U.S. patent application number 16/506079 was filed with the patent office on 2019-10-31 for system and method for production of on-demand proteins in a portable unit for point of care delivery.
The applicant listed for this patent is University of Maryland, Baltimore County. Invention is credited to Douglas Frey, Xudong GE, Yordan KOSTOV, Govind RAO, Leah TOLOSA.
Application Number | 20190330586 16/506079 |
Document ID | / |
Family ID | 56553907 |
Filed Date | 2019-10-31 |
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United States Patent
Application |
20190330586 |
Kind Code |
A1 |
RAO; Govind ; et
al. |
October 31, 2019 |
SYSTEM AND METHOD FOR PRODUCTION OF ON-DEMAND PROTEINS IN A
PORTABLE UNIT FOR POINT OF CARE DELIVERY
Abstract
A portable and mobile bioprocessing system and method for
protein manufacturing that is compact, integrated and suited for
on-demand production of any type of proteins and for delivery of
the produced proteins to patients or for assay purposes. The
portable system and method can also be used for efficient on-demand
production of any type of protein with point-of-care delivery.
Inventors: |
RAO; Govind; (Ellicott city,
MD) ; KOSTOV; Yordan; (Columbia, MD) ; TOLOSA;
Leah; (Columbia, MD) ; GE; Xudong; (Woodstock,
MD) ; Frey; Douglas; (Ellicott City, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Maryland, Baltimore County |
Baltimore |
MD |
US |
|
|
Family ID: |
56553907 |
Appl. No.: |
16/506079 |
Filed: |
July 9, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15966609 |
Apr 30, 2018 |
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16506079 |
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15095305 |
Apr 11, 2016 |
9982227 |
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15966609 |
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13823911 |
Jun 28, 2013 |
9388373 |
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PCT/US2012/028358 |
Mar 8, 2012 |
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15095305 |
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61450191 |
Mar 8, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 47/12 20130101;
C12M 47/10 20130101; C12M 41/12 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12M 1/34 20060101 C12M001/34 |
Goverment Interests
GOVERNMENT RIGHTS IN INVENTION
[0002] This invention was made with government support under Grant
Number N66001-13-C-4023 awarded by the Defense Advanced Research
Projects Agency (DARPA). The government has certain rights in the
invention.
Claims
1. A composition comprising an on-demand cell-free synthesized
protein and a buffer, wherein the composition is free of
stabilizers, oxidation/reduction agents, and/or other excipients,
and wherein the composition is delivered in vivo to a patient in
need of such protein within one hour to two weeks after
purification of the cell-free synthesized protein.
2. The composition of claim 1, wherein the composition is delivered
continuously or as a bolus to a subject in need of the protein
comprised therein.
3. The composition of claim 1, wherein the on-demand cell-free
synthesized protein is produced in a portable bioprocessing system
comprising: a production module for producing the on-demand
cell-free synthesized protein, and a purification module for
receiving the protein from the production module and for purifying
the protein from reagents, wherein the purification module
comprises: a chromatography component for receiving the protein
from the production module and for outputting purified protein; and
a diafiltration component for receiving the purified protein from
the chromatography component and for outputting further purified
protein, wherein the diafiltration component comprises a product
section for receiving the purified protein from the chromatography
component and a buffer section for receiving a buffer solution.
4. The composition of claim 1, wherein the composition is
refrigerated at an above-freezing temperature from 0 to 6.degree.
C. after purification.
5. The composition of claim 3, wherein the production module
comprises a reaction mixture comprising a nucleotide template for
production of a protein; monomers for the protein to be
synthesized, co-factors, enzymes and reagents that are necessary
for the synthesis.
6. The composition of claim 5, wherein the enzymes and reagents
that are necessary for the synthesis are ribosomes, tRNA,
polymerases and transcriptional factors.
7. The composition of claim 3, wherein the production module
comprises a bioreactor for cell-free protein expression.
8. The composition of claim 5, wherein the reaction mixture is
maintained at a pH between pH 6-9 during production of the
on-demand cell-free synthesized proteins.
9. The composition of claim 5, wherein the reaction mixture is
maintained at a temperature between 20.degree. C. and 40.degree. C.
during production of the on-demand cell-free synthesized
protein.
10. The composition of claim 3, wherein the diafiltration component
further comprises: a first substrate; a second substrate; and a
diafiltration membrane positioned between the first and second
substrates.
11. The composition of claim 10, wherein the diafiltration
component comprises a flow cell.
12. The composition of claim 3, wherein the production module
comprises at least one sensor for monitoring at least one of
conductivity, temperature, pH, oxygen and CO.sub.2.
13. The composition of claim 3, wherein the production module and
purification module each comprise a heating and cooling element for
controlling the temperature of solution inside the production
module and purification module.
14. The composition of claim 3, further comprising a fluid storage
and dispensing module for storing solutions used by the production
module for protein expression and for storing waste product
produced by the purification module.
15. The composition of claim 14, wherein the fluid storage and
dispensing module also stores purified protein output by the
purification module.
16. The composition of claim 3, wherein the portable bioprocessing
system further comprises a processor for controlling and/or
monitoring at least one of the production module and the
purification module.
17. The composition of claim 3, wherein a therapeutic dose and
potency/activity of the on-demand cell-free synthesized protein is
determined by expression time in the production module.
18. The composition of claim 1, wherein the composition can be
delivered directly to a patient.
19. The composition of claim 3, wherein a therapeutic dose and
potency/activity of the on-demand cell-free synthesized protein is
determined by the holding time in the production module.
20. The composition of claim 1, wherein potency/activity of the
on-demand cell-free synthesized protein is at least 55% or more of
the initial activity for at least 3 days at temperature from about
0.degree. C. to about 30.degree. C.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S. patent
application Ser. No. 15/966,609 filed on Apr. 30, 2018, which is a
continuation of U.S. patent application Ser. No. 15/095,305 filed
on Apr. 11, 2016, now U.S. Pat. No. 9,982,227, which is a
continuation-in-part application of U.S. patent application Ser.
No. 13/823,911, filed on Jun. 28, 2013, now U.S. Pat. No.
9,388,373, which in turn was a 371 application of PCT Application
No. PCT/US2012/028358, filed on Mar. 8, 2012, which in turn claims
priority to U.S. Provisional Application Ser. No. 61/450,191, filed
Mar. 8, 2011, the contents of all is hereby incorporated by
reference herein for all purposes.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates to protein manufacturing and,
more particularly, to an integrated and compact bioprocessing
system for on-demand production or manufacturing of proteins for
point-of-care delivery.
Background of the Related Art
[0004] The time it takes for a new drug to reach the market is 8-10
years at a cost approaching $1.2 billion. Many of these new drug
entities are referred to as biologics (e.g., a protein used as a
drug or therapeutic). These are molecules produced by living cells
in vitro using cell culture and fermentation technologies.
Stringent process control is required since changes in culture
conditions can lead to, for example, altered glycosylation
profiles, which can then drastically change the drug's
pharmacokinetics, efficacy and immunogenicity. Therefore, much
effort towards FDA approval is devoted to the development of
documented and robust manufacturing processes that will produce
safe and efficacious biologics of consistent quality. These are
collectively referred to as good manufacturing processes (GMP). The
goal is to arrive at a process that is well defined and
reproducible, and that leads to products that meet pre-determined
characteristics of quality, identity, purity, safety and
efficacy.
[0005] Biologics are currently produced in a centralized
manufacturing facility with large scale (>10,000 liters) cell
cultures, and with the necessary large volume separation,
purification, formulation, packaging, and distribution
infrastructure (e.g. a typical Merck, Pfizer or Genentech plant).
The time period from a cell bank to the final delivery of the
therapeutic vial is on the order of 6-8 weeks under ideal
conditions and produces batches of around 10 Kg bulk protein. As
shown in FIG. 1, the process itself is complex. FIG. 1 shows a
typical flow sheet for the manufacturing of protein biologics--both
for proteins that are expressed intracellularly and proteins
expressed extracellularly. Every step needs to be individually
developed, scaled-up, optimized and validated in a manufacturing
setting. The final product will also have an expiration date and is
either shipped lyophilized or via a cold chain, which must also be
documented.
[0006] Thus, there is a need for production of biological medicines
in real-time and/or on-demand to provide therapeutic proteins in
remote locations. Also, there is a need for a system and method of
preparing therapeutic proteins with increased activity with the
possibility of reduced amount of preservation, enhancing and/or
stabilizing excipients because of the immediate and/or timely use
of such therapeutic proteins.
[0007] A similar opportunity exists for proteins in non-therapeutic
applications. By their nature, proteins are unstable and require
preservation by a variety of methods to preserve their activity.
They are widely used in the diagnostics industry, in food and
cosmetics. In these cases as well, the ability to manufacture fresh
protein on-demand would be of great benefit.
SUMMARY OF THE INVENTION
[0008] The present invention provides for an integrated and compact
bioprocessing system and method for the production of proteins with
increased activity with the possibility of reduced amount of
preservation, enhancing and/or stabilizing excipients because of
the immediate and/or timely use of therapeutic proteins and
diagnostic proteins, where a critical assay is to be performed.
[0009] In one aspect, the present invention provides a portable and
compact bioprocessing system for the production of proteins for
point-of-care delivery.
[0010] In another aspect, the present invention provides for an
integrated and compact bioprocessing system for protein expression
and purification.
[0011] In yet another aspect, the present invention provides for a
method for on-demand production and delivery of a therapeutic
protein to a patient.
[0012] In a still further aspect, the present invention provides
for a portable system and method for on-demand production of a
therapeutic protein prepared in a composition with reduced amounts
of stabilizers, oxidation/reduction agents and/or other
excipients.
[0013] In another aspect, the present invention provides for a
portable system and method for on-demand production of a
therapeutic protein, wherein the therapeutic protein exhibits
increased potency due to the timely synthesis and substantially
immediate delivery of protein. Preferably, the newly synthesized
proteins are delivered to a patient within one hour, to one day, to
two weeks. Preferably any refrigeration is at a temperature above
freezing from 0 to 6.degree. C. Any freezing of the proteins is
preferably a single event with temperatures ranging from about
-2.degree. C. to about -10.degree. C.
[0014] In yet another aspect, the present invention provides for a
portable system and method for on-demand production of a
therapeutic protein, wherein the produced therapeutic protein can
be delivered continuously or as a bolus as it is produced and as it
happens physiologically, where the body produces needed proteins
over an extended time in vivo and when needed.
[0015] A still further aspect, the present invention provides for a
freshly synthesized protein in a composition, wherein the freshly
synthesized protein is synthesized on-demand and exhibits increased
activity, and wherein a buffering composition comprising the
freshly synthesized protein is essentially lacking a
stabilizer.
[0016] To achieve at least the above aspects, in whole or in part,
there is provided a bioprocessing system comprising a production
module for producing a protein and a purification module for
receiving the protein from the production module and for purifying
the protein from reagents. The bioprocessing system may further
comprise a processor for controlling and/or monitoring at least the
production module and/or the purification module. The processor is
communicatively connected to at least the production module and/or
purification module to control the timing, temperature and other
parameters necessary for optimizing the production and purification
of the synthesized proteins to provide a sufficient amount of or a
therapeutic dosage of the synthesized protein. Such length of time
in the production module and/or purification module may be used to
affect the potency and/or activity of the synthesized protein.
[0017] To achieve at least the above aspects, in whole or in part,
there is also provided a bioprocessing system, comprising a reactor
for protein expression, a membrane chromatography component for
receiving and purifying protein output by the reactor and a
diafiltration component for receiving purified protein from the
membrane chromatography and for further purifying the purified
protein.
[0018] Additional advantages, aspects, and features of the
invention will be set forth in part in the description which
follows and in part will become apparent to those having ordinary
skill in the art upon examination of the following or may be
learned from practice of the invention. The aspects and advantages
of the invention may be realized and attained as particularly
pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The invention will be described in detail with reference to
the following drawings in which like reference numerals refer to
like elements wherein:
[0020] FIG. 1 shows a typical flow sheet for the manufacturing of
protein biologics, both for proteins that are expressed
intracellularly and proteins expressed extracellularly.
[0021] FIG. 2 is a block diagram that illustrates the principles of
operation of one preferred embodiment of the present invention.
[0022] FIG. 3 is a schematic diagram of a bioprocessing system, in
accordance with another preferred embodiment of the present
invention.
[0023] FIG. 4 is a schematic diagram of a microscale bioprocessing
system, in accordance with another embodiment of the present
invention.
[0024] FIG. 5 is a side schematic view of a membrane chromatography
component that can be used in the systems of FIGS. 3 and 4, in
accordance with the present invention.
[0025] FIG. 6A is a top plan view of a microfluidic diafiltration
component that can be used in the systems of FIGS. 3 and 4, in
accordance with the present invention.
[0026] FIG. 6B is a schematic cross-sectional view of the
equilibrium chamber of FIG. 6A looking along the cross-section line
A-A of FIG. 6A.
[0027] FIG. 6C is a bottom plan view of the equilibrium chamber of
FIG. 6A.
[0028] FIG. 7 is a perspective schematic view of another
microfluidic diafiltration component that can be used in systems of
FIGS. 3 and 4, in accordance with the present invention.
[0029] FIG. 8 is a diagram showing the main steps in in vivo
protein expression.
[0030] FIG. 9A shows results for the expression of G-CSF from
different runs (Run #3) and showing the reproducibility.
[0031] FIG. 9B shows results for the expression of G-CSF from
different runs (Run #4) and showing the reproducibility.
[0032] FIG. 10A shows the quantified values for Run#3 of FIG.
9A.
[0033] FIG. 10B shows the quantified values for Run#4 of FIG.
9B.
[0034] FIG. 11 shows the potency results of recombinant human G-CSF
from Run #3 and Run #4.
[0035] FIG. 12 shows the Western blot of quadruplicate run of EPO
in dialysis cassettes prior to purification.
[0036] FIG. 13 A shows EPO expression in stirred tank bioreactor
and FIG. 13 B shows Streptokinase expression in stirred tank
bioreactor.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0037] The present invention is particularly suited for the
on-demand manufacturing of therapeutic proteins (either cell-based
or cell-free) that are suitable for direct delivery to a patient.
Therefore, the present invention will be primarily described and
illustrated in connection with the manufacturing of therapeutic
proteins. However, the present invention can also be used to
manufacture any type of protein, including toxic proteins, proteins
with radiolabeled amino acids, unnatural amino acids, etc. Further,
the present invention is particularly suited for the on-demand
manufacturing of proteins using cell-free expression, and thus the
present invention will be described primarily in the context of
cell-free protein expression. However, the present invention can
also be used in connection with cell-based protein expression.
[0038] FIG. 2 is a block diagram that illustrates the principles of
operation of one preferred embodiment of the present invention. The
bioprocessing system 100 includes a production module 200, a
purification module 300 and a fluid storage/dispensing module 400
that are fluidly coupled via coupling components 500. A processor
600 may be in electrical communication with one or more of the
production module 200, purification module 300, coupling components
500 and fluid storage/dispensing module 400 for controlling and
monitoring the operation of the system 100.
[0039] The fluid storage/dispensing module 400 is adapted to store
the solutions needed for the production of a protein. The fluid
storage/dispensing module 400 may also include containers for
storing any waste product produced during the production of the
protein. The fluid storage/dispensing module 400 may be temperature
controlled, if needed, to maintain the solutions at a required
temperature.
[0040] The production module 200 is adapted to receive the
solutions required for production of a protein, such as a
therapeutic protein, from the fluid storage/dispensing chamber via
coupling components 500. The production module 200 may suitably
include a bioreactor adapted for maintaining living cells that
incorporates non-invasive optical chemical sensing technology for
monitoring culture parameters (e.g., pH, oxygen, optical density,
fluorescence, absorbance, redox, temperature, etc.), such as the
bioreactors and optical chemical sensing technology illustrated and
described in commonly assigned and related U.S. Pat. Nos. 6,673,532
and 7,041,493, as well as co-pending commonly assigned and related
patent application Ser. No. 12/991,947, whose disclosures are
incorporated by reference herein in their entirety. These types of
bioreactors are particularly suited for cell-based production of
therapeutic proteins. Alternatively, the production module 200 may
suitably include a stirred mini-reactor such as, for example, the
BioGenie Minibioreactor sold by Scientific Bioprocessing, Inc.,
that is adapted for the cell-free production of a protein, and that
are also equipped with sensors for monitoring reaction parameters
(e.g., pH, oxygen, optical density, fluorescence, absorbance,
redox, temperature, etc.).
[0041] The production module 200 as illustrated in FIG. 2 is
designed for batch mode protein production where all the components
for protein production (DNA, cell-free lysate, reaction buffer,
etc.) are combined in a single step and then delivered to the
purification module via coupling components 500 at the end of the
reaction (3-6 hours). The production module 200 may also be a cup
with a dialysis membrane bottom or a dialysis cassette with
dialysis membrane on both sides. The cup or cassette will be
surrounded by a dialysis buffer to remove reaction waste products
such as inorganic phosphate, but also to maintain the concentration
of nutrients such as amino acids and creatine phosphate. The
solutions for protein production are delivered to the dialysis cup
or the dialysis cassette and the surrounding dialysis buffer from
the fluid storage/dispensing chamber 400 via coupling components
500.
[0042] After the reaction is complete, the raw product is then
transferred to the purification module 300 via coupling components
500. The purification module 300 contains the necessary
purification components for purifying the protein from the
reagents. The purification module 300 can include, for example,
chromatography components and dialyses components for purifying the
biologic.
[0043] The production module 200 and the purification module 300
may each include sensors for monitoring reaction parameters and/or
product quality parameters. The parameters monitored can include,
but are not limited to, conductivity, temperature, pH, oxygen and
CO.sub.2. The sensors may be any type of invasive sensor known in
the art for monitoring these parameters, where the sensors are in
contact with the process fluid. In addition, the sensors may be
non-invasive optical chemical sensors, such as those described in
U.S. Pat. Nos. 6,673,532 and 7,041,493, and U.S. patent application
Ser. No. 12/991,947. In addition, spectrometers known in the art
can be used in the production module 200 and/or the purification
module 300 to monitor the product stream and/or the inputs to each
module. The parameters measured by such spectrometers can include,
but are not limited to, absorbance, fluorescence, Raman scattering,
circular dichroism and infrared spectral characteristics.
[0044] FIG. 3 is a schematic diagram of a bioprocessing system 700,
in accordance with another preferred embodiment of the present
invention. The system 700 is particularly suited for the cell-free
production of proteins and will be described in this context.
[0045] The system 700 includes a reactor 210, in which protein
expression takes place, a membrane chromatography component 310, a
diafiltration component 320 and a fluid storage/dispensing module
400. The reactor 210 preferably includes a heating and cooling
element 220, suitably a thermoelectric cooler, for controlling the
temperature of the solution 230 inside the reactor 210. The reactor
also preferably includes sensors 240 and 250 for monitoring
parameters in the reactor solution 230, such as pH, oxygen, redox,
conductivity or any other parameter that can be measured with
existing sensors. The sensors 240 and 250 can be implemented with
any type of sensor known in the art for measuring the desired
parameters. However, the sensors 240 and 250 are preferably
non-invasive optical chemical sensors.
[0046] The system 700 also includes a processor 600 that is in
communication with one or more of the reactor 210, optoelectronics
270, membrane chromatography component 310, diafiltration component
320, fluid storage/dispensing module 400 and pumps 520A and 520B
for controlling and/or monitoring the operation of the system
700.
[0047] Optoelectronics 270 are provided for exciting the optical
chemical sensors 240 and 250 with excitation light 242 and 244,
respectively, and for receiving and detecting emission light 246
and 248 from the optical chemical sensors 240 and 250,
respectively. As discussed above, commonly assigned and related
U.S. Pat. Nos. 6,673,532 and 7,041,493, as well as co-pending
commonly assigned and related U.S. patent application Ser. No.
12/991,947 describe in more detail how non-invasive optical
chemical sensing technology can be used to monitor parameters.
[0048] In FIG. 3, two optical chemical sensors 240 and 250 are
shown, and are preferably adapted to measure pH and dissolved
oxygen, respectively. However any number of optical chemical
sensors (including only one) may be used depending on the number
and type of parameters being measured. Optoelectronics 270 include
optical excitation sources (not shown) for generating the
excitation light 242 and 244, as well as photodetectors (not shown)
for detecting the emission light 246 and 248 from the optical
chemical sensors 240 and 250. The type of optical excitation source
or sources are the types used in optoelectronics. Any combination
of optical excitation sources and optical chemical sensors may be
used, depending on the number and types of parameters being
measured. Examples of optical excitation sources that can be used
included in optoelectronics 270 include, but are not limited to,
light emitting diodes and laser diodes. Alternatively, the
optoelectronics 270 may just be used to measure optical properties
of the reactor contents in their entirety absent any sensors.
[0049] Further, for each optical chemical sensor 240 and 250, two
possible placements on the reactor 210 are shown. The two possible
placements for optical chemical sensor 240 are shown as 240A and
240B. The two possible placements for optical chemical sensor 250
are shown as 250A and 250B. The use of other non-contact sensors
(i.e. Raman, contact free conductivity sensors etc) is also
possible in this context.
[0050] In the "A" placement (240A and 250A), the optical chemical
sensors 240A and 250A are positioned inside the reactor 210 on a
reactor wall 260. With this placement, the optical chemical sensors
240A and 250A are in physical contact with the solution 230, and
the reactor wall 260 on which the optical chemical sensors 240A and
250A are placed is optically transparent to the excitation light
242 and 244, so that the excitation light can reach the optical
chemical sensors 240A and 250A.
[0051] In the "B" placement (240B and 250B), the optical chemical
sensors 240B and 250B are positioned outside the reactor 210 on
reactor wall 260. With this placement, the thickness of the reactor
wall 260 is sufficiently small so as to allow the analytes that are
being measured to diffuse through the reactor wall 260 and contact
the optical chemical sensors 240B and 250B. Alternatively, the
portions of the reactor wall 260 on which the optical chemical
sensors 240B and 250B are attached can be replaced with barrier
membranes 249A and 249B that are adapted to allow the analytes
being measured to diffuse through so that they come in contact with
optical chemical sensors 240B and 250B. The use of barrier
membranes and thin reactor walls to effectuate diffusion of the
analytes of interest through a container wall to optical chemical
sensors is described in more detail in commonly assigned and
related U.S. patent application Ser. No. 13/378,033, which is
incorporated herein by reference in its entirety.
[0052] In the FIG. 3 embodiment, the fluid storage/dispensing
module 400 preferably includes a buffer solution container 410 for
holding buffer solution, an mRNA/DNA solution container 420 for
holding mRNA/DNA solution, a reaction solution container 430 for
holding reaction solution, a waste storage container 440 for
holding waste solution and a product storage container 450 for
holding the purified protein. In operation, reaction solution,
mRNA/DNA solution and buffer solution are directed to reactor 210
via conduits 510A, 510B, 510C and pump 520A.
[0053] After the reaction in the reactor 210, the raw product is
directed to membrane chromatography component 310 via conduit 510E
and pump 520B for purification of the protein from the reagents.
Membrane chromatography component 310 may suitably include a
cylindrically shaped housing which contains porous membrane layers
(preferably at least 10 porous membrane layers), where the
individual membranes consist of an appropriate polymer, such as
polymethacrylate, that has been chemically functionalized with a
ligand, such as a diethylaminoethyl (DEAE), a quaternary amine (Q),
or a carboxymethyl (CM) ligand for the case of ion-exchange
chromatography, or a phenyl or butyl ligand for the case of
hydrophobic interaction chromatography, or a mercaptoethylpyridine
(MEP) ligand for the case of mixed mode chromatography. One
preferred embodiment of the membrane chromatography component 310
will be discussed in more detail below in connection with FIG. 5.
Waste from the membrane chromatography process is directed to waste
storage container 440 via conduit 510F. The purified product is
directed to diafiltration component 320 for dialysis via conduit
510G and pump 520C.
[0054] Membrane chromatography component 310 may also include one
or more sensors 312 for monitoring product quality parameters, such
as conductivity, temperature, pH, oxygen, CO.sub.2, absorbance,
fluorescence, Raman, circular dichroism and infrared spectral
characteristics. The sensors 312 may be any type of invasive or
noninvasive sensor known in the art for measuring these parameters
including, but not limited to, spectrometers. In addition, the
sensors may be non-invasive optical chemical sensors, such as those
described in U.S. Pat. Nos. 6,673,532 and 7,041,493, and U.S.
patent application Ser. No. 12/991,947. In addition, membrane
chromatography component 310 preferably includes a heating and
cooling element 314, suitably a thermoelectric cooler, for
controlling the temperature of the solution (raw product) inside
the membrane chromatography component 310.
[0055] The diafiltration component 320 may suitably include a
hydrophilic polymeric membrane for use as a separation mode for
dialysis for separating proteins in a diluent liquid on the basis
of differences in their ability to pass through a membrane or in
the alternative for diafiltration to simply exchange the buffer
solutions. Such hydrophilic polymeric membrane may include, but not
limited to, polyethersulfone, a cellulosic, or a polyvinylidene
fluoride (PVDF) membrane with a well-defined pore structure that
yields a desired molecular weight cut-off (MWCO) value in the range
of 10 k to 200 k Da as appropriate for a given application. The
final protein that comes out of the diafiltration component 320 is
directed to product storage container 450 via conduit 510H. The
waste product produced from the dialysis process in the
diafiltration component 320 is directed to waste storage container
440 via conduit 510I.
[0056] Diafiltration component 320 may also include one or more
sensors 322 for monitoring product quality parameters, such as
conductivity, temperature, pH, oxygen, CO.sub.2, absorbance,
fluorescence, Raman, circular dichroism and infrared spectral
characteristics. The sensors 322 may be any type of invasive or
noninvasive sensor known in the art for measuring these parameters
including, but not limited to, spectrometers. In addition, the
sensors may be non-invasive optical chemical sensors, such as those
described in U.S. Pat. Nos. 6,673,532 and 7,041,493, and U.S.
patent application Ser. No. 12/991,947.
[0057] In addition, diafiltration component 320 preferably includes
a heating and cooling element 316, suitably a thermoelectric
cooler, for controlling the temperature of the solution (raw
product) inside the membrane chromatography component 320.
[0058] In addition to the pumps 520A, 520B and 520C, any number of
valves or other hydraulic components, such as additional pumps, may
be used throughout the system 700 to assist in controlling the flow
of solution/product between the various components of the system
700.
[0059] The present invention is particularly suited to
miniaturization by using micropumps and microfluidic technology.
FIG. 4 is a schematic diagram of a microscale bioprocessing system
800, in accordance with another embodiment of the present
invention. The system 800 includes many of the same components of
the system 700 of FIG. 3, and common elements are labeled with
common element numbers.
[0060] The system 800 contains a fluid storage/dispensing module
400 that includes a buffer solution container 410 for holding
buffer solution, an mRNA/DNA solution container 420 for holding
mRNA/DNA solution, a reaction solution container 430 for holding
reaction solution, a waste storage container 440 for holding waste
solution and a product storage container 450 for holding the
purified protein. The system 800 also includes a reactor 210, a
membrane chromatography component 310, a diafiltration component
820, a processor 600, optical chemical sensors 840 chosen and
positioned to monitor finished product quality parameters, such as,
for example, conductivity, redox, pH, UV spectrum and protein
concentration, and optoelectronics 830 for providing optical
excitation light and for detecting emission light from the optical
chemical sensors 840. The optoelectronics 830 may also just be used
to measure the optical properties of the finished product absent
any sensors.
[0061] The reactor 210 can be of any size, but in the microscale
embodiment of FIG. 4, it preferably has a volume capacity of less
than approximately 50 milliliters, and more preferably
approximately 20 milliliters or less, in order to keep the system
800 relatively compact. The reactor 210 may be implemented, for
example, with the BioGenie minibioreactor system manufactured by
Scientific Bioprocessing, Inc.
[0062] Micropumps 850A and 850B and conduits 510A-510I direct
solution to the various components in a manner similar to pumps
520A, 520B and conduits 510A-510I in the system 700 of FIG. 3.
Although not shown in FIG. 4, the reactor 210 contains optical
chemical sensors and optoelectronics for monitoring parameters in
the reactor solution 230 in a manner similar to system 700 of FIG.
3. The micropumps 850A and 850B may be implemented with any type of
micropump known in the art such as, for example, the mp5 micropump
or the mp6 micropump manufactured by Bartels Mikrotechnik.
[0063] The housing lid 850 may contain a display, such as an LCD
display 860, that connects to the processor 600 and that can
provide information about the system 800, such as, for example,
diagnostic information, reaction parameters and/or finished product
quality parameters, such as, for example, conductivity, redox, pH,
UV spectrum and protein concentration.
[0064] The processor 600 in FIGS. 2, 3 and 4 may be implemented
with a general purpose desktop computer or a general purpose laptop
computer. In addition, the processor may be implemented with a
tablet computer or smartphone, such as iOS or Android-based tablets
and smartphones. However, processor 600 can also be implemented
with a special purpose computer, programmed microprocessor or
microcontroller and peripheral integrated circuit elements, ASICs
or other integrated circuits, hardwired electronic or logic
circuits such as discrete element circuits, programmable logic
devices such as FPGA, PLD, PLA or PAL or the like. In general, any
device on which a finite state machine capable of executing code
for implementing the functionality described herein can be used to
implement the processor 600.
[0065] FIG. 5 shows a membrane chromatography component 310 that
can be used in systems 700 and 800, in accordance with one
preferred embodiment of the present invention. The membrane
chromatography component 310 includes a housing 2000 and porous
membrane layers 2010 (preferably at least 10 porous membrane
layers). As discussed above, the individual porous membrane layers
2010 preferably consist of an appropriate polymer, such as
polymethacrylate, that has been chemically functionalized with a
ligand, such as a diethylaminoethyl (DEAE), a quaternary amine (Q),
or a carboxymethyl (CM) ligand for the case of ion-exchange
chromatography, or a phenyl or butyl ligand for the case of
hydrophobic interaction chromatography, or a mercaptoethylpyridine
(MEP) ligand for the case of mixed mode chromatography.
[0066] The membrane chromatography component 310 can be of any
size, but in the microscale embodiment of FIG. 4, it preferably has
a volume capacity of less than approximately 100 milliliters, and
more preferably less than approximately 5 milliliters, in order to
keep the system 800 relatively compact. The membrane chromatography
component 310 may be implemented, for example, with a
Sartobind.RTM. Q SingelSep Nano manufactured by Sartorius Stedim
Biotech, which has a bed volume of 1 ml and a membrane area of 36
cm.sup.2.
[0067] Raw product from reactor 210 is mixed with elution buffer
solution via three-way valve 2015, and the mixture enters the
membrane chromatography component 310 via inlet 2020. Purified
product and waste exits via the outlet 2030. Three-way valve 2040
directs the purified product to the diafiltration component
320/900/1100 and directs the waste to waste storage 440.
[0068] FIGS. 6A-6C show a diafiltration component 900 that can be
used in systems 700 and 800, in accordance with one preferred
embodiment of the present invention. The diafiltration component
900 includes serpentine-shaped product and buffer sections 910 and
920, respectively. The diafiltration component 900 of FIGS. 6A-6C
include a product section 910 that is a serpentine-shaped channel
formed on a first substrate 1000. Similarly, the buffer section 920
is a channel formed on a second substrate 1010 with the same
serpentine shape as the product section 910. A diafiltration
membrane 930 is sandwiched between the first and second substrates
1000 and 1010, such that the serpentine-shaped channels that form
the product and buffer sections 910 and 910 substantially overlap
each other. The substrates 1000 and 1010 are attached to each
other, with the diafiltration membrane 930 sandwiched between them,
with any adhesive known in the art.
[0069] In the diafiltration component 900 of FIGS. 6A-6C, a
diafiltration buffer solution flows through the serpentine-shaped
product section 920 and purified product from the membrane
chromatography component 310 flows through the serpentine-shaped
product section 910. Diffusion takes place from the product section
910 to the counterpart, similarly shaped buffer section 920 via the
diafiltration membrane 930.
[0070] The purified product from the membrane chromatography
component 310 enters the product section 910 via inlet buffer
reservoir 1020 and inlet 1030. The diafiltered product exits the
product section 910 via outlet 1040 and outlet buffer reservoir
1050. Diafiltration buffer enters the buffer section 920 via inlet
1060 and exits the buffer section via outlet 1070. The
diafiltration buffer is chosen to facilitate the transfer of
components through the diafiltration membrane 930, and could be,
for example, 25 millimolar phosphoric acid titrated to pH 7 with
sodium hydroxide, or 25 millimolar citric acid tritrated to pH 5
with sodium hydroxide.
[0071] The inlet and outlet buffer reservoirs 1020 and 1050 are
optionally used in order to dampen the back-and-forth oscillating
flow, if needed. A makeup buffer solution is preferably added to
the diafiltered product via the outlet buffer reservoir 1050 in
order to replace the fluid that was that passed through the
diafiltration membrane 930 with an equivalent volume of a different
type of buffer, thereby transferring the protein of interest to the
makeup buffer. Alternatively, the volume of the makeup buffer added
via the outlet buffer reservoir 1050 can be less than the volume of
fluid that has passed through the diafiltration membrane 930, in
which case the diafiltration component 900 accomplishes both buffer
exchange and protein concentration.
[0072] As discussed above, diafiltration membrane 930 may suitably
be a hydrophilic polymeric membrane, such as a polyethersulfone, a
cellulosic, or a polyvinylidene fluoride (PVDF) membrane with a
well-defined pore structure that yields a desired molecular weight
cut-off (MWCO) value in the range of 10 k to 200 kDa as appropriate
for a given application.
[0073] FIG. 7 shows a diafiltration component 1100 in accordance
with another embodiment of the present invention. The diafiltration
component 1100 may be used in system 700 or system 800 of FIGS. 3
and 4, respectively. The diafiltration component 1100 includes a
buffer section 1120, and a product section 1110 that comprises
tubing 1112 that is passed through the buffer section 1120. The
tubing 1112 that makes up the product section 1110 can be any type
of tubing known in the art that can function as the dialysis
membrane 1140 between the product 1115 in the product section 1110
and the buffer 1130 in the buffer section 1120.
[0074] The tubing 1112 is preferably flexible so that a larger
amount of tubing can be placed inside the solvent section 1120. The
more tubing 1112 is present in the buffer section 1120, the more
diffusion can take place between the tubing 1112 and the buffer
1130 due to the larger tubing surface area in contact with the
buffer 1130. End portions 1140 and 1150 of the diafiltration
component 1100 contain openings 1160 for the tubing 1112 to enter
and exit the diafiltration component 1100. The end portions 1140
and 1150 also contain an inlet 1170 for receiving diafiltration
buffer solution, and an outlet 1180 for expelling used
diafiltration buffer solution (waste). Although the diafiltration
component 1100 is shown as rectangularly-shaped, it can be any
other shape, such as cylindrically-shaped. Further, the
diafiltration component 1100 can suitably be a flow cell that has
been modified to pass the tubing 1112 through the buffer section
1120.
[0075] Protein Expression in In Vivo and Cell-Free Systems
[0076] A protein is expressed in three main steps: replication,
transcription and translation, as shown in FIG. 8. DNA multiplies
to make multiple copies by a process called replication.
Transcription occurs when the double-stranded DNA is unwound to
allow the binding of RNA polymerase producing messenger RNA (mRNA).
Transcription is regulated at various levels by activators and
repressors, and also by chromatin structure in eukaryotes. In
prokaryotes, no special post-transcriptional modification of mRNA
is required. However, in eukaryotes, mRNA is further processed to
remove introns (splicing), to add a `cap` (M7 methyl-guanosine) at
the 5' end and to add multiple adenosine ribonucleotides at the 3'
end of mRNA to generate a poly(A) tail. The modified mRNA is then
translated.
[0077] The translation or protein synthesis is also a multi-step
process with Initiation, Elongation and Termination steps and is
similar in both prokaryotes and eukaryotes. The difference is that
in eukaryotes, proteins may undergo post-translational
modifications, such as phosphorylation or glycosylation. The
translation process requires cellular components such as ribosomes,
transfer RNAs (tRNA), mRNA and protein factors as well as small
molecules like amino acids, ATP, GTP and other cofactors.
[0078] The difference between in vivo and in vitro (cell-free)
protein expression is that in cell-free expression, the cell wall
and the nuclei are no longer present.
[0079] Cell-Free Protein Expression from an Engineer's
Perspective
[0080] To obtain the cell extract for cell-free protein expression,
cells (E. coli, wheat germ, mammalian cells) are subjected to cell
lysis followed by separation of the cell wall and nuclear DNA. The
desired protein is synthesized by adding a DNA or mRNA template
into the cell extract together with a reaction mix comprising of
biological extracts and/or defined reagents. The reaction mix is
comprised of amino acids, nucleotides, co-factors, enzymes and
other reagents that are necessary for the synthesis, e.g.
ribosomes, tRNA, polymerases, transcriptional factors, etc. When
DNA is used as template (i.e. linked reaction), it is first
transcribed to mRNA. Alternatively mRNA could also be used directly
for translation.
[0081] The template for cell-free protein synthesis can be either
mRNA or DNA. Translation of stabilized mRNA or combined
transcription and translation converts stored information into a
desired protein. The combined system, generally utilized in E. coli
systems, continuously generates mRNA from a DNA template with a
recognizable promoter. Either endogenous RNA polymerase is used, or
an exogenous phage RNA polymerase, typically T7 or SP6, is added
directly to the reaction mixture. Alternatively, mRNA can be
continually amplified by inserting the message into a template for
QB replicase, an RNA dependent RNA polymerase. Purified mRNA is
generally stabilized by chemical modification before it is added to
the reaction mixture. Nucleases can be removed from extracts to
help stabilize mRNA levels. The template can encode for any
particular gene of interest.
[0082] Salts, particularly those that are biologically relevant,
such as manganese, potassium or ammonium, may also be added. The pH
of the reaction is generally run between pH 6-9. The temperature of
the reaction is generally between 20.degree. C. and 40.degree. C.
These ranges may be extended.
[0083] In addition to the above components such as cell-free
extract, genetic template, and amino acids, other materials
specifically required for protein synthesis may be added to the
reaction. These materials may include salts, polymeric compounds,
cyclic AMP, inhibitors for protein or nucleic acid degrading
enzymes, inhibitors or regulators of protein synthesis,
oxidation/reduction adjusters, non-denaturing surfactants, buffer
components, spermine, spermidine, etc.
[0084] The salts preferably include potassium, magnesium, ammonium
and manganese salts of acetic acid or sulfuric acid, and some of
these may have amino acids as a counter anion. The polymeric
compounds may be polyethylene glycol, dextran, diethyl aminoethyl
dextran, quaternary aminoethyl and aminoethyl dextran, etc. The
oxidation/reduction adjuster may be dithiothreitol (DTT), ascorbic
acid, glutathione and/or their oxides. Further DTT may be used as a
stabilizer to stabilize enzymes and other proteins, especially if
some enzymes and proteins possess free sulfhydryl groups. Also, a
non-denaturing surfactant such as Triton X-100 may be used at a
concentration of 0-0.5 M. Spermine and spermidine may be used for
improving protein synthetic ability, and cAMP may be used as a gene
expression regulator.
[0085] Synthesized product is usually accumulated in the reactor
within the production module, and then is isolated and purified
according to the methods of the present invention for protein
purification after completion of the system operation. The amount
of protein produced in a translation reaction can be measured in
various fashions. One method relies on the availability of an assay
that measures the activity of the particular protein being
translated. Examples of assays for measuring protein activity are a
luciferase assay system and a chloramphenicol acetyl transferase
assay system. These assays measure the amount of functionally
active protein produced from the translation reaction. Importantly,
activity assays will not measure full length protein that is
inactive due to improper protein folding or lack of other post
translational modifications necessary for protein activity. As used
herein, the term "activity" refers to a functional activity or
activities of a peptide or portion thereof associated with a
full-length (complete) protein. Functional activities include, but
are not limited to, catalytic or enzymatic activity, antigenicity
(ability to bind or compete with a polypeptide for binding to an
anti-polypeptide antibody), immunogenicity, ability to form
multimers, and the ability to specifically bind to a receptor or
ligand for the polypeptide. Preferably, the activity of produced
proteins retain at least 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95% or
more of the initial activity for at least 3 days at a temperature
from about 0.degree. C. to 30.degree. C.
[0086] Another method of measuring the amount of protein produced
in a combined in vitro transcription and translation reactions is
to perform the reactions using a known quantity of radiolabeled
amino acid such as .sup.35S-methionine or .sup.14C-leucine and
subsequently measuring the amount of radiolabeled amino acid
incorporated into the newly translated protein. Incorporation
assays will measure the amount of radiolabeled amino acids in all
proteins produced in an in vitro translation reaction including
truncated protein products.
[0087] In another study, expression of a fusion protein consisting
of murine GM-CSF (granulocyte macrophage colony stimulating factor)
and a scFv antibody, in reactor systems such as thin film, bubble
column and Eppendorf tube without membrane, were studied, producing
protein up to >500 .mu.g/ml protein with significant amount of
precipitated protein (apprx eq.50%). Recently, rhGM-CF was
expressed in a 100 L stirred tank reactor expressing protein up to
700 mg/L which was subsequently purified with DEAE resin,
tangential flow filtration membrane (3 kD cut off) and Sephacryl
S-100 size exclusion chromatography with 99% purity and 65%
recovery. Cell-free expression has not only been successful in the
expression of bacterial proteins, but also successfully produced
glycoproteins like human choriogonadotropin (hCG) and envelope
glycoprotein (gp120) of human immunodeficiency virus type-1 (HIV-1)
in hybridoma cell extract (HF10B4).
[0088] For protein purification, people have relied on column
chromatography traditionally, but in recent years membrane
chromatography has emerged as an additional aid in this field,
eliminating column chromatography at specific steps like capture
and polishing of protein at final step with overall cost reduction
up to 65%. Column chromatography is still useful for gradient
purification of proteins, but membrane chromatography could also be
studied by relying on the fact that step elution of protein and
removal of the impurities could be done at different buffer
conditions.
[0089] The chart below compares cell-free and in vivo protein
expression systems.
TABLE-US-00001 In vivo Cell free Biological cell required No cell,
but cellular machinery is required Time consuming process Time
effective process Toxic protein may be difficult to express Toxic
protein could be expressed Multiple steps in purification required
Relatively less number of steps required Higher fraction of
misfolded protein along Reduced levels of misfolded protein
reported, with folded protein along with folded protein but
precipitated Higher endotoxins challenge Relatively less endotoxins
challenge Higher amount of impurities in crude protein Relatively
less impurities, enhancing capture causing challenges in capture
step and increasing yield of the protein Established scale up Has
significant potential to scale up Protein expression up to g/l
Protein expression up to mg/l
[0090] Biomolecules for Protein Expression
[0091] The following biomolecules are preferably used for protein
expression. To carry out a protein expression reaction, energy
components and amino acids are supplied externally and may include,
but not limited to the following components:
a. A genetic template for the target protein (mRNA or DNA)
expression; b. T7 RNA polymerases for mRNA transcription; c. 9
Translation factors (initiation, elongation and termination); d. 20
aminoacyl-tRNA synthetases (ARSes) for esterification of a specific
amino acid to form an aminoacyl-tRNA; e. Methionyl-tRNA
transformylase transfers hydroxymethyl-, formyl-groups; f. Creatine
kinase converts ATP to ADP; g. Myokinase catalyzes the inter
conversion of adenine nucleotides; h. Pyrophosphatase are acid
anhydride hydrolases that act upon diphosphate bonds; i. 4
nucleoside triphosphates (ATP, GTP, CTP, TTP) for DNA formation; j.
Creatine phosphate which serves as a reserve of high-energy
phosphates for rapid mobilization; k.
10-formyl-5,6,7,8-tetrahydrofolate for the formylation of the
methionyl initiator tRNA (fMet-tRNA); l. 20 amino acids for protein
synthesis; m. Ribosomes for polypeptide translation; n. 46 tRNAs in
protein synthesis; and o. Cellular components which assist in
proper protein folding.
[0092] Some of the proteins that may be expressed by the present
invention for on-demand production may include, but not limited to,
adrenocorticotropic hormone peptides, adrenomedullin peptides,
allatostatin peptides, amylin peptides, amyloid beta-protein
fragment peptides, angiotensin peptides, antibiotic peptides,
antigenic polypeptides, anti-microbial peptides, apoptosis related
peptides, atrial natriuretic peptides, bag cell peptides, bombesin
peptides, bone GLA peptides, bradykinin peptides, brain natriuretic
peptides, C-peptides, C-type natriuretic peptides, calcitonin
peptides, calcitonin gene related peptides, CART peptides,
casomorphin peptides, chemotactic peptides, cholecystokinin
peptides, colony-stimulating factor peptides, corticortropin
releasing factor peptides, cortistatin peptides, cytokine peptides,
dermorphin peptides, dynorphin peptides, endorphin peptides,
endothelin peptides, ETa receptor antagonist peptides, ETh receptor
antagonist peptides, enkephalin peptides, fibronectin peptides,
galanin peptides, gastrin peptides, glucagon peptides, Gn-RH
associated peptides, growth factor peptides, growth hormone
peptides, GTP-binding protein fragment peptides, guanylin peptides,
inhibin peptides, insulin peptides, interleukin peptides, laminin
peptides, leptin peptides, leucokinin peptides, luteinizing
hormone-releasing hormone peptides, mastoparan peptides, mast cell
degranulating peptides, melanocyte stimulating hormone peptides,
morphiceptin peptides, motilin peptides, neuro-peptides,
neuropeptide Y peptides, neurotropic factor peptides, orexin
peptides, opioid peptides, oxytocin peptides, PACAP peptides,
pancreastatin peptides, pancreatic polypeptides, parathyroid
hormone peptides, parathyroid hormone-related peptides, peptide T
peptides, prolactin-releasing peptides, peptide YY peptides, renin
substrate peptides, secretin peptides, somatostatin peptides,
substance P peptides, tachykinin peptides, thyrotropin-releasing
hormone peptides, toxin peptides, vasoactive intestinal peptides,
vasopressin peptides, and virus related peptides.
[0093] Conventional and Non-Conventional Method of GBP
Production
[0094] The systems and methods of the present invention can be
used, for example, for the cell-free expression and purification of
glucose binding protein (GBP). Glucose is a major carbon and energy
source in cellular metabolism of animal body and in bioprocess
industry. Glucose is not always beneficial in bioprocesses, it
could also be detrimental in bacterial culture leading to
self-lysis of cells by formation of acetate in Krebs cycle and
reducing the pH of the culture. Thus, fast and efficient
concentration detection of glucose is desired.
[0095] Glucose binding protein is a protein which could bind to
glucose and serve this purpose by acting as a biosensor. A
biosensor is an analytical device used for the detection of an
analyte that combines a biological component with a physicochemical
detector component. GBP is such a biosensor, where GBP binds with
glucose and binding is analyzed using fluorescence intensity and
the corresponding signal is compared with standard glucose signal
to estimate concentration of unknown sample. GBP is a monomeric
periplasmic protein with molecular weight of 34 kD (kilo Dalton)
and is synthesized in the cytoplasm of E. coli.
[0096] In the conventional method, GBP (L225C mutant) is produced
in multiple steps, pre-inoculation of E. coli mutants in Luria
Bertani (LB) broth, culturing, harvesting, cell washing, osmotic
shock, labeling, liquid chromatography and dialysis. All these
steps are time consuming (around 4 days) and cumbersome. The
present invention enables a non-conventional cell free expression
of GBP where expression is faster and the resulting protein is
relatively pure. This protein would preferably be labeled using a
fluorophore called acrylodan
(6-Acryloyl-2-dimethylaminonaphthalene) and purified by D15 (DEAE)
chromatography membrane. The protein would preferably further be
concentrated and dialyzed against 5 mM tris-HCl, pH 7.5.
[0097] Cell Free Method of Production of Granulocyte Colony
Stimulating Factor (G-CSF) and Pharmaceutical Analog Fligrastim
[0098] For proof of principle, G-CSF also known as the
pharmaceutical analog Filgrastim is used as a model therapeutic
protein. Notably, the same method holds for any therapeutic protein
for administration at the point-of-care. Filgrastim is used to
stimulate the production of granulocytes (a type of white blood
cell) in patients undergoing cancer therapy with specific drugs
that are known to cause low white blood cell counts.
[0099] Cell-free protein synthesis system was tested for G-CSF
protein expression, the DNA used as the template in the system was
G-CSF plasmid: 80 .mu.g (concentration: 0.47 .mu.g/.mu.L) in
combination with lyophilized CHO lysate in an amount of 1 mL,
Gadd34-Myc plasmid 8 ug (@ 0.4 ug/uL)=20 uL)), Thermo Reaction Mix
(5.times.): 400 uL, nuclease free water: balance to 2 mL, and
1.times.CHO dialysis buffer at 25 mL. Six batches were run with a
total batch volume for each batch of about 2 mL and the process
took about 6 hours at a temperature of about 30.degree. C. The
bioreactor used was SLIDE-A-LYZER.TM. dialysis cassette (10 kDa
cutoff: 3 mL).
[0100] About 1.98 mL of harvested product was subjected to
purification in an IMAC spin column with loading buffer: 10 mM
imidazole, wash buffer 1:10 mM imidazole, wash buffer 2:30 mM
imiadazole and elution buffer: 150 mM imidazole. Notably PBS
buffer, without DTT, was used in the purification process. The
fractions were collected from triplicate runs where G-CSF was
expressed over a six hour period in the presently claimed cell-free
system. The G-CSF was purified using a His-tagged affinity column.
The data show the remarkable consistency of the expression and
purification of the target product.
[0101] FIGS. 9A and 9B show Western Blot results with an Anti-G-CSF
antibody. The pellets discussed in both figures were washed once
with 500 .mu.L of PBS and solubilized in 1500 .mu.L of PBS with 1%
Tween-20 and 1.5% Triton X-100. The "H" represents the Harvest, the
"P" is the Pellet and the "E" is the Elute. The elution column
shows a clear band representing G-CSF for all three runs showing
the remarkable consistency of the expression and purification of
the target product. The far right column shows the G-CSF standard
that was also run. FIG. 9A shows the results of Run #3 and FIG. 9B
shows the results of Run #4. Clearly the results are consistent and
provide evidence that the process of the present invention is
reproducible.
[0102] FIG. 10 A shows the quantified values of the harvested
proteins and purified protein of Run #3 of FIG. 9A and FIG. 10B
show the values of Run#4 of FIG. 9B.
[0103] Further it was shown that proteins produced in the on-demand
system of the present invention provide for improved and increased
potency relative to freeze/thaw data of the prior art method and it
is evident that using a freeze/thaw cycle impacts activity. As
shown in FIG. 11, in the results of separate runs of 3 and 4, it
can be seen that the freshly made G-CSF has potency twice that of
the reconstituted lyophilized standard. The same molecule loses its
potency after just one freeze-thaw cycle approaching that of a
boiled control. These data prove our assertion that administering a
freshly made therapeutic protein (at most, refrigerated for a few
days) provides maximal potency and has the additional advantage of
no additives. This is a significant and surprising improvement over
the current paradigm of biologics production and delivery.
[0104] In another example, the therapeutic protein Erythropoetin
(EPO, used to stimulate red blood cell production in the human
body) was produced in the cell free system. FIG. 12 shows the same
consistency of expression in 4 separate batches as evidenced by the
bands on the Western blot.
[0105] As the cell proliferation-based activity assay shows in the
top panel of FIG. 13A, the EPO in the extract is more active
compared to controls. It should be noted that the EPO tested from
the cell free process was diluted 100-fold, so the activity in the
extract was in excess of 100,000 units/mL. Given the typical EPO
dosage is between 50-100 units/mL/kg, it appears that 1 mL product
contains sufficient EPO to dose 10 adults. These data show the
remarkable potential of point-of-care manufacturing as the freshly
expressed protein shows very high activity. This is likely due to
virtually no "aging" of the protein that normally takes place in
conventionally manufactured proteins (i.e. deamidation, oxidation,
aggregation etc.).
[0106] Streptokinase was also produced successfully in a cell free
stirred tank bioreactor. A representative sample of activity at two
harvest times is shown in FIG. 13B, lower panel. As can be seen,
the harvest time may be used to pick a desired activity in the case
of Streptokinase, several dosing regimens are in use clinically and
the harvest can be timed to conform to the desired dose,
eliminating any dilution for the final delivery to obtain the
correct dose. As can be seen, active EPO was produced and had
extremely high activity (samples 32K14 and 36K14 were diluted 100
fold for the assay). For Streptokinase, lysate was harvested at two
time points and activity measured. This approach may be used to
determine the dose needed for delivery to the patient.
* * * * *