integration of the
functionality of at least two elements of the circuitry into an
integrated microfluidic element plays an important role.
[0020] In a preferred embodiment the circuitry of the filtration
system comprises an optical measuring element for obtaining
information about the concentration of components contained in the
fluid solution. This optical measuring element preferably comprises
a cuvette or the like through which the fluid sample flows during
the concentration process and which is transparent in such a manner
that an optical measuring of parameters related to the
concentration of one or more components contained in the fluid
solution can be performed.
[0021] In another preferred embodiment the circuitry comprises a
measuring element for obtaining information about the viscosity of
the fluid sample and/or at least a valve element and/or a capillary
channel element and/or a hollow fiber element wherein these
elements are also members of the group of the elements of the
circuitry. The measuring element for obtaining information about
the viscosity of the fluid sample usually consists of at least two
pressure sensors whose results can be used to determine the
viscosity of the fluid sample using the Hagen-Poiseuille equation.
During the development of the invention investigations have been
performed which show that there is a linear dependency between the
pressure difference at two points in the fluid circuitry and the
viscosity of the solution flowing through the circuitry.
[0022] The tangential flow filtration element of the circuitry
comprises a feed inlet, a retentate outlet, a permeate outlet and a
semipermeable membrane which is capable of separating the fluid
sample into a retentate stream and a permeate stream upon passage
of the fluid sample into the tangential flow filtration element
through the feed inlet. For the concentration process tangential
flow filtration elements can be used which are commercially
available from a couple of manufacturers. An important criterion
for choosing a fluid filtration element for a microfluidic flow
filtration system to process even small volumes of a solution is
the contribution of the tangential flow filtration element to the
minimal working volume. So, the surface area of the membrane and
the volume of the tangential flow filtration element are important.
The contribution to the minimal working volume of the flow
filtration element should be as small as possible, preferably the
minimal working volume of the TFF-element should be at most 1000
.mu.l, preferably at most 500 .mu.l or further preferably at most
100 .mu.l, particularly preferably at most 20 .mu.l.
[0023] It is quite clear for a person skilled in the art that the
element for pumping is a pump which is integrated in the circuitry.
Here the known pumps can be used. For example, the pumping element
can comprise a syringe or two or more syringes to assure a
continuous flow of the fluid through the circuitry, alternatively
piston pumps or peristaltic pumps or gear pumps can be used.
[0024] Preferably the element for obtaining information about the
properties of the fluid is an element for obtaining information
about the pressure and the flow within the circuitry particularly
preferably one or more pressure sensors.
[0025] Preferably, also one or more regulator elements for
regulating the flow through the circuitry are integrated within the
circuitry. Such regulator elements can be valves, adjustable
pumping elements, and/or pressure regulators or the like.
[0026] In a preferred embodiment the microfluidic flow filtration
system is used for solutions in which the contained component is
protein. Especially in the field of research where only small
amounts of the components contained in the fluid are available for
e.g. process development purposes, there is a need for an improved
TFF system to perform e.g. concentration experiments with very
small amounts of these compounds (e.g. less than 100 or even less
than 20 mg) under conditions resembling the technical process
conditions with regard to e.g. mechanical stress. Although in some
cases it is possible to supply such proteins in a sufficiently
large amount such production requires a large effort of time and
materials and leads to an extraordinary expense.
[0027] It is known that the concentration factor depends on the
ratio of the sample volume and the minimal working volume of the
circuitry or the microfluidic flow filtration system. So, if only a
small amount of fluid sample is available and the desired
concentration factor is in range of 2 to 100 or more, the minimal
working volume of the circuitry has to be significantly reduced.
Preferably the minimal working volume is at most 1 ml, further
preferably at most 500 .mu.l. In some cases the minimal working
volume of the circuitry is at most 200 .mu.l, particularly
preferably at most 100 .mu.l. Particularly, if the concentration
factor is determined to be more than 50, the small amounts of the
minimal working volume as mentioned before are preferred.
[0028] In a preferred embodiment the working volume of the
circuitry without the reservoir is of special interest. Preferably
this minimal working volume should be at most 900 .mu.l, further
preferably at most 500 .mu.l and also further preferably at most
350 .mu.l. Particularly preferably is a minimal working volume of
the circuitry of at most 200 .mu.l, 150 .mu.l, 120 .mu.l, 100 .mu.l
or 90 .mu.l. Such small minimal working volume is preferred if the
predetermined concentration ratio is more than 50 or 100.
[0029] To arrange a micro flow filtration system with small minimal
working volume the internal diameter of the conduits used in the
circuitry is at most 1.5 mm, preferably at most 1 mm. For minimal
working volumes of less than 200 .mu.l conduits which have an
internal diameter of at most 0.7 mm or particularly preferably of
at most 0.3 mm are used. The conduits are preferably channels or
passages in an element of the circuitry. They can also be a pipe or
a tubing. These conduits connect the integrated microfluidic
element of the circuitry with the other elements of the circuitry.
Therefore, a rigid pipe as a conduit can be used.
[0030] In a preferred embodiment of the flow filtration system
according to the invention the system comprises a second reservoir
which is located outside the circuitry but which is in fluid
connection with the circuitry. The second reservoir has a volume
which is substantially larger than the volume of the circuitry
itself or of a first reservoir element in the circuitry if present.
So, the second external reservoir would be the main reservoir for
the fluid solution containing the component to be concentrated.
Preferably this second reservoir outside the circuitry has a volume
which is at least 10 times larger than the volume of the reservoir
element of the circuitry or of the circuitry itself, particularly
preferably at least 20 times larger.
[0031] The system according to the invention can be used for
concentrating a component contained in a fluid sample like a
protein or a molecule or the like. Optionally the system can also
be used in a diafiltration mode. In this case the external second
reservoir contains a solution with a buffer that should be used to
exchange a buffer in the solution contained in the circuitry which
also contains the desired component. So, an exchange of the buffer
can easily be performed. It is evident that a diafiltration mode
can also be supplied using only one reservoir in the circuitry. In
such case, the circuitry is filled with the fluid sample and the
reservoir is filled with the buffer solution.
[0032] In the field of biotechnology and process technology there
are a lot of applications which require the determination of the
concentration of components of the fluid sample, the formation of
aggregates within the fluid sample and the viscosity of the fluid
sample using a flow through method. A typical example is the
requirement that the concentration of proteins contained in the
fluid sample should be measured online during the concentration
process. So, the actual fluid parameters, i.e. viscosity,
concentration, and aggregate formation have to be determined during
the process. Based on these online-measured parameters, the system
can be controlled respectively. Essential process parameters for
the concentration and purification process will change during the
process. This leads to the resulting knowledge that the
concentration and the rate of aggregate formation of the proteins
and the viscosity of the solution have to be measured in real time
so that an optimal concentration process can be performed by
adjusting the relevant process parameters.
[0033] In the development of the subject matter of the invention it
has been identified that for the determination of the concentration
using photometric methods can be applied. For this photometry
technique preferably a cuvette is used through which the fluid
sample flows without diluting the sample. For an optical
measurement of the absorption a light source is used which is
transmitting light through the cuvette. In a preferred embodiment
light crosses the cuvette perpendicular to the flow direction.
Performing the photometric measurement using the cuvette preferably
an ultraviolet radiation is used to determine the concentration of
compounds contained in the sample (e.g. of proteins). For this
online determination of the concentration good results could be
achieved using a radiation of light with a wave length of 280
nm.+-.10 nm.
[0034] For measuring the appearance of aggregates in the sample
fluid, the ratio between the absorption values at the wavelength of
280.+-.10 nm and at a wave length larger than 290 nm (e.g. 320-330
nm) is preferred.
[0035] In addition, the viscosity of the fluid sample can be
measured using a pressure sensing element comprising two pressure
sensors at the inlet and at the outlet of the capillary,
respectively. These pressure sensors detect a difference of
pressures between the pressure sensor at the inlet and the pressure
sensor at the outlet during the flow of the sample through the
cuvette. This measurement is performed using a known geometry of
the cuvette and a predetermined (constant) flux or flow through
rate. Based on the change of the pressure during the flow through
the cuvette, the viscosity of the fluid sample can be calculated
based on well-known equations like the Hagen-Poiseuille equation.
The viscosity is proportional to the measured pressure
difference.
[0036] In a preferred embodiment, the integrated microfluidic
element according to the invention comprises and combines these two
functionalities of the two pressure sensors and the cuvette. So,
the integrated microfluidic element can be used to determine the
viscosity, the concentration of the compound, and the formation of
aggregates in the fluid sample in real time and online in one
element. In addition to this advantage the minimal working volume
of this integrated microfluidic element is substantially smaller
than using three separate components and conduits for connecting
these components.
[0037] In the context of the invention it was recognized that
measuring the viscosity and the concentration of the sample fluid
in the fluidic circuitry in parallel and within the same
microfluidic element leads to a couple of difficulties. To
determine the viscosity a pressure drop along a capillary has to be
detected. In typical lab-scale systems or
industrial-production-scale-systems such measurements are not
possible due to the large volumes of the fluid samples to be
transported through the fluidic system and due to the large
diameters of the tubes and fluid conduits. So, the overall pressure
and the pressure differences within such systems are too low to
allow a precise viscosity determination based on pressure drop
measurements. During the invention it was recognized that for a
precise viscosity measurement the capillary has to be very small in
diameter (smaller than 0.5 mm) and preferably with a relatively
short length (less than 200 mm), as the diameter has the largest
influence on the pressure drop. Therefore, variances in the channel
diameter have a significant impact on the precision of the
measurement of the pressure drop. To determine a high variation of
the viscosity a high pressure range must be handled in the system.
Typical pressures that occur in such channels are in the range of
100.000 Pa to 500.000 Pa, preferably up to 1.000.000 Pa. Therefore,
the channel has to be stable with respect to high pressures. So,
the material of the channel, which is preferably a capillary, is
preferably metal. Investigations of the inventors have shown that
also glass is a suitable material for this purpose, especially if
the walls of the glass channels have a sufficient thickness.
Fluidic channels made of glass, like it is used for cuvettes for
example, can be produced with a very high precision and therefore
reduce variances in the channel diameter which is positive with
respect to the pressure measurement.
[0038] On the other hand the relatively small diameter shows
additional advantages for the optical measurement in a transparent
capillary when measuring the concentration. Especially in the case
of high concentrations to be determined the small diameters have a
positive effect because the optical density of the sample
significantly increases with increasing sample concentrations. This
causes high absorption values that are very complicated to be
measured. To reduce the absorption measured through the channel or
cuvette and to simplify the absorption measurement, the optical
path length of the light beam passing through the sample have to be
reduced. The reduction of the cuvettes diameter reduces the optical
path length and therefore reduces the absorption to be measured.
Therefore, a suitable material for a transparent channel or
capillary is glass. To fulfill the requirements regarding the
pressure in the channel or capillary with respect to the viscosity
measurement, the walls have to have a sufficient thickness to be
stable enough. Despite the thickness of the walls an optical
measurement of the concentration remains possible with a reduced
optical path length.
[0039] Although channels with small diameters tends to be blocked
or clogged by aggregates typically formed in filtration processed
it was realized that a transparent channel made of glass having a
small diameter can be used for measuring the viscosity via pressure
drop measurement and for optically measuring the concentration in
the sample. So, such transparent capillary, in combination with the
two pressure sensors, can be used in an integrated microfluidic
element.
[0040] In another alternative or cumulative preferred embodiment,
the integrated microfluidic element according to the invention
comprises and combines the functionality of the micro tangential
flow filtration element (TFF-element) and at least one element for
obtaining information about the properties of the fluid sample
within the circuitry. Preferably, elements for obtaining
information about the properties of the fluid sample within the
circuitry are pressure sensors to measure the pressure of the fluid
sample in the circuitry and, based thereon, can be used to
determine the viscosity of the fluid sample. So, in this preferred
embodiment a micro TFF-element is combined with at least one
pressure sensor, further preferred with two pressure sensors.
[0041] Preferably the TFF-element comprises a TFF-housing which can
be a filter cassette. The TFF-housing is part of the housing of the
integrated microfluidic element. In this case the TFF-housing is
integrated in the housing of the microfluidic element. Preferably
the membrane of the TFF-element is disposable. The membrane can be
exchanged in the case that the membrane is clogged-up. If the
efficiency of the TFF-element is dropped to a predetermined
threshold value an exchange of the membrane becomes necessary. To
design the TFF-element with a disposable membrane has the advantage
that the filter cassette itself (TFF-housing) can remain unchanged
in the fluid circuitry so that the connections to the connected
conduits do not have to be touched especially not to be opened.
Further, if the TFF-element is integrated in the microfluidic
element together with two pressure sensors, the sensors remain at
their location, especially in their position in the housing of the
microfluidic element. So, neither the sensors themselves nor their
electrical connections are influenced if only the membrane of the
TFF-element is exchanged in case of clogging. Further the size and
especially the volume of the fluidic channels of a TFF-element
comprising pressure sensors can be reduced because the channels
connecting the filter cassette and the pressure sensors can be
short.
[0042] In a further preferred embodiment this integrated
microfluidic element also contains the functionality of the
pressure regulator element so that in addition the pressure in the
circuitry can be controlled and adjusted. So, not only the
functionality of two elements is integrated within the integrated
microfluidic element according to the invention, but the
functionality of three (or even more) elements.
[0043] In another preferred embodiment of the integrated
microfluidic element according to the invention, it comprises the
functionality of a capillary or a capillary channel and of the two
pressure sensors.
[0044] The invention is illustrated in more detail hereafter based
on particular embodiments shown in the figures. The technical
features shown therein can be used individually or in combination
to create preferred embodiments of the invention. The described
embodiments do not represent any limitation of the invention
defined in its generality.
[0045] FIG. 1 shows a schematic view of a filtration system;
[0046] FIG. 2 shows a flow filtration circuitry according to the
invention with an integrated microfluidic element;
[0047] FIG. 3 shows another schematic view of a fluidic circuitry
for the determination of viscosity according to the invention;
[0048] FIG. 4 shows an integrated microfluidic element comprising
the functionality of two pressure sensors and a measuring element
for detecting the concentration and viscosity;
[0049] FIG. 5a, b shows two embodiments of another microfluidic
element comprising the functionality of measuring the pressure in
the circuitry and filtering the fluid sample.
[0050] FIG. 1 shows a state-of-the-art microfluidic flow filtration
system 100 having a circuitry 1. The circuitry 1 comprises a
reservoir element which is a reservoir 2, a container or a tank
containing the fluid sample, an element for pumping which is a pump
3, three pressure sensors 4, 5, 39, a cuvette 6, a tangential flow
filtration element 7, a pressure regulator 8 and a valve 9 which is
a T-shaped conjunction to withdraw fluid from the circuitry 1. Each
functionality of the circuitry 1 is implemented by one single
element like the reservoir 2, the pressure sensors 4, 5 or 39 or
the cuvette 6. Because each of the elements and also the conduits
connecting these elements have a contribution to the minimal
working volume of the circuitry, the minimal working volume is
relatively large. In the state of the art the minimal working
volume of the fluid circuitry 1 is at least approximately 20 ml.
Normally the minimal working volume is in the range of some 100
ml.
[0051] FIG. 2 shows a micro fluid filtration system 100 according
to the invention with a circuitry 1 for and by a plurality of
conduits 24. The fluid circuitry 1 shown in FIG. 2 also comprises a
reservoir 2, a pump 3 which is implemented by a 4-port-valve
(valving apparatus) 10 and two syringes 11 that serve as a piston
pump.
[0052] The micro tangential flow filtration element 7 comprises a
feed inlet 12, a retentate outlet 13, a permeate outlet 14 and a
semipermeable membrane 15. The membrane 15 is capable of separating
the fluid sample into a retentate stream and a permeate stream upon
passage of the fluid sample into the tangential flow filtration
element 7 through the feed inlet 12. The permeate stream withdrawn
from the circuitry 1 via the permeate outlet 14 is collected in a
permeate chamber 16. The permeate chamber can be located on a
balance 17 to weight the amount of the permeate stream and to
control the flow through the membrane 15 and to measure the amount
of withdrawn fluid. The retentate stream flows through conduit 24,
through the reservoir 2, the valving apparatus 10, the integrated
microfluidic element 20 into the TFF-element 7. This circuitry is
called retentate circuitry in which the microfluidic element 20 is
located.
[0053] The circuitry 1 according to the invention also comprises a
valve 9 with a T-shaped conjunction and an outlet port 18. The
outlet port 18 is used to withdraw fluid from the circuitry 1,
particularly to withdraw the concentrated fluid at the end of the
concentration process. The fluid is conducted to a collection
reservoir 29.
[0054] The pressure regulator 8 is a regulator element for
regulating the pressure (and thereby the fluid flow) in the fluid
circuitry 1. The pressure regulator 8 is controlled by a control
unit 19 which is fed by the pressure values measured within the
circuitry 1. These pressure values are detected by pressure sensor
39 and at least one pressure sensor which is integrated within the
integrated microfluidic element 20.
[0055] The integrated microfluidic element 20 defines a so-called
volume element which is a separate and discrete element. The
microfluidic element 20 has a volume in which fluid of the fluid
circuitry is contained during its flow through the microfluidic
element 20. The volume of element 20 is at most 25% of the working
volume of the complete fluid circuitry 1. It could be shown that
the microfluidic element 20 is one of the major elements of the
fluid circuitry. Therefore, reducing its volume has a direct and
positive influence to the complete fluid circuitry and its minimal
working volume. So, preferably the working volume of the
microfluidic element 20 is at most 20% of the minimal working
volume, further preferably at most 15%. It can also be shown that
the positive influence is increased if the volume of the
microfluidic element 20 is at most 10% of the minimal working
volume. During investigations within the frame of the invention
positive effects of the volume of the volume element 20 have been
determined if the volume of the microfluidic element 20 is at most
400 .mu.l, preferably at most 50 .mu.l. Nevertheless this allows
processing of small fluid samples and in case of a concentration
mode achieving high concentration rates.
[0056] The circuitry 1 comprises the integrated microfluidic
element 20 instead of the separated elements of the pressure
sensors and the cuvette (which has here the functionality of a
capillary with a different diameter compared to the diameter of the
conduits before and after the integrated pressure sensors,
respectively) which are signed by the reference numbers 4, 5 and 6
respectively in FIG. 1. In the embodiment shown in FIG. 2 the
integrated microfluidic element 20 is a viscosity module 21 capable
to measure the viscosity of the fluid sample contained in the
circuitry 1. The dimensions of the viscosity module 21 are
significantly reduced with respect to the overall dimensions of the
separated elements of two pressure sensors and a cuvette. An
important role plays the fact that the conduits 24 between the
elements can be shortened with so that the minimal working volume
of the circuitry 1 can be reduced in total.
[0057] FIG. 3 shows a schematic principal view of a reduced
circuitry 1 which comprises a reservoir 2, a pump 3, a pressure
regulator 8 in form of a hose clamp 22 and a viscosity module 21
which is an integrated microfluidic element 20 or an microfluidic
module. The microfluidic element 20 comprises the functionality of
two pressure sensors and a cuvette. The viscosity module 21 further
allows to determine the concentration and aggregation of compounds
contained in the fluid sample by an optical measurement using the
integrated transparent capillary 28 providing a cuvette function
and to determine the viscosity by measuring a pressure gradient or
difference using two pressure sensing elements, for example in form
of pressure sensing modules 26, 27.
[0058] The viscosity module 21 shown in FIG. 3 has two tube
fittings 23 for connecting to the conduits 24 which are tubes 25 in
this example. Between the two pressure sensing modules 26, 27 the
capillary 28 is arranged which is directly connected to the
pressure sensing modules 26, 27.
[0059] The volume or minimal working volume of the viscosity module
21 is formed by the effective volume of (or in) the pressure
sensing modules 26, 27 and by the effective volume of the capillary
28. To vary the volume of the viscosity module 21 (being the
integrated microfluidic element 20) the volume of the capillary 28
or the volume of the fluidic connection to the pressure sensing
modules 26 can be changed.
[0060] To reduce the minimal working volume of the viscosity module
21 and so also the minimal working volume of the circuitry 1 and to
enable viscosity determination, the internal diameter of the
capillary 28 is preferably in a range between 100 .mu.m and 500
.mu.m. Particularly preferred is an internal diameter of the
capillary 28 between 100 .mu.m and 250 .mu.m. The internal diameter
is understood as the diameter of the capillary 28 if the capillary
has a circular cross section. If the capillary is not round, the
internal diameter is to be understood as the dimension which is
parallel to the optical measurement direction (arrow 34). So, a
radiation or light beam which is transmitted by a source 31 passes
through the capillary 28 along the internal diameter and is
received by an optical detector 32. The width of the capillary
which is perpendicular to the optical measurement distance 34 is
not relevant for the optical measurement (as long as it is not too
small to allow the light beam to pass through the cuvette).
[0061] According to the invention the capillary 28 is preferably
implemented in such a manner that the fluidic resistance of the
capillary 28 and the fluidic length are adjusted in a way that
establishes a pressure gradient along the capillary 28 which is
substantially high. A pressure gradient or pressure difference is
understood as substantially high if the pressure gradient along the
capillary is at least in the range of approximately 0.05 bar per
mPa sec (milli Pascal second).
[0062] FIG. 4 shows a detailed view of an integrated microfluidic
element 20 according to the invention. The microfluidic element 20
is a viscosity module 21 which comprises the functions of two
pressure sensing modules 26, 27 and the function of a cuvette 6
which is integrated as a capillary 28.
[0063] The upper picture in FIG. 4 shows a top view of the
viscosity module 21. It is clearly shown that the width w of the
transparent capillary 28 is wider than the width of the connecting
conduits 24. This arrangement is used to calculate the viscosity on
basis of measuring a pressure difference with the two pressure
sensing modules 26, 27.
[0064] The lower picture of FIG. 4 shows a side view of the
viscosity module 21. It is clearly shown that the height h of the
cuvette element 6 (capillary 28) is smaller than the height of the
connecting conduits 24 (which are preferably pipes) and the
fittings 23 respectively. An optical measuring unit 30 comprises a
light emitting source 31 arranged above the capillary 28. The light
emitting source 31 can be every source emitting an electromagnetic
radiation which can be for example visible light or invisible light
like ultraviolet light. A respective optical detector 32 is
arranged below the capillary 28, preferably below the viscosity
module 21, so that a radiation transmitted from the optical source
31 along the optical measurement direction 34 passes through the
transparent capillary 28 of the viscosity module 21 and reaches the
detector 32. This allows an online, real-time monitoring of the
concentration of components or aggregates contained in the solution
which flows in an unidirectional manner through the circuitry 1 and
the viscosity module 21.
[0065] The FIGS. 5a, b show two other embodiments of an integrated
microfluidic element 40 which provides and contains the
functionality of the tangential flow filtration element 7 and of
the two pressure sensors 5 and 39 shown in FIG. 1 or FIG. 2,
respectively.
[0066] The upper picture of FIG. 5a shows a schematic
cross-sectional side view of the first embodiment of the integrated
microfluidic element 40 which includes the functions of the flow
filtration element 7 and of two pressure sensors 5, 39 according to
FIG. 2. The integrated microfluidic element 40 comprises a housing
50 in which a capillary channel 44 is formed. The capillary channel
also comprises the feed inlet 12, the retentate outlet 13 and the
permeate outlet 14. The housing 50 of the microfluidic element 40
forms a TFF-housing 51 of the tangential flow filtration element 7.
In a preferred embodiment the TFF-housing 51 can be part of the
housing 50 of the microfluidic element 40. The integrated
microfluidic element 40 comprises two pressure sensing modules 26,
27 each located at an end of the filtration element 41. The
filtration element 41 comprises the feed inlet 12 followed by a
filtration chamber 42 containing the membrane 15 which is located
above a support structure 43. The membrane 15 is sealed by a
sealing 45 so that fluid flowing through the permeate outlet 14
have to pass the membrane 15. Preferably the membrane 15 of the
tangential flow filtration element 7 is disposable. So, in case of
clogging or after a predetermined process time the efficiency of
the membrane may be reduced. Then, only the membrane has to be
exchanged. The TFF-element 7 and the microfluidic element 40 remain
unchanged. Especially the connections to connecting conduits do not
have to be exchanged or renewed. Additionally, replacing the
membrane 15 only does not influence the pressure sensing modules
26, 27. Only the sealing 45 sealing the membrane 15 to the housing
50 will also be renewed.
[0067] The permeate outlet 14 of the filtration chamber 42 is
located at the end of the chamber 42 which is essentially
perpendicular to the flow direction. At the end of the chamber 42
also the retentate outlet 13 is positioned so that a part of a
fluid flow through the filtration element 41 leaves the chamber at
this end. In flow direction before and behind the filtration
chamber 42 a channel 44 is implemented in the microfluidic element
40. In this channel 44 the two pressure sensing modules 26 and 27
respectively are arranged. So, the pressure difference between the
two pressure sensing modules 26, 27 can be used to calculate the
transmembrane pressure in the microfluidic element 40. The
filtration chamber 42 can further be complemented with a turbulence
promotor.
[0068] The lower picture in FIG. 5a shows a cross-sectional top
view along the line A-A of the upper picture. It is clearly seen
that the capillary channels 44 at the two ends of the microfluidic
element are relatively small. In the area of the pressure sensing
modules 26, 27 the capillary is widened. The filtration chamber 42
is further widened in respect to the capillary channel 44 and the
sensing modules 26, 27. Between the sensing modules 26, 27 and the
filtration chamber 42 the capillary channels have their (normal)
width.
[0069] FIG. 5b shows another embodiment of an integrated
microfluidic element 40 comprising a filtration element 41 and two
pressure sensing modules 26, 27. The upper picture shows a cross
sectional side view of the integrated microfluidic element 40; the
lower picture shows a cross-sectional top view along the line A-A.
The difference between the two embodiments in the FIGS. 5a and 5b
is that in the embodiment shown in FIG. 5b the feed inlet 12 and
the retentate outlet 13 are located at the upper side of the
microfluidic element 40. So, the flow is deflected two times during
passage of the microfluidic element 40. The pressure sensing
modules 26 and 27 are located at the feed inlet 12 and at the
retentate outlet 13, respectively, so that the pressure of the
fluid is measured before and after the fluid passes the filtration
chamber 42.
[0070] The construction of the filtration element 41 of FIG. 5b
itself is similar to the construction of the filtration element 41
according to FIG. 5a with respect to the arrangement of the
membrane 15 and the permeate outlet 14. The cross sectional top
view clearly shows that the capillary channel 44 is also widened in
the area of the pressure sensing module 26, 27.
[0071] The two embodiments of the microfluidic element 40 shown in
the FIGS. 5a and 5b have the advantage that the construction of the
element is cheap and easy to perform. Due to the fact that only
small pieces have to be arranged together, the pieces can be
manufactured with a high accuracy so that very small volumes of the
filtration chamber 42 can be achieved. Further, due to the not
needed fittings and conduits between the filtration element 41 and
the pressure sensing modules 26, 27 the minimal working volume can
be reduced further.
[0072] So, using these alternative embodiments of an integrated
microfluidic element or module 40, which contains at least two
functionalities of the elements comprised in a fluidic circuitry,
especially for concentration or purification of components
contained within the fluid sample within this circuitry, allows to
reduce the minimal working volume of the circuitry 1. Merging the
functionality of at least two circuitry elements results in a
compact component or module with a small and reduced minimal
working volume that is optimized for concentrating small amounts of
fluid, preferably of less than 20 ml.
* * * * *