WO2001058501A1

WO2001058501A1 - Apparatus and process for removal of carbon dioxide in a bioreactor system

Info

Publication number: WO2001058501A1
Application number: PCT/US2001/003947
Authority: WO
Inventors: Randal M. Wenthold
Original assignee: Minntech Corporation
Priority date: 2000-02-09
Filing date: 2001-02-07
Publication date: 2001-08-16
Also published as: AU4314301A

Abstract

A hydrophobic hollow fiber membrane filter (10) defining a bundle of hydrophobic hollow fiber membranes (42) disposed within the interior chamber (34) is disclosed. The membranes (42) are in fluid communication with a recirculating bioreactor system wherein soluable carbon dioxide may be removed from the living solution (60) by applying a vacuum (72) to the device. The hydrophobic hollow fiber membrane filter (10) may be installed in an external piping recirculation loop (66) where living solution (60) may be introduced via a mechanical pump into a cylindrical access channel (50, 52). A pH sensor (70) indicates the living solution in the tank has moved outside the pH set-point, a vacuum (72) is initiated and dissolved carbon dioxide may be removed across the hydrophobic filter (10) in the recirculation loop (66). A control circuit (68) initiates vacuum (72) on the vent filter (64) to remove carbon dioxide from the mixing tank (78) headspace.

Description

APPARATUS AND PROCESS FOR REMOVAL OF CARBON DIOXIDE

IN A BIOREACTOR SYSTEM

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a device and method for carbon dioxide removal and pH control in a bioreactor system. More particularly, this invention relates to a hollow fiber membrane device that provides pH control in a bioreactor system by removing carbon dioxide from the living solution and a method of removal of C02 from solution.

2. Description of the Related Art Numerous biochemical processes are known wherein a microorganism is propagated in a suitable culture medium, either for the purpose of growing large quantities of the microorganism for some particular purpose or use, or for recovering products generated by the living microorganism. The cells to be cultured are viable, growing or non-growing, prokaryotic and eukaryotic cells such as bacteria, virus, yeast, plant, animal and human cells. Bioreactor systems for continuous processing of microorganisms grown in suspension are currently favored for economic and technical reasons. However, continuous bioreactor processing -presents a variety of problems including the uniform supply of oxygen to all living organisms; difficulty in controlling pH gradients; and removal of gaseous waste products, in particular carbon dioxide . Conventional art bioreactor systems may include batch systems, fed-batch systems and perfusion systems. Batch systems typically include a mixing tank with an agitator device. A fed-batch system includes the addition of a feed stream into the mixing tank. A perfusion type system is a fed-batch system with the addition of a product recirculation stream, which recirculates back to the mixing tank. Oxygen is provided by a variety of means described below and pH and p02 measurement probes are utilized in the system. The use of hydrophobic filters to remove carbon dioxide from the living solution in these systems is unknown.

Many existing bioreactors have been designed on the principles originally developed for microbial culture. These fermentors may be aerated by gas overlay and/or sparged air provided through an open pipe or perforated ring at the bottom of the compartment or by a separate oxygenation device. Blade impellers, sail impellers or floating stainless steel mesh stirrers provided agitation to increase oxygen transfer from the gas overlay. One of the problems associated with these systems is the buildup of waste by-products and little control over pH.

Consequently, conventional bioreactor systems rely heavily on the introduction of buffers to control the pH of the living solution. However, some buffers such as sodium bicarbonate liberate carbon dioxide into the system exacerbating the problem of pH. Another problem with these types of buffers is the excess salts introduced into the system. While most cell cultures will utilize a small portion of the salts, the majority of the salts introduced into the system are not consumed by the cells. The build-up of salts in solution may result in cellular toxicity.

Other systems include the use of hydrophobic membranes made of polypropylene that are formed as a porous hollow fiber membranes. The hydrophobic membrane may be looped around a carrier that is slowly moved through the culture to produce a membrane stirrer causing oxygen transfer. However, associated problems include the build-up of cells on membrane walls and dead zones of cells that starve due to the lack of oxygen.

U.S. Pat. No. 4,661,468 discloses a bioreactor system in which the cell culture is grown on an organic tubular membrane support. However, the growth of cells in such a system impedes the transfer of nutrients and gases resulting in biofouling of the system caused by dead cells and excess waste product build-up and the formation of pH gradients within the system.

Other conventional devices utilize indirect gas transfer such as gas-permeable membranes. For example, U.S. Pat. No. 4,764,471 discloses a nutrient medium continuously flowed through the length of a cylindrical, spirally-wound, ultrafiltration membrane device. However, similar problems of waste product build-up, formation of pH gradients and the growth of cells on the membrane wall which impedes the transfer of nutrients including oxygen exist .

Problems associated with currently available bioreactor systems include the lack of uniform oxygen supply to living organisms throughout the bioreactor system; build-up of dead cells throughout the bioreactor system; removal of waste products, in particular carbon dioxide; and pH control.

SUMMARY OF THE INVENTION It is an object of the apparatus and process of the present invention to overcome the above-mentioned problems by providing a filter which improves the utilization of oxygen by all cells in the living solution.

It is a further object of the apparatus and process of the present invention to minimize or eliminate the build-up of dead cells throughout the bioreactor system.

It is a further object of the apparatus and process of the present invention to remove waste products, in particular carbon dioxide, thereby providing pH control in the bioreactor system.

In accordance with the principles of the present invention, these and other objects are achieved by a process for removing carbon dioxide from a bioreactor system comprising providing a bioreactor mixing tank into which living solution is introduced; providing a hollow fiber membrane filter defining an interior chamber in fluid communication with the bioreactor mixing tank, said filter including a vacuum access port, a bundle of hollow fiber membranes disposed within the interior chamber, each of said hollow fiber membranes defining an interior lumen therewithin; causing the living solution to flow through the lumens of the hollow fiber membranes; removing carbon dioxide from the living solution by applying a vacuum to the vacuum access port; and recirculating the living solution into the bioreactor mixing tank.

These and other objects, features, and advantages of this invention will become apparent from the following detailed description when taken in conjunction with the accompanying drawings which illustrate, by way of example, the principles of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is an exploded view of the apparatus in accordance with the present invention;

FIGURE 2 is a cross-section of the apparatus in accordance with the present invention;

FIGURE 3A is a depiction of a hollow fiber membrane as it is utilized in the process of a conventional bioreactor system;

FIGURE 3B is a depiction of a hollow fiber membrane as it is utilized in the process of the present invention; FIGURE 4 is a schematic illustration of a conventional laboratory, scale-up type bioreactor system;

FIGURE 5 is a schematic illustration of a perfusion type bioreactor system utilizing the apparatus and process of the present invention;

FIGURE 6 is a schematic illustration of a fed batch bioreactor system utilizing the apparatus and process of the present invention;

FIGURE 7 is a schematic illustration of a batch type bioreactor system utilizing the apparatus and process of the present invention;

FIGURE 8 is a graphical representation depicting the removal of waste products at low gas levels in accordance with an apparatus of the present invention having a surface area of 950 cm²;

FIGURE 9 is a graphical representation depicting the removal of waste products at high gas levels in accordance with an apparatus of the present invention having a surface area of 950 cm²; FIGURE 10 is a graphical representation depicting the removal of waste products at low gas levels in accordance with an apparatus of the present invention having a surface area of 2300 cm²;

FIGURE 11 is a graphical representation depicting the removal of waste products at high gas levels in accordance with an apparatus of the present invention having a surface area of 2300 cm²;

FIGURE 12 is a graphical representation depicting the removal of waste products at low gas levels in accordance with an apparatus of the present invention having a surface area of

6000 cm²;

FIGURE 13 is a graphical representation depicting the removal of waste products at high gas levels in accordance with an apparatus of the present invention having a surface area of 6000 cm².

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The apparatus and process of the present invention will be more particularly described hereinbelow with reference to the accompanying drawings, where like reference to numerals designate corresponding elements in the various figures. Referring to FIGURES 1 and 2, the apparatus 10 embodying the present invention includes casing 12, bundle of hollow fibers 14, first sealing end 16, and second sealing end 18. First end cap 20 and second end cap 22, and vacuum access ports 24, 26 with cover caps 28 may be provided as desired.

Casing 12 may have any cross-sectional configuration, for example square, circular, octagonal, triangular, etc., but for purposes of description herein, casing 12 is preferably circular in cross-section. Casing 12 and vacuum access ports 24, 26, are preferably integrally molded to form a unitary piece. Casing 12 may be solid or may have perforations therewithin. If casing 12 is manufactured with perforations then in operation, apparatus 10 would be placed within a solid filter housing, which is known in the art .

Casing 12, first end cap 20, second end cap 22, vacuum access ports 24, 26, and cover caps 28 may be made of any sturdy fluid impermeable material, for example stainless steal, aluminum, copper, zinc, thermoplastic polymers such as polyethylene, polycarbonate, polyvinylchloride and polyethylene-terepthalate or glass. Preferably, a suitable material for casing 12, first end cap 20, second end cap 22, vacuum access ports 24, 26, and cover caps 28 is polycarbonate or polypropylene .

Casing 12 has first casing end 30 and second casing end 32 defining internal cylindrical chamber. Positioned within internal cylindrical chamber 34 is at least one bundle of microporous hollow fiber membranes 14 which includes individual microporous hollow fibers rectilinearly aligned with one another. The longitudinal ends 36a, 36b of the hollow fiber bundle 14 are impermeably potted within a potting compound 38.

The hollow fiber bundle 14 is retained within the chamber 34 defined by the casing 12 with the longitudinal ends 36a, 36b of the fiber bundle 14 projecting past the longitudinal ends 30, 32 of casing 12. The manufacture of hollow fibers 14 is widely understood and a wide variety of hollow fibers 14 may be purchased from a number of different sources. In general, hollow fibers 42 having an outer diameter 54 as small as about 260 microns and a wall thickness 56 as small as about 20 microns may be manufactured from a variety of hydrophobic materials including polypropylene, polysulfones, polyvinyli ene fluoride, teflon, cellulose esters, polyethylene, polytetrafloroethylene and other suitable materials. In the apparatus in accordance with the present invention the preferred polymer is polypropylene. The membrane may also be coated with any biocompatible material or hydrophobic polymer such as teflon to increase the hydrophobic nature of the membrane .

Selection of the preferred size of the individual hollow fibers 14 is generally dependent upon a number of factors including the particular living solution to be filtered, the desired waste products to be removed, the desired filtration efficiency, etc. The porosity of the membrane should be sized so as to exclude solids and cells above a predetermined size or molecular weight. In the preferred embodiment, the inside diameter 40 of the individual hollow fibers 42 is about 220 microns to about 2000 microns. The external surface area of the hollow fiber bundle available for filtration is substantially

92.9 cm² to substantially 371,600 cm². The porosity of the individual hollow fiber membranes 42 is substantially about 0.001 micron to substantially about 10 microns.

Hollow fiber membranes suitable for use in the present invention may be symmetric as disclosed in U.S. Pat. No. 4,055,696 (available from Mitsubishi Rayon Co., Ltd., Tokyo, Japan) or asymmetric as disclosed in U.S. Pat. No. 5,762,798 (available from Minntech Corporation, Minneapolis, MN) . The preferred membrane is disclosed in U.S. Pat. No. 4,055,696.

In accordance with generally accepted practices in the manufacture of hollow fiber filter devices 10, the packing density of the individual hollow fibers 42 within cylindrical chamber 34 should be such that the area occupied by the individual hollow fibers 42 is about 30% to 75%, more preferably 40% to 50%, and most preferably 40% to 45% of the casing cross-sectional area available for fibers. A packing density of greater than about 75% significantly interferes with the ability to sealingly pot the longitudinal ends 36a, 36b of the hollow fiber bundle 14 while a packing density of less than about 40% decreases the filtration capacity of the hollow fiber filter apparatus 10 without any corresponding benefits.

The longitudinal ends 36a, 36b of the hollow fiber bundle 14 are sealingly potted with a suitable potting compound 38 which occupies the interstitial void volume between the individual hollow fibers 42 and the annular channel 44 formed by casing 10. The potting compound 38 functions to (i) prevent fluid from passing through the interstitial void space 46 between the hollow fibers 42; and (ii) hold the hollow filter fibers together in a bundle. Selection of a suitable potting compound 38 depends upon several variables including the particular living solution to be filtered, the material from which the hollow fibers 42 are manufactured, and the material from which casing 12 is constructed. The compound 38 must possess sufficient initial fluidity to permit penetration of the potting compound 38 into the interstitial void volume between the individual hollow fibers 42 while resulting in a solid plug which is impermeable to the fluid being filtered. Most importantly, because the living solution to be filtered will come into contact with the potting compound 38, it is imperative that the potting compound 38 be biocompatible and not toxic to living organisms. A number of suitable potting compounds are well known and include such curable resins as polyurethanes, unsaturated polyesters, and silicones.

The longitudinal ends 36a, 36b of the potted hollow fiber bundle 14 are cut to open the longitudinal ends 36a, 36b of the hollow fiber lumens 48.

First and second end caps 20, 22 are fixedly attached, preferably by welding, to casing 10. End caps 20, 22 define cylindrical access channels 50, 52 which permit access of the living solution to be filtered to lumens 48 of hollow fiber membranes 42.

Referring to FIGURE 3A, conventional art filters used in bioreactor systems introduce the living solution 60 into the interstitial void space 46 between the hollow fiber membranes 42. Nutrient solutions or oxygen flows through the lumens 48 of the hollow fiber membranes 42. The disadvantage of these conventional art filters and process as previously stated is multifold. First, the majority of these filters utilize hydrophilic membrane and the cells embed themselves into the pores on the outer surfaces 58 of the hollow fiber membranes 42. This prevents proper utilization of oxygen by other cells present in the interstitial void space 46 by blocking oxygen transfer. Lack of oxygen will eventually cause cell death with resultant bio-fouling of the membrane and the living solution. Bio-fouling, lack of oxygen in the system and resultant increases in carbon dioxide cause the formation of pH gradients in the system. This in turn, causes additional cell death and additional bio-fouling.

Referring to FIGURES 3A and 4, the conventional hollow fiber membrane system described above is illustrated in a simple laboratory, scale-up type bioreactor system. Cell culture solution is inoculated into the hollow fiber membrane device into the interstitial spaces surrounding the individual hollow fiber membranes. Nutrients and air or oxygen are pumped into the lumens of the hollow fiber membranes where it diffuses into the cell culture solution. As oxygen is transferred to cells, they multiply and grow into the interior walls of the hollow fiber lumens. As described previously, this type of system presents a variety of problems including inefficient utilization of oxygen by cells located further away from the wall of the hollow fiber membrane, membrane bio- fouling, and the build-up of dead cells and waste products in solution.

Referring to FIGURE 3B, the process in accordance with the present invention introduces the living solution to be filtered into the lumens 48 of hydrophobic hollow fiber membranes 42. The use of a hydrophobic membrane ensures that the cells will not embed themselves into the membrane preventing transfer of gases.

In use, living solution 60 is introduced via a mechanical pump into cylindrical access channel 50 and flows through lumens 48. Port 26 is capped with cover 28 -while a vacuum of from 0.5 mmHg to 760 mmHg is applied to inlet 24, which removes carbon dioxide from solution. The living solution is pumped through the bioreactor system and the process in accordance with the present invention is repeated. The process may utilize the apparatus in accordance with the present invention in a cross-flow, also known as a tangential, configuration. The precise configuration will depend upon cell size, cell culture type and/or bioreactor size.

Preferably, a cross-flow or tangential configuration is desirable over an in-line system because a cross-flow configuration provides a dynamic system for gas removal whereby the fluid to be filtered is recirculated. An in-line system is a static system wherein dissolved gases cannot be entirely removed for the purposes described herein.

The removal of carbon dioxide from the system enables the operator to regulate pH precisely without relying on the introduction of buffer solutions. In addition, the hydrophobic nature of the hollow fiber membrane 42 as well as the pore size of the membranes 42 minimizes and/or eliminates living cells from embedding into the pores of the hollow fibers 42. This ensures the proper and uniform utilization of oxygen throughout the system. Lastly, proper and uniform utilization of oxygen throughout the system reduces cellular death and minimizes debris and waste build-up in the system.

Referring generally to FIGURES 5 through 7, the apparatus and process in accordance with the present invention may be used in perfusion type bioreactor operations, pilot or lab scale systems, batch type bioreactor systems or fed batch systems .

FIGURE 5 depicts a perfusion type bioreactor operation utilizing the apparatus and method of the present invention. Feed medium is introduced into a mixing tank bioreactor system by piping located at the top of the tank 62. A bacteria rejecting filter of from about 0.1 to 0.2 microns as well as a viral rejection filter of approximately 0.05 microns are situated between the feed line and the mixing tank to purify the feed solution delivered to the cell culture. A hydrophobic vent filter 64 is located proximally and adjacent to the mixing tank to vent the mixing tank of C0₂ in the headspace above the liquid. A hydrophobic filter in accordance with the present invention 10 is installed in an external piping recirculation loop on the mixing tank 66 to remove soluble carbon dioxide from the living cell solution. The hydrophobic filter in accordance with the present invention 10 and the hydrophobic vent filter 64 are in sensory communication via control circuit 68. When pH sensor 70 indicates the cell solution in the mixing tank has moved outside the set-point, vacuum 72 will be initiated and dissolved C0₂ will be removed across the hydrophobic filter in the recirculation loop. In addition, control circuit 68 will initiate vacuum on vent filter 64 to remove C0₂ from the mixing tank headspace. Another hydrophobic filter 74 is installed on a product recirculation loop to the mixing tank. A dissolved oxygen sensor is installed which controls the introduction of additional air or oxygen across the hydrophobic filter and into the mixing tank solution as needed. An additional bacteria filter and a limited viral filter are installed in the product piping line to ensure further protection of the cell culture from contamination. FIGURE 6 depicts a fed batch bioreactor system utilizing the apparatus and process of the present invention. Feed medium enters into the mixing tank bioreactor system by piping located at the top of the tank 62. A bacteria rejecting filter of from about 0.1 to 0.2 microns as well as a viral rejection filter of approximately 0.05 microns are situated between the feed line and the mixing tank to purify the feed solution delivered to the cell culture. A hydrophobic filter in accordance with the present invention 10 is installed in an external piping recirculation loop on the mixing tank 66 to remove soluble carbon dioxide from the living cell solution. A hydrophobic vent filter 64 is located proximally and adjacent to the mixing tank to vent the mixing tank of C0₂ in the headspace above the liquid. The hydrophobic filter in accordance with the present invention 10 and the hydrophobic vent filter 64 are in sensory communication via control circuit 68. When pH sensor 70 indicates the cell solution in the mixing tank has moved outside the set-point, vacuum 72 will be initiated and dissolved C0₂ will be removed across the hydrophobic filter in the recirculation loop. In addition, control circuit 68 will initiate vacuum on vent filter 64 to remove C0₂ from the mixing tank headspace. Another hydrophobic filter 74 is installed on a product recirculation loop to the mixing tank. A dissolved oxygen sensor 74 is installed which controls the introduction of additional air or oxygen across the hydrophobic filter and into the mixing tank living solution as needed.

FIGURE 7 illustrates a batch type bioreactor system utilizing the apparatus and process of the present invention.

A hydrophobic filter in accordance with the present invention 10 is installed in an external piping recirculation loop on the mixing tank 66 to remove soluble carbon dioxide from the living cell solution. A hydrophobic vent filter 64 is located proximally and adjacent to the mixing tank to vent the mixing tank of C0₂ in the headspace above the liquid. The hydrophobic filter in accordance with the present invention 10 and the hydrophobic vent filter 64 are in sensory communication via control circuit 68. When pH sensor 70 indicates the cell solution in the mixing tank has moved outside the set-point, vacuum 72 will be initiated and dissolved C0₂ will be removed across the hydrophobic filter in the recirculation loop. In addition, control circuit 68 will initiate vacuum on vent filter 64 to remove C0₂ from the mixing tank headspace. Hydrophobic filter 74 is installed on a product recirculation loop distal to the mixing tank. A dissolved oxygen sensor 74 is installed which controls the introduction of additional air or oxygen across the hydrophobic filter and into the mixing tank living solution as needed. EXAMPLE 1

FIGURE 8 graphically illustrates the removal of carbon dioxide in accordance with the process of the present invention. A device containing 950 cm² of hollow fibers was tested at low gas levels. Carbon dioxide was introduced into water and inlet gas partial pressures of 155 and 172 were recorded. Water was pumped through the device at flow rates of 0.1 liters per minute and 0.75 liters per minute and a vacuum of 700 mmHg was applied. Outlet gas partial pressures of carbon dioxide of 38 and 123 were recorded. The device reduced carbon dioxide in solution by 75.5% and 28.5%, respectively. EXAMPLE 2 FIGURE 9 graphically illustrates the removal of carbon dioxide in a device containing 950 cm² of hollow fiber membranes at high gas levels. Carbon dioxide was introduced into water and inlet gas partial pressures of 492 and 500 were recorded. Water was pumped through the device at flow rates of 0.1 liters per minute and 0.75 liters per minute and a vacuum of 700 mmHg was applied. Outlet gas partial pressures of carbon dioxide of 100 and 361 were recorded. The device reduced carbon dioxide in solution by 79.7% and 27.8%, respectively . EXAMPLE 3

FIGURE 10 graphically illustrates the removal of carbon dioxide in a device in accordance with the present invention containing 2300 cm² of hollow fiber membranes at low gas levels . Carbon dioxide was introduced into water and inlet gas partial pressures of 69 and 151 were recorded. Water was pumped through the device at flow rates of 0.3 liters per minute and 1.9 liters per minute and a vacuum of 700 mmHg was applied. Outlet gas partial pressures of carbon dioxide of 8 and 102 were recorded. The device reduced carbon dioxide in solution by 88.4% and 32.4%, respectively. EXAMPLE 4

FIGURE 11 graphically illustrates the removal of carbon dioxide in a device containing 2300 cm² of hollow fiber membranes at high gas levels. Carbon dioxide was introduced into water and inlet gas partial pressures of 580 and 601 were recorded. Water was pumped through the device at flow rates of 0.3 liters per minute and 3.0 liters per minute and a vacuum of 700 mmHg was applied. Outlet gas partial pressures of carbon dioxide of 135 and 442 were recorded. The device reduced carbon dioxide in solution by 76.7% and 26.5%, respectively. EXAMPLE 5 FIGURE 12 graphically illustrates the removal of carbon dioxide in a device containing 6000 cm² of hollow fiber membranes at low gas levels. Carbon dioxide was introduced into water and inlet gas partial pressures of 112 and 136 were recorded. Water was pumped through the device at flow rates of 0.8 liters per minute and 6.1 liters per minute and a vacuum of 700 mmHg was applied. Outlet gas partial pressures of carbon dioxide of 30 and 94 were recorded. The device reduced carbon dioxide in solution by 73.2% and 30.9%, respectively. EXAMPLE 6

FIGURE 13 graphically illustrates the removal of carbon dioxide in a device containing 6000 cm² of hollow fiber membranes at high gas levels. Carbon dioxide was introduced into water and inlet gas partial pressures of 519 and 558 were recorded. Water was pumped through the device at flow rates of 0.8 liters per minute and 6.1 liters per minute and a vacuum of 700 mmHg was applied. Outlet gas partial pressures of carbon dioxide of 91 and 353 were recorded. The device reduced carbon dioxide in solution by 82.5% and 36.7%, respectively .

Although the description of the preferred embodiment has been presented, it is contemplated that various changes may be made to the apparatus and/or process of the present invention and such would be changes of form, not substance, and could be made without deviating from the spirit of the present invention. It is therefore desired that the present embodiment be considered in all respects as illustrative, not restrictive, and that reference be made to the appended claims rather than to the foregoing description to indicate the scope of the invention.

Claims

I CLAIM :

1. A process for removing carbon dioxide from a bioreactor system comprising;

(a) providing a bioreactor mixing tank into which living solution is introduced;

(b) providing a hollow fiber membrane filter defining an interior chamber in fluid communication with the bioreactor mixing tank, said filter including a vacuum access port, a bundle of hollow fiber membranes disposed within the interior chamber, each of said hollow fiber membranes defining an interior lumen therewithin;

(c) causing the living solution to flow through the lumens of the hollow fiber membranes;

(d) removing carbon dioxide from the living solution by applying a vacuum to the vacuum access port- -and

(e) recirculating the living solution into the bioreactor mixing tank.

2. The process according to claim 1 wherein removing carbon dioxide from the living solution further comprises a removal efficiency rate of from about 28.5% to about 88.4%.

3. The process according to claim 1 wherein removing carbon dioxide from the living solution by applying a vacuum to the vacuum access port further comprises applying a vacuum pressure of from about 0.5 mmHg to about 760 mmHg.

4. The process according to claim 1 wherein recirculating the living solution into the bioreactor mixing tank further comprises recirculating the living solution in a cross-flow configuration.

5. A device for removing carbon dioxide f om a bioreactor system, said device comprising a hollow fiber membrane filter.

6. The device of Claim 5 wherein said hollow fiber membrane filter further comprises:

(a) a filter housing defining an interior chamber,

(b) at least one vacuum access port operably coupled to said housing,

(c) a bundle of hollow fiber membranes disposed within the interior chamber of said filter housing, each of said hollow fiber membranes defining an interior lumen therewithin.

7. The hollow fiber membrane filter of Claim 6 wherein said hollow fiber membranes comprise a hydrophobic material.

8. The hollow fiber membrane filter of Claim 7 wherein the hydrophobic material is selected from a group consisting of polypropylene, polysulfones, polyvinylidene fluoride, teflon, cellulose esters, polyethylene, and polytetrafloroethylene.

9. The hollow fiber membrane filter of Claim 6 wherein the hollow fiber membranes have an outer diameter of about 260 microns.

10. The hollow fiber membrane device of Claim 6 wherein the hollow fiber membranes have a wall thickness from about 20 microns .

11. The hollow fiber membrane device of Claim 6 wherein said hollow fiber membranes are coated with a biocompatible material.

12. The hollow fiber membrane device of Claim 6 wherein said hollow fiber membranes are coated with teflon.

13. The hollow fiber membrane device of Claim 6 wherein the hollow fiber membranes have an inside diameter of from about 220 microns to about 2000 microns.

14. The hollow fiber membrane device of Claim 6 wherein the hollow fiber bundle has an external surface area of from about 92.9 cm² to about

371,600 cm².

15. The hollow fiber membrane device of Claim 6 wherein the hollow fiber membranes have a pore size of from about 0.001 microns to about 10 microns.

16. The hollow fiber membrane device of Claim 6 wherein the hollow fiber membranes have a packing density of from about 30% to about 75%.

17. The hollow fiber membrane device of Claim 6 wherein the hollow fiber membrane is symmetric or asymmetric.