U.S. patent application number 13/144967 was filed with the patent office on 2012-08-16 for optimization of separation for viscous suspensions.
This patent application is currently assigned to SMARTFLOW TECHNOLOGIES, INC.. Invention is credited to James A. Kacmar, Henry B. Kopf.
Application Number | 20120205311 13/144967 |
Document ID | / |
Family ID | 42542592 |
Filed Date | 2012-08-16 |
United States Patent
Application |
20120205311 |
Kind Code |
A9 |
Kopf; Henry B. ; et
al. |
August 16, 2012 |
OPTIMIZATION OF SEPARATION FOR VISCOUS SUSPENSIONS
Abstract
The present invention relates to methods and systems for
optimization of dilution of a viscous starting material to isolate
and/or concentrate the product of interest from the starting source
material such that the process minimizes the volume of diluent and
the total volume of the waste stream generated during the process
as well as maximizing the yield of desired product. The system
employs cross-flow filtration modules with sub-channels that are
equidistant to the inlet and outlet of said modules and such
modules are characterized by optimal channel height, optimal
transmembrane pressure, etc., which are selected in order to
achieve the best combination of product quality and production
yield.
Inventors: |
Kopf; Henry B.; (Cary,
NC) ; Kacmar; James A.; (Apex, NC) |
Assignee: |
SMARTFLOW TECHNOLOGIES,
INC.
APEX
NC
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20110309018 A1 |
December 22, 2011 |
|
|
Family ID: |
42542592 |
Appl. No.: |
13/144967 |
Filed: |
January 21, 2010 |
PCT Filed: |
January 21, 2010 |
PCT NO: |
PCT/US10/21626 PCKC 00 |
371 Date: |
September 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61146142 |
Jan 21, 2009 |
|
|
|
61148959 |
Jan 31, 2009 |
|
|
|
Current U.S.
Class: |
210/639 ;
210/195.2; 210/645 |
Current CPC
Class: |
B01D 61/147 20130101;
B01D 29/56 20130101; B01D 2315/10 20130101; C07H 1/08 20130101;
B01D 61/58 20130101; B01D 2317/022 20130101; B01D 61/145 20130101;
B01D 2315/16 20130101; B01D 2317/025 20130101; B01D 61/142
20130101; B01D 63/082 20130101 |
Class at
Publication: |
210/639 ;
210/645; 210/195.2 |
International
Class: |
B01D 61/58 20060101
B01D061/58; B01D 65/00 20060101 B01D065/00 |
Claims
1. A method for purifying one or more target substances from a
viscous source material, said process comprising: contacting the
viscous source material with a diluent in an amount sufficient to
reduce the viscosity of the viscous source material and form a
continuous stream of diluted source material, wherein the diluent
is contained in a separated vessel from the viscous source
material; flowing the diluted source material into a recirculation
loop of a first cross-flow filter apparatus; diafiltering the
diluted source material with sufficient diafiltration buffer
retained in a buffer reservoir so as to recover the desired yield
of the target substance by passing said target substance into the
first permeate fluid; flowing the first permeate fluid containing
the target substance to a end product vessel; flowing out the first
retentate solution from the recirculating liquid of the first
cross-flow filter into a second cross-flow filter unit, wherein the
flow rate of the first retentate solution is at the same flow rate
as the diluted source material being fed into the recirculation
loop of the first cross-flow filter apparatus; diafiltering the
flow of retentate into the second cross-flow filter unit with
sufficient diafiltration buffer so as to recover the desired yield
of the target substance by passing said target substance into the
second permeate fluid; flowing the second permeate fluid containing
the target substance to the end product vessel; concentrating the
first and second retentate fluid by flowing same to a third
cross-flow filter apparatus communicatively connected with the
second cross-flow filter unit, wherein the volume of the third
retentate fluid is reduced to the approximate volume of the
undiluted source material or less thereby forming a waste stream
for further use; recirculating the third permeate fluid back to the
diluent vessel for reuse; concentrating the first and second
permeate fluid by flowing same to a fourth cross-flow filter
apparatus communicatively connected to the end product vessel
wherein target substance is concentrated and diafiltration buffer
is removed in fourth permeate stream and recirculated for
reuse.
2. The method of claim 1, wherein the amount of buffer introduced
into the is conserved and available for reuse.
3. The method of claim 1, wherein the diluent is returned to
diluent vessel simultaneously with the concentration of the produce
and the return of the buffer to the buffer reservoir.
4. The method of claim 1, wherein the cross-flow filters comprises:
a multilaminate array of sheet members of generally rectangular and
generally planar shape with main top and bottom surfaces, wherein
the sheet members include in sequence in the array a first
retentate sheet, a first filter sheet, a permeate sheet, and second
filter sheet, and a second retentate sheet, wherein each of the
sheet members in the array has at least one inlet basin opening at
one end thereof, and at least one outlet base opening at an
opposite end thereof, with at least one permeate passage opening at
longitudinal side margin portions of the sheet members; each of the
first and second retentate sheets having at least one channel
opening therein, wherein each channel opening extends
longitudinally between the inlet and outlet basin openings of the
sheets in the array and is open through the entire thickness of the
retentate sheet, and with each of the first and second retentate
sheets being bonded to an adjacent filter sheet about peripheral
and side portions thereof, with their basin openings and permeate
passage openings and register with one another, and arranged to
permit flow of filtrate through the channel openings of the
retentate sheet between the inlet and outlet basin openings to
permit permeate flow through the filter sheet to the permeate sheet
to the permeate passage openings; and the cross-flow filters
comprising a unitary article of inter-bonded sheet members.
5. The method of claim 1, wherein the viscous source material is a
cell mass and target substance is a protein or fatty acid.
6. The method of claim 4, wherein the cross-flow filtration module
comprises channel height and length of the retentate sheet for
optimal production yield.
7. A system comprising: a first reservoir constructed and arranged
for holding a diluent solution, and for selectively flowing liquid
into and out of said first reservoir; a second reservoir
constructed and arranged for holding a starting material, and for
selectively flowing liquid into and out of said second reservoir, a
first cross-flow filtration apparatus for separating liquids into
permeate and retentate streams, provided with means for flowing
liquid in from the first and second reservoir and permeate and
retentate streams out of said first cross-flow filtration
apparatus; a second cross-flow filtration apparatus for receiving
retentate from the first cross-flow filtration apparatus and
separating liquids into permeate and retentate streams, provided
with means for flowing liquid in and permeate and retentate streams
out of said second cross-flow filtration apparatus; a third
reservoir constructed and arranged for holding a buffer, and for
selectively flowing liquid into the first and second cross-flow
filtration apparatus and out of said third reservoir and; a third
cross-flow filtration apparatus for separating liquids into
permeate and retentate streams, provided with means for flowing
liquid in and permeate and retentate streams out of said third
cross-flow filtration apparatus; a product reservoir constructed
and arranged for holding the isolated product received as permeate
from the first and second cross-flow filtration apparatus, and for
selectively flowing liquid into and out of the product
reservoir.
8. The system of claim 7, wherein the second reservoir can be the
cell culture reservoir such as a fermentor or culture bag;
9. A method to optimize dilution, separation and concentration of a
desired endproduct from a viscous material, the method comprising:
diluting a cell suspension with different dilution amounts and
rates to determine the minimal amount of dilution to maximize the
amount of endproduct recovered.
10. The method of claim 9, further comprising determining the
amount of diafiltration buffer to separate permeate from cells
suspension, wherein the permeate includes the desired
endproduct.
11. The method of claim 10, further comprising determining the
level of concentration of the permeate to recover optimal amount of
desired endproduct.
12. The method of claim 11, further comprising determining the
level of concentration of the retentate to recapture cell material
to the concentration of the original cell suspension.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 61/146,142 filed on Jan. 21, 2009 and
U.S. Provisional Patent Application Ser. No. 61/148,959, filed on
Jan. 31, 2009, the contents of which are incorporated herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of Invention
[0003] The present invention relates to separation of viscous
source materials, and more particularly, to methods and systems for
optimization of dilution of a viscous starting material to isolate
and/or concentrate the product of interest from the starting source
material such that the process minimizes the volume of diluent and
the total volume of the waste stream generated as well as
maximizing the yield of desired product.
[0004] 2. Discussion of Related Technology
[0005] Throughout the world more and more companies are looking to
recover value added products from a wide variety of starting
materials including plants, roots, root crops, grains, flowers,
animal tissue, cell cultures comprising yeast, algal, bacteria, or
fungi species, milk, milk products, fruits and fruit juices.
Additional companies are looking to extract value added products
from solid and liquid waste streams such as mill and grain wash
waters and fermentation bio-mass. One such waste stream will be the
bio-mass from bio-fuel production which after production of fuels
such as diesel and alcohol will still be rich in plant proteins,
sugars and carbohydrates. Another such waste stream will be
cellular bio-mass used for protein and essential fatty acids
production from wild and/or recombinant yeast, algae, bacteria,
larvae or fungi species.
[0006] A common practice for dry or solid starting materials is to
solubilize starting materials in a solvent such as aqueous and
organic solvents so that the valuable component becomes soluble in
the solvent. The solution is then processed by one or more of the
known techniques of filtration, precipitation, extraction,
chromatography and centrifugation to separate the valuable
components from the starting material and solvent. As a result of
growth in demand for naturally derived products companies are
increasing production of these products. Production costs and
environmental issues such as the release of contaminated liquid
waste streams have pressured companies to extract more of the final
product from the starting material and to minimize the use of
solvents by preparing larger more viscous process streams.
[0007] In recent years the science of cell culture has also
endeavored to increase production of cell derived products such as
antibiotics, vaccine and therapeutic compounds by increasing the
density of the cell cultures utilized to produce these highly
valuable materials. Increased cell density can be a highly
beneficial as it allows for the increased production of the final
product in the same space as a less dense cell culture. It would
seem that doubling the concentration of a cell culture forming a
viscous material should yield twice as much final product without
any substantial increase in fermentation facility costs.
[0008] However, it has been found that all of these highly viscous
materials are far more difficult to process, such that, even though
the cell culture is five (5) times denser the yield of final
product is only 50% greater because the viscosity of the material
prevents the separation of the desired target molecule from the
mass of cellular materials. In the case of extracts of solid phase
material such as plants and animal tissue the problem is the same
such that the viscous materials clog filters and block
chromatography columns as well as not separating efficiently under
normal centrifugal forces. One way of describing the problem is to
say that although larger crowds would contain more people able to
buy a particular good or service it is harder to get the people
with money through the stores doors due to the congestion caused by
the crowd itself.
[0009] Although it would appear that a simple dilution of the
viscous material would solve the problem, this creates at least
four additional problems: 1) the cost of the diluent which can be
highly expensive in the case of diluents for pharmaceutical
intended for human injection, 2) disposal of the higher volume of
the waste stream, i.e. the original volume plus the volume of
diluent, 3) the cost of the necessary tanks and mixing equipment in
order to dilute the starting material, and 4) additional
purification costs for the diluted final product.
[0010] As important as these problems are the single most important
point is to have the highest percentage of yield so that the
initial purpose of processing higher density materials is not
negated by problems with recovery of the desired product. Thus, it
would be advantageous to provide a method and system that provides
higher yields from high density materials.
SUMMARY OF THE INVENTION
[0011] The present invention solves all of the aforementioned
problems in a simple, inline, space efficient and continuous
process that lowers costs and maximizes yield.
[0012] The present invention relates to the method and apparatus
necessary to dilute a viscous starting material to isolate and/or
concentrate the product of interest from the starting material such
that the process minimizes the volume of diluent and the total
volume of the waste stream generated as well as maximizing the
yield of desired product.
[0013] One such method employs one or more cross-flow filter units
and their associated pumps, pipes and tanks. It is also a further
embodiment of this invention that the further purification of the
target of interest can be accomplished by complimentary
purification techniques such as chromatography all as one unit of
operation.
[0014] An extremely beneficial element of this method is that the
process can be readily modeled and optimized on the laboratory
scale, with volumes as small as 0.5 L or in a continuous flow of 1
liter per minute for example. This is extremely important in the
pharmaceutical market as large volumes of highly specific
therapeutic proteins are neither inexpensive nor readily available.
The separation methods of the present invention are envisioned in
batch mode, continuous, or semi continuous mode.
[0015] One aspect of the present invention relates to a process for
purifying one or more target substances from a viscous source
material, the process comprising:
[0016] contacting the viscous source material with a diluent in an
amount sufficient to reduce the viscosity of the viscous source
material and form a continuous stream of diluted source material,
wherein the diluent is contained in a separated vessel from the
viscous source material;
[0017] flowing the diluted source material into a recirculation
loop of a first cross-flow filter apparatus;
[0018] diafiltering the diluted source material with sufficient
diafiltration buffer so as to recover the desired yield of the
target substance by passing said target substance into the first
permeate fluid;
[0019] flowing the first permeate fluid containing the target
substance to a end product vessel;
[0020] flowing out the first retentate solution from the
recirculating liquid of the first cross-flow filter into a second
cross-flow filter unit, wherein the flow rate of the first
retentate solution is at the same flow rate as the diluted source
material being fed into the recirculation loop of the first
cross-flow filter apparatus;
[0021] diafiltering the flow of retentate into the second
cross-flow filter unit with sufficient diafiltration buffer so as
to recover the desired yield of the target substance by passing
said target substance into the second permeate fluid;
[0022] flowing the second permeate fluid containing the target
substance to the end product vessel;
[0023] concentrating the first and second retentate fluid by
flowing same to a third cross-flow filter apparatus communicatively
connected with the second cross-flow filter unit, wherein the
volume of the third retentate fluid is reduced to the approximate
volume of the undiluted source material or less thereby forming a
waste stream for further use;
[0024] recirculating the third permeate fluid back to the diluent
vessel for reuse;
[0025] concentrating the first and second permeate fluid by flowing
same to a fourth cross-flow filter apparatus communicatively
connected to the end product vessel wherein target substance is
concentrated and diafiltration buffer is removed in fourth permeate
stream and recirculated for reuse.
[0026] In another aspect, the present invention provides for a
method of for separating a target substance, the method
comprising:
[0027] providing a diluent to a first reservoir;
[0028] providing a starting source material to a second
reservoir:
[0029] providing a buffer to a third reservoir;
[0030] flowing a portion of the starting material with a portion of
diluent to form a mixture and flowing the mixture to a first
cross-flow filtration apparatus; [0031] recirculating the mixture
of diluent and starting material in the first cross-flow filtration
apparatus in a flow path adapted for: [0032] diafiltering the
mixture; [0033] permeating the target substance through the
membrane; [0034] selectively flowing a portion of the retentate of
the first cross-flow filtration apparatus to a second cross-flow
filtration apparatus;
[0035] recirculating the retentate of the first cross-flow
filtration apparatus across to a second cross-flow filtration
apparatus in a flow path adapted for: [0036] selectively flowing a
portion of the retentate out of the second cross-flow filtration
apparatus as a concentrate; [0037] selectively flowing the permeate
to a product reservoir; and [0038] capturing the permeate of the
first cross-flow filtration apparatus in the product reservoir.
[0039] Optionally, the permeate fluid of the first cross-flow
filtration apparatus in the product reservoir can be recirculated
across a third cross-flow filtration apparatus in a flow path
adapted for: [0040] concentrating the molecule of interest in the
product reservoir; [0041] permeating the target substance free
liquid into the third reservoir; selectively flowing the liquid in
the third reservoir into the first cross-flow filtration apparatus
as the diafiltration buffer.
[0042] In a still further aspect, the present invention provides
for a system comprising:
[0043] a first reservoir constructed and arranged for holding a
diluent solution, and for selectively flowing liquid into and out
of said first reservoir;
[0044] a second reservoir constructed and arranged for holding a
starting material, and for selectively flowing liquid into and out
of said second reservoir, the second reservoir can be the cell
culture reservoir such as a fermentor or culture bag;
[0045] a first cross-flow filtration apparatus for separating
liquids into permeate and retentate streams, provided with means
for flowing liquid in and permeate and retentate streams out of
said first cross-flow filtration apparatus;
[0046] a second cross-flow filtration apparatus for separating
liquids into permeate and retentate streams, provided with means
for flowing liquid in and permeate and retentate streams out of
said second cross-flow filtration apparatus;
[0047] a third reservoir constructed and arranged for holding a
buffer, and for selectively flowing liquid into and out of said
third reservoir;
[0048] a third cross-flow filtration apparatus for separating
liquids into permeate and retentate streams, provided with means
for flowing liquid in and permeate and retentate streams out of
said third cross-flow filtration apparatus;
[0049] a product reservoir constructed and arranged for holding the
isolated product, and for selectively flowing liquid into and out
of said fourth reservoir; and
[0050] conduit, valve and pump means constructed and arranged for:
[0051] providing an initial volume of diluent to the first
reservoir; [0052] providing an initial volume of buffer to the
third reservoir; [0053] selectively flowing a portion of the
starting material with a portion of diluent to form a mixture and
flowing the mixture to the first cross-flow filtration apparatus;
[0054] recirculating the mixture of diluent and starting material
in the first cross-flow filtration apparatus in a flow path adapted
for: [0055] diafiltering the mixture; [0056] permeating the target
substance through the membrane; [0057] selectively flowing a
portion of the retentate of the first cross-flow filtration
apparatus to the second cross-flow filtration apparatus; [0058]
recirculating the retentate of the first cross-flow filtration
apparatus across the second cross-flow filtration apparatus in a
flow path adapted for: [0059] selectively flowing a portion of the
retentate out of the second cross-flow; [0060] filtration apparatus
as a concentrate; [0061] selectively flowing the permeate to the
first reservoir; [0062] capturing the permeate of the first
cross-flow filtration apparatus in the product reservoir and
recirculating the permeate fluid of the first cross-flow filtration
apparatus in the product reservoir across the third cross-flow
filtration apparatus in a flow path adapted for: [0063]
concentrating the molecule of interest in the product reservoir;
[0064] permeating the target substance free liquid into the third
reservoir; [0065] selectively flowing the liquid in the third
reservoir into the first cross-flow filtration apparatus as the
diafiltration buffer.
[0066] Yet another aspect of the invention provides for a process
for isolation of a desirable product from a viscous starting
mixture; the process comprising the steps of: [0067] diluting the
starting mixture with a minimum amount of diluent necessary for
effecting passage of the target substance through a first
cross-flow filter membrane; [0068] continually diafiltering the
diluted material on the first cross-flow filter membrane with
sufficient diafiltration volumes of buffer to achieve the desired
yield of product in the permeate; and [0069] concentrating the
permeate on a second cross-flow filter membrane to recover the
diluent for recycling while simultaneously concentrating the
permeate fluid containing the product of interest on the second
cross-flow filter membrane, such that the product is
concentrated.
[0070] Importantly, the product-free permeate is utilized and
recycled as the diafiltration buffer such that at the end of the
process, the product has been isolated from the viscous starting
mixture and concentrated into a smaller volume, i.e. less than the
volume of the undiluted starting material. Further any remaining
starting material is returned to the initial undiluted viscous
volume, or a lower volume, and no buffers where consumed other than
the initial volumes utilized to start the process.
[0071] The present system and method may be carried out to effect a
separation selected from the group consisting of: separating insect
cell culture fluid into its constituent parts; separating viral
culture fluid into its constituent parts; separating an
immunoglobulin from an immunoglobulin-containing culture of
bacteria, yeast, algal, fungus, insect cells, or animal cells;
separating an immunoglobulin from serum; separating a clotting
factor from a clotting factor-containing culture of bacteria,
yeast, fungus, insect cells, or animal cells; separating a protein
from a protein-containing culture of bacteria, yeast, fungus,
insect cells, or animal cells; separating an antigen from an
antigen-containing culture of bacteria, yeast, fungus, insect
cells, or animal cells; separating an antigen from a viral culture
containing same; separating a hormone from a hormone-containing
culture of bacteria, yeast, fungus, insect cells, or animal cells;
separating essential fatty acids from a fatty acid containing
culture of bacteria, yeast, algal, fungus, insect cells, larva or
animal cells; separating a glycoprotein from a viral culture;
and/or separating a glycoprotein from a glycoprotein-containing
culture of bacteria, yeast, fungus, insect cells, or animal
cells.
[0072] Other aspects and advantages of the invention will be more
fully apparent from the ensuing disclosure and appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0073] FIG. 1 shows a general scheme of an isolation system
according to one embodiment of the invention, using a cross-flow
filtration based apparatus.
[0074] FIG. 2A shows stacking of permeate and retentate sheets in
the cross-flow filter of the present invention; B shows the sheet
members P/F/P according to another embodiment of the invention; and
C shows plan view of retentate sheet of the present invention.
[0075] FIG. 3 shows the system components for diluting the source
material comprising the desired product.
[0076] FIG. 4 shows the system components for moving the diluted
source material through a cross-flow filtration stack and the
addition of buffer to the cross-flow filtration wherein the
permeate from the first and second cross-flow filtration stacks is
moved to a product reservoir.
[0077] FIG. 5 shows the system components for further concentration
of the permeate in the product reservoir wherein the buffer in the
permeate is separated and moved back to the buffer reservoir for
reuse.
[0078] FIG. 6 show the recapturing of the diluent in the retentate
and the separation of waste cells.
[0079] FIG. 7 shows the full components of the system as described
in FIGS. 3, 4, 5 and 6.
[0080] FIG. 8 shows the system for passage of the product away from
the diluted cell suspension.
[0081] FIG. 9 shows the separation of product from the cell
suspension by constant diafiltration of the cells.
[0082] FIG. 10 shows the components required to concentrate
diafiltered retentate of FIG. 8 to show the return of cell mass to
original volume of undiluted cell mass.
[0083] FIG. 11 shows the component necessary to separate the
product from cells by constant volume diafiltration while
simultaneously concentrating the product.
DETAILED DESCRIPTION OF THE INVENTION
[0084] In the description of the present invention, certain terms
are used as defined below.
[0085] A "source material or starting material" as used herein
refers to a viscous mixture containing solid and liquid materials
such as mill and grain wash waters, culture medium and fermentation
bio-mass. The source or substance material are often complex
mixtures or solutions containing many biological molecules such as
proteins, antibodies, essential fatty acids, hormones, and viruses
as well as small molecules such as salts, sugars, lipids, etc.
Examples of source or substance material that may contain valuable
biological substances amenable to the purification method of the
invention include, but are not limited to, a culture supernatant
from a bioreactor, a homogenized cell suspension, plasma, plasma
fractions, milk, colostrum and cheese whey.
[0086] "Essential fatty acids (EFAs)," as used herein, means
Omega-3 Fatty Acids and Omega-6 Fatty Acid. EFAs are given the
title `essential` not only because they are critical in promoting
overall health, but because they cannot be manufactured by the
body; therefore, it is essential that intake is through diet. EFAs
are considered to be long chain polyunsaturated fatty acids
(PUFAs). PUFAs of importance include, but are not limited to,
docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA),
alpha-linolenic acid (ALA), gamma-linolenic acid (GLA),
docosapentaenoic acid (DPA), arachidonic acid
(all-cis-5,8,11,14-eicosatetraenoic acid; AA) and stearidonic acid
(cis-6,9,12,15-octadecatetraenoic acid; SDA).
[0087] "Cross-flow filtration module" refers herein to a type of
filter module or filter cassette that comprises a porous filter
element across a surface of which the liquid medium to be filtered
is flowed in a tangential flow fashion, for permeation through the
filter element of selected component(s) of the liquid medium and
include hollow fibers, spiral wound nodules, ceramic filters,
cassette filters, plate and frame filters etc.
[0088] In a cross-flow filtration module employed in the present
invention, the shear force exerted on the filter element
(separation membrane surface) by the flow of the liquid medium
serves to oppose accumulation of solids on the surface of the
filter element. Useful cross-flow filters include microfiltration,
ultrafiltration, nanofiltration and reverse osmosis filter systems.
The cross-flow filters can be used in parallel or series flow path
stacked in a single housing or multi-element housing arranged as a
single or multiple loop system.
[0089] A preferred cross-flow filter system comprises a
multiplicity of filter sheets (filtration membranes) in an
operative stacked arrangement, e.g., wherein filter sheets
alternate with permeate and retentate sheets, and as a liquid to be
filtered flows across the filter sheets, impermeate
(non-permeating) species, e.g., solids or high-molecular-weight
species of diameter larger than the filter sheet's pore size(s),
are retained and enter the retentate flow, and the liquid along
with any permeate species diffuse through the filter sheet and
enter the permeate flow (See FIG. 2B). In a preferred embodiment of
the present invention, such cross-flow filtration module comprises
a permeate collection and discharge arrangement, a feed inlet, a
retentate outlet, and multiple fluid-flow sub-channels that may for
example be equidistant to the inlet and the outlet as shown in
FIGS. 2A and C.
[0090] Cross-flow filtration modules and cross-flow filter
cassettes useful in practice of the present invention are
commercially available from Smartflow Technologies Inc, (Cary,
N.C.), and are variously described in the following United States
patents: U.S. Pat. No. 4,867,876, "Filter Plate, Filter Plate
Element, and Filter Comprising Same", issued Sep. 19, 1989; U.S.
Pat. No. 4,882,050, same title, issued Nov. 21, 1989; U.S. Pat. No.
5,034,124, same title, issued Sep. 11, 1990; U.S. Pat. No.
5,049,268, same title, issued Sep. 17, 1991; U.S. Pat. No.
5,232,589, "Filter Element and Support", issued Aug. 3, 1993; U.S.
Pat. No. 5,342,517, "Filter Cassette Article," issued Aug. 30,
1994; U.S. Pat. No. 5,593,580, same title, issued Jan. 14, 1997;
and U.S. Pat. No. 5,868,930, same title, issued Feb. 9, 1999; the
disclosures of all of which are hereby incorporated herein by
reference in their respective entireties.
[0091] Briefly, a preferred cross-flow filter cassette of the
present invention is a stacked cassette filter assembly, as shown
in FIG. 2A, in which the base sequence of retentate sheet (R),
filter sheet (F), permeate sheet (P), filter sheet (F), and
retentate sheet (R) may be repeated in the sequence of sheets in
the filter assembly as desired, e.g., in a repetitive sequence of
retentate sheet (R), filter sheet (F), retentate sheet (R), filter
sheet (F), permeate sheet (P), filter sheet (F), retentate sheet
(R), filter sheet (F), permeate sheet (P), filter sheet (F),
retentate sheet (R), filter sheet (F), retentate sheet (R). Thus,
the filter cassette of a desired total mass transfer area is
readily formed from a stack of the repetitive sequences. In all
repetitive sequences, except for a single unit sequence, the
following relationship is observed: where X is the number of filter
sheets, 0.5X-1 is the number of interior retentate sheets, and 0.5X
is the number of permeate sheets, with two outer retentate sheets
being provided at the outer extremities of the stacked sheet
array.
[0092] The filter sheets, and the retentate and permeate sheets
employed therewith, may be formed of any suitable materials of
construction, including, for example, polymers, such as
polypropylene, polyethylene, polysulfone, polyethersulfone,
polyetherimide, polyimide, polyvinylchloride, polyester, etc.;
nylon, silicone, urethane, regenerated cellulose, polycarbonate,
cellulose acetate, cellulose triacetate, cellulose nitrate, mixed
esters of cellulose, etc.; ceramics, e.g., oxides of silicon,
zirconium, and/or aluminum; metals such as stainless steel;
polymeric fluorocarbons such as polytetrafluoroethylene; and
compatible alloys, mixtures and composites of such materials.
[0093] Preferably, the filter sheets, retentate and permeate sheets
are made of materials which are adapted to accommodate high
temperatures and chemical sterilants, so that the interior surfaces
of the filter may be steam sterilized and/or chemically sanitized
for regeneration and reuse, as "steam-in-place" and/or
"sterilizable in situ" structures, respectively. Steam
sterilization typically may be carried out at temperatures on the
order of from about 121.degree. C. to about 130.degree. C., at
steam pressures of 15-30 psi, and at a sterilization exposure time
typically on the order of from about 15 minutes to about 2 hours,
or even longer. Alternatively, the entire cassette structure may be
formed of materials which render the cassette article disposable in
character.
[0094] In one particular aspect, the present invention relates to a
filtration cassette comprising a multilaminate array of sheet
members of generally rectangular and generally planar shape with
main top and bottom surfaces, wherein the sheet members include in
sequence in the array a first retentate sheet, a first filter
sheet, a permeate sheet, and second filter sheet, and a second
retentate sheet, wherein each of the sheet members in the array has
at least one inlet basin opening at one end thereof, and at least
one outlet base opening at an opposite end thereof, with at least
one permeate passage opening at longitudinal side margin portions
of the sheet members;
[0095] each of the first and second retentate sheets having at
least one channel opening therein, wherein each channel opening
extends longitudinally between the inlet and outlet basin openings
of the sheets in the array and is open through the entire thickness
of the retentate sheet, and with each of the first and second
retentate sheets being bonded to an adjacent filter sheet about
peripheral and side portions thereof, with their basin openings and
permeate passage openings and register with one another, and
arranged to permit flow of filtrate through the channel openings of
the retentate sheet between the inlet and outlet basin openings to
permit permeate flow through the filter sheet to the permeate sheet
to the permeate passage openings;
[0096] the filtration cassette comprising a unitary article of
inter-bonded sheet members.
[0097] In another embodiment, the present invention relates to a
filtration cassette comprising a multilaminate array of sheet
members of generally rectangular and generally planar shape with
main top and bottom surfaces, wherein the sheet members include in
sequence in said array a first retentate sheet, a first filter
sheet, a permeate sheet, a second filter sheet, and a second
retentate sheet, wherein each of the sheet members in said array
has at least one inlet basin opening at one end thereof, and at
least one outlet basin opening at an opposite end thereof, with at
least one permeate passage opening at a longitudinal side margin
portion of the sheet members;
[0098] each of the first and second retentate sheets having at
least one channel opening therein, extending longitudinally between
the inlet and outlet basin openings of the sheets in the array, and
being bonded (e.g., compression bonded) to an adjacent filter sheet
about peripheral end and side portions thereof, with their basin
openings and permeate passage openings in register with one another
and the filtrate passage openings of each of the retentate sheets
being circumscribingly bonded to the adjacent filter sheet, and
with a central portion of each of the retentate sheets and adjacent
filter sheets being unbonded to permit permeate contacting the
retentate sheet to flow through the filter sheet to the permeate
sheet; and
[0099] each of the filter sheets being secured at its peripheral
portions on a face thereof opposite the retentate sheet, to the
permeate sheet.
[0100] In yet another embodiment, the present invention relates to
a filtration cassette comprising a multilaminate array of sheet
members of generally rectangular and generally planar shape with
main top and bottom surfaces, wherein the sheet members
include:
[0101] a first retentate sheet of suitable material, e.g.
polysulfone, polyethersulfone, polycarbonate, urethane, silicone,
or other material of construction, having (i) at least one
longitudinally extending rib or partition element, such partition
element(s) when provided in multiple configuration being
transversely spaced-apart from one another and being of
substantially the same height and substantially parallel to one
another to define a single or a series of channels between the
partitions, extending longitudinally between the respective inlet
and outlet basin openings of associated filter elements and
permeate sheet members, on both faces thereof, (ii) filtrate
passage openings at side portions of the sheets, and (iii) the
retentate sheet aligned to the first sheet of filter material at
respective end and side portions thereof, with the basin openings
and filtrate passage openings of the associated sheet members in
register with one another and the filtrate passage opening of the
retentate sheet member being circumscribingly compressed against
the first sheet of filter material, and with a central portion of
the first sheet of filter material and the retentate sheet member
being unbonded to permit permeate contacting the retentate sheet
member to flow through the first sheet member of filter material to
the permeate sheet member;
[0102] a first sheet member of filter material having (i) at least
one basin opening, of a suitable shape, e.g., polygonal,
semicircular, oval or sector shape, at each of opposite end
portions of the sheet member defining respective inlet and outlet
passages, and (ii) at least one filtrate passage opening at the
side portions of the sheet member, wherein the first sheet member
of filter material is bonded to the permeate sheet member at their
respective end and side portions, with their basin openings and
filtrate passage openings in register with one another and the
basin openings being circumscribingly bonded at respective end
portions of the first sheet member of filter material and the
permeate sheet member, and with a central portion of the first
sheet member of filter material and the permeate sheet member being
unbonded so as to define a central portion permeate channel of the
permeate sheet communicating with the filtrate passages in the
first sheet member of filter material and in the permeate sheet
member;
[0103] a permeate sheet member, having (i) multiple basin openings
of suitable shape at each of opposite end portions of the sheet
member defining respective inlet and outlet passages, and (ii)
filtrate passage openings at the side portions of the sheet
member;
[0104] a second sheet member of filter material having (i) at least
one basin opening at each of opposite end portions of the sheet
member defining respective inlet and outlet passages, and (ii) at
least one filtrate passage opening at the side portions of the
sheet member, wherein the second sheet member of filter material is
compression sealed to the retentate sheet member at their
respective end and side portions, with their basin openings and
filtrate passage openings in register with one another and the
filtrate passage opening of the retentate sheet member being
compression sealed to the second sheet member of filter material,
and with a central portion of the second sheet member of filter
material and the retentate sheet member being unbonded to permit
permeate contacting the retentate sheet member to flow through the
second sheet member of filter material; and
[0105] a second retentate sheet member of suitable material, e.g.
polysulfone, polyethersulfone, polycarbonate, urethane, silicone,
having (i) at least one longitudinally extending rib or partition
element, provided that when multiple partition elements are
employed, the partition elements are transversely spaced-apart from
one another, such partition elements being of substantially the
same height and substantially parallel to one another, to define a
single channel or a series of channels between the partitions,
extending longitudinally between the respective inlet and outlet
basin openings of the filter elements and permeate sheet members,
on both faces thereof, (ii) filtrate passage openings at the side
portions of the sheet member, and (iii) the retentate sheet
compression sealed to the second sheet of filter material at
respective end and side portions thereof, with their basin openings
and filtrate passage openings in register with one another and the
filtrate passage opening of the retentate sheet member being
compression sealed to the second sheet member of filter material,
and with a central portion of the first sheet member of filter
material and the retentate sheet member being unbonded to permit
permeate contacting the retentate sheet member to flow through the
second sheet member of filter material to the permeate sheet
member.
[0106] The end plates used with the cassette articles of the
invention to form a unitary filter assembly may be formed of any
suitable materials of construction, including, for example,
stainless steel or other suitable metal, or polymers such as
polypropylene, polysulfone, and polyetherimide.
[0107] Specifically, the present invention employs cross-flow
filtration modules with sub-channels that are equidistant to the
inlet and outlet of said modules such as shown in FIG. 2A and 2C
(retentate sheet). Moreover, said cross-flow filtration modules are
characterized by optimal channel height, optimal transmembrane
pressure, optimal membrane pore size and pore structure, optimal
membrane chemistry, etc., which are selected in order to achieve
the best combination of product quality and production yield.
[0108] For example, shear at the surface of the membrane is
critical in minimizing gel layer formation, but excessive shear is
deleterious in the following three key aspects: (1) excessive shear
increases energy consumption, (2) excess shear interferes with
diffusion at the membrane surface, upon which separation process
directly depends, (3) excessive shear can deprive certain compounds
of their bioactivities. It is therefore desirable to maintain shear
within an optimal range.
[0109] Furthermore, it is possible to optimize the separate
processes with cross-flow filtration modules of variable channel
velocities but of uniform channel heights, given the fact that most
commercial cross-flow modules are only available in a single
channel height. When the channel height of a cross-flow filtration
module is known, shear is directly proportional to channel velocity
of such module for the same solution passing by.
[0110] In the literature, numerous techniques have been proposed to
effect the separation of target substances using membrane
separations with addition of foreign substances such as acid, base,
salt and solvents. In contrast to these chemical additives-based
methods, the methodology of the present invention permits a target
substance to be separated from an input fluid by the simplest
mechanical means. In the use of cross-flow filtration modules of
the type described in the aforementioned patents, the specificity
and speed of a desired separation is effected by a) fluid
distribution in the cross-flow module, b) channel height of the
cross flow module, c) channel length, d) shear rate, e) membrane
pore structure, f) membrane structure, g) membrane chemistry, h)
trans-membrane pressure, and i) pressure drop, which is a function
of channel length, velocity and solution viscosity.
[0111] The approaches by others involving various additives and
manipulations of transmembrane pressure appear to be predicated on
overcoming problems created by poor distribution of flow within the
cross-flow module. It is not to say that the addition of salts and
solvents do not have a place in separation but without proper flow
distribution the membrane separation cannot be optimally operated
nor will cleaning techniques be fully beneficial. It will be
appreciated, based on the disclosure herein that numerous
heretofore expensive or difficult separations are rendered far
simpler and more economical by employing the techniques described
herein.
[0112] Thus, the invention relates in another aspect to optimizing
the membrane separation process, comprising:
[0113] selecting a cross-flow membrane module wherein the distance
from the inlet port to the outlet port is equidistant from the
inlet to outlet for each sub-channel of the device, i.e., each
sub-channel is of a same dimensional character;
[0114] selecting an optimal channel height;
[0115] selecting an optimal shear rate and/or channel velocity;
[0116] selecting an optimal transmembrane pressure;
[0117] selecting an optimal membrane pore size;
[0118] selecting an optimal temperature;
[0119] selecting an optimal channel length; and
[0120] selecting an optimal pressure drop which is the composite of
[0121] the optimal channel height; [0122] the optimal shear rate
and/or channel velocity; [0123] optimal channel length; and [0124]
the viscosity of the solution being filtered.
[0125] Selecting a channel height can be performed mathematically
or empirically by trial and error. In most cell fermentation
applications, trial and error has been more appropriate due to the
fact that the viscosity of the cell broth or product solution is
rarely known, the cell count and cell viability are highly
variable, and the solution is frequently non-Newtowian. The
objective of channel selection is to minimize channel height with
three critical stipulations: first, the channel must be
sufficiently high to allow the unrestricted passage of any larger
material such as clumped cells; second, the channel should not
cause excessive pressure drop and loss of linear efficiency; and
third, the channel should be sufficiently high as to allow the
proper angle of attack for substances to encounter the membrane
pore and pass through the pore. The optimal channel height is
dependent on the length and viscosity of the solution.
[0126] Several notable observations have been made in initial
trials and process scale-up, as discussed below.
[0127] For cell suspensions having an optical density (OD) of 2 to
500, and a path length of 6 to 12 inches, start with a channel
height between 0.4 to 0.75 mm. If the inlet pressure is above 15
PSIG at a velocity of 2.0 M/sec, then the channel is too thin.
[0128] For cell suspensions having an optical density (OD) of 2 to
500, and a path length of 6 to 12 inches, start with a channel
height between 0.4 to 0.75 mm. If the inlet pressure is below 5
PSIG at a velocity of 2.0 M/sec the channel is too high.
[0129] For cell suspensions having an optical density (OD) of 2 to
500, and a path length of 25 to 40 inches, start with a channel
height between 0.7 to 1.0 mm. If the inlet pressure is above 15
PSIG at a velocity of 2.0 M/sec, the channel is too thin.
[0130] For cell suspensions having an optical density (OD) of 2 to
500, and a path length of 25 to 40 inches, start with a channel
height between 0.7 to 1.0 mm. If the inlet pressure is below 5 PSIG
at a velocity of 2.0 M/sec, the channel is too high.
[0131] Shear at the surface of the membrane is critical in
minimizing gel layer formation, but excess shear is deleterious in
at least three key aspects: first, it increases energy consumption
costs; second, excess shear and the resulting pressure has been
demonstrated to interfere with separations which appear to be based
on diffusion at the membrane surface; and third, shear can result
in damage to cells and impairment of the bioactivity of certain
compounds. It is apparent that the benefits of shear are readily
observed within a specific range for each process and that shear
rates outside that range are highly destructive.
[0132] Before progressing in the explication of the optimization
process, it must be pointed out that the shear stability of the
substances in solution or suspension is a key element in shear
optimization. Only through accurately calculating and charting the
specific shear rates utilized during optimization can the true
benefits of shear optimization become apparent. In concentration
processes, it is graphically clear that the higher the shear, the
higher the membrane flux, with two striking observations.
[0133] First, there is a minimum shear value that minimizes the
gel-layer formation. This minimum shear can be best demonstrated
for any specific solution by first running the device at an
excessively high shear rate and then systematically lowering the
shear incrementally until the resultant flux decay of each
incremental reduction in shear is disproportional to the reduction
in shear. Given the repeated observation during cross-flow
concentration applications that increasing the shear increases the
flux, the maximum flux for solutions is clearly governed by the law
of diminishing returns, where at some point increases in shear
provide lower increases in flux.
[0134] For concentration applications, it can be stated that there
is a minimum shear required to keep the membrane clean through
minimizing the gel-layer formation, as well as a maximum shear
which is determined by the cost of energy required to marginally
increase flux.
[0135] For separation applications it can be stated that there is a
minimum shear required to minimize the gel-layer formation and
allow the passage of a target substance, as well as a maximum shear
that interferes with the passage of a target substance, even though
the higher shear results in higher water flux rates.
[0136] Furthermore, it is possible to develop processes based on
channel velocity, given that most cross-flow end users tend to work
with a single channel height based on past experiences, and the
predominance of cross-flow modules that are only available in a
single channel height.
[0137] When working with a single device of uniform height, shear
is directly proportional to channel velocity for the same solution.
In concentration applications, the end user should install a flow
meter on the permeate port and record the maximum flux obtained at
reasonable cross-flow velocities between 1 and 4 M/sec for devices
with channel heights between 0.5 mm and 1.0 mm. In separation
applications, the end user should assay the passage of the target
material(s) at cross-flow velocities between 0.5 and 2.5 M/sec for
devices with channel heights between 0.5 mm and 1.5 mm.
[0138] The optimization of transmembrane pressure (TMP) can only be
performed after the appropriate tangential velocity has been
determined. Transmembrane pressure is calculated as TMP=(inlet
pressure+outlet pressure)/2-permeate pressure. It is imperative
that the tangential velocity (flow rate) be monitored during the
optimization of transmembrane pressure, since increasing the
pressure normally decreases the output of most pumps due to
slippage. The objective of the optimization of transmembrane
pressure is to define the correlation of transmembrane pressure to
permeate flow rate. The normal relationship is a traditional bell
curve. A graph of transmembrane pressure versus permeate flow rate
should resemble a bell curve. Increases in transmembrane pressure
cause increases in the permeate rate until a maximum is reached,
and thereafter further increases in transmembrane pressure result
in decreases in the permeate rate. The reason for this result is
that the decreasing flow rate, resulting from higher transmembrane
pressures, is the result of gel layer and/or membrane
compression.
[0139] The procedure is set out below:
[0140] (1) Operate the system in total recycle mode at the optimum
tangential velocity for sufficient time, typically fifteen minutes,
for any gel layer to accumulate.
[0141] (2) Measure the permeate rate. This is the Base Rate.
[0142] (3) Increase the transmembrane pressure by 3 PSIG and
measure the permeate rate immediately and after five minutes at the
new transmembrane pressure. Compare the permeate rates to the base
rate. If the rates have increased go to Step 4. If the rate
decreases go to step 5.
[0143] (4) Repeat steps 2 and 3 until the permeate rate no longer
increases during each step or does not hold that increase for five
minutes.
[0144] (5) The optimum transmembrane pressure is the last pressure
reading where the increase in pressure result in an increase in
permeate rate.
[0145] In separation applications, the end user should assay the
passage of the target material(s) at TMP's between 2 and 15 PSIG
where the cross-flow velocity is optimized between 0.5 and 2.5
M/sec for devices with channel heights between 0.5 mm and 1.5
mm.
[0146] Selecting and optimizing the channel length has been totally
impractical if not an impossible task until the advent of the
stacked cross-flow filtration units as described herein. The
inherent difficulty of optimizing the channel length in prior art
devices has been three-fold: first, the devices such as spirals
were designed to maximize membrane utilization based on the width
that membranes could be cast rather than any other factor; second,
increases in channel length for devices such as cassettes resulted
in enormous increases in pressure drop due to the predetermined
channel geometry imposed by the retentate screen; and third, plate
and frame devices, such as for example Pleidae by Rhodia, France,
use fixed molded plates which are manufactured in a single length
and cannot be changed without manufacturing a new mold.
[0147] The present invention eliminates these prior art
restrictions by providing the ability to select the channel length
by utilization of an infinitely variable retentate sheet that is
cut to length from an appropriately manufactured film, selected
from a variety of standard or starting point thicknesses. Likewise,
the membrane sheets and permeate sheets are cut to matching lengths
and laminated into a stacked cassette.
[0148] There undoubtedly are many ways of selecting the optimum
membrane for any given process, yet it appears the most reliable
method of using membranes is to consider the manufacturer's
specified pore size as a theoretical starting point which then is
modified by the solution and the operating conditions. As a result
of numerous trials, a practical parameter has been determined and
termed the coefficient of rejection.
Coefficient of Rejection (CRV)
[0149] Membranes have a rejection characteristic (value) that is
first order and is defined by the size, charge and shape of the
pore. For simplicity the CRV, coefficient of rejection value, is
the stated pore size provided by the manufacturer. In purifying a
product of interest the CRV of a membrane is more important for
separation applications as compared to concentration applications.
The rules below specifically relate to separation applications.
These effects will vary in concentration applications.
[0150] The CRV of a membrane is subject to the velocity of the
tangential flow operation. Empirical evidence suggests that the
neutral point of any membrane can occur in two zones, the first
zone being the point at which the transmembrane pressure and/or
shear compress the gel layer and the CRV increases, and the second
zone occurring where the TMP and velocity minimize the shear and
the CRV decreases. The neutral point (NP) is defined as the point
where a membrane freely passes particles 0.5 times the stated pore
size, NP=0.5(Pore Size).
Therefore:
[0151] the effective CRV of a typical micro porous membrane is
greater than the pore size, for velocities greater than 1.5 M/sec
and less than 3.0 M/sec.; and
[0152] the effective CRV of a typical ultrafiltration membrane is
greater than the pore size, for velocities greater than 1.5 and
less than 3.0 M/sec.
[0153] Example: A 0.3 .mu. particle may freely pass a 0.45 .mu.
polymeric membrane when the velocity is between 1.5 and 4.0 M/sec
but not for velocities between 0.5 and 1.5 M/sec or 4.5 and 12
M/sec.
[0154] Example: A 45,000 MW protein may freely pass a 0.2 .mu.
membrane for velocities of 0 to 1.0 M/sec but be significantly
retained when the velocity is increased above 1.5 M/sec. In the
same experiment, it was documented that protein passage was above
90% for velocities between 0.8 and 1.5 M/sec and 25% for a velocity
of 2.0 M/sec. Additionally, this same protein had 65% membrane
transmission through a 100,000 MW membrane at velocity of 1.0
M/sec.
Further,
[0155] the CRV of a membrane is proportional to the molarity of the
solution;
[0156] the greater the solute concentration, the greater the CRV;
and
[0157] the lower the solute concentration, the smaller the CRV.
[0158] Thus, a membrane may have a stated pore size of 500,000 MW
but will retain proteins of 110,000 MW in cell suspension with an
OD over 100 and pass the same 110,000 MW protein when the OD is
less than 50.
[0159] The process can be developed and optimized by empirical
testing of undiluted and/or diluted volumes of starting source
material to measure the percent of target molecule passed into the
permeate fluid. Two testing methodologies can be employed
including:
[0160] 1) Concentrate the undiluted and/or diluted material as much
as possible, from 1 to 10X for example, collect and assay samples
of the retentate fluid and the permeate fluid simultaneously
collected at various points in the concentration process such as
start, 2X, 3X , 5X, 7X and 10X, divide the assayed level of target
substance measured in the permeate fluid by the assayed level of
target substance in the retentate sample that was taken at the same
point in time and multiply by 100 in order to express the result as
percent passage of the target material.
[0161] 2) Continuously diafiltering the undiluted and/or diluted
material against multiple volumes, from 1 to 10X for example,
collect and assay samples of the retentate fluid and the permeate
fluid simultaneously collected at various points in the
diafiltration process such as start, 2X, 3X , 5X, 7X and 10X,
divide the assayed level of target substance measured in the
permeate fluid by the assayed level of target substance in the
retentate sample that was taken at the same point in time and
multiply by 100 in order to express the result as percent passage
of the target material.
[0162] The data from these two processes will indicate several key
factors which will provide a total isolation process as described
herein:
[0163] a) The appropriate dilution of the starting material that
results in good passage of the desired product away from the
starting material.
[0164] b) The number of diafiltration volumes necessary to achieve
an acceptable yield.
[0165] c) The degree of concentration to which the starting
material can be concentrated.
[0166] d) The membrane performance of the tested membranes at the
operating conditions utilized in the testing.
[0167] e) Optimization of the membrane performance.
[0168] A succinct description of the process would be to start the
isolation of a desirable product from a viscous mixture by diluting
the starting mixture the minimum amount necessary to effect good
passage of the target substance through a separating membrane,
followed by continually diafiltering the diluted material on said
separating membrane with sufficient diafiltration volumes to
achieve the desired yield, then to concentrate the diluted mixture
on a second membrane device to recover the diluent for recycling
while simultaneously concentrating the permeate fluid, containing
the product of interest that was in the mixture, on the separating
membrane, such that the product is concentrated. Then the
product-free permeate is utilized and recycled as the diafiltration
buffer such that at the end of the process, the product has been
isolated from the viscous mixture and concentrated into a smaller
volume, i.e. less than the volume of the undiluted starting
material. Further any remaining starting material is returned to
the initial undiluted viscous volume, or a lower volume, and no
buffers where consumed other than the initial volumes utilized to
start the process.
[0169] Another way to understand the invention is to look at how
the fluid flows through the various steps mathematically:
[0170] The terminal retentate flow (TRF), in liters per hour, for
the starting material concentration step (SMCS) is approximately
equal to the starting volume (SV) of the starting material, in
liters, divided by the desired processing time (DPT), hours. TRF
(LPH)=SV (L)/DPT (h)
[0171] In preferred embodiments of the apparatus, the terminal
retentate flow derived from the starting material concentration
step (SMCS) can be changed to a fraction of the starting volume
(SV) flow rate by decreasing the volume of starting material in
order to lower the waste stream or to concentrate the remaining dry
matter in the starting volume (SV) as this fluid stream may be a
valuable by-product. One such example would be to utilize the
invention to isolate one or more proteins and/or carbohydrates from
a plant material such as soy, potato, tobacco and milk where the
starting material less the protein or carbohydrate had residual
value as a bulk protein or additive to a third product such as soy
flour, milk powder, and fish feeds etc. The reduced volume would
lower the cost of either drying or transporting the liquid
stream.
[0172] The feed flow rate (FF), in liters per hour, into the
separating filter apparatus (SFA) is equal to the terminal
retentate flow rate (TRF) where no concentration of the starting
volume is desired. FF (LPH)=TRF (LPH)=SV (L)/DPT (h)
[0173] If for example the terminal retentate flow rate (TRF), in
liters per hour, is to be one-half (1/2) of the starting solution
flow rate (PF) when there is no dilution of the starting solution,
then the equation is simply modified. FF (LPH)=PF (LPH)=2xRF
(LPH)=0.5x(SV (L)/DPT (h))
[0174] Further, the feed flow rate (FF), in liters per hour, into
the separating filter apparatus (SFA) is equal to the sum of the
diluting fluid flow rate (DF) plus the product flow rate (PF). FF
(LPH)=DF (LPH)+PF (LPH)
[0175] The retentate fluid flow (RFF), liters per hour, from the
separating filter apparatus (SFA) into the starting material
concentration step (SMCS) is equal to the feed flow rate (FF) when
the feed flow rate is neither diluted or concentrated by the
separating filter apparatus (SFA).
[0176] The diluting fluid flow rate (DF) is equal to the desired
initial dilution for the product flow rate. If for example, it was
determined that the product of interest could be separated when the
starting material was diluted with an equal volume of buffer than
the equation would be DF=PF wherein we could say that FF
(LPH)=2x(SV (L)/DPT (h)).
[0177] If for example, it was determined that the product of
interest could be separated when the starting material was diluted
with two equal volumes of buffer than the equation would be DF=2xPF
wherein FF=3xPF=3x(SV/DPT).
[0178] If for example, it was determined that the product of
interest could be separated when the starting material was first
concentrated in half before entering the separating filter
apparatus (SFA) than the equation would be FF=0.5xPF.
[0179] If for example, the starting volume (SV) was to be
concentrated in half within the separating filter apparatus (SFA)
before initiating the diafiltration fluid flow than the equation
would be TRF=0.5xPF.
[0180] The permeate fluid from the starting material concentration
apparatus (PCA) replaces the diluting fluid flow rate (DF) that is
utilized to dilute the starting volume as necessary. The equation
for this relationship is PCA=DF.
[0181] The flow rate of diafiltration buffer (DFB) into the
separating filter apparatus (SFA) is determined by the number of
diafiltration volumes necessary to pass the target molecule into
the permeate stream of the separating filter apparatus (SFA) in
order to recover the desired yield of the target molecule. In the
case where the diluting flow rate, expressed as DF, resulted in a
process where the target substance passed freely into the permeate
stream than the following table can be utilized to determine the
yield of the target substance based on the number of diafiltration
volumes.
TABLE-US-00001 Solute Recovery vs. Volume Replacement Recovery of
Target Molecule in the Permeate Fluid i.e. when passage is Volume
Replacement unrestricted, 0% rejection 0 0 1 50% 2 75% 3 87.5% 5
96.9% 7 98.7% 10 99.8%
[0182] In the case where the feed flow rate (FF) is to undergo
diafiltration without being concentrated or diluted in the
separating filter apparatus (SFA) than the feed flow rate will
equal the retentate fluid flow (RFF) to the starting material
concentration step (SMCS) and the permeate rate (PSA) of the
separating filter apparatus is equal to the flow rate of
diafiltration buffer (DFB) into the separating filter apparatus
(SFA).
[0183] If for example, the process is determined to require a five
(5) fold diafiltration of the feed flow rate (FF) in order to
obtain a yield of 96.9%, as shown in the table, than the equation
can be expressed as DFB=5xFF .
[0184] The permeate flow rate of the product concentration
apparatus (PCA) needs to replace the permeate fluid discharged from
the separating filter apparatus (SFA) as permeate flow rate of the
separating filter apparatus is equal to the diafiltration buffer
(DFB) flow rate such that the equation is PCA=DFB.
[0185] In preferred embodiments of the apparatus, it maybe
advantageous to intermittently harvest the concentrated product
from the product reservoir to avoid prolonged exposure to the shear
forces of the concentrating membrane apparatus or simply to avoid
product degradation over time as a result of varied biological
and/or chemical effects.
[0186] FIG. 1 shows an arrangement of reservoirs and cross-flow
filtration units that is representative of one embodiment,
understanding that a system may include from one to multiple
reservoirs and cross-flow filtration units, the present system
comprising:
[0187] A first reservoir 1 constructed and arranged for holding a
diluent solution, and for selectively flowing liquid into and out
of said first reservoir;
[0188] A second reservoir 2 constructed and arranged for holding a
viscous starting source material, and for selectively flowing
liquid into and out of said second reservoir, the second reservoir
preferably is a cell culture reservoir such as a fermentor or
culture bag; wherein the first and second reservoir are
communicatively connected to a channel 3 for delivering components
of the first and second reservoir and combining therein for
delivery to at least one cross-flow filtration unit positioned
downstream of the combining channel;
[0189] A first cross-flow filtration apparatus 4 for separating
liquids into permeate and retentate streams, provided with means
for flowing liquid in and permeate and retentate streams out of
said first cross-flow filtration apparatus, wherein the permeate
includes at least the target of choice and can be directed to a end
product reservoir 6 and wherein the retentate comprises cell mass
and/or culture material for movement downstream or recirculation
into the first cross-flow filtration unit;
[0190] A second cross-flow filtration apparatus 5 communicatively
connected to the first cross-flow filtration unit and retentate
stream leaving therefrom, wherein the second cross-flow filtration
unit is used for separating the retentate stream into permeate and
retentate streams and provided with means for flowing liquid in and
permeate and retentate streams out of said second cross-flow
filtration apparatus, wherein the permeate includes at least the
target of choice and can be directed to the end product reservoir 6
and wherein the retentate comprises cell mass and/or culture
material for movement downstream or recirculation into the second
cross-flow filtration unit;
[0191] A third reservoir 7 constructed and arranged for holding a
diafiltration buffer, and for selectively flowing liquid into and
out of said third reservoir; wherein the buffer is deliverable,
though a channel system 10, to the first and second cross-flow
filtration units and for mixing with the input stream therein;
[0192] A third cross-flow filtration apparatus 8 for separating
retentate stream from the second cross-flow filtration apparatus
into permeate and retentate streams, provided with means for
flowing liquid in and permeate and retentate streams out of said
third cross-flow filtration apparatus, wherein the third cross-flow
filtration unit is communicatively connected to the retentate
stream of the first and/or second cross-flow filtration unit;
wherein the dilution buffer is removed via the permeate stream for
optional recirculation into dilution buffer reservoir 1 and the
retentate stream which includes waste cells can be optionally used
for multiple purposes including further separation of additional
target molecules or used in feed products for animals, both
terrestrial and aquatic.
[0193] The end product reservoir 6 constructed and arranged for
holding the isolated end product, and for selectively flowing
liquid into and out of said end product reservoir; wherein the end
product is removed directly from the end product reservoir or in
the alternative directed through a separation cross-flow filtration
unit 9 for separation of end product from at least the
diafiltration buffering solution, wherein the diafiltration
buffering solution can be directed to the buffer reservoir 7 for
reuse in the system.
[0194] The system further comprises conduit, valve and pump means
constructed and arranged to move liquid and slurries from different
reservoirs to cross-flow filters. In preferred embodiments of the
apparatus, the reservoirs are provided with thermal jackets to
maintain appropriate process temperatures.
[0195] An illustrative example is provided using the system of
OPTISEP.RTM. filtration modules for processing Pichia pastoris. The
present example can be used to separate expressed proteins from
high cell density P. pastoris cell culture, wherein the starting
concentration of 50% solids is able to provide a recovery of
95%+.
[0196] The process comprises diluting the Pichia so that it is
readily filtered (step 1), then filtering the diluted material in a
first OPTISEP.RTM. filter module via diafiltration so as to
separate the product from the feed stock (step 2), while
simultaneously: a) concentrating the permeate on a second
OPTISEP.RTM. filter module which both concentrates the product and
recycles the diafiltration buffer (step 3) and b) concentrating the
retentate of the first OPTISEP.RTM. filter module with a third
OPTISEP.RTM. filter module recovering the diluent and returning the
feed stock to its original volume or less (step 4).
[0197] Typical P. pastoris fermentations can reach a wet cell
weight of 50 to 60%. At these high solid concentrations, the
culture typically must be diluted to permit effective passage
during filtration. The dilution step 1 is depicted in FIG. 3 and
includes the following observations and or parameters:
[0198] The cell culture is diluted to a predetermined concentration
with diluent.
[0199] The flow rate of cell culture fluid into the diluent is
equal to the volume of cell culture fluid divided by the desired
processing time.
[0200] Increasing the amount of diluent increases the effective
separation of product from the cell suspension.
[0201] Increasing the amount of diluent increases the flux rate of
the membrane.
[0202] Increasing the amount of diluent decreases the operating
pressure.
[0203] Increasing the amount of diluent increases the total volume
of liquid to be processed.
[0204] Step 2, once the culture is diluted; the cells are separated
from the product in solution using an OPTISEP filter module with a
microfiltration (MF) membrane. In this process, the product passes
through the MF membrane into the MF permeate by continuous
diafiltration, as labeled in step 2 (FIG. 4) the cells remain in
the recirculation loop, i.e. the retentate fluid. The MF membrane
capacity is increased by adding more membrane area to the filter
holder and/or more recirculation loops. The attached depiction of
FIG. 4 shows two (2) recirculation loops in series with one filter
holder in each loop. Because the diafiltration is a steady state
diafiltration, the volume entering the loop (i.e. the feed rate) is
equal to the volume leaving the loop (i.e. the bleed rate) and the
volume of permeate leaving the loop (i.e. permeate rate) is equal
to the volume of diafiltration buffer entering the system (i.e. the
diafiltration rate.) Therefore, the concentrations of the cells
entering, leaving, and inside the recirculation loop are constant
at the optimal concentration set in the dilution step (step 1). The
concentrations of the molecules that pass through the membrane such
as the product are reduced. The source of the diafiltration buffer
is described in the third step. The concentration step 2 includes
the following observations and/or parameters:
[0205] The flow rate into the MF stage is equal the flow rate out
of the MF stage.
[0206] The permeate rate out of the MF stage equals the flow rate
of diafiltration buffer into the MF stage.
[0207] Increasing the diafiltration factor will increase the
product yield.
[0208] The third step (Step 3, FIG. 5) is the concentration of the
product derived from the MF permeate fluid as well as generating
the diafiltration buffer. Utilizing a second OPTISEP filter module
containing an ultrafiltration (UF) membrane, the permeate fluid of
the MF membrane containing the product protein is concentrated. The
product is concentrated in the retentate of this filter as depicted
in FIG. 5. The UF permeate is recycled back to step 2 as the
diafiltration buffer. This recycling dramatically lowers the waste
produced from the system and decreases the operating expenses
through the virtual elimination of buffers normally required for
diafiltration. The concentration of the product includes the
observations and/or parameters:
[0209] MF permeate containing the product is concentrated.
[0210] The rate of concentration is equal to the rate of the
diafiltration of step 2.
[0211] Product can be continually harvested from the product
retention loop or the product vessel if desired.
[0212] The fourth and final step is the concentration of the cells
back to their original concentration or a higher concentration
using the third OPTISEP filter module with a UF membrane, step 4
(FIG. 6). By concentrating the cells, the volume of cell waste is
decreased. The recycle of the permeate dramatically lowers the
waste produced from the system and decreases the operating expenses
through the virtual elimination of the diluent needed to lower the
concentration of the original fermentation broth. In certain
situations this step could be accomplished with an MF filter such
that the number of diafiltration required in step 2 would be
reduced and the permeate flow paths would be altered slightly from
the attached slides. The concentration of the cells back to the
original concentration includes the following observations and/or
parameters:
[0213] The diluted cell broth is concentrated back to the original
cell concentration or greater.
[0214] The permeate fluid of the concentration is recycled back to
be reused as a diluent.
[0215] The final volume of cell paste can be less than the volume
of the fermentor.
[0216] The final volume of cell paste can be chemically and/or heat
treated in line for discharge.
[0217] One advantage of separating the overall process into these 4
distinct unit operations is that each unit operation can be
studied, understood, and optimized independently. Then the
optimized parameters can be implemented when designing the large
scale design.
[0218] FIG. 7 shows the entire process without the various visual
keys.
[0219] Optimization of the process includes the following
experiments as outlined in FIGS. 8, 9, 10 and 11 including;
EXPERIMENT 1
[0220] The purpose of experiment 1 is to demonstrate the passage of
the product away from the diluted cell suspension. Experiment 1 is
performed at various dilution rates to optimize product recovery,
minimize the rate of dilution and maximize the membrane performance
in liters per meter square per hour (LMH). The starting material is
diluted in a batch mode using different levels of diluent to
determine the appropriate dilution and the product is separated
from the diluted starting material via constant volume
diafiltration. The level of diafiltration is determined.
EXPERIMENT 2
[0221] The purpose of experiments 2 is to concentrate the permeate
of experiment 1. The permeate of experiment 1 contains the product
which was separated from the cells by constant volume diafiltration
of the cells. The permeate of the separation is concentrated in
order to demonstrate recovery of the product and number of passes
through MF for acceptable product concentration.
EXPERIMENT 3
[0222] The purpose of experiments 3 is to concentrate the
diafiltered retentate of experiment 1 in order to demonstrate the
ability to return the cell mass to the original undiluted volume or
less. In other words the purpose of experiment 3 is to show the
feasibility of reducing the volume of the process waste stream as
well as the ability to recover the diluent. The diluted cellular
material is concentrated to the original starting volume or less
and number of passes through MF for acceptable concentration.
[0223] Another embodiment for optimization comprises performing
experiments 1, 2 and 3 utilizing three (3) different filtration
steps (MF, UF of the MF permeate, and UF of cells); followed by a
experiment 4 (FIG. 11) which is the simultaneous operation of the
MF separation and the UF concentration of the MF permeate
fluid.
EXPERIMENT 4
[0224] The purpose of Experiment 4 is the separation of the product
from the cells by constant volume diafiltration while
simultaneously concentrating the product such that the product is
concentrated and the permeate of the product concentration is
recycled as the diafiltration buffer. The diluted starting material
is simultaneously separated and the product harvested via one MF
and one UF membrane working simultaneously.
* * * * *