U.S. patent application number 09/829217 was filed with the patent office on 2001-10-25 for culture device and method.
Invention is credited to Kopf, Henry B..
Application Number | 20010034058 09/829217 |
Document ID | / |
Family ID | 22771473 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010034058 |
Kind Code |
A1 |
Kopf, Henry B. |
October 25, 2001 |
Culture device and method
Abstract
A culturing system and method particularly useful for producing
cellular products such as viral pathogens of cells. It includes a
mass transfer culture segment, stacked filter plates to adjust the
medium composition, and a product removal and concentration
segment. The mass transfer culture segment utilizes changed
directional flow of the medium to maximize cell growth and
production of product. The stacked filter plates allow addition of
sterile fresh medium and removal of growth inhibitory
substances.
Inventors: |
Kopf, Henry B.; (Cary,
NC) |
Correspondence
Address: |
Steven J. Hultquist
Intellectual Property/Technology Law
P.O. Box 14329
Research Triangle Park
NC
27709
US
|
Family ID: |
22771473 |
Appl. No.: |
09/829217 |
Filed: |
April 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09829217 |
Apr 9, 2001 |
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09397291 |
Sep 15, 1999 |
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6214574 |
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09397291 |
Sep 15, 1999 |
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09307932 |
May 10, 1999 |
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6127141 |
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09307932 |
May 10, 1999 |
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07207655 |
Jun 21, 1988 |
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6022742 |
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07207655 |
Jun 21, 1988 |
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06936486 |
Nov 26, 1986 |
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4885087 |
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Current U.S.
Class: |
435/293.1 ;
435/235.1 |
Current CPC
Class: |
B01D 2321/32 20130101;
B01D 2321/2066 20130101; B01D 2321/2025 20130101; B01D 61/32
20130101; Y10S 435/818 20130101; B01D 63/084 20130101; B01D 2321/16
20130101; B01D 2321/168 20130101; C12M 29/12 20130101; B01D 65/08
20130101; B01D 2321/2083 20130101; C12M 29/16 20130101 |
Class at
Publication: |
435/293.1 ;
435/235.1 |
International
Class: |
C12M 001/00 |
Claims
What is claimed is:
1. A culturing system, comprising: (a) a medium reservoir
containing a growth medium; (b) a tangential flow growth device
connected to the medium reservoir, said tangential flow growth
device having a medium flow control means; (c) a pump system having
discharge and inlet ports for pumping the medium from the medium
reservoir to the tangential flow growth device; (d) a sterile
barrier tangential flow membrane device connected to the tangential
flow growth device; (e) a means for monitoring medium conditions;
and (f) a means for harvesting cells grown in the tangential flow
growth device, said harvest means openably and closably connected
to the tangential flow growth device;
2. A culturing system according to claim 1, wherein said cells
produce infective viruses and wherein said means for harvesting
cells comprises a vessel, and said vessel is connected to a source
of chemical capable of inactivating said viruses to render them
uninfective.
3. A culturing system according to claim 1, wherein the tangential
flow growth device comprises a mass transfer culture system.
4. A culturing system according to claim 3 wherein said medium flow
control means comprises flow ports designated A, B, C, and D, for
connecting: (a) in a first configuration, port A in flow
communication with port B, and port C in flow communication with
port D; and (b) in a second configuration, port A in flow
communication with port D, and port B in flow communication with
port C; and wherein said medium reservoir has inlet and outlet
ports; and wherein said mass transfer culture system has an
elongated mass transfer chamber having first and second inlets at
opposite ends of the chamber and conduits connecting the pump
discharge port to said port C, the pump inlet to the reservoir
outlet port, the reservoir inlet port to the port A, the chamber
first inlet to the port B and the chamber second inlet to the port
D.
5. A culturing system according to claim 3, wherein the sterile
barrier tangential flow membrane device comprises a stacked plate
filter system.
6. A culturing system according to claim 5, wherein said stacked
plate filter system comprises: (a) a support including a
circumscribing frame with an array of spaced-apart and
substantially parallelly aligned ribs extending between and joined
at their opposite ends to said frame, so that the ribs and frame
form a series of corresponding substantially parallel filtrate flow
channels, and openings in said frame in liquid flow communication
with said filtrate flow channels for egress of filtrate from said
filtrate flow channels through said frame openings; (b) a first
filter sheet continuously secured along its margins to a first face
of said frame; and (c) a second filter sheet continuously secured
along its margins to a second face of said frame; (d) the first and
second filter sheets together with the frame defining an enclosed
interior volume comprising said filtrate flow channels separated by
said ribs; (e) whereby filtrate entering said enclosed liquid
volume through said first and second filter sheets may flow in said
filtrate flow channels and be discharged from said filter element
through said frame openings in liquid flow communication with said
filtrate flow channels.
7. A culturing system according to claim 5, wherein said stacked
plate filter system comprises a stacked plate filter for use with a
disposable sheet filter element, said stacked plate filter
comprising: (a) a first generally planar and rectangular filter
plate having a substantially flat bottom surface, and a top surface
with an unwardly extending wall circumscribingly bounding a flow
channel of generally rectangular shape with a liquid inlet port at
a medial part of a first side of said flow channel and a liquid
outlet port at a medial part of a second side of said flow channel
opposite said first side thereof, said liquid inlet port being
joined in liquid flow communication with a liquid feed trough
extending transversely across said first side of said flow channel,
and said liquid outlet port being joined in liquid flow
communication with a liquid collection trough extending
transversely across said second side of said flow channel, with a
plurality of spaced-apart partitions extending upwardly from the
floor of said flow channel between said liquid feed trough and said
liquid collection trough, said partitions being of lesser height
than said wall circumscribing said flow channel and substantially
parallel to each other to define a series of sub-channels extending
longitudinally between said liquid feed trough and said liquid
collection trough, said liquid feed trough being of progressively
decreasing depth from its medial portion, in communication with
said liquid inlet port, to its marginal extremities, and said
liquid collection trough being of progressively decreasing depth
from its medial portion, in communication with said liquid outlet
port, to its marginal extremities; (b) a foraminous support of
generally rectangular shape supportively reposable at a first face
thereof on said partitions of said first filter plate; (c) a second
filter plate structurally identical to said first plate member,
positioned in inverted relations ship to said first plate member
such that said circumscribingly bounding walls of said first and
second filter plates are in abutting contact with one another, with
said foraminous support betwen said first and second filter plates
and supported by the respective partitions thereof; and (d) filter
sheets on either side of the foraminous support, interposed between
the foraminous support and the supporting partitions of the
respective first and second filter plates.
8. A culturing system according to claim 5, wherein said stacked
filter plate system comprises a filter plate having a generally
planar and rectangular shape with a substantially flat bottom
surface, and a top surface with an upwardly extending wall
circumscribingly bounding a flow channel of generally rectangular
shape, with a liquid inlet port at a medial part of a first side of
said flow channel and a liquid outlet port at a medial part of a
second side of said flow channel opposite said first side thereof,
said liquid inlet port being joined in liquid flow communication
with a liquid feed trough extending transversely across said first
side of flow channel, and said liquid outlet port being joined in
liquid flow communication with a liquid collection trough extending
transversely across said second side of said flow channel, with a
plurality of spaced-apart partitions extending upwardly from the
floor of said flow channel between said liquid feed trough and
liquid collection trough, said partitions being of lesser height
than said wall circumscribing said flow channel and substantially
parallel to one another to define a series of sub-channels
extending longitudinally between said liquid feed trough and said
liquid collection trough, said liquid feed trough being of
progressively decreasing depth from its medial portion, in
communication with said liquid inlet port, to its marginal
extremities, and said liquid collection trough being of
progressively decreasing depth from its medial portion, in
communication with said liquid outlet port, to its marginal
extremities.
9. A culturing system according to claim 3, wherein the medium flow
control means comprises a switchable f low control means for
controlling the direction of flow of medium through the mass
transfer culture system.
10. A culturing system according to claim 9, wherein the growth
medium is capable of growing host cells for viral pathogens and the
means for harvesting cells comprises a virus removal, concentration
and lysis system.
11. A culturing system according to claim 1, further comprising a
culturing system control means for automatically controlling medium
flow.
12. A culturing system according to claim 5, further comprising a
means for nutrient exchange into and out of the growth medium.
13. A method of growing cell cultures using a closed culturing
system to produce a cell product, comprising the steps of: (a)
inoculating a mass transfer culture system having a switchable flow
control means with cells capable of producing a desired cell
product; (b) exposing the cells to a flowing cell growth medium;
(c) periodically changing direction of flow of the cell growth
medium with the switchable f low control means; (d) adjusting
medium components to optimize cell product production by means of a
sterile barrier tangential flow membrane device; and (e)
filtratingly concentrating the cell product by removal of liquid
from the medium without removing cell product from the microbial
culturing system; and (f) removing low molecular weight inhibitory
substances by membrane dialysis of the flowing cell growth
medium.
14. A method according to claim 13, wherein said mass transfer
culture system comprises flow ports designated A, B, C, and D, for
connecting: (a) in a first configuration, port A in flow
communication with port B, and port C in flow communication with
port D; and (b) in a second configuration, port A in flow
communication with port D, and port B in flow communication with
port C; and wherein said mass transfer culturing system comprises a
first pump having discharge and inlet ports; an elongated mass
transfer chamber having first and second inlets at opposite ends of
the chamber; a reservoir having inlet and out ports; and conduits
connecting the pump discharge port to said port C, the pump inlet
to the reservoir outlet port, the reservoir inlet port to the port
A, the chamber first inlet to the port B and the chamber second
inlet to the port D.
15. A method for growing cell cultures to produce a cell product
according to claim 13, wherein the cell product comprises a
cellular pathogen.
16. A method for growing cell culture to produce a cell product
according to claim 15, wherein the cellular pathogen is a
virus.
17. A method for growing cell cultures to produce a cell product
according to claim 15, further comprising treating of the cell
product to destroy pathogenicity after the cell product is
filtratingly concentrated.
18. A method according to claim 13, wherein said cell product is
HIV virus.
19. A method according to claim 13, wherein said cell product is a
virus.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates to a device and method for culturing
pathogenic microorganisms and viruses to conserve laboratory space
and avoid contamination of the culture and release of pathogens
into the environment. In particular, this device pertains to a
device and method for obtaining large volumes of pathogenic viruses
such as the AIDS virus in a closed culture system.
[0003] 2. Description of the Related Art
[0004] Pathogenic animal viruses, such as the human
immunodeficiency virus (HIV) the rabies and herpes viruses, and
pathogenic bacteria such as Neisseria meningiditis and Mycobacteria
avium must be studied with extreme precaution to avoid spread of
the virus and contamination of workers and research areas. In the
following discussion of viruses, it is understood that other
pathogens may be handled in analogous fashion. The problem is that
in order to study these viruses, large quantities of viruses and
large volumes of virus extracts must be prepared and isolated from
growth media and contaminating cells, microbes and debris. Although
other pathogens, such as pathogenic bacteria or yeasts do not
usually require such large volumes of cell growth as are required
for viruses to obtain sufficient material for study, the cells must
also be cultured in quantity and handled with great care to avoid
worker exposure and accidental release of the organisms.
[0005] Microbial cellular pathogens of animals such as cellular
viruses generally must be cultured in growing animal cell cultures.
The standard method to culture such cells is by the use of roller
bottles. Roller bottles are coated on the inside by a layer of
solid or semi-solid growth substrate bathed by a nutrient medium on
which the cells are grown. The cell growth in roller bottles and
similar growth vessels, and thus, the viral yield, are limited by
the the internal surface area of the glass or plastic bottle and by
the nutrients in the nutrient medium. To obtain the large scale
viral yields that are necessary to study viruses, the number of
roller bottles needed may often fill shelves that extend from wall
to wall and floor to ceiling in several growth rooms. This requires
that large areas of laboratory space be allocated for incubation
facilities and often necessitates construction and/or room
conversion and attendant delays for those beginning to do research
in this area.
[0006] The processes of preparing the large number of roller
bottles, inoculating them with infected cells, incubating them
under the appropriate conditions, extracting the viruses from the
individual bottles, concentrating the viral suspensions, and lysing
the viruses to obtain non-pathogenic viral extracts for research
also are time-consuming and expensive. The numerous complicated
manual manipulations required in these procedures allow many
opportunities for mistakes to occur and problems to develop. Errors
by the laboratory workers may result in unwarranted experimental
conclusions and/or increased expenses and delays occasioned by the
repeated experiments.
[0007] The multiple experimental steps involving opening of the
roller bottles and the multiple number of roller bottles required
increases the likelihood that some of the human host cells will be
contaminated by undesirable microorganisms or viruses that may kill
the host cells. This may decrease viral production of the system
being studied or complicate extraction of the desired virus
components from the culture.
[0008] The large volumes of viral suspensions produced in the
existing culture systems necessitates a massive time-consuming
effort of viral harvesting. The dilute viral suspensions that are
obtained from the prior culture systems are difficult to treat
effectively to lyse the viruses until the suspension has been
concentrated. Thus, in a typical procedure, these large volumes of
infective viruses must be placed in centrifuge tubes. The
supernatant fluid must be decanted, with the possibility of
resuspension of the infective particles, and the pellet must then
be treated to lyse the viruses.
[0009] Thus, the numerous manual procedures that are required by
the prior art increases the possibility of escape of some of the
pathogenic virus particles from the culture system and their
release from the laboratory into the environment. It is of the
highest priority that the release of pathogenic viruses such as the
AIDS virus be avoided.
[0010] Mass transfer operations are often used to attempt to solve
some of the problems of growing a large number of cells that are
associated with culture systems such as roller bottles. In the mass
transfer chamber, a biological medium may be on one side of a mass
transfer surface element and the medium to which or from which mass
transfer is to be effected is disposed on the opposite side of the
element. Counterflow of the two media past each other effects
diffusional and/or osmotic mass transfer. Problems in the prior
art, which include reduction of mass transfer after prolonged
operation of a mass transfer element, longitudinal decrease of mass
transfer efficiency due to fouling of mass transfer surfaces, and
presence of undesirable micro-environments and areas of
preferential cell growth, are substantially avoided with the cell
culturing system disclosed in my copending patent application U.S.
Ser. No. 06/936,486 filed Nov. 26, 1986, the entire disclosure of
which is hereby incorporated by reference. This system may be used
to culture cells continuously on cellular or microbead substrates
with a minimum of risk.
[0011] In the culture of host cells to produce progeny viruses,
even with optimized mass cell culturing systems, there remain
problems of handling the culture fluid containing the viruses that
is being circulated through the system. Inhibitory substances of
various sizes must be removed from the media without removing the
viruses to allow cell growth to continue and viral concentration to
increase. There also must be a way to add new medium in order to
allow cell growth to continue without increasing the volume of the
culture medium that contains the virus. These purposes may be
accomplished by use of the stacked filter train disclosed in my
copending patent application U.S. Ser. No. 07/104,177, filed Oct.
2, 1987, the entire disclosure of which is hereby incorporated by
reference, which allows media to be added or wastes to be withdrawn
from a cell culture system without contamination of the system or
the operator.
[0012] Even with the prior art technology, the problems remain of
finding a method of culture of the cells to maximize virus yield;
an arrangement of culture vessel apparatus and stacked filter
train(s) to allow optimal recovery of viruses without excessive
contamination by metabolites, medium components, cell debris, or
other unwanted materials; and most importantly, a means by which
viruses may be concentrated and lysed without causing them to be
removed from the culture system to avoid all handling of the
pathogenic viruses after the initial inoculation of the system.
[0013] Accordingly, it is an object of the present invention to
provide an improved method and apparatus for effecting increased
cell growth and pathogen yield in which release of infective
pathogens is avoided.
[0014] It is another object of the invention to provided an
improved method and apparatus for effecting increased cell growth
and pathogen yield in which the infective pathogens are not handled
by laboratory personnel after the original inoculation with the
infected cells.
[0015] It is another object of the invention to provide an improved
method and apparatus for effecting increased cell growth and
pathogen yield by aseptic removal of spent medium components.
[0016] It is another object of the invention to provide an improved
method and apparatus for effecting increased growth and pathogen
yield by aseptic addition of nutrients.
[0017] It is a further object of the invention to provide a method
and apparatus for effecting increased pathogen yield that occupies
a minimum of laboratory space and does not require construction of
special rooms or buildings.
[0018] It is another object of the invention to provide a method
and apparatus for concentrating pathogen suspensions such as viral
suspensions for subsequent lysis without requiring handling large
volumes of infective pathogens.
[0019] It is another object of the invention to provide a
cost-reducing method and apparatus for effecting increased cell
growth and pathogen yield.
[0020] It is a further object of the invention to provide a sealed
virus-tight, unified, preassembled apparatus for aseptically
growing pathogens, monitoring and adjusting medium components,
harvesting the pathogens from the medium, rendering the pathogens
harmless and obtaining unhazardous pathogens or pathogen
components.
[0021] Other objects and advantages of the invention will be more
fully apparent from the following disclosure and appended
claims.
SUMMARY OF THE INVENTION
[0022] In a broad aspect, the invention relates to a microbial
culturing system closed by sterile barriers, comprising:
[0023] (a) a medium reservoir containing a medium for growing
cells;
[0024] (b) a tangential flow growth device connected to the medium
reservoir, said tangential flow growth device having a medium flow
control means;
[0025] (c) a pump system having discharge and inlet ports for
pumping the medium from the medium reservoir to the tangential flow
growth device;
[0026] (d) a sterile barrier tangential flow membrane device
connected to the tangential flow growth device;
[0027] (e) a means for monitoring medium conditions; and
[0028] (f) a means for harvesting culture product openably and
close ably connected to the tangential flow growth device. In this
aspect of the invention as well as the others discussed herein, the
term "microbial" comprises viruses as well as bacteria and other
microbes. Similarly, because the system may be used to culture
non-pathogens as well as pathogens, the term "pathogens" refers to
the microbial product whether pathogen or non-pathogen unless the
context indicates otherwise.
[0029] In particular, the invention relates to a closed microbial
pathogen culturing system, comprising:
[0030] (a) a medium reservoir containing a growth medium,
[0031] (b) a mass transfer culture system connected to the medium
reservoir, said mass transfer culture system having switchable flow
control means for controlling the direction of flow of medium
through the mass transfer culture system;
[0032] (c) a pump system having discharge and inlet ports for
pumping the medium from the medium reservoir to the mass transfer
culture system;
[0033] (d) a stacked plate filter system capable of solids
filtration and connected to the mass transfer culture system;
[0034] (e) a means for nutrient exchange into and out of the
medium;
[0035] (f) a means for sampling and monitoring components of the
medium;
[0036] (g) a means for harvesting culture product openably and
closably connected to the mass transfer culture system; and
[0037] (h) a culturing system control means for automatically
controlling medium flow.
[0038] The preferred method of the invention for growing cell
cultures using a closed microbial culturing system comprises the
steps of:
[0039] (a) inoculating a mass transfer culture system having a
switchable flow control means with cells capable of producing a
desired cell product;
[0040] (b) exposing the cells to a flowing cell growth medium;
[0041] (c) periodically changing direction of flow of the cell
growth medium with the switchable flow control means;
[0042] (d) adjusting medium components to optimize cell product
production by means of a sterile barrier tangential flow membrane
device; and
[0043] (e) filtratingly concentrating the cell product by removal
of liquid from the medium without removing cell product from the
microbial culturing system.
[0044] In the mass transfer culture system of the preferred
embodiment of the invention, a four-way valve is used to control
the direction of flow of the medium. Thus the circulating medium
may be reversed in direction of flow without the need for changes
in the inlet and outlet ports. The change in the direction of flow
causes a better mixing of the extracapillary volume, which aids in
the diffusion of the nutrients required for growth, to the cells,
and in the diffusion of growth inhibitory substances away from the
cells. The four-way valve may be a manually operated device
switched periodically, or more appropriately, an automated device
switched by a timed signal from a remote controller. Four-way
valves suitable for use in the invention are currently available
from Quality Controls (Tilton, N.H.) and Alpha Larval (Kenosha,
Wis.).
[0045] Tangential flow devices used in the invention for growing
cells and for adjustment of medium component concentrations
comprise nonrestrictive sterile barriers for the metabolites of
cell growth. In addition, they have a low coefficient for
absorption and adsorption of the cellular metabolites.
[0046] In addition to the particular preferred embodiments of the
mass transfer culture system and the stacked plate filter system
discussed below, the tangential flow devices may comprise a mass
transfer culture system utilizing a hollow fiber device as marketed
by Amicon Corporation (Danvers, Mass.) or Microgon Corp. (Laguna
Hills, Calif.) or plate and frame devices such as Minitan.RTM. or
Pellicon Cassette.RTM. (Millipore Corp., Bedford, Mass.). Thus, a
hollow fiber device such as the stainless steel Microgon.RTM.
equipped with a 0.2 micron hydrophilic membrane may be used for
small to medium volume (1000 ml) applications. A typical tangential
flow device for cell culture will include a filter having 0.2
micron diameter or smaller pores, configured to assure continued
operation over the lifetime of the culture for the purposes of
sampling, for example, for glucose and lactic acid or collection of
expressed proteins, IgG or growth hormones, and replenishment of
the growth medium without loss of sterility in the recirculating
loop.
[0047] A preferred filter system component that may be used in this
invention is disclosed in U.S. patent application Ser. No.
07/104,177, referred to above and comprises stacked filter plates
forming a cross-f low filter and is capable of substantially
uniform transverse distribution of inflowing liquid from a feed
port and highly uniform liquid cross-flow across the full
transverse extent of the flow channel. Each filter plate has on the
inlet side, a transverse liquid feed trough and on the outlet side,
a liquid collection trough. Between the liquid feed trough and the
liquid collection trough is a plurality of parallel partitions that
define subchannels and are of a lesser height than a wall that
circumscribes the flow channel that is between the two troughs.
[0048] Filter plates as disclosed above may be utilized in stacked
pairs to form enclosed flow channels within which efficient
filtration may occur. A first plate of the stacked pair is paired
with a second plate that is a mirror image of the first plate
positioned in an inverted relationship to the first plate such that
the respective circumscribingly bounding walls of the first and
second plates are in abuttingly sealing contact with one another.
Between the adjacent stacked plates is placed a foraminous support
of the general rectangular dimensions of the flow channel, with
filter sheet elements between the foraminous support and each of
the paired plates. The foraminous support functions to positionally
retain the filter on either side thereof and to accommodate the
interior flow of solids-depleted liquid toward the filtrate
collection means associated with the filter plate.
[0049] In operation of the stacked filter plate assembly, liquid is
introduced via the liquid inlet port. The liquid enters the liquid
feed trough and is laterally distributed from the medial portion of
the feed trough into the subchannels and toward its outer
extremities in a highly uniform flow over the full areal extent of
the sheet filter elements. This structure results in an increased
solids filtration capacity and extended operation time and thus a
higher microbial or virus yield may be obtained before the filter
must be regenerated or changed.
[0050] In lieu of the foraminous support, a filter element
comprising a support may be employed that includes a circumscribing
frame with an array of spaced-apart and substantially parallelly
aligned ribs extending and joined at their opposite ends to the
frame so that the ribs form a series of correspondingly
substantially parallel filter plate flow channels. Openings in the
frame allow liquid flow communication with the filtrate flow
channels for egress of filtrate from the filtrate flow channels. A
first filter sheet is secured to one face of the frame and a second
filter sheet is secured to the second face of the frame. Together
the filter sheets and the frame define an enclosed interior volume
comprising the filtrate flow channels separated by the ribs.
Filtrate enters the enclosed liquid volume of the filter through
the first and second filter sheets and is able to flow in the
filtrate flow channels and be discharged from the filter element
through the frame openings. Employment of a series of filter trains
allows washing of the cells, desalting of the medium, and finally,
concentration of the viruses (dewatering).
[0051] The filter element may be formed as a conventional wire
screen, a sintered metal plate or any other construction providing
the requisite supportive function for the filter sheets and
accommodating flow between the filter sheets toward the liquid
filtrate collection and discharge means.
[0052] The preferred mass transfer culture system to be used in
this invention has a switchable f low control means comprising a
four-way valve enabling the direction of fluid flow to be reversed
by activation of the valve. Such a mass transfer culture system is
disclosed in my pending patent application (Ser. No. 06,936,486)
and is discussed below as used in the pathogen culturing system of
the invention disclosed herein. Growth of host cells occurs in one
mass transfer medium in the hollow fiber membrane while fresh
culture medium comprises a second mass transfer medium.
[0053] In use with the invention, one or more mass transfer
chambers connected to a pump are linearly connected by means of
tubing to one or more stacked plate filters and to a viral harvest
means. The mass transfer chambers are inoculated with a cell
culture. The cells are either previously infected with a pathogen
or are inoculated after placement in the chambers. The cells are
bathed with fresh medium from the medium reservoir. As metabolic
waste products are accumulated in the spent medium, they are
removed by a stacked plate filter.
[0054] When the pathogen concentration has reached the desired
level, and the viral suspension has been concentrated such as
through use of a stacked filter plate, the concentrated suspension
is allowed to enter the viral lysis chamber by means of a valve.
Appropriate lysing agent is aseptically added to the viral
suspension and after incubation to ensure complete lysis, the
inactivated pathogen components may be removed from the system. It
may not be necessary to use this lysis step when non-pathogens are
grown in this culturing system.
[0055] The entire culturing system may be constructed to occupy a
space of 4.times.4.times.6 feet to allow harvest of about 10.sup.8
to 10.sup.10 cells per ml. Due to the compact and efficient nature
of the invention, the following yield is obtained with the
invention. An appropriate volume starting is approximately 100
liters of medium, depending on the type of cells to be grown and
the efficiency of medium utilization of cells. This volume has the
same pathogen production capability as about 1000 roller bottles
having one liter of medium each. Such roller bottles may, for
example, produce 10.sup.6 cells per ml or 10.sup.9 cells per
bottle. In the culture system of the invention, 100 liters of
medium are added to the system and about 99 liters of fluid are
removed prior to addition of the concentrated pathogen suspension
to the harvest vessel. The final pathogen producing cell density
may be about 10.sup.10-10.sup.12 pathogens per ml in the final
1-liter harvest vessel. The host cells are thus somewhat more
productive per volume of medium used in the culture system of the
invention than in the prior culture systems. More significantly, a
substantially higher pathogen concentration may be obtained using
the invention without the costly, hazardous, time-consuming and
space-consuming roller bottle system. The result with the invention
is increased personnel efficiency and safety at decreased cost in
about one one-thousandth of the laboratory space.
[0056] Other aspects and features of the invention will be more
fully apparent from the description set forth hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1 is a schematic diagram of a mass transfer system
component that may be used in the invention.
[0058] FIG. 2 is a schematic diagram of the four-way valve in a
first position.
[0059] FIG. 3 is a schematic diagram of the four-way valve in a
second position.
[0060] FIG. 4 is another schematic diagram of a mass transfer
system component that may be used in the invention.
[0061] FIG. 5 is a top plan view of a filter plate component of the
invention.
[0062] FIG. 6 is a sectional elevation view of the filter plate of
FIG. 5, taken along line A-A thereof.
[0063] FIG. 7 is a sectional elevational view of the filter plate
of FIG. 5, taken along line B-B thereof.
[0064] FIG. 8 is a top plan view of a foraminous support suitable
for use with paired plates in the stack plate fliter assembly
component of the invention.
[0065] FIG. 9 is an edge elevation view of the foraminous support
of FIG. 8.
[0066] FIG. 10 is an exploded perspective view of a stacked plate
filter assembly component of the invention.
[0067] FIG. 11 is a transverse sectional elevation view of a
stacked plate filter assembly component of the invention.
[0068] FIG. 12 is a plan view of a support for a unitary filter
element that may be used in the stacked filter assembly component
of the invention.
[0069] FIG. 13 is an edge elevation view of a unitary filter
element of FIG. 12.
[0070] FIG. 14 is an exploded perspective view of the unitary
filter element of FIG. 13.
[0071] FIG. 15 is a schematic drawing of the microbial pathogen
culturing system.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
THEREOF
[0072] The present invention comprises a pathogen culturing system
in which a pathogen such as a cellular virus may be grown to high
concentrations in a closed system using a mass transfer culture
system and a series of stacked filter plates. After the appropriate
number of pathogens has been produced, the pathogen may be
separated from the host cells, growth medium constituents and
unwanted growth products, and then be concentrated and treated to
lyse the pathogen without opening the sytem or risking release of
the pathogen.
[0073] FIGS. 1-4 depict more detailed features of the mass transfer
culture system. FIGS. 5-14 show details of the stacked plate filter
system. The whole pathogen culturing system of the invention is
shown schematically in FIG. 15.
[0074] Medium Reservoir and Pump
[0075] The medium reservoir 1 is preferably a vessel of stainless
steel or other durable easily sterilizable material capable of
holding 100 to 1000 liters of medium. Such reservoir may be of a
type commercially available from the Walker Stainless Equipment,
New Lisbon, Wis. As shown in FIG. 1, a variable speed pump 2 is
connected to the medium reservoir 1 and mass transfer culture
system 3. The pump 2 may comprise a peristaltic pump as shown in
FIG. 1, such as is commercially available from Cole-Palmer Company,
Chicago, Ill., or a variable speed gear pump, such as the
MICROPUMP.RTM. gear pump, commercially available from the Micropump
Corporation, Concord, Calif. A positive displacement lobe pump may
also be used (Waukesha Pump Co., Waukesha, Wis.).
[0076] The medium reservoir 1 may be equipped with stirring or
agitation means to promote uniform distribution of medium
components, such as a magnetic stirrer, an internal agitator, or a
bottom mounted, magnetically coupled agitator as is made by APCO
Technologies (Vancouver, Wash.). As shown in FIG. 1, the medium
reservoir 1 is disposed on a magnetic stirrer device 6 which
provides agitation of the contents of the reservoir 1. The magnetic
stirrer 6 may suitably be of a type having a variable speed, to
provide a varying level of agitation in the reservoir 1 depending
on the density and suspension characteristics of the nutrient
medium contained therein. The medium reservoir may also be provided
with a medium pH monitoring and adjustment means or other means for
adjustment of medium components or conditions of incubation
according to means and devices known in the art. The temperature of
the medium reservoir 1 may be varied depending on the desired cell
growth conditions. Medium reservoir(s) may be associated with a
temperature control unit, may be placed in a controlled temperature
environment or may be left at ambient temperature.
[0077] Mass Transfer Culture System
[0078] A mass transfer culture system 3 is shown in FIG. 1 that may
be employed for cell growth processes and for desalting and
harvesting of cellular products including viruses and utilizing a
hollow fiber membrane 4 of conventional type. The hollow fiber
membrane 4 provides a first set of interior passages in constituent
hollow fibers 5 through which a first medium, supplied from medium
reservoir 1, is flowed. The internal diameter of individual fibers
in the bundle may for example be from about 0.25 to about 1 mm,
with pores for diffusional and/or osmolytic transfer of nutrient
species, in the wall of the tubular fibers, of about 0.2 micron
diameter. Such bundles are typically potted at their ends in
urethane or epoxy resins, so that they may be suitably headered to
accommodate flow through the interior passages of the hollow
fibers, without leakage into the interstitial passages. The
nutrient thus is flowed longitudinally through the interior
passages of the hollow fibers, and nutrient species transfuse
through the fiber walls to the cellular culture contained in the
interstitial passages of the bundle. Illustrative hollow fiber
membrane mass transfer elements suited to the practice of the
invention are made by A/G Technologies, Needam, Mass., and CD
Medical Corporation, Miami, Fla.
[0079] The hollow fiber membrane 4 is connected at one end,
designated E-1, by conduit 7 to a port designated B, which is one
of four such ports, the others being designated A, C, and D, of a
four way valve 8. The conduit 7 may be flexible, elastic silicone
tubing, or may be formed of a more rigid material such as 316
stainless steel tubing. The opposite end of the hollow fiber
membrane 4, designated E-2, is connected by conduit 9 to port D of
the 4-way valve 8. A 4-way LL valve 8 generally useful in the broad
practice of the invention is manufactured by Quality Controls
Company, Tilton, N.H.
[0080] Pump 2 is connected on its discharge side by conduit 10 to
port C of the 4-way valve 8. The inlet side of pump 2 is connected
by conduit 11 to the outlet port of a reservoir 1 containing the
nutrient medium for the culture system. The inlet end of the
reservoir 1 is connected through another conduit 12 to port A of
the 4-way valve 8.
[0081] The mass transfer chamber 3 comprising the hollow fiber
membrane 4 also features a first port 3 in proximity to inlet E-1,
and a second port 14 proximate to inlet E-2. These ports may be
provided with suitable closure means, or may be joined with
suitable flow circuitry, as hereinafter described in greater
detail, for circulation of the cellular medium contained in the
hollow fiber membrane 4.
[0082] 4-way valve 8 has two positions. In a first position, shown
schematically in FIG. 2, port C is connected to port D and port B
is connected to port A. In this mode, the nutrient broth in conduit
11 from reservoir 1 is flowed by pump 2 into conduit 10, from which
it enters port C, discharges through port D into conduit 9 and
enters the hollow fiber membrane 4 at its inlet designated E-2. At
the same time the contacted nutrient broth exits through the
opposite end, designated as inlet E-2, of the mass transfer chamber
3 and passes through ports B and A of 4-way valve 8 to be returned
through conduit 12 to the inlet of reservoir 1, from which the
nutrient medium is circulated in the previously described
manner.
[0083] When the 4-way valve 8 is in a second position (FIG. 3),
port C is connected to port B and thus the valve discharges the
nutrient broth into conduit 7 from which it enters the mass
transfer chamber 3 containing the hollow fiber membrane 4, at the
inlet designated E-1, passes through the interior passages of the
hollow fibers and exits at the opposite end of the mass transfer
chamber 3, designated E-2, for return in conduit 9 through ports D
and A of 4-way valve 8 and conduit 12 to the reservoir 1.
[0084] FIG. 3 further shows the 4-way valve 8 in a first position,
in which the interior element 23 of the valve 8 is oriented so that
valve ports B and A are in flow communication with one another, and
ports C and D are in flow communication with each other. By a
90.degree. rotation of the valve element 23, the configuration
shown in FIG. 4, designated as the second position, is achieved. In
this position, valve ports B and C are in fluid communication with
one another, while flow communication is likewise established
between ports A and D. Alternation of the valve position between
the respective configurations shown in FIGS. 3 and 4 effects a
cyclic alternating switching of the nutrient medium flow to the
respective ends of the mass transfer chamber 3 containing hollow
fiber membrane 4.
[0085] Switching of the 4-way valve 8 may be accomplished either
manually or through an automatic actuator such as is schematically
illustrated in FIG. 1, in which the valve 8 is controlled by a
suitable automatic controller 15, operatively connected to the
valve assembly by control signal wires 16. The continued operation
of the system with either manual or automatic repositioning of the
valve 8 results in a balanced delivery of nutrients to the mass
transfer chamber alternately from its respective ends and also
enhances the transport of metabolic wastes away from the cells.
Further, the increased agitation incident to the switching of
nutrient flows results in a more homogeneous environment with
respect to other metabolic parameters.
[0086] The mass transfer culture system may also be provided at
each of the ports 13 and 14, with two valves in series. At the
first port 13, the valve assembly 17 comprises a first valve 18
contiguous to the port, and a second valve 19 connected in series
with the first valve 18, as shown. A similar construction is
employed at second port 14, where the double valve assembly 20
comprises a first valve 21 and a second outer valve 22.
[0087] The double valve arrangement described in the preceding
paragraph is highly preferred in practice, since it permits
cellular inoculation of the interstitial passages in the hollow
fiber bundle, as well as withdrawal of the product cell culture
from the interstitial passages, in a manner enabling complete
sterility of the apparatus to be maintained. In the prior practice
of utilizing ported hollow fiber cell growth chambers, where only
one valve has been employed on each port, the introduction to or
withdrawal from the mass transfer chamber of the cellular medium
results in loss of sterility of the valve's interior surfaces,
raising the potential for contamination of the mass transfer
chamber, unless the apparatus is thereupon fully shut down, and the
valves removed and autoclaved. Although some attempts have been
made in the past to sterilize single-valved ports by directing
super-heated steam against the valve body structure, such mode of
sterilization does not reach the interior surfaces of the valve,
which must be closed to retain the contents in the mass transfer
chamber.
[0088] By contrast, the double valve arrangement shown permits
ready sterilization of the valve assembly in a manner which
preserves the sterility of the associated apparatus, by the simple
expedient of opening the outermost valve (19, or 22) while the
corresponding inner valve (18, or 21) is kept closed, and
superheated steam, or other sterilant, is directed into the opened
outer valve, to effect complete sterilization thereof. Thus, a
sterile barrier is maintained at the mass transfer chamber 3 even
during continuous operation, and without the necessity of shutting
down the apparatus for autoclaving of the valve elements.
[0089] Concurrently, as shown in FIG. 4, with flow of the first
mass transfer medium, a second mass transfer medium stored in
second reservoir 24, may be withdrawn in conduit 25, passed to
peristaltic pump 26, and discharged into conduit 27 from which it
enters the mass transfer chamber 3 at port 28 and longitudinally
flows through the interstitial passages in the hollow fiber bundle.
After such longitudinal flow, in which the second medium is
contacted with the first mass transfer medium concurrently flowed
through the interior passages of the hollow fiber bundle, the
contacted second medium is discharged from the mass transfer
chamber in port 29 and flowed in conduit 30 back to second
reservoir 24. A sterile barrier at the mass transfer chamber may be
provided by double valve assemblies 31 and 32, as shown on the
third and fourth ports, 33 and 34, respectively, of the mass
transfer chamber 3 as discussed in detail above with respect to the
first reservoir 1. (The analogous valves in these valve assemblies
are designated by the same numbers in FIG. 4.)
[0090] Although the mass transfer chamber 3 has been described with
specific reference to a hollow fiber bundle as the mass transfer
surface element, it is within the purview of the invention to
utilize other mass transfer elements, such as planer membranes,
through which mass transfer may be effected, it being further
understood that the number of specific passages within the mass
transfer chamber and the number of mass transfer chambers employed
may be varied widely depending on the specific mass transfer media
and application employed. Use of multiple mass transfer chambers is
described in pending application Ser. No. 06/936,486, referred to
above.
[0091] Stacked Plate Filter System
[0092] The stacked filter plate system component of the invention
comprises one or more filters similar to those disclosed in my
earlier patent application. FIG. 5 shows an illustrative filter
plate in plan view. FIG. 6 shows a sectional elevation view of the
FIG. 1 plate, taken along line A-A thereof, and FIG. 7 is a
sectional elevation view of the FIG. 5 plate, taken along line B-B
thereof.
[0093] Each filter comprises pairs of filter plates, each plate
member 35 having a generally planar and rectangular shape with a
substantially flat bottom surface 36 as shown in FIGS. 5-7. The top
surface 37 of the plate has an upwardly extending wall 38
circumscribingly bounding a generally retangular flow channel
39.
[0094] The flow channel 39 of each filter plate has a liquid inlet
port 40 at a medial part on a first side 41 of the flow channel 39
and a liquid outlet port 42 at a medial part on a second side 43 of
the flow channel 39 opposite the first side 41. The liquid inlet
port 40 is joined in liquid flow communication with a liquid feed
trough 44 extending transversely across the first side 41 of the
flow channel 39, and the liquid outlet port 42 is joined in liquid
flow communication with a liquid collection trough 45 extending
transversely across the second side 43 of the flow channel 39.
[0095] There are a plurality of spaced-apart partitions 46a, 46b,
46c, 46d and 46e, extending upwardly from the floor 47 of the flow
channel 39 between the liquid feed trough 44 and the liquid
collection trough 45. The partitions 46a-46e are of a lesser height
than the wall 38 circumscribing the flow channel 39 and are
substantially parallel to one another to define a series of
sub-channels extending longitudinally between the liquid feed
trough 44 and the liquid collection trough 45.
[0096] The liquid feed trough 44 and the liquid collection trough
45 each decrease in depth from their respective medial portions in
communication with the liquid inlet port 40 and outlet port 42,
respectively, to their marginal extremities 48 and 49, and 50 and
51, respectively.
[0097] The outer circumscribing wall 38 may as shown be formed with
integral cylindrical flanges 52, 53, 54, and 55, each of which
circumscribes a circular opening in the periphery of the plate to
accommodate the positioning of the plate on spaced-apart rods, as
hereinafter shown with reference to FIG. 10 hereof.
[0098] At the medial portions of the first and second sides of the
plate, there are provided respective oblong openings 56 and 57 to
accommodated the liquid feed and liquid withdrawal conduits which
are employed to introduce liquid to and remove liquid from the flow
channels defined by adjacently paired stacked plates. Such feed and
discharge liquid conduits are more fully shown and described with
reference to FIG. 6 herein. The respective liquid feed and
discharge conduits are suitably formed with spaced-apart
perforations therein which permit egress or ingress of liquid into
or out of the flow channel via the above-described respective
liquid inlet and outlet ports of the plate. In order to assure
positive sealing of the flow channels and adjacently positioned
plates relative to the liquid feed and discharge conduits, the
liquid inlet and outlet ports of the plate are suitably provided
with gasket elements 58 and 59 as shown in FIG. 1, at the bottom
surface 36 of the plate.
[0099] As an example of plate dimensional characteristics for an
illustrative embodiment of the invention, a filter plate suitable
of filtration of aqueous biomass suspensions may be generally of
square shape as shown in FIG. 1 with sides on the order of about 6
inches long, and with feed and collection troughs 44 and 45 which
are each 2 millimeters deep at their medial portions, continously
decreasing to a depth of 1.5 millimeters at their respective
extremities (peripheral portions 48 and 49 of feed trough 44, and
peripheral portions 50 and 51 of collection trough 45). The
transverse dimension (width) of each of the sub-channels defined by
the partition walls 46a-e is approximately 2 centimeters.
[0100] The details of the plate construction are shown in FIG. 6
with respect to the structural features of the liquid inlet port 40
and liquid outlet port 42. The filter plate may be provided with a
circumscribing main wall 38 and an interior circumscribing wall 60
of lesser height than the main wall. Between these respective walls
is formed a circumscribing channel (see FIGS. 2 and 3), into which
suitable openings 61 and 62 may communicate as shown in FIGS. 5 and
7. These respective openings are usefully employed as filtrate
(permeate) flow channels to convey or drain the solids-depleted
filtered liquid or other permeate from the stacked plate
assembly.
[0101] Openings 61 and 62 may also be usefully employed as gas flow
openings to assist in draining the stacked plate filter upon
cessation of normal operation for regeneration. Thus, when the
filter is shut down, gas from a suitable supply source (not shown)
may be introduced in openings 61 and/or 62 to pressurize the flow
channel 39 to a sufficient extent where the same can be drained of
accumulated biomass suspension upon the termination of normal
liquid flows through the system. Similarly, these respective
openings may be employed for introduction and egress of steam for
steam sterilization of the system or for flowing a chemical
sterilant through the flow channel 39 prior to initiation or
re-initiation of normal filtration operation.
[0102] Further, because the edges of the foraminous support are
disposed in the channel between bounding walls 60 and 38, as shown
in FIG. 11, described more fully hereinafter, it is also possible
to utilize openings 61 and 62 as respective secondary fluid inlet
and discharge passages, for flowing a secondary fluid through the
foraminous support for mass transfer contacting of the liquid
introduced into the flow channel 39 from inlet port 40 and
discharged from the flow channel in outlet port 42. For such
purpose, it may be advantageous to "block" the channel between
bounding walls 60 and 38, at symmetrically opposed regions, as
shown in FIG. 5, where channel blocking segment 63 is disposed in
the channel along the side thereof containing opening 61, and
channel blocking segment 64 is similarly disposed in the channel
proximate to opening 62. With such arrangement, fluid entering in
opening 61 is diverted downwardly in the channel as shown in the
drawing and across the lower portion of the channel as shown until
it encounters the channel blocking element 64. Subsequently, when
the fluid so introduced is issued from the edges of the foraminous
support into the opposite portion of the channel as shown, it flows
to outlet opening 62.
[0103] Openings 61 and 62 may be appropriately sealed between
adjacent plates by provision of suitable gasket means 65 and 66,
respectively, at the flat bottom surface 36 of the plate, as shown
in dotted line representation in FIG. 5.
[0104] FIGS. 8 and 9 show respective top plan and edge views of an
illustrative foraminous support element for the stacked plate
filter assembly. The foraminous support 67 is simply a support
element of generally rectangular shape which is supportively
reposable at a first face 68 thereof on the partitions 46a-46e and
the circumscribing wall 60 of the plate element, with a first
filter sheet, e.g. a filter paper sheet, therebetween.
[0105] The foraminous support 67 is likewise supportively reposable
at a second face 69 thereof on the partitions and inner bounding
wall of a complementary filter plate paired with the filter plate
against which the first face 68 of the support is reposed. The
second face 69 of the foraminous support 67 likewise has a sheet
filter element between its surf ace and the partitions of the
adjacent plate member.
[0106] The foraminous support 67 is formed with a plurality of
longitudinally extending interior liquid flow channels 70 and a
plurality of transversely extending interior liquid flow channels
71, wherein the longitudinal and transverse channels criss-cross
one another to establish an extended interconnected network for
liquid flow through the interior of the support element.
Concurrently connecting the internal liquid flow network with the
top and bottom foraminous support surfaces 68 and 69 on which
sheets of filter paper or other filtration sheet members are
mounted, is an array of surface openings 72. Thus, when a sheet of
filter paper is provided for example on the top surface 69 of the
foraminous support 67, the liquid (permeate) component of the
solids-liquid suspension passes through the filter paper and
openings 72 into the interior liquid flow network comprising
channels 70 and 71, for flow through the foraminous support 67 to
the edge regions thereof, where the solids-depleted liquid filtrate
issues from the support into the channel between bounding walls 38
and 24 and may be removed via openings 61 and 62.
[0107] FIG. 11 is a transverse sectional elevation view of a
stacked plate filter assembly component of the invention, showing
the arrangement of the constituent parts thereof in which the
identical complementary upper and lower plates are mated to one
another. To insure positive sealing, suitable gaskets (not shown)
may be interposed (e.g., in opposing grooves) between the abutting
top surfaces of the respective opposed bounding walls 38. A lower
filter sheet 73 is disposed between the lower surface 68 of the
foraminous support 67 and the partition bearing surface of the
lower filter plate. Likewise, an upper filter sheet 74 is
interposed between the top surface 69 of the foraminous support 67
and the partition bearing surfaces of the upper filter plate.
[0108] By this arrangement, there is formed a series of
sub-channels 75-80 between the upper filter sheet 74 and the upper
filter plate, while correspondingly a series of sub-channels 81-86
are formed between the lower filter sheet 73 and the lower filter
plate, with the sub-channels being longitudinally bounded by the
respective partition walls 46a-46e, as shown.
[0109] Although the foraminous support 67 has been shown as a
structural element of mat-like form, the function of the support is
merely to retain the filter sheet positionally on either side
thereof and to accommodate the interior flow of solids-depleted
liquid toward the filtrate (permeate) collection means associated
with the filter plate.
[0110] The filter plates and foraminous support may be formed of
any suitable materials of construction, including plastics such as
polypropylene, polyethylene, polysulfone, polyimides, etc.;
ceramics; metals such as stainless steels; and polymeric
fluorocarbons such as polytetrafluoroethylene. Preferably the
materials used are capable of withstanding sterilization for
regeneration and reuse such as by high-temperatures, steam
sterilization and/or chemical sanitization. Thus, the foraminous
support may comprise a sintered ceramic material, e.g., of alumina,
zirconia, etc., having an internal network of interconnected voids
with an average void passage diameter on the order of about 1
micron. Such support may have a total void space on the order of
from about 50 to about 90% by volume, e.g., about 80% voids.
Further, it is to be recognized that such sintered ceramic plate
may be glazed or otherwise treated on selected portions of its
surface to render it liquid impermeable in such regions. Thus, the
sintered ceramic plate could be selectively glazed to provide for
flow through the interior thereof of a second fluid, e.g., a
dialysis fluid for desalting of proteins, amino acids, and/or other
biological substances being contacted with the filter sheets
supported on such sintered plate.
[0111] FIG. 10 shows an exploded, perspective view of a stacked
plate filter according to the present invention, as disposed on a
base comprising a mounting plate 87 having vertically upwardly
extending rods 88-91 at its respective corner portions. Mounted on
the base as a lowermost element of the stack, is a filter plate 92
of the type shown in FIGS. 5-7. The respective rods 88, 90, and 91
extend through the circular openings in the plate which are
surrounded by the respective cylindrical flanges 93, 94, and 95 (a
similar flanged opening, not visible in this view, is provided for
rod 89). The liquid feed conduit 96 for the filter extends through
an opening in the base mounting plate 87 and through the liquid
inlet opening 97 of the plate member, so that when filter plate 92
is in position, the liquid feed opening 98 is in register with the
liquid inlet opening 97 and liquid inlet port 99 of the filter
plate.
[0112] In like manner, the liquid withdrawal conduit 100 extends
through a corresponding opening in the base plate 87 and liquid
outlet openings 101 whereby the liquid discharge opening 102 in
conduit 100 is brought into register with liquid outlet port 103
when the bottom filter plate 92 is properly positioned.
[0113] Reposed on the upper bearing surfaces of the partition walls
104 of the bottom filter plate 92 is a filter sheet 105. The filter
sheet 105 may be a paper filter sheet, comprising a non-woven web
of cellulosic fibers, or any other replaceable or disposable
filtration medium commonly provided in sheet form and which is
readily cut or otherwise shaped to the form required in the filter
of the present invention. A particularly advantageous filter sheet
in filter systems of the type envisioned by the present invention
are polysulfone filter sheets which are readily commercially
available.
[0114] Overlying the filter sheet 105 is the foraminous support
106, which is of the form illustratively shown and described with
reference to FIGS. 8-9 herein. Overlying the foraminous support 106
is filter sheet 107, which may be identical in shape and
construction to filter sheet 105.
[0115] Overlying the upper filter sheet 107 is a filter plate 108
according to the present invention, of identical construction to
lower plate 92 but positionally inverted with respect to the lower
plate 92, to form interior sub-channels for liquid flow which are
configured as shown in FIG. 11 when the stacked filter plate
assembly of FIG. 10 is fully mated with respect to its constituent
elements.
[0116] As shown, the upper filter plate 108 is configured with
openings 109 and 110 communicating with the circumscribing channel
surrounding the main flow channel on the plate. Opening 109 in this
configuration is closed by a suitable plug, while opening 110 has a
fluid introduction passage 111 in flow communication therewith, for
feeding of a second liquid, e.g., dialysate solution, into the
circumscribing channel (the direction of liquid feeding being
indicated by the arrow P). From the circumscribing channel, the
liquid enters the foraminous support through the edge openings 112
thereof and flows therethrough to the opposite side of the lower
filter plate for discharge through openings 113 and 114 and out of
the system through the fluid discharge passage 115 in the direction
indicated by arrow Q. Circumscribing channel opening 114 of the
lower filter plate is closed by a suitable plug in this
arrangement. The stacked filter plate assembly may be retained on
the rods 88-91 by suitable mechanical fasteners, such as washers
116-119 and respective lock-nuts 120-123. For such purpose, the
rods 88-91 are suitably configured with threaded outer
surfaces.
[0117] FIG. 12 is a plan view of a support for a unitary filter
element that may be used in the invention. The support 124 includes
a circumscribing frame 125 formed by the respective side portions
126-129. The circumscribing frame 125 is associated with an array
of spaced-apart and substantially parallelly aligned ribs 130-134
extending between and joined at their opposite ends to the frame
125 (sides 128 and 129, respectively). The ribs 130-134 and frame
125 thus corporately form a series of corresponding substantially
parallel filtrate flow channels 135-140 as shown. Openings 141 are
provided in the frame 125 in liquid flow communication with the
filtrate flow channels 135-140 for egress of filtrate from the
filtrate flow channels 135-140 through the frame openings 141.
[0118] FIG. 13 is an edge elevational view of the filter element
comprising the support shown in FIG. 12. FIG. 14 is an exploded
perspective view of the unitary filter element whose edge
elevational view is shown in FIG. 13. The unitary filter element
features a first filter sheet 142 which is continously secured
along its margins to a first face of the frame 125. Likewise, a
second filter sheet 143 is continously secured along its margins to
a second face of the frame. When thus assembled, the first and
second filter sheets together with the frame 125 define an enclosed
interior volume comprising the filtrate channels separated by the
ribs. Accordingly, filtrate entering the enclosed liquid volume
through the first and second filter sheets, i.e., by permeation of
liquid through the filter sheets, may flow in the filtrate flow
channels and be discharged from the filter elements through the
frame openings 141 which are in liquid communication with the
filtrate flow channels 135-140.
[0119] The above-described unitary filter element may suitably be
constructed and employed for short term filtration operation, e.g.,
on the order of about 6 months, following which the filter element
may be discarded and replaced with a corresponding new element.
[0120] The unitary filter element may be formed of any suitable
materials, such as for example polysulfone, polyvinylidene
difluoride, polypropylene, nitrocellulose, polyethylene, and the
like, as may be useful in the desired end use filtration
application. The first and second filter sheets may be continously
secured along their margins to the respective first and second
faces of the frame by any suitable joining or attachment method,
including, but not limited to, ultrasonic welding, heat sealing,
solvent welding, and adhesive bonding, as well as mechanical
affixation.
[0121] It will be apparent from the preceding description that any
number of paired filter plates, with interposed support element and
filter sheets, may be assembled to form a cross-flow filter
depending on the desired purpose of the culture system. The number
of stacked filter plates in a specific filter system will be
largely determined by space requirements and constraints, allowable
pressure drop in the system, solids concentration and volumetric
flow rate of the liquid to be filtered, and the filtration
efficiency of the specific filter sheets employed.
[0122] In an embodiment having the dimensions for the filter plates
previously described in connection with FIGS. 5-7 hereof, a
superficial velocity of aqueous biomass suspension in the range of
1.5 meters per second through the flow channel defined between
adjacent paired plates is readily accommodated, at a volumetric
feed rate of approximately 1 liter of aqueous biomass suspension
per minute in the flow channel, without any significant
maldistribution of the liquid flow therein.
[0123] Incorporation of the stacked filter assembly comprising
filter plates into the culture system of the present invention
provides filtration that is highly hydraulically uniform in
operation, without the existence of operational tendencies toward
flow anomalies, such as bypassing, channeling, and "dead space"
formations, which are found in stacked plate filters of the prior
art.
[0124] A preferred embodiment of the entire pathogen culturing
system of the invention is shown schematically in FIG. 15. As
indicated below, various components of the pathogen culturing
system shown in this figure may be modified or used in more than
one form to enable the desired system functions to be obtained.
Larger scale cell culture efforts require additional filtration and
growth capability for the added volume being handled. Different
media or types of cells cultured may also require different types
or numbers of filter systems to enable waste product removal,
optimal cell growth and product harvest.
[0125] Referring now to FIG. 15, cells are grown in a tangential
flow growth device 144 such as a hollow fiber or plate and frame
device or a mass transfer culture system 3. Cells may be either of
the suspension or exchange dependent type. The membrane porosity in
the tangential flow device is selected by consideration of the
growth requirements of the cell type. For example, for a cell type,
which (a) grows anchored to the membrane and produces a product of
160,000 molecular weight, (b) produces and reacts to a coexpressed
inhibitory substance that does not pass any molecular filter, a
larger pore membrane such as one having a 0.2 micron pore size may
be used. A growth chamber for a second cell type which (a) is an
anchorage cell producing a 26,000 molecular weight product and no
inhibitory substance and (b) growing in a serum-free medium, would
better be equipped with a 10,000 molecular weight membrane.
[0126] Tangential flow growth device 144 as shown in FIG. 15 may
have two or four permeate ports 145a or 145b. When there are only
two permeate ports 145a they are used to connect the tangential
flow growth device 144 to auxiliary reversible pump 146 for
recirculation of the medium in the extracapillary space of the
tangential flow growth device 144 in a permeate recirculation loop.
This recirculation may be operated with or counter to the current
in the main stream for better mixing. Valves or stopcocks 147 allow
the medium flow to be stopped or started.
[0127] A second pair of permeate ports 145b allows substances to be
added to the system from the auxiliary reservoir 148, for example,
to cause the cells in the tangential flow growth device 144 to have
increased yield or to produce a factor or byproduct, which may then
be separated by ultrafilter 149.
[0128] The auxiliary reservoir 148 has a vent or extra port 150 for
pressure release and a sterile filter 151 of a dead end type placed
between auxiliary pump 152 and a valve or stopcock 153. The
auxiliary pump 152 and reservoir 148 may be as simple as a standard
syringe for the addition of supplements directly to the
extracapillary space of the tangential flow growth device 144. The
ultrafiltration scheme employing untrafilter 154 allows the
separation and concentration of both (1) suspension culture cells
with any extracellular products retained by the given porosity of
fibers used in the tangential flow growth device 144 and/or (2)
extracellular products from anchorage dependent cells with some
stuffed cells. A valve or stopcock 155 allows the tangential flow
growth device 144 to be connected to the ultrafilter 154. A sterile
dead end filter 156 is placed between the valve or stopcock 155 and
ultrafilter 154, unless the cells being grown are the product to be
harvested as when the product is intracellular (for example, rabies
virus). Ultrafilter 154 may be a microporous filter and/or an
ultrafilter for separating and concentrating the extracapillary
volume as required. An auxiliary pump 157 allows recirculating of
the concentrate loop. An auxiliary reservoir 158 is for
concentrating and separating the cellular byproducts. Another
auxiliary reservoir 159 allows dialysis or cell washing or
detergent treatment of a product such as a pathogen. An additional
port 160 may be used for a vent.
[0129] Tangential flow growth device 144 may comprise multiple
chambers that may be serially connected but preferably are in
parallel because each chamber receives the same quality of nutrient
supply when the chambers are in parallel. As discussed in detail
above, tangential flow growth device 144 is attached to tubing 161
at each end that is of a size and manufacture appropriate for
sterile production and flow. A four-way valve 8 connected to tubing
2 enables adjustment of medium flow direction through the
tangential flow growth device 144.
[0130] Monitoring chambers 162 may comprise multiple ports and/or
multiple chambers that concurrently monitor system parameters of
parts of the system such as temperature, pH, PO.sub.2, glucose,
etc. Such monitoring may also be performed at the main reservoir
163. This monitoring may be implemented by computerization as has
been developed for other systems to measure and adjust system
conditions and may be used to compare different valves of
particular parameters at different monitoring chambers 162 within
the system.
[0131] A sterile barrier tangential flow membrane device 164, which
is nonrestrictive for flow or pressure as well as having a low
coefficient of absorption and adsorption for extracellular products
is connected to the tangential flow growth device by means of
tubing 161. The sterile barrier tangential flow membrane device 164
may comprise semipermeable membranes in the form of hollow fibers
or the stacked plate filter system discussed above.
[0132] The stacked plate device can be comprised of sequential
chambers of different membrane types to effect different membrane
separations in a discrete system. For example, the stacked plate
device may comprise a membrane for ammonia removal, and a
subsequent membrane for immunoglobulin removal would be highly
useful in immunoglobulin production for diagnostic kits. In other
words, the sterile barrier tangential flow membrane device 164 can
be comprised of multiple chambers each of which is independent of
the other and has its own specific membrane type and permeate ports
165. All of the chambers of 164 are housed in a singular unit
having only one inlet and one outlet port. The flow through the
chambers would be in a serpentine path created by the installation
of another base plate 87 (FIG. 10) between each of the chambers of
different membrane types.
[0133] Two permeate ports 165 are shown on the sterile barrier
tangential flow device 164 in FIG. 15. One of these permeate ports
165 is connected to auxiliary nutrient tank 166. A valve or
stopcock 167 allows media or extra nutrient to be pumped by
auxiliary pump 168 from the nutrient tank 166 through sterile
filter 169, a dead end type filter of the appropriate size. Port
170 allows further media or nutrient adidition to nutrient tank
166.
[0134] The second permeate port 165 on the sterile barrier
tangential flow device 164 is connected to ultrafilter 171 in FIG.
15. Ultrafilter 171 allows separation and concentration of an
extracellular product that is secreted by the cells and that passed
through the pores of devices 144 and 164. Valve or stopcock 172
controls connection of ultrafilter 171 to the system. Sterile
filters 151, 156, 169 and 173 are back up protective devices which
aid in the maintenance of a sterile environment. These devices
should not pose a barrier to the desired product but only to
contaminants. These devices cannot replace good sterile techniques.
Obviously, where good sterile technique is used these filters are
not necessary. Auxiliary tank 174 allows concentration of
extracellular products such as IgG, hormones and HIV virus, and
auxiliary pump 175 is used to recirculate spent medium in the
concentration loop. Auxiliary reservoir 176 connected to auxiliary
tank 174 allows dialysis of concentrated extracellular metabolites
and viruses and has a port 177 useful to add dialysate or to vent
the system.
[0135] Preferably an open structure of 0.2 micron membranes is used
for tangential flow growth device 144 and sterile barrier
tangential flow membrane device 164. Finer membranes (molecular
weight) may be placed at these locations if this does not restrict
growth of the cells being grown. In addition, multiple chambers can
be placed at the location of each tangential flow device 144 and
164 shown in FIG. 15 to be linked in parallel with each other.
[0136] The filters in the microbial pathogen culturing system of
the invention allows inhibitory substances and non-essential cell
by-products to be filtered out of the medium while retaining
cell-stimulatory substances. Alternatively, or in addition, the
spent medium may be dialyzed against a medium containing
concentrated glucose and desirable salts using the stacked plate
filter of the previous invention. Media is circulated from main
reservoir 163 through the main system by recirculating pump 178. A
gas permeator chamber 179 is utilized for replenishment of oxygen,
for pH control by utilizing carbon dioxide, or for the addition of
other required gas or gases. The main reservoir may be provided
with one or more sterile vents 180 and ports 181.
[0137] Main valve 182 allows the culture system to be backpressured
as required to enhance mass transfer. Valves, stopcocks or clamps
183 may be used to isolate the tangential flow growth device 144
from the rest of the system.
[0138] Removal of Medium Components
[0139] The cell production of viruses and thus, density of viruses
in the medium may be increased by removal of inhibitory substances,
such as cellular metabolites, from the culture medium. Removal of
the spent medium from a microbial pathogen culture system may be
accomplished by a number of methods. In many systems now used,
removal is accomplished by simply opening a valve. Disadvantages of
such a system are that (1) contaminants may come into the culture
system through the opening; (2) useful medium components and
desirable end products of the system are removed from it; and (3)
pathogenic viruses are prematurely released from the system with
resulting exposure of laboratory personnel to the hazard. To avoid
some or all of these problems, a series of filters may be provided
as a filter barrier when the valve is opened. A filter barrier of
about a 0.2 u pore size still allows desirable soluble end products
and viruses out of the system but does not allow bacteria in or
out, or allow out any particulate end products such as cell walls
that have a size greater than 0.2 u. Use of a smaller filter
(molecular sieve) allows end products such as urea, ammonia,
creatinine and small carbohydrates to escape but retains viruses
and cells. Filters with pore sizes between these primary types
allow finer discrimination between particular sizes of medium
components.
[0140] Lysing Chamber
[0141] The reduced volume of concentrated viral suspension is
transferred under sealed conditions in previously connected sterile
tubing from the filter to the lysing chamber or harvest vessel.
[0142] The means for harvesting the culture product preferably
comprises a harvest vessel 174 as is shown in FIG. 15 that is
leak-proofly connected to the mass transfer culture system by a
harvest line. During growth of the cells and until the desired time
of harvest the harvest line connecting the vessel to the mass
transfer culture system is closed by valve 185. When the harvest
line is opened by a valve means, culture medium containing
pathogens, spent medium and waste products begins to flow toward
the reservoir. Prior to the opening of valve 185, the previously
used filter system for solids filtration and means for nutrient
exchange may be used to remove excess medium and undesirable
components prior to entry of the medium into the harvest line.
Alternatively, filtration and nutrient exchange means may be
provided on the harvest line so that medium is treated prior to
flowing into the harvest vessel. Detergent or other
pathogen-destroying substance is then added aseptically to the
treated, reduced-in-volume, pathogen-containing medium in the
harvest vessel by means of a valve on a line 186 connected to
reservoir of the pathogen destroying substance (not shown).
Treatment regimes for pathogenic viruses or other pathogens using
detergents or other substances to destroy all pathogenic activity
are well known. Thus one standard treatment comprises addition of
Non-ident P40.TM. Sigma Chemical, St. Louis, Mo. according to
standard manufacturer's instructions. Following the appropriate
treatment, the outlet port of the vessel may be opened to remove
the unhazardous pathogen product for its intended use.
[0143] All of the steps for growing viruses, between the initial
inoculation of the mass transfer culture tube and the removal of
the viral particles from the last filter are done under sealed
conditions with all viable viruses being completely contained
within the system. When the system is opened, the laboratory worker
and the laboratory are only exposed to noninfective viral
particles. Thus laboratory contamination and workers exposure to
infective viruses are minimized. The sealed pathogen culturing
system into which cell culture nutrients may be aseptically added
and from which undersirable end products may be removed while
increasing cell production of the pathogens within the system,
allows high concentrations of pathogen to be obtained in a small
confined laboratory area without opening the system or removing
pathogens. All sampling, monitoring, and medium adjustments may be
performed automatically and aseptically. When the appropriate
amount of time has elapsed to yield the desired pathogen
concentration, the system may be manually or automatically changed
from the cell growth to the product harvest phase by appropriate
valve means adjustments.
[0144] The features and advantages of the present invention will be
more clearly understood by reference to the following examples,
which are not to be construed as limiting the invention.
EXAMPLE I
[0145] Cell Growth.
[0146] A hollow fiber mass transfer culture system is constructed
as described in U.S. patent application Ser. No. 06/936,486. This
system is sterilized with hypochlorite solution and rinsed with
sterile deionized water using standard procedures. The bioreactor
is then filled with cell culture medium, RPMI 1640 (Rochester Park
Memorial Institute medium) plus 5% fetal calf serum, and incubated
for 48 hours at 37 degrees C in a 5% carbon dioxide, no humidity
environment, as a check for sterility and system equilibration.
[0147] Using standard cell culture techniques, an inoculum of cell
type EL-4-IL-2 is prepared from stock cultures and inoculated into
the extracapillary space of the hollow fiber bioreactor. The system
is again incubated for 20 days with media being changed as required
in accordance with prior practice for this cell type. The four-way
LL valve is switched each half hour during the working day and once
each night, usually about midnight, taking care to use alternate
positioning on successive nights.
[0148] Resulting cell growth in the bioreactor as determined by
visual observation of turbidity of the liquid containing the
suspension culture is equally dispersed throughout the growth
chamber after incubation as described.
[0149] Other cell types, including BALB-3T3 and Mycoplasma
galasepticum, grown in standard media as described in the
above-cited patent application, and observed microscopically also
are found to be uniformly dispersed throughout the growth chamber.
use of sterile barrier tangential flow devices (Microgon, Inc.,
Laguna Hills, Calif.) as well as dual valve arrangements allowed a
sterile environment to be maintained over extended time periods in
spite of numerous nutrient media exchanges and sample
withdrawals.
EXAMPLE II
[0150] Culture System Inoculation.
[0151] The mass transfer culture tube is inoculated with a
concentrated suspension of virus-infected cells. In the preferred
embodiment three to ten ml of a suspension having about 10.sup.6
cells per ml is added aseptically by using a syringe to inject
inoculum through a septum at port 189. Alternatively, port 189
comprises a double valve assembly as described above (see
discussion of double valve assemblies 17 and 20) and the culture
inoculum is introduced aseptically following sterilization of the
outer valve. It is clear that a wide range of volumes and
concentrations of cells may be used, with the ideal inoculum
providing a sufficient number of cells to multiply and produce
large numbers of progeny viruses but not so many cells as will be
overburden and clog the system. Inoculum preparation and aseptic
handling of cell cultures is well known in the art and is not part
of the invention.
EXAMPLE III
[0152] Harvest of Progeny Viruses.
[0153] As an example of viral harvest, a viral suspension is washed
of medium components by using the stacked filter plate components
of the invention. Stacked filter plates may be assembled with one
or more filter papers that will allow differential separations by
size, molecular weight, ionic charge, hydrophilicity,
hydrophobicity, antigen antibody complexing, or other membrane
separable characteristics. In the sequential chambers of the flat
plate device is a membrane capable of passing low molecular weight
substances having an ionic charge such as urea and ammonia. A
suitable filter is the Charge Mosaic.TM. (TOSOH, Tokyo, Japan). In
a second chamber is a filter paper that will allow proteins and
other macromolecules but not viruses to pass through. Suitable
filters include the MX-100K.TM. (Membrex, Garfield, N.J.), and the
TSK-1000K.TM. (TOSOH, Tokyo, Japan). In the third chamber is a
membrane capable of sterilizing nutrient media additions. A
suitable filter is Durapore.RTM. (Millipore, Bedford, Mass.). Other
sequential chambers may be added as deemed appropriate for
particular applications and functions, such as adding glucose,
removing creatinin, or harvesting immunoglobulins.
[0154] To obtain progeny viruses for harvesting, 100 liters of
medium (RPMI 1640, Example I) plus 5% fetal calf serum is placed in
the main reservoir 163 of the tangential flow growth device 144.
The tangential flow growth device 144 is inoculated with an
HIV-infected cell line through port 189 (Example II). The culture
is allowed to grow until the consumption of nutrients has ceased.
The incubation time depends on environmental parameters and
inoculum size and viability but may be in the order of four days
for a suitable progeny concentration to be attained. At the
termination of incubation, valve 172 is opened. The virus-rich
medium is transferred by pumping action of the system
(recirculating pump 178) and partial closure of main valve 182
causes the liquid to be forced through sterile barrier tangential
flow device 164 into auxiliary tank 174, so that essentially all of
the 100-liter liquid volume is transferred to tank 174. While valve
185 remains closed (it is closed during the culture growth and
incubation period) and valve 188 is open, auxiliary pump 175 is
turned on and valve 172 is closed. The ports on ultrafilter 171 are
opened so that liquid separated from the viruses is pumped through
ultrafilter 171 and out of the system. The product viruses remain
in the system and are increasingly concentrated until they are
suspended in about 5 liters of liquid. Valve 188 is then closed so
that the remaining 5 liters of liquid are transferred into vessel
184. Port 186 is then opened and detergent or other
virus-destroying chemical is added at the manufacturer's
recommended concentration. Vessel 184 contains a magnetic stirring
bar or may be otherwise equipped for agitation of the contents to
mix the detergent into the virus suspension as appropriate for the
virus treatment. After the virus particles are rendered harmless,
port 187 is opened to remove the uninfective end-product for any
desired experimentation or use.
[0155] The detergent treatment or other pathogen treatment may
alternatively be done in auxiliary tank 174 without withdrawing the
liquid to vessel 184, but removal of the liquid to vessel 184
allows the remaining growth portion of the system to continue being
used or to be treated or cleaned for other uses. Because of the
multiple chamber potential of tangential flow sterile barrier
device 164, the functions of ultrafilter 171, auxiliary tank 174
and auxiliary pump 175 may be accomplished by tangential flow
sterile barrier device 164 in batching operations where continuous
culture is not desired.
[0156] While the invention has been described with reference to
specific embodiments thereof, it will be appreciated that numerous
variations, modifications, and embodiments are possible, and
accordingly, all such variations, modifications, and embodiments
are to be regarded as being within the spirit and scope of the
invention.
* * * * *