Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 1 of 166 PageID #:
`6024
`
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`

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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 21 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 22 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 23 of 166 PageID #:
`6046
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 24 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 25 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 26 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 27 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 28 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 29 of 166 PageID #:
`6052
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 30 of 166 PageID #:
`6053
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`

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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 31 of 166 PageID #:
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`

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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 32 of 166 PageID #:
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`

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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 33 of 166 PageID #:
`6056
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 34 of 166 PageID #:
`6057
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 35 of 166 PageID #:
`6058
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 36 of 166 PageID #:
`6059
`
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`

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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 37 of 166 PageID #:
`6060
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`

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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 38 of 166 PageID #:
`6061
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 39 of 166 PageID #:
`6062
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 40 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 41 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 42 of 166 PageID #:
`6065
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 43 of 166 PageID #:
`6066
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`

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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 44 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 45 of 166 PageID #:
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`6071
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`6074
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`6080
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 71 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 72 of 166 PageID #:
`6095
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 73 of 166 PageID #:
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 74 of 166 PageID #:
`6097
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 75 of 166 PageID #:
`6098
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 86 of 166 PageID #:
`6109
`A Kinetic Analysis of Hybridoma Growth
`and Metabolism in Batch and Continuous
`Suspension Culture: Effect of Nutrient
`Concentration, Dilution Rate, and pH
`
`W. M. Miller*, H. W. Blanch, and C. R. Wilke
`Department of Chemical Engineering, University of California,
`Berkeley, California 94720
`
`Accepted for publication November 30, 1987
`
`Hybridomas are finding increased use for the produc-
`tion of a wide variety of monoclonal antibodies. Under-
`standing the roles of physiological and environmental
`factors on the growth and metabolism of mammalian
`cells is a prerequisite for the development of rational
`scale-up procedures. A n SP2/0-derived mouse hybri-
`doma has been employed i n the present work as a
`model system for hybridoma suspension culture. In pre-
`liminary shake flask studies to determine the effect of
`glucose and glutamine, it was found that the specific
`growth rate, the glucose and glutamine metabolic quo-
`tients, and the cumulative specific antibody production
`rate were independent of glucose concentration over
`the range commonly employed in cell cultures. Only the
`specific rate of glutamine uptake was found to depend
`on glutamine concentration. The cells were grown in
`continuous culture at constant pH and oxygen concen-
`tration at a variety of dilution rates. Specific substrate
`consumption rates and product formation rates were
`determined from the steady state concentrations. The
`specific glucose uptake rate deviated from the mainte-
`nance energy model' at low specific growth rates, proba-
`bly due to changes i n the metabolic pathways of the
`cells. Antibody production was not growth-associated;
`and higher specific antibody production rates were ob-
`tained at lower specific growth rates. The effect of pH
`on the metabolic quotients was also determined. An
`optimum i n viable cell concentration was obtained
`between pH 7.1 and 7.4. The viable cell number and
`viability decreased dramatically at pH 6.8. At pH 7.7 the
`viable cell concentration initially decreased, but then re-
`covered to values typical of pH 7.1-7.4. Higher specific
`nutrient consumption rates were found at the extreme
`pH values; however, glucose consumption was inhib-
`ited at low pH. The pH history also influenced the behav-
`ior at a given pH. Higher antibody metabolic quotients
`were obtained at the extreme pH values. Together with
`the effect of specific growth rate, this suggests higher
`antibody production under environmental or nutritional
`stress.
`* Present address: Department of Chemical Engineering, Northwestern
`University. Evanston, IL 60208.
`
`INTRODUCTION
`Monoclonal antibodies (MAbs) produced by hybridomas
`have an expanding market for use in diagnostic and chemi-
`cal assays, as well as for affinity separation of other valu-
`able fermentation products and for therapeutic uses.' Mi-
`crobial production of MAbs is desirable because mam-
`malian cells grow more slowly, are more sensitive to shear,
`and require more expensive media than bacteria or yeast.
`Functional antibodies have been expressed in yeast ' and
`b a ~ t e r i a , ~ but the large number of different antibodies re-
`quired makes cloning less attractive than for less complex
`products such as insulin or human growth hormone. Micro-
`bial antibodies may not be identical to those made by hy-
`bridomas' and efforts to amplify product formation rates in
`mammalian cells are progressing.6 Thus it is likely that
`MAbs will be produced in cell culture for the foreseeable
`future. Before promising applications can be efficiently
`commercialized, however, basic information must be ob-
`tained on the environmental and physiological factors that
`affect cell growth and metabolism.
`Several general reviews on mammalian cell culture tech-
`nology have appeared during the past few
`The
`status of hybridoma production has been recently reviewed
`by Randerson." Many of the techniques proposed to in-
`crease antibody production employ various forms of cell
`immobilization. This precludes obtaining representative
`cell samples and often results in metabolite concentration
`gradients. The uniform cell and metabolite concentrations
`characteristic of suspension culture facilitate the modelling
`of cell growth and metabolism. The status of suspension
`culture for mammalian cells has been reviewed by Katinger
`and Scheirer. " Hybridomas have been studied in suspen-
`I' Hybridomas
`sion culture by a number of investigators. I'
`have been investigated in continuous suspension culture by
`Fazekas de St. Groth'? and at Celltech.''.'' Similar studies
`
`Biotechnology and Bioengineering, Vol. 32, Pp. 947-965 (1988)
`0 1988 John Wiley & Sons, Inc.
`
`CCC 0006-3592/88/080947- 19$04.00
`
`JA00004546
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`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 87 of 166 PageID #:
`6110
`MATERIALS AND METHODS
`have been also conducted using other mammalian
`Most of these studies covered a limited range of dilution
`rates and/or provided limited data on nutrient and by-
`product concentrations.
`Batch and continuous suspension cultures have been em-
`ployed in the present work to determine the effects of the
`major nutrients, dilution rate, and pH on the growth and
`metabolism of an SP2iO-derived mouse hybridoma line.
`Steady-state viable cell, total cell, glucose, lactate, anti-
`body, glutamine, and ammonia concentrations were ob-
`tained over a wide range of dilution rates (0.31-1.32 day-’)
`and culture pH (6.8-7.7) at constant dissolved oxygen
`concentration; and were used to calculate the metabolic
`quotients. The cellular responses to changes in culture con-
`ditions were also obtained.
`Glucose and glutamine are the major carbon and energy
`sources in most cell culture media and both nutrients are
`required for cell growth. Glutamine metabolism can provide
`
`30-65% of the energy for mammalian cell g r ~ w t h . ~ ~ , * ~ The
`metabolic fates of these major nutrients are illustrated in
`Shake Flask Cultures
`Figure 1. The proportion of each nutrient consumed by the
`different pathways depends on the metabolic state of the
`Cells were inoculated into 25 mL of complete medium
`to give an initial concentration of -3 X lo4 viable
`cells. The metabolic byproduct ammonia has been shown
`to inhibit cell growth in culture.’3325 Lactate can also inhibit
`cells/mL. The 200-mL polystyrene bottles (Coming) were
`cell growth,26 although Reuveny et. al.I3 found that addi-
`placed on a shaker (-70 rpm) and equilibrated with 7%
`tion of as much as 2.5 g/L lactate can stimulate the growth
`CO, in air in a 37°C incubator. Samples (1.lmL) were
`of some hybridomas.
`taken daily.
`
`Cell Line and Medium
`(provided by G. Lewis and
`Cell line AB2-143.2’’
`J . Goodman, University of California, San Francisco CA)
`is an SP2/0-derived mouse hybridoma that produces an
`IgG2a antibody to benzene-arsonate. The cells were grown
`in Dulbecco’s Modified Eagle’s Medium (with bicarbonate
`buffer) supplemented with 10% fetal bovine serum (Hy-
`clone) and 1% each of lOOX MEM nonessential amino
`acids and 11 g/L sodium pyruvate (all except serum from
`Gibco). Initial glucose and glutamine concentrations are
`given below for shake flask studies. For 1-L suspension
`cultures the initial (and feed) concentrations were 22mM
`glucose and 4.8mM glutamine. No antibiotics were used
`and periodic mycoplasma samples were negative.
`
`Pentose Phosphate
`
`I
`
`GLUCOSE
`
`& WATER
`
`LACTATE
`
`T
`CYCLE 0
`
`CELL MASS
`
`Glutamate 1
`
`AMMONIA
`
`GLUTAMINE
`
`Figure 1.
`Summary of the metabolic pathways for the major carbon and energy
`sources for mammalian cell culture.
`
`948
`
`BIOTECHNOLOGY AND BIOENGINEERING, VOL. 32
`
`OBER 1988
`
`JA00004547
`
`

`

`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 88 of 166 PageID #:
`6111
`Suspension Cultures
`Determination of Specific Growth Rate and
`Metabolic Quotients
`A 1-L glass reactor (Pegasus) was used with 600 mL
`The experiments described below were carried out in
`working volume. Agitation was provided by a 5-cm axial
`flow turbine operating at - 150 rpm. Oxygen transport was
`batch or constant-volume, continuous-flow (with sterile
`feed) reactors. A material balance around the reactor yields
`via surface aeration and the partial pressure was controlled
`at 80 ?5 mm Hg by varying the oxygen concentration in
`the following equations for cell growth:
`the headspace. Temperature was maintained at 37.0 2 0.2"C
`dn/dt = paPpn - Dn = pn, - Dn
`(1)
`with a circulating water bath. The pH was controlled at
`dn,/dt = pn, - k,n, - Dn,
`7.1 k0.1 by addition of 1M NaOH in batch experiments.
`(2)
`Base (0.5M NaHCO,) addition was also used for automatic
`p = pap,(n/nJ = (n/n,) [don n)/dt + Dl
`(3)
`pH control of the continuous suspension cultures, but the
`k, = p - D
`CO, concentration in the headspace was also adjusted to
`(4)
`at steady state
`optimize control and minimize base addition at each pH.
`
`The distinction between p and papp is based on the assump-
`Samples ( 5 mI, including purge) were taken twice daily.
`tion that only viable cells can divide. The difference be-
`A multichannel peristaltic pump (Gilson) was used for
`tween p and papp is especially important when the viability
`medium addition and product removal in continuous cul-
`is low. The specific metabolic quotients for substrate and
`ture. Product was removed from the surface at a higher
`oxygen consumption and product formation were obtained
`rate than the feed to maintain the liquid level at the height
`from:
`of the outlet line. Silicone tubing was used for the feed
`line because inhibition had previously been observed when
`medical-grade PVC was used.2s
`
`q, = [D(s, - s) - ds/dt]/n,
`qo* = [K,aG, - COJ - d(Co,)/dfl/n,
`q A b = [D(Ab) -I- d(Ab)/dtI/n,
`Note that these quantities are per viable cell.
`
`( 5 )
`(6)
`(7)
`
`Sample Analyses
`The cell sample was diluted 1: 1 with 0.16% trypan blue
`in normal saline and counted on a hemacytometer; nonvi-
`able cells stained blue. An average of two (shake flask and
`batch experiments) or four (continuous experiments) deter-
`minations was used to calculate the viable cell concentration
`and percent viability. A minimum of four hemacytometer
`fields and 250 viable cells (or all nine fields for slides with
`less than 250 viable cells) were counted per determination.
`The remainder of each sample was centrifuged to remove
`the cells, preserved with sodium azide and frozen for later
`analysis. Glucose was measured using a clinical glucose
`analyzer; lactate was determined by an enzymatic assay;
`and ammonia was measured with an ion-selective elec-
`trode. Samples were reacted with o-phthaldialdehyde to
`form fluorescent amino acid derivatives. Glutamine was
`separated from the other amino acids via HPLC using an
`RP-18 column with gradient elution from 25% methanol
`(the balance was O.1M sodium acetate, pH 6.8) to 80%
`methanol over 25 min.
`Antibody was determined using a sandwich ELlSA as-
`say in 96-well microtiter plates. The benzene-arsonate
`antigen was conjugated to bovine serum albumin and ad-
`sorbed to the wells. Six duplicate dilutions were used for
`each standard or sample evaluated. Alkaline phosphatase
`conjugated to goat anti-mouse IgG was used to detect the
`bound antibody.
`The dissolved oxygen concentration was measured with
`a polarographic oxygen electrode (Ingold). Medium satu-
`rated with air was estimated to have an oxygen concentra-
`tion 194pM. 29,30 The volumetric mass transfer coefficient
`KL a was experimentally determined in sterile medium by
`following the increase (or decrease) in oxygen concentra-
`tion when air (or nitrogen) was passed through the reactor
`headspace.
`
`RESULTS AND DISCUSSION
`
`Batch Culture
`Typical batch growth curves are shown in Figure 2(a).
`There is an initial period of exponential growth followed
`by a decline in viable cell concentration and a plateau in
`total cell concentration. The peak in the viable cell count
`corresponds to the time at which the glutamine has been
`exhausted [Fig. 2(b)], which suggests that glutamine is the
`limiting nutrient for this medium. Glucose consumption
`and the complementary production of lactate ceased about
`24 h later. The apparent molar yield of lactate from glu-
`cose was about 1.5 (75% of the theoretical maximum).
`Antibody production also continued after the maximum in
`viable cell concentration [Fig. 2(c)]. The cumulative
`specific antibody production rate declined somewhat be-
`fore reaching a constant value of 22 X
`pg/cell/day.
`The maintenance energy model' for nutrient consump-
`tion may be written as:
`4 s = E.L/y:,, = P/Y,,, + me
`(8)
`Data for the glucose metabolic quotient [Fig. 2(d)] ap-
`pear to fit this model with yglucose = 2.0 X 10' cells/mmol
`and mglucose = 1.2 X
`mmol/cell/day. This value of
`mgfucose is - 15% of the value for qglucov at pmax = 1.3 day-'.
`There is more scatter in the glutamine metabolic quotient
`data.
`A generally employed model for product formation is:
`q A b = ffp + P
`
`(9)
`
`MILLER, BLAN
`
`ND WILKE: KINETIC ANALYSIS OF HYBRIDOMA
`
`949
`
`JA00004548
`
`

`

`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 89 of 166 PageID #:
`6112
`
`35 T
`
`T 6
`
`C
`E
`L L
`O L
`G s
`I
`rn
`L
`
`2,
`
`Specific
`growth rate
`
`3 0
`
`25
`C
`0
`N 20
`C
`
`rn
`M
`
`15
`
`10
`
`5
`
`100
`
`20
`
`0
`
`E
`6
`2o c
`e
`I
`I
`/
`10 d
`
`0
`
`1
`
`2
`
`4
`3
`TIME (days)
`
`0
`
`5
`
`6
`
`7
`
`
`
`-
`
`50
`
`--
`+
`Antibody
`rng
`--
`
`\
`\
`
`40
`
`I
`I
`E
`
`30
`
`C q f
`
`I
`I
`d
`a
`Y
`
`--
`
`20
`
`/
`-7
`
`10
`
`/ /
`
`I
`
`A
`
`0
`
`1,’
`
`:
`
`Glucose
`rnrnol
`
`Y
`
`- 8
`
`-_ 7
`
`-- 6
`
`/
`
`/ ,
`
`= -- 5
`
`-- 4
`
`\
`
`-- 3
`,
`-- 2
`-- 1
`
`1
`
`Glutamine
`rnrnol
`
`r
`
`O
`
`
`
`(C)
`Figure 2. Hybridoma batch growth in a I-L reactor with dissolved oxygen and pH control: (a) cell concentration and specific growth rate vs.
`culture time; (b) glucose, glutamine and lactate concentrations vs. culture time; (c) antibody concentration and average specific antibody pro-
`duction rate vs. culture time; (d) antibody (squares), glutamine (triangles) and glucose (circles) metabolic quotients vs. specific growth rate.
`
`For totally growth-associated products /3 is zero, and for
`non-growth-associated products a is zero. The data for the
`antibody metabolic quotient in Figure 2(d) and the cumula-
`tive data in Figure 2(c) suggest that antibody production is
`partially growth-associated. The scatter in the metabolic
`quotients is not unexpected for batch data, as culture con-
`ditions are constantly changing, and precludes one from
`obtaining reliable values for the constants in eq. (8) and (9).
`
`Effect of Varying Glucose and Glutamine
`Concentration
`A series of batch experiments was carried out in shake
`flasks to determine the effect of different initial glucose
`
`and glutamine concentrations on cell growth and metabo-
`lism. Shake flask growth and nutrient concentration curves
`(data not shown) were essentially the same as those ob-
`tained in a 1-L reactor; however, the shake flask glucose
`consumption ceased earlier.
`The viable cell concentration curves and metabolite pro-
`files are shown in Figure 3 for different glucose and glu-
`tamine concentrations. There was a significant increase in
`maximal cell concentration when the glucose concentration
`was increased from 5mM to 13mM [Fig. 3(a)]. A further
`increase to 21mM glucose extended the duration of the sta-
`tionary phase, but did not significantly increase the maximal
`cell concentration. Increasing the glutamine concentration
`from 3.0 to 7.6mM at 21mM glucose also resulted in an
`
`950
`
`BIOTECHNOLOGY AND BIOENGINEERING, VOL. 32
`
`OBER 1988
`
`JA00004549
`
`

`

`Case 1:18-cv-01363-CFC Document 48-13 Filed 12/11/18 Page 90 of 166 PageID #:
`6113
`
`/
`
`\\
`
`5 rnM
`
`i
`
`0
`
`A
`
`6.0
`
`C
`E
`L L 5.5
`O L
`G s
`I
`m
`L
`
`5.0
`
`4.5
`
`4.5 1-
`
`4.0 4
`0
`
`I
`6
`
`5
`
`1
`
`2
`
`3
`TIME (days)
`
`4
`
`(a)
`
`0 Glutamme-
`0 Glucose
`
`0
`
`1
`
`2
`
`3
`
`5
`4
`TIME (days)
`(b)
`
`6
`
`7
`
`8
`
`9
`
`
`0
`N
`
`\
`
`\
`
`8
`
`7
`
`6
`
`5
`
`4
`
`3
`
`2
`
`1
`
`0
`
`1
`
`2
`
`3
`TIME (days)
`
`4
`
`(c)
`
`5
`
`6
`
`0
`
`1
`
`2
`
`3
`
`6
`
`7
`
`8
`
`9
`
`
`
`5
`4
`TIME (days)
`( 4
`
`25
`
`21 mM
`
`c 20
`0
`N
`C 1 5
`
`m
`M 10
`
`5
`
`0
`0
`
`Figure 3. Effect of glucose and glutamine concentrations on hybridoma growth and metabolism in shake flask culture: (a) viable cell concentra-
`tion vs. culture time as a function of initial glucose concentration in medium containing 5.0mM glutamine; (b) viable cell concentration vs. culture
`time as a function of initial glutamine concentration in medium containing 21mM glucose; (c) glucose and glutamine concentration profiles as a
`function of initial glucose concentration in medium containing 5.0mM glutamine; (d) glucose and glutamine concentration profiles ah a function of
`initial glutamine concentration in medium containing 21mM glucose.
`
`extended stationary phase without a significant increase in
`maximal cell concentration [Fig. 3(b)]. For both nutrients
`the growth curves for different concentrations initially fall
`on the same line. This suggests that the specific growth
`rate is not sensitive to differences in nutrient concentra-
`tion within the range evaluated. Low and Harbour3' ob-
`tained similar results for the effect of glucose on the
`maximum cell concentration and growth rate for two
`hybridoma cell lines.
`By varying the glucose conce