WO1995004813A1

WO1995004813A1 - Hollow fiber bioreactor system with improved nutrient oxygenation

Info

Publication number: WO1995004813A1
Application number: PCT/US1994/008849
Authority: WO
Inventors: Randal A. Goffe; David M. Lowrey; Yuan Shi
Original assignee: Unisyn Technologies, Inc.
Priority date: 1993-08-06
Filing date: 1994-08-04
Publication date: 1995-02-16
Also published as: AU7520894A

Abstract

An improvement in small scale hollow fiber bioreactor systems is described. Small scale bioreactor systems provide a hollow fiber surface area of from 0.2 to 500 square feet. Oxygenation in such systems is improved by the inclusion of a hollow fiber oxygenator (OXY-1) which sparges oxygen containing gas into the nutrient.

Description

HOLLOW FIBER BIOREACTOR SYSTEM WITH IMPROVED NUTRIENT OXYGENATION

This application is a continuation of United States application Serial No. 08/102,776 filed 6 August 1993.

FIELD OF INVENTION

This invention provides an improvement in the oxygenation in small scale bioreactors.

BACKGROUND OF THE INVENTION

Various large scale hollow fiber bioreactors for the culture of various anchorage dependent and suspended cells are known. See, e.g., U.S. patents 3,821,087, 4,220,725, 4,804,628, 4,889,912 and 5,126,938.

More recently small scale hollow fiber bioreactors which have a hollow fiber membrane surface area of from less than about 1900 square feet have been designed and used for hybridoma cell culture to produce monoclonal antibodies. One such small scale bioreactor, the "Micro Mouse" (™) having a membrane surface area of 1.5 square feet, is described in copending United States patent application Serial No. 07/774,828 assigned to UniSyn Technologies, Inc.

Small scale hollow fiber hybridoma cell culture as typically performed yields about 100 mg to about 1000 mg of monoclonal antibodies per hollow fiber cartridge. Actual productivity is directly dependent upon the specific hybridoma cell line employed. At this scale of biomanufacturing by cell culture means, the market place is very price sensitive. Small scale bioreactors have therefore been engineered to be as simple as possible, while retaining good productivity.

Two main methods for oxygenation are now used to accommodate cell culture in small scale hollow fiber bioreactors:

(i) simple diffusion of gas through silicone tubing carrying the recirculating media; and

(ii) sparging gas directly into the media reservoir.

While the former is highly efficient, the entire cell culture system has to be placed in a CO2 incubator, thus limiting its use and the possibility of incorporating features for process control and automation.

The sparging approach goes some way toward liberating the cell culture system from the confines of an incubator, but it is much less efficient than diffusion through silicone tubing. Furthermore, direct sparging does not address the fact that the supply of nutrients and of oxygen to the cell culture are inseparably linked because both are contained in the media. Also of concern is the fact that direct sparging of gas into the media reservoir may result in bubbles that pass into the bioreactor and cause deleterious effects. Even the media flow rate has to be restricted to a relatively narrow range above which bubbles are more likely to be entrained into the bioreactor thus limiting the ability of the user to vary the parameters independently. A variety of approaches have been proposed to deal with the short comings of the direct sparging approach, while taking advantage of a system requiring no incubator, and producing an affordable product. One such system is the BioFarm 2000 developed by Arcos Engineering, which employs three small scale cell culture bioreactors (1.5 square feet cartridges) .

SUMMARY OF THE INVENTION

This invention provides a small scale bioreactor system of improved efficiency. The system of the invention includes a microporous hollow fiber oxygenator through which media passes prior to introduction into the lumens of the bioreactor hollow fibers. Oxygen containing gas is sparged from the pores of the oxygenator hollow fibers into the media as it passes through the oxygenator. Antibody production is substantially greater than that observed in a similar diffusion based system.

This invention accordingly provides:

(i) Small scale cell culture systems that operate outside of an incubator, the performance of which is at least equivalent to that of a diffusion-based system requiring an incubator, (ii) Relatively independent control over process parameters, oxygenation level and supply of nutrients by perfusion of media.

(iii) The operation of a plurality of bioreactors as a means of scaling up lot sizes, or as a means of culturing multiple cell lines simultaneously. DESCRIPTION OF THE FIGURES

Figure 1 is a schematic drawing of one form of oxygenator useful in the invention, i.e., the OXY-1 oxygenator available from UniSyn Technologies, Inc., 14272 Franklin Avenue, Suite 106, Tustin, California 92680.

Figure 2 is a schematic drawing of one form αf bioreactor useful in the invention, i.e., the BR170 bioreactor available from UniSyn Technologies, Inc.

Figure 3 is a schematic view of one embodiment of a commercial small scale bioreactor system, specifically a Micro Mouse system, available from UniSyn Technologies, Inc. fitted with a UniSyn OXY-1 oxygenator.

Figure 4 shows the configuration of the Arcos BioFarm 2000 unit available from UniSyn fitted with the OXY-1 oxygenator.

Figures 5, 6, 7, and 8 depict data from a 26-day run in which the 3C11 hybridoma cell line was cultured in a Micro Mouse BR170 bioreactor fitted with silicone tubing for the diffusion of oxygen containing gas into the circulating media (run 1) and fitted with a OXY-1 hollow fiber oxygenator (run 2) .

Figure 5 is a plot of cumulative glucose consumption in grams as a function of time. Solid squares ■ indicate run 1; partially filled squares |^* indicate run 2.

Figure 6 is a plot of cumulative lactate production in grams as a function of time. Solid squares ■£ indicate run 1; partially filled squares r« indicate run 2. Figure 7 is a plot of cumulative MAb (monoclonal antibody) in milligrams (mg) against time. Solid squares H indicate run 1; partially filled squares PH^" indicate run 2.

Figure 8 is a plot of monoclonal antibody (MAb) collected at each harvest (mg/mL/day) for the duration of the experiment. Solid squares ■■ indicate run 1; partially filled squares | r] indicate run 2.

Figures 9A, 9B and 9C show comparative cell culture data for experiments using a BioFarm 2000 hollow fiber bioreactor system equipped with an OXY-1 oxygenator. Cumulative antibody production, glucose uptake and lactate production over the course of the experiment are shown. Solid squares VH indicate run 1; partially filled squares

indicate run 2. GENERAL DESCRIPTION OF THE INVENTION

The invention generally comprises a bioreactor system including a hollow fiber bioreactor, means for passing an oxygenated nutrient through the lumens of the hollow fibers in said bioreactor and means for oxygenating the nutrient prior to introduction into said lumens of said bioreactor hollow fibers.

An important aspect of the invention resides in the provision in such a system of a hollow fiber oxygenator.

The Hollow Fiber Oxygenator

Commercially available hollow fiber oxygenators are appropriate for use in the invention. The UniSyn OXY-1 oxygenator depicted by Figure 1 is preferred. As Figure 1 shows, the OXY-1 device 1 comprises a jacket 10 having end caps 11 and 12 fitted with nutrient inlet and outlet ports 13 and 14. The jacket 10 contains an assembly of generally parallel hollow fibers 15 surrounded by an extracapillary space (ECS) 16. The hollow fibers in the bundle are each separated by a substantially equal distance and take the general form of a woven mat with bundle spacing maintained with threads 17 interwoven between the fibers. A controlled gas mixture, e.g., of air, CO2 and or oxygen, is introduced through port 13 into the lumens of the oxygenator hollow fibers. Gas mixture is removed through port 14.

The hollow fibers of the oxygenator are formed from microporous polyethylene or another hydrophobic polymer to provide a high surface area liquid gas interface. The gas mixture is sparged from the oxygenator hollow fibers into the media which flows counter-currently through the extracapillary space. The Hollow Fiber Bioreactor

Referring to Figure 2, the bioreactor 19 includes a jacket 20, headers 21 and 22 and a hollow fiber bundle 23.

The hollow fiber bundle 23 is separated by space 24 from the interior wall of the jacket 20. Oxygenated nutrient media is introduced into the bioreactor through port 25 in header 22 and removed through port 26 in header 21. Appropriate ports 27 and 28 are provided for the introduction of fresh nutrient and withdrawal of product from the extracapillary space in the bioreactor.

The hollow fiber bioreactor 19 preferably includes hollow fibers formed from polymethylmethacrylate, cellulose, cellulose acetate or similar polymers having a molecular weight cutoff range of about 1,000 to about 150,000 daltons. Description of One Form of

The System of the Invention Figure 3 schematically illustrates one form of the invention. This system includes a media reservoir bottle 29 provided with an outlet means 30 connected to peristaltic pump 31 through media flow line 32. Effluent from the pump 31 passes through the line 32 and the filter 33 and thence to the hollow fiber oxygenator 34.

As shown in the Figure, the oxygenator 34 includes a manifold port 35 for the introduction of air or a mixture of air and oxygen or carbon dioxide. Gas may be withdrawn from the extracapillary space through the port 36.

Air is introduced into the system through the line 37 and carbon dioxide is introduced into the system through line 38. The air and carbon dioxide streams are combined at "Y" 39 and introduced into the oxygenator manifold port 35 through the line 40.

The media passes from the oxygenator 34 through the line 41 into the hollow fiber bioreactor 44 in which the hollow fiber membrane surface area preferably is from 0.2 to 500 square feet.

In the preferred practice of the invention, a UniSyn BR170 bioreactor assembly as depicted by Figure 2 having a hollow fiber surface area of about 1.5 square feet is utilized.

Oxygenated media from the line 43 passes through the lumens of the hollow fibers in the bioreactor 44 and thereafter through the line 45 for return to the media reservoir 29 through the line 47 or for withdrawal from the system through line 48. EXAMPLE I

This example entails two 26 day runs to culture the 3C11 hybridoma cell line in a UniSyn Micro Mouse BR170 small scale bioreactor in a system generally depicted by Figure 3. In run 1, the bioreactor was equipped with a silicone tubing for oxygen mass transfer whereas in run 2, the bioreactor was fitted with a UniSyn OXY-1 oxygenator as shown by Figure 2.

Run 1 was mounted in a C0 incubator for maintaining a constant temperature (37^βC) and a constant pH (from 7.0 to 7.2) in the bioreactor system by setting the ratio of 7% CO₂ to 93% air in incubator. For run 2, the bioreactor system was mounted in an oven for maintaining constant temperature (37°C) . Pre-mixed gas with CO2 and air was passed through the oxygenator at a rate of about 50 to 70 mL/min counter-current to the direction of the loop recirculating medium in the intracapillary space (ICS) of the bioreactor. To control the pH in the range from 7.0 to 7.4, the ratio of CO₂ to air can be adjusted by using two gas flow meters.

To illustrate the effect of oxygen mass transfer on cell metabolism, a high cell density, 8 x 10⁸ cells with the viability of 97% was inoculated into the extracapillary space (ECS) of each bioreactor by using two sterile, 10 ml syringes with attached needles. One syringe will contain 10 ml of ECS medium and the cells. The second syringe is empty and used to collect the medium displaced during the inoculation. The standard operating procedure for the Micro Mouse bioreactor system was followed for daily maintenance of cell culture. The process parameters in the cell culture systems were set up to monitor glucose uptake, lactate production, NH₃ production, and MAb production. Since one cannot directly estimate the cell growth with time in the hollow fiber bioreactor, the MAb production may be regarded as a major influence reflecting the effect of oxygen mass transfer using either the oxygenator or silicone tubing. Harvesting of MAb from ECS of the bioreactor was done at a frequency of 3 times/week and a volume of 10 ml/each. MAb production was analyzed by using radial immunodiffusion assay, and glucose, lactate, and NH₃ sampled from ICS of the bioreactor were analyzed by using a Kodak Ektachem Analyzer. The medium in ICS of the bioreactor was replaced by the fresh medium when either the glucose concentration fell below 1.5 g/L or lactate concentration was higher than 20 mM in medium.

Figure 5 shows the cumulative glucose consumption for both runs 1 and 2. Significantly higher cell metabolism occurred in run 2 compared to run l, suggesting that the oxygenator helps to reduce the resistance of oxygen mass transfer. The relatively high dissolved oxygen concentration in the ICS medium results in more efficient cell growth. A limited oxygen supply may lower the cell metabolism is lowered even though the glucose and other nutrient can be adequately supplied by perfussion from ICS to ECS of the bioreactor.

As a result, monoclonal antibody (MAb) production shown in run 2 is increased relative to that in the control (run 1) . As Figure 5 shows, the glucose uptake is significantly increased as compared with the data obtained from run 1. It appears that the oxygenator increases the availability of oxygen to the cells. This relatively high dissolved oxygen concentration in the medium facilitates more efficient cell metabolism at nutrients such as glucose than in run 1,

As Figure 6 shows, the cumulative lactate production for runs 1 and 2. More efficient cell growth is evidenced by a comparison of the curve for the bioreactor with the oxygenator and the curve for the bioreactor with silicone tubing.

Figure 7 is a plot of the cumulative MAb production in milligrams against time.

After about day 5, cumulative antibody production remains consistently higher for run 2.

Good economics and the biopharmaceuticals market dictate that bioreactors should produce relatively large quantities of MAb in the shortest possible time. The run 2 data accords with that objective.

EXAMPLE II

This Example compares the results of two experiments conducted in the UniSyn Technologies, Inc. BioFarm 2000 cell culture system. The components of this system are apparent from the Figure 4 schematic.

The BioFarm is a benchtop hollow fiber cell culture system for the continuous production of mammalian cell culture products. The system is reusable and supplied with a flowpath which can be used up to 3 times before replacement. They system provides several automated features such as gas control, pH control, temperature control and oxygen supplementation. The system can use 1-3 hollow fiber cartridges which are configured with parallel flowpath so that different cell lines can be used in each bioreactor. Bioreactors of 10,000, 30,000, or 70,000 molecular weight cutoff (MWCO) are utilized. Cells are innoculated into the 13 ml extracapillary space (ECS) of the hollow fiber bioreactor(s) and proliferate to tissue-like densities. Products secreted by the cells (e.g., monoclonal antibodies, viruses, antigens) are too large to cross the pores of the membranes. These products accumulate and concentrate in the ECS where they can be aseptically harvested via the ECS ports with sterile syringes. Product harvesting is typically done three time per week. As the product is removed it is simultaneously replaced with fresh complete medium to maintain a productive culture environment.

Nutrient media, residing in a one liter bottle, circulates through the lumen of the hollow fibers by means of a peristalic pump. This allows diffusive exchange of low molecular weight nutrients (e.g., glucose, oxygen) and metabolic wastes (e.g. , lactate, ammonia) to occur between the recirculating stream and the cells. Fresh media is continuously replaced and waste media removed by the instrument based on a value set by the operator. Recirculating media is usually a commercially available formulation (e.g., DMEM, RPMI 1640) which contains no serum whereas the ECS media consists of the same media formulation with serum added. Typically, the ECS media is the same formulation used for the cells in standard cell culture. The lumen flow is controlled by pinch valves so that media flows through only one bioreactor at a time. In a three bioreactor configuration for instance, this flow control permits lumenal media for one minute in every three for each bioreactor.

The BioFarm has a unique gas control system which circulates an operator controlled gas mixture of air, air/C02 or air/oxygen to allow optimal gas control.

An oxygenator cartridge (OXY-1) is used to sparge media with the gas mixture. Due to the rapid nature and high capacity of gas exchange with the OXY-1 oxygenator, fluctuation in pH due to cell growth is eliminated and optimal oxygen delivery to the cells is achieved. The gas mix flow into the media is directly proportional to the selected lumen media recirculation rate.

In a first experiment, gases required to maintain healthy cell growth is sparged into the intracapillary space by an OXY-1 oxygenator. A second run was performed with no oxygenator cartridge but with gas sparging means to provide the necessary cell culture gases. These two cell culture experiments were otherwise identical in all respects with the exception that the experiment with the OXY-1 cartridge also contained 5% fetal bovine serum (FBS) in the intracapillary space media to provide a slight positive effect on cultures.

The OXY-1 cartridge provides 1 sq. ft. of surface area of microporous polyethylene for gas/media diffusion which is an approximately 10-fold increase over the available surface area without the cartridge. A mixture of CO₂ and air (10% v/v CO₂) is sparged through the cartridge. Three, 10,000 mol wt. cutoff, cellulosic hollow fiber cartridges were inoculated with 10⁸ cells. Media was fed at 120 ml per day initially and gradually increased to 960 ml per day. The extracapillary space was harvested three times a week (10 ml per cartridge) beginning at day 4 with DMEM media containing 20% v/v FBS.

Figures 9A, 9B and 9C show the differences in cumulative antibody production, glucose uptake rate and lactate concentration with and without the oxygenator. Nearly a five-fold increase in antibody production was achieved by the addition of an OXY-1 oxygenator to the BioFarm 2000. Similar increases in glucose consumption and lactate production were noted.

This experiment indicates that addition of an oxygenator to the BioFarm 2000 small scale hollow fiber bioreactor instrument, when nutrient supply is not the limiting factor, dramatically increases its performance.

Claims

CLAIMS:

1. In a hollow fiber bioreactor system including at least one bioreactor, said bioreactor having a plurality of hollow fibers said hollow fibers having lumens, means for passing a nutrient medium through the lumens of the hollow fibers in said at least one bioreactor and means for oxygenating the nutrient medium prior to introduction into said lumens, wherein the improvement comprises a hollow fiber oxygenator, said hollow fiber oxygenator comprising a shell, a hollow fiber bundle in said shell, extracapillary space between the fibers of said bundle and between said bundle and the interior wall of said shell, means for the passage of nutrient medium through said extracapillary space of said hollow fibers and means for passing an oxygen containing gas through the lumens of said fibers to sparge said gas into said nutrient medium, said hollow fibers in said hollow fiber bundle being formed from a microporous hydrophobic synthetic resin.

2. A hollow fiber bioreactor system as defined by claim 1 in which said hollow fibers in said hollow fiber bundle are formed from microporous polyethylene.

3. A hollow fiber bioreactor system as defined by claim 1 or claim 2 in which the surface area of said hollow fibers in said bioreactor is less than 1900 square feet.

4. A hollow fiber bioreactor system as defined by claim 1 or claim 2 in which the surface area of said hollow fibers in said bioreactor is from 0.2 to 500 square feet. 5. A hollow fiber bioreactor system as defined by claim 1 or claim 2 in which the surface area of said hollow fibers in said bioreactor is 1.

5 square feet.

6. A hollow fiber bioreactor system as defined by claim 1, further including a plurality of said at least one hollow fiber bioreactor arranged for parallel operation, wherein said hollow fiber oxygenator provides oxygenated nutrient medium simultaneously to said plurality of bioreactors.

7. A hollow fiber bioreactor system comprising:

(i) a plurality of hollow fiber bioreactors, said bioreactors having a plurality of hollow fibers, said hollow fibers having lumens arranged for operation in parallel; each of said bioreactors having a hollow fiber membrane surface of from 0.2 to 500 square feet;

(ii) means for passing a nutrient medium simultaneously through the lumens of the hollow fibers in each of said plurality of hollow fiber bioreactors;

(iii) a single means for oxygenating said nutrient medium prior to introduction of said medium into the lumens of said hollow fibers of said plurality of bioreactors, said means for oxygenating comprising a hollow fiber oxygenator, the hollow fibers in said oxygenator being formed from a microporous synthetic resin.