Biotechnol. frog. 1990, 6, 142- 148
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Structural Features of Nonionic Polyglycol Polymer Molecules Responsible for the Protective Effect in Sparged Animal Cell Bioreactors David W. Murhammert*$and Charles F. Goochee*’§ Department of Chemical Engineering, University of Houston, Houston, Texas 77204-4792 and Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025
T h e nonionic surfactant Pluronic F-68 poly01 is commonly used to protect cultured animal cells from the detrimental effects of sparging. In this study we investigated the structural features of the Pluronic F-68 molecule responsible for this protective behavior. Poly(oxyethy1ene)-poly(oxypropy1ene) block copolymer polyols of various molecular weights and percentages of hydrophobe (poly(oxypropylene)), including both Pluronic and reverse Pluronic polyols, were considered. The potential toxicity of these agents was examined in the absence of sparging (i.e., in spinner flasks) by using the attachment-independent Sf9 insect cell line as a model system. Each poly01 resulted in one of three distinct types of behavior in these spinner flask experiments: (1)cells lysed a t a n exponential rate, (2) inhibition of cell growth (Le., no net cell growth), or (3) uninhibited cell growth. I t was then shown that all of the Pluronic and reverse Pluronic polyols that did not inhibit cell growth provided protection from sparging in the bioreactors used in this study; thus, finding a poly01 that protected cells was synonymous with finding one that did not inhibit cell growth. The ability of these poly01s to protect animal cells in sparged bioreactors was found to correlate well with the hydrophilic-lipophilic balance (HLB). Those polyols with the largest HLB values were found t o be protective agents. These poly(oxyethy1ene)-poly(oxypropy1ene) poly01s were also shown to be more effective protective agents than pure poly(oxyethy1ene); thus, the presence of the hydrophobe (poly(oxypropy1ene)) is important in their ability t o serve as protective agents.
Introduction There is an increasing need for growing animal cells in large-scale bioreactors to produce proteins of therapeutic and diagnostic value. In large-scale animal cell bioreactors, sparging is the most practical method of supplying oxygen to the culture medium. Unfortunately, sparging can damage mammalian cells (Kilburn and Webb, 1968; Handa et al., 1987) and insect cells (Murhammer and Goochee, 1988). The specific mechanism of sparging damage is unknown, although it appears to involve events near the sparger and/or near the medium surface (Handa-Corrigan et al., 1989; Tramper et al., 1987). Addition of the nonionic block copolymer Pluronic F68 to the culture medium protects mammalian cells (Kilburn and Webb, 1968; Handa et al., 1987; HandaCorrigan, 1989; Passini and Goochee, 1989; Reuveny et al., 1986; Radlett et al., 1971) and insect cells (Murhammer and Goochee, 1988; Maiorella et al., 1988) from the detrimental effects of sparging. Pluronic F-68 is a nonionic block copolymer with an average molecular weight of 8400, consisting of a center block of poly(oxypropy1ene) (20% by weight) and blocks of poly(oxyethy1ene) a t both ends (Figure 1A; Table I) (Stanton, 1957; Swim and Parker,
* Author to whom correspondence should be addressed. +
University of Houston.
* Current Address:
Department of Chemical and Biochemical Engineering, The University of Iowa, Iowa City, IA 52242. Stanford University.
*
8756-7938/90/3006-0142$02.50/0
Polyoxyethylene Block
Polyoxypropylene Block
Polyoxyethylene Block
Block
Block
H O -(CH,-CHOj,
Block
(B)
H - (CH,- CH3,
- (0- CH, - CHJx-OH
Polyoxyethylene Block
Hydrocarbon Block
(C)
Figure 1. (A) Structure of Pluronic polyols. (B) Structure of reverse Pluronic polyols. (C) Structure of Plurafac linear alcohol ethoxylates.
1960; Schmolka, 1972, 1977). The specific mechanism of Pluronic F-68 protection is unclear. Handa et al. (1987, 1989) hypothesized that the protection is due to the formation of a stable foam layer on the medium surface that
0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990,Vol. 6, No. 2
Table I. Properties of the Pluronic Polyols Tested and Their Effect on Cell Growth in 50-mL Spinner Flasks and Airlift Bioreactors.
143
Table 11. Properties of the Reverse Pluronic Polyols Tested and Their Effect on Cell Growth in 50-mL Spinner Flasks and Airlift Bioreactors
wt % hydrophobe HLB av poly01 pluronic (poly(oxypropy1ene)) range polyol molec wt Exponential Cell Lysis in Spinner Flask Studies 2000 90 1-7 L-61 4400 90 1-7 L-121 4950 70 7-12 P-103 5750 70 7-12 P-123 12-18 P-84 4200 60 5900 60 12-18 P-104
reverse av poly01 wt % hydrophobe HLB Pluronic poly01 molec wt (poly(oxypropy1ene)) range Exponential Cell Lysis in Spinner Flask Studies 25 R-2 3100 80 1-7
Inhibition of Cell Growth in Spinner Flask Studies 1900 50 12-18 6600 50 12-18
Uninhibited Cell Growth in Spinner Flask Studies and Protection in Airlift Bioreactor 1950 50 12-18 10 R-5 4550 20 18-23 10 R-8
P-65 P-105
Uninhibited Cell Growth in Spinner Flask Studies and Protection in Airlift Bioreactor L-35 1900 50 18-23 F-127 12600 30 18-23 20 >24 F-38 4700 20 >24 F-68 8400 14600 20 >24 F-108 a The medium was supplemented with 0.2% (w/v) of the respective polyol. The "HLB" value is the hydrophilic-lipophilic balance as discussed in the text. The letters L, P, and F in the Pluronic poly01 names represent liquid, paste, and flake, respectively. The first 2 digits of a 3-digit poly01 number, or the first digit of a 2-digit poly01 number, are indicative of the hydrophobe (poly(oxypropylene)) molecular weight, e.g., 6,8,12 for hydrophobe molecular weights of 1750, 2250, and 4000, respectively. The last digit in the poly01 number represents approximately l/lOth the percentage of hydrophile (poly(oxyethy1ene)) in the molecule. (Data were compiled from (Schmolka, 1972, 1977; BASF Publications, 1987, 1988).)
the cells do not penetrate; thus, the cells are not subject to damage at the medium surface. We have hypothesized that an interaction of Pluronic F-68 with the cell membrane plays an important role in the protective effect (Murhammer and Goochee, 1988). Pluronic F-68 is just one of many related Pluronic (Figure 1A) and reverse Pluronic polyols (Figure 1B). The Pluronic and reverse Pluronic poly01 families contain polymers with a wide variety of molecular weights and percentages of hydrophobe (poly(oxypropy1ene))(Figure 1A,B; Tables I and 11). In this study we examined many Pluronic and reverse Pluronic polyols to determine the structural features of the Pluronic F-68 molecule responsible for its protective effect. We investigated the effects of the relative sizes, relative positions, and molecular weights of the poly(oxypropy1ene) and poly(oxyethy1ene) blocks on the ability of a given poly01 molecule to serve as a protective agent. Poly(oxyethy1ene) was then tested to determine if the hydrophobic portion of the Pluronic F68 molecule is necessary for its protective effect. In addition, we studied the possibility of using a diblock surfactant of the Plurafac family (Figure IC) as a protective agent. Plurafacs contain a saturated hydrocarbon chain for the hydrophobe and poly(oxyethy1ene) as the hydrophile.
Materials and Methods Cells, Medium, and Protective Agents. The cell line used in this study, Spodoptera frugiperda Sf9 insect cells, was generously provided by Max Summers, Department of Entomology, Texas A&M University. These cells are also available from the American Type Culture Collection (catalog no. CRL 1711). The cells were maintained in 25- and 75-cm2 tissue culture flasks (Corning Glass Works, Corning, NY). Cells grown in 50-mL spinner flasks (Model 1967-00050, Bellco Biotechnology, Vineland, NJ) a t 27 "C and 100 rpm were used to seed the spinner flasks
Inhibition of Cell Growth in Spinner Flask Studies 3600 60 7-12 25 R-4 4250 50 7-12 25 R-5 8550 20 12-18 25 R-8
The medium was supplemented with 0.2% (w/v) of the respective polyol. The "HLB" value is the hydrophilic-lipophilic balance as discussed in the text. The first 2 digits of a reverse poly01 number is approximately 1 % of the hydrophobe (poly(oxypropy1ene)) molecular weight. The "R" is indicative of a reverse Pluronic polyol. The last digit in the poly01 number represents approximately 1/ 10th the percentage of hydrophile (poly(oxyethy1ene)) in the molecule. (Data were compiled from (Schmolka, 1972,1977; BASF Publications, 1987, 1988).)
and bioreactors. Cells were grown in TNM-FH medium (Summers and Smith, 1987) supplemented with 50 wg/ mL gentamycin sulfate (Sigma Chemical Co., St. Louis, MO), 2.5 pg/mL Fungizone (Gibco Laboratories, Grand Island, NY) and 5% heat-inactivated (45 min a t 56 OC) fetal bovine serum (Hyclone, Logan, UT). The osmolality and pH of the medium were 315 f 10 mmol/kg and 6.2 f 0.1, respectively. Commercial-grade Pluronic polyols, reverse Pluronic polyols, and Plurafacs were generously provided by the BASF Corp. (Parsippany, NJ). Plurafacs A-38 and A-39 have average molecular weights of approximately 1450 and 2600, respectively, and contain 20% and 10% saturated hydrocarbon, respectively (Heffner and Holland, personal communication). The hydrophilielipophilic balance (Griffin, 1949, 1954, 1955, 1956; Becher and Birkmeier, 1964) is 20 and 24 for Plurafacs A-38 and A-39, respectively (BASF publication, 1988). Poly(ethy1ene oxide-propylene oxide) of average molecular weight 8750 (ratio of ethylene oxide to propylene oxide of 5 to 1) [PEO:PPO (5:1)] was purchased from Polysciences, Inc. (Warrington, PA). This substance has the same general structure as the Pluronic polyols (see Figure 1A) and is very similar to Pluronic F-68 in percentage of hydrophobe and total molecular weight. Poly(oxyethy1ene) (also called poly(ethy1ene glycol)), average molecular weight 8000, was purchased from Sigma. Poly(oxyethylene),average molecular weight 2000, was purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Dow Corning medical-grade antifoam C was generously provided by Dow Corning Corp. (Midland, MI). Solutions (10% (w/v)) of the Pluronics, reverse Pluronics, Plurafacs, and poly(oxyethylene) were sterilized by autoclaving (15 min at 121 "C). These solutions were used to supplement medium with 0.2% (w/v) of the respective agent to determine the effect on cell growth in the absence of sparging (Le., in 50-mL spinner flasks maintained at 27 "C and 100 rpm). The solutions (also sterilized by autoclaving) prepared for use in the sparged bioreactor experiments (in both the bioreactors and the spinner flask controls) contained 0.1% (v/v) antifoam C in addition to 10% (w/v) of the respective Pluronic, reverse Pluronic, Plurafac, or poly(oxyethy1ene). These solutions were used to supplement the medium in the sparged bioreactor experiments
Biotechnol. Prog., 1990, Vol. 6, No. 2
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Table 111. Summary of the Protective Effect (as Given by “PDT Ratio”) of Various Additives in Sparged Bioreactorss
additive Pluronic F-68 (0.1%) F-68 (0.2%) F-68 (G.l%) F-68 (0.2%) F-38 (0.2%) F-108 (0.2%) F-127 (0.2%) L-35 (0.2%) PE0:PPO (5:l) (0.2%) 10 R-8 (0.2%) 10 R-5 (0.2%) MW = 8000 (0.2%) MW = 8000 (0.2%) MW = 2000 (0.2%) MW = 2000 (0.2%)
bioreactor Polyols and airlift airlift agitated agitated airlift airlift airlift airlift airlift
PDT ratiob foam formation PE0:PPO (5:l) 1.26 f 0.33‘ excessive 1.15 f 0.31‘ moderate 1.31 f 0.50‘ none 1.03, 1.42 none 1.12 moderate 1.29 excessive 1.15 excessive 1.10 slight 0.99 slight
Reverse Pluronic Polyols airlift 0.97 airlift 1.23, 1.25 Poly(oxyethy1ene) airlift d agitated 1.15, 1.31 airlift e agitated f
moderate none to slight
IO7
i
-Q-
-. 103
Uninhibited cell growth Inhibition of cell growth
3 0
50
100
150
200
250
Hours in culture
moderate none moderate none
’The degree of foam formation on the medium surface is indicated as “excessive” (foaming results in a loss of cells late in the run thus decreasing the maximum cell density in the bioreactor when compared to that obtained in the spinner flask control), “moderate” (a foam layer on the culture surface approximately 1-3 cm thick, resulting in minimal loss of cells), ”slight” (a foam layer on the culture surface is clearly present but is less than 1 cm thick), and -none’’ (no noticeable foam layer is present). Data were obtained in the Applikon agitated, sparged bioreactor and the Ventrex airlift bioreactor. The medium in both the bioreactor and control spinner flask was supplemented with 5% FBS, antibiotics, 20 ppm antifoam c, and the indicated concentration of the potential protective agent. “PDT ratio” is the ratio of population doubling time obtained in the given sparged bioreactor to that obtained in the spinner flask control. Values given with 95% confidence levels of the mean based on three runs. Minimal to no protection, Le., one run resulted in no net cell growth, while two runs resulted in exponential cell death. e No protection, Le., two runs with exponential cell death. f Variable protection, two runs with no net cell growth and runs with PDT ratios of 1.21 and 1.48. with 0.2% (w/v) of the respective agent and 20 ppm antifoam. Cell Counts and Population Doubling Time. Cell counts were measured with a Coulter multisizer. All particles in the range of approximately 9-25 pm were considered as cells. Since there was some minor cell clumping, the numbers obtained with the Coulter multisizer are probably slightly low. The population doubling times were calculated from a least-squares fit of the growth curve data (counts from Coulter multisizer) in the exponential growth region. Agitated, Sparged 3-L Bioreactor and Airlift Bioreactor. A 3-L water-jacketed bioreactor (operating volume of approximately 1.5 L) with a scoping (Le., nonvortexing) marine impeller and an air-inlet pipe with a sparger was used (Applikon, Foster City, CA). The sparger consists of seven holes, each with a diameter of approximately 1 mm, on the underside of a tube situated directly below the impeller. An air pump was used to supply air to the bioreactor a t approximately 100 mL/ min. (i.e., a sparging rate of approximately 0.07 VVM). The dissolved oxygen concentration was maintained above 50% (but under 100%) of air saturation by the addition of oxygen (extra dry grade) as needed. The gases were sterilized by passing through a Millex-FG,, 0.2-pm filter unit (Millipore Corp., Bedford, MA) prior to introduction into the bioreactor. Due to the effectiveness of the bicarbonate buffer system in the medium, pH control was not necessary. Temperature and the agitation rate were maintained at 27 “C and 200 rpm, respectively. A t this
Figure 2. Three different types of cell growth curves obtained in 50-mL spinner flasks with medium supplemented with 0.2% (w/v) of the respective Pluronic polyol, reverse Pluronic polyol, Plurafac, or poly(oxyethy1ene) as summarized in Tables I a n d I1 and discussed in the text.
agitation rate, bubbles rose in the bioreactor without breakup by the impeller. Disposable airlift bioreactors (Cellift model) were generously provided by Ventrex Laboratories (Portland, ME). The operating volume of these bioreactors was approximately 570 mL. The gas was introduced into the bioreactor through a distributor (pore size
