WO1995027040A1

WO1995027040A1 - Culture media additives for hollow fiber bioreactors

Info

Publication number: WO1995027040A1
Application number: PCT/US1994/003613
Authority: WO
Inventors: Randal A. Goffe
Original assignee: Unisyn Technologies, Inc.
Priority date: 1994-04-01
Filing date: 1994-04-01
Publication date: 1995-10-12
Also published as: JPH08511173A; CA2163618A1; AU7371394A; WO1995027041A1; NO954854D0; AU6766994A; NO954854L; EP0711339A1

Abstract

A hollow fiber bioreactor culture medium which contains a stabilized hemoglobin or a perfluorochemical emulsion is described. The culture medium mediates oxygen demand and utilization. Efficiency of the bioreactor is enhanced.

Description


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   CULTURE MEDIA ADDITIVES
FOR HOLLOW FIBER BIOREACTORS 
FIELD OF INVENTION
This invention relates to improved cell culture media and to the use of such media in the operation of hollow fiber bioreactors. 



   BACKGROUND OF THE INVENTION 
Hollow fiber bioreactors commonly comprise a hollow fiber bundle positioned in a casing. Cells are cultured in the extracapillary space within the casing. Nutrient culture media is circulated through the lumens of the hollow fibers. The prior art is essentially devoid of information regarding culture media modifications which concomitantly enhance oxygen utilization and bioreactor efficiency. 



   SUMMARY OF THE INVENTION
This invention provides cell culture media which contain additives effective to mediate oxygen demand and utilization with consequent improvement in hollow fiber bioreactor efficiency. 



   One embodiment of the invention includes cell culture media containing blood substitutes effective to enhance not only the uptake of oxygen at the oxygenator but also oxygen release and carbon dioxide uptake in the intracapillary circulation loop of the bioreactor. This embodiment of the invention limits the need for carbon dioxide control of media pH. An advantage of this embodiment is that upon reaching reasonable confluence, pH control by use of a carbon dioxide--air mixture can be phased out or replaced by air or pure oxygen. 



   Another embodiment of the invention entails the use of blood substitutes as media additives only in the earliest stage (the inoculum expansion phase) of a long term hollow fiber cell culture operation. 

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  This embodiment of the invention stimulates rapid expansion of viable cells to   quickly   attain confluence. Upon reaching reasonable confluence, such additives are excluded from the media whereupon cell replication rates decline and the metabolic production phase ensues. 



   BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a curve which represents cell production rate in milligrams over time with continuous use of a media additive of this invention. 



   Figure 2 is a curve which represents cell production rate in milligrams over time with short term use of a media additive of this invention. 



   Figure 3 is a schematic illustration of the commercially available bioreactor system utilized to perform of Example I. 



   Figure 4 illustrates the effect of a commercial stabilized bovine hemoglobin (ER1) (0.1% w/v) on glucose uptake rate (GUR). 



   Figure 5 illustrates the effect of a commercial stabilized bovine hemoglobin (ER1) (0.1% w/v) on the ratio of lactate production rate (LPR) to glucose uptake rate (GUR). 



   Figure 6 illustrates the effect of a stabilized bovine hemoglobin (ER1) (0.1% w/v) on cumulative IgG1 production. 



   DETAILED DESCRIPTION OF THE INVENTION
Natural hemoglobin, albeit an oxygen carrier is unstable outside the erythrocyte. Hemoglobin decomposition products are both inefficient oxygen carriers and toxic to the kidneys. 



   This invention entails the use as nutrient media additives of various stabilized hemoglobins 

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 and perfluorochemical emulsions are known to be useful as human blood substitutes. Stabilized hemoglobins and perfluorochemical emulsions useful in the invention are commercially available from the following companies under the tradenames or trademarks indicated:
Biorelease Technologies, Inc. ER-1 (chemically stabilized hemoglobin)
Salem, New Hampshire
Enzon Corporation PEG-hemoglobin (hemoglobin stabilized by reacting with polyethylene glycol)
Piscataway, New Jersey
Somatogen, Inc. rHBl-1 (crosslinked recombinant human hemoglobin)
Boulder, Colorado
Biopure Corporation Formula   One TM   (buffer stabilized hemoglobin)
Boston, Massachusetts
HemaGen/PFC   OxyfluorTM   (perfluorochemical emulsion)
St.

   Louis, Missouri 
This invention provides cell culture media which contain from 0.0001 to 2.0%, preferably from 0.001 to 0.5 percent w/v of a stabilized hemoglobin or a perfluorochemical emulsion. A particularly preferred culture medium contains between 0.001 to 0.1% (w/v) of a stabilized hemoglobin or perfluorochemical emulsion. 



   As Figures 1 and 2 show, the media additives of this invention may be used continuously or only for 

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 a short term, i.e., only during the inoculum expansion phase. Continuous use enables rapid production of relatively large amounts of both cells and biomolecules. Optimal performance may be short lived. 



   Short term use of media containing the additives of the invention enables rapid expansion of the cell inoculum during the inoculum expansion phase followed by long-term, optimal production of biomolecules in the metabolic production phase in the absence of such additives. 



   EXAMPLE I
A 30 day cell culture experiment using 3C11 hybridoma (American Type Culture Collection (ATCC) Accession No. HB8511) is described. The product of interest in the cell culture harvest is a mouse monoclonal antibody IgG1 quantified by radial immunodiffusion assay (RID). 



   Run 1 used a commercially available UniSyn Micro   MouseTM   small scale bioreactor system with a UniSyn (UniSyn Technologies, Inc. , 14272 Franklin Avenue, Tustin, CA 92680) BR 110 bioreactor cartridge (1.5 ft.2 cellulose hollow fibers) fitted with a UniSyn OXY-1 oxygenator (1.0 ft.2, 0.2  m pore size polyethylene fibers) as shown by Figure 3. 



  This system was mounted in an oven for maintaining 

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 constant temperature (37 C). 0.1% (w/v)   ErythrogenTM   (a stabilized form of bovine   hemoglobin,   ER1) purchased from Biorelease Technologies, Inc. , Salem, NH, was added to cell culture media described below. 



  This was recirculated through the fiber lumens of both the bioreactor and oxygenator cartridges and back to the media reservoir. The media flow path is shown by arrows in Figure 3. Pre-mixed C02 and air was passed through the extracapillary space (ECS) of the OXY-1 at a rate of about 75 mL (std)/min counter-current to the direction of the media recirculation loop. The media pH was controlled in the range from 7.0 to 7.4 by adjusting C02 and air flow rate, respectively. 



   As a reference, a similar UniSyn Micro   MouseTM   BR 110 small scale bioreactor with a UniSyn OXY-1 oxygenator was employed in run 2 without bovine hemoglobin added in cell culture media. Run 2 was assembled in an oven for maintaining constant temperature (37 C). Pre-mixed air and C02 was passed through the ECS of OXY-1 oxygenator at a rate of about 70 mL (std) /min. counter-current to the direction of flow in the media recirculation loop. 



  To control the pH between 7.0 to 7.4, the ratio of   C02 to   air was adjusted by using valves on two gas flow meters. 

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   In Run 3 a UniSyn Micro   MouseTM   BR 110 small scale bioreactor with silicone tubing for oxygen mass transfer by diffusion instead of an OXY-1 . oxygenator was used. This bioreactor was mounted in a C02 incubator for maintaining a constant temperature (37 C). Constant pH (between 7.0 to 7.4) in the media was established by setting the ratio of 7% C02 to 93% air in the C02 incubator. 



   Media Conditions:
Run 1 -
Intracapillary space (ICS) - 5% FBS +
4 mM glutamine + 100K unit penicillin/100 mg streptomycin +
1.03g ER1 in 1000 mL DMEM (Dulbecco's
Modified Gagle's high glucose (4.5 g/L) basal medium). ECS - 20% FBS +
4 mM glutamine + 100K unit penicillin/100 mg streptomycin in
1000 mL DMEM
Runs 2 & 3 -
ICS - 5% FBS + 4 mM glutamine + 100K unit penicillin/100 mg streptomycin in 1000 mL DMEM. ECS - the same as that used for run 1. 



   An inoculum containing 5 x 108 3C11 cells (89% viability) was syringed into the ECS of the BR 110 

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 bioreactor by using two sterile, 10 mL syringes with 18 gauge needles. One syringe contained 5 mL ECS media and cells. The second syringe was empty and used to collect the media displaced during the inoculation. Both run 2 and run 3 were inoculated with 4.5 x 108 3C11 (97% viability) in the ECS using the method described above. A standard operating procedure for daily maintenance of cell culture for the Micro   MouseTM   bioreactor system was followed for all three runs. 



   The process parameters in the cell culture system were set up to monitor the glucose uptake, lactate production, NH3 production, and monoclonal antibody (MAb) production. Glucose,   lactate,.and   NH3 sampled from ICS of the BR 110 bioreactor were analyzed by using Kodak Ektachem analyzer. 



  Harvesting of MAb from ECS of the BR 110 bioreactor was typically done at a frequency of three times/week and a volume of 10 mL/each throughout the run. The media used in the ICS of the BR 110 was replaced by the fresh media either when the glucose concentration fell below 1.5 g/L or when lactate concentration was higher than 20 mM in the media. 



  Because the cell growth with time cannot be directly estimated in the hollow fiber bioreactor, the glucose uptake rate (GUR) and lactate production 

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 rate (LPR) can be used as a metabolic finger print. 



  This approach is deemed valid   because   the specific GUR appears to be generally constant in the exponential growth phase of cells. Therefore, higher GUR in the experiment (run 1) relative to the controls (runs 2 & 3), under the same operating conditions, can be used as an indication of increased cell growth and/or productivity. 



   Figure 4 shows the GUR obtained from all three experimental runs. Due to the batch operation, the GUR appears to change periodically between media replacements. The GUR for run 1 appears to be significantly higher for the first 15 days than the other two runs. A relatively high dissolved oxygen concentration (resulting from the recirculating hemoglobin) gives cells a chance to utilize more glucose. While measurements were not made, it is postulated that transport of C02 away from the bioreactor (by hemoglobin), and back to the oxygenator (i.e., gas exchanger unit), is also occurring simultaneously. 



   After 15 days from the start of the cell culture experiment, the cell mass in run 1 was so great that it became difficult to harvest and sample from the ECS ports. 

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   The ratio of LPR to GUR reflects the relative aerobic condition of the cell culture. Figure 4 shows the lowest ratio in run 1 compared to run 2 and run 3. This data indicate that the lactate production has been effectively reduced by improving the oxygen supply, i.e., glucose is metabolized more efficiently to C02 and water. 



   Figure 5 shows the differences in cumulative antibody production for the three runs. Nearly a two-fold increase in IgG1 production was achieved by the addition of 0.1% (w/v) bovine hemoglobin to the BR 110/OXY-1 system (i.e., run 1 compared with run 2). A similar conclusion is drawn when comparing run 1 with run 3. Again, Figure 3 shows that after day 15 antibody productivity fell off dramatically as the over crowded cells began to die. 



  This conclusion is supported by the GUR trace for run 1 in Figure 3 between days 15-30. 



   The optimal concentration of a particular blood substitute in a hollow fiber bioreactor culture medium is influenced by various factors including (i) binding oxygen release efficiency, (ii) degree of stabilization, (iii) concentration effect on media viscosity, (iv) relative cytotoxicity, and (v) oxygen partial pressure. 

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   Table 1 reports selected data from the use of a plurality of concentrations of stabilized hemoglobin (ER-1) in a medium and a bioreactor system as described in Example I. 



   Table 1 
Effect of ER1* Concentration on
Cumulative IgG1 Production 
Percentage of Stabilized 
Bovine Hemoglobin, ER1* (w/v) in ICS 
 EMI10.1 
 
<tb> 
<tb> ** <SEP> 0.1 <SEP> 0.01 <SEP> 0.000
<tb> Day <SEP> 5 <SEP> 25.3 <SEP> mg <SEP> 30.6 <SEP> mg <SEP> 7.8 <SEP> mg
<tb> Day <SEP> 15 <SEP> 126.2 <SEP> mg <SEP> 72.4 <SEP> mg <SEP> 35.1 <SEP> mg
<tb> Day <SEP> 30 <SEP> 177.2 <SEP> mg <SEP> 99.4 <SEP> mg <SEP> 117.8 <SEP> mg
<tb> (Day <SEP> 29)
<tb> 
 * A form of stabilized hemoglobin (ErythrogenTM) used in Example II. 



  ** Time in Culture.

Claims

CLAIMS: 1. A cell culture medium comprising a stabilized hemoglobin or a perfluorochemical emulsion.

2. A cell culture medium as defined by claim 1 containing 0.001 to 0.1% w/v of a stabilized hemoglobin or a perfluorochemical emulsion.

3. A cell culture composition as defined by claim 1 or claim 2 in which said stabilized hemoglobin-is stabilized bovine hemoglobin.

4. A cell culture composition as defined by claim 1 and claim 2 in which said stabilized hemoglobin is crosslinked recombinant human hemoglobin.

5. In a method for the culture of cells in a hollow fiber bioreactor in which cell nutrient medium is circulated through the lumens of the hollow fibers in said bioreactor, the improvement which comprises circulating through said hollow fiber lumens a nutrient medium which contains a stabilized hemoglobin or a perfluorochemical emulsion.

6. A method as defined by claim 5 in which said nutrient medium is circulated through said lumens only during the inoculum expansion phase of said cells cultured in said bioreactor. <Desc/Clms Page number 12>

7. A method as defined by claim 5 or claim 6 in which said culture medium contains 0.001 to 0.1% (w/v) of stabilized bovine hemoglobin.

8. A method as defined by claim 5 or claim 6 in which the pH of said nutrient medium is controlled by the introduction of carbon dioxide or a mixture of carbon dioxide and air into said lumens of said hollow fibers.

9. In a method for the culture of cells in a hollow fiber bioreactor in which cell nutrient medium is circulated through the lumens of the hollow fibers in said bioreactor, the improvement which comprises controlling the pH of said medium by the introduction of carbon dioxide or a mixture of carbon dioxide and air into said lumens and thereafter terminating said carbon dioxide introduction and controlling pH in the medium by air or pure oxygen.