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`EXHIBIT 10
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`Case 1:18-cv-01363-CFC Document 85-3 Filed 03/22/19 Page 2 of 260 PageID #:
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`JOURNAL OF BIOSCIENCE AND BIOENGINEERING
`Vol. 100, No. 5, 502–510. 2005
`DOI: 10.1263/jbb.100.502
`
`© 2005, The Society for Biotechnology, Japan
`
`Development of a Fed-Batch Culture Process for Enhanced
`Production of Recombinant Human Antithrombin
`by Chinese Hamster Ovary Cells
`
`Shinobu Kuwae,1* Toyoo Ohda,1 Hiroshi Tamashima,1
`Hideo Miki,1 and Kaoru Kobayashi1
`
`Protein Research Laboratory, Pharmaceutical Research Unit, Mitsubishi Pharma Corporation,
`2-25-1 Shodai-ohtani, Hirakata, Osaka 573-1153, Japan1
`
`Received 30 March 2005/Accepted 14 July 2005
`
`Antithrombin is a serine protease inhibitor that inactivates several coagulation proteases, pri-
`marily thrombin and factor Xa. The Chinese hamster ovary (CHO) cell line transfected with a
`vector expressing recombinant human antithrombin (rAT) and a selectable marker, glutamine
`synthetase (GS), was cultivated in a 2-l fed-batch culture process using serum-free, glutamine-free
`medium. To maximize the rAT yield, effects of culture pH, balanced amino acid feeding, and an
`increased glutamate concentration on cell metabolism and rAT production were investigated.
`When cells were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the maximum cell density and maxi-
`mum lactate concentration decreased with decreasing pH. The highest production level of rAT
`was obtained at culture pH 6.8 due to the extended culture lifetime. Compared to the imbalanced
`amino acid feeding at culture pH 6.8, the balanced amino acid feeding increased the amount of
`rAT activity by 30% as a result of an increased viable cell number. A decrease in the specific glu-
`cose consumption rate (q
`Glc) with increasing culture time was observed in all the above-mentioned
`experiments, while the glucose concentration was maintained above 0.7 g l–1. In addition, a de-
`crease in the specific rAT production rate (q
`rAT) was observed after the depletion of lactate in the
`late cultivation stage. Taken together, these results suggest that the reduced availability of cellular
`energy caused by the decrease in q
`Glc and depletion of lactate led to the decrease in q
`rAT. This de-
`crease in q
`rAT was partially prevented by increasing the residual glutamate concentration from
`1 mM to 7 mM, thus resulting in an additional 30% increase in the amount of rAT activity. The
`optimized fed-batch culture process yielded 1.0 g l–1 rAT at 287 h of cultivation.
`
`[Key words: Chinese hamster ovary (CHO) cells, glutamine synthetase, fed-batch culture, antithrombin, glutamate,
`specific glucose consumption rate]
`
`Antithrombin (AT) is a serine protease inhibitor that in-
`activates a number of enzymes of the coagulation cascade,
`particularly thrombin, factor Xa and factor IXa; therefore, it
`plays a crucial role in the regulation of blood coagulation
`(1, 2). Human AT is a single-chain glycoprotein with a mo-
`lecular weight of 58 kDa, and it contains 432 amino acids,
`three disulfide bridges and four carbohydrate side chains
`that account for 15% of the total mass (3, 4). The concentra-
`tion of AT in normal plasma is about 150 mg l –1 (5). AT
`pharmaceutical products are used in therapy by the intrave-
`nous route to manage acute thrombotic episodes and to treat
`patients suffering from an AT deficiency. These products
`have been produced by the fractionation of plasma from
`blood donors. However, since there is a potential risk of
`contaminating the products with blood-derived pathogens,
`the production of AT by recombinant DNA technology is
`desired. To replace plasma-derived AT (pAT) with recombi-
`
`* Corresponding author. e-mail: Kuwae.Shinobu@mc.m-pharma.co.jp
`phone: +81-(0)72-856-9295 fax: +81-(0)72-864-2341
`
`nant AT (rAT), reducing the cost of manufacturing rAT is
`necessary to meet commercialization challenges.
`Chinese hamster ovary (CHO) cells are the most com-
`monly used eukaryotic expression hosts for the production
`of therapeutic glycoproteins in the biotechnology industry
`(6). Biologically active human AT has been expressed in
`CHO cells (7), but the reported expression level was insuffi-
`cient (< 120 mg l–1) (8) to overcome the commercialization
`challenges.
`To obtain a high yield and a cost-effective process, the
`choice of expression system and culturing method are of
`great importance. A glutamine synthetase (GS) gene expres-
`sion system is one of the most used systems for the produc-
`tion of recombinant proteins in mammalian cells. GS cata-
`lyses the formation of glutamine from glutamate and ammo-
`nia. In the GS expression system, which uses GS as a select-
`able marker, a week promoter (e.g., SV40) is usually used
`for the expression of the GS enzyme, while a strong human
`cytomegalovirus (hCMV) promoter is used to drive the prod-
`uct gene expression (9–11). This enables the selection of
`
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`CHO CELL FED-BATCH CULTURE FOR ANTITHROMBIN PRODUCTION
`
`503
`
`rare high-producing transfectants where the gene of interest
`has integrated into transcriptionally active sites within the
`host cell genome. The transfectants that can survive using
`the weakly transcribed GS gene in glutamine-free medium
`containing a GS inhibitor, methionine sulphoximine (MSX),
`should produce a relatively high level of the recombinant
`protein due to the strong promoter. In fact, the GS system
`requires fewer copies of the gene per cell, typically 1–30
`copies, to obtain an efficient production level than the dihy-
`drofolate reductase (DHFR) expression system, which usu-
`ally requires several hundred copies of the gene (12–14).
`One of the additional advantages of the GS expression sys-
`tem is that it mediates a low level of toxic ammonia ac-
`cumulation during cultivation, because the cells that grow
`in glutamine-free medium consume ammonia to synthesize
`glutamine from glutamate (15, 16). Ammonia is one of the
`major inhibitory substances accumulating in cell culture and
`is mainly a by-product of glutamine metabolism and the
`chemical decomposition of glutamine in the medium (17).
`There are other possible strategies to address the problem of
`ammonia accumulation in batch and fed-batch cell culture
`processes, which include maintaining the residual glutamine
`concentration at a low level (18–20) and replacing glu-
`tamine with glutamine-based dipeptides (21). However, the
`former strategy, which proved that a reduced ammonia ac-
`cumulation led to a high cell density and a high product
`yield, generally requires a high degree of instrumentation to
`control the residual glutamine concentration at an optimal
`level. The latter strategy uses more expensive substrates
`than glutamate, which is used in the GS expression system.
`The culture of GS-transfected cells does not require such a
`high degree of instrumentation or expensive substrates to
`reduce ammonia accumulation during cultivation. It is also
`interesting to note that the cell growth using glutamate in
`glutamine-free medium reduces the accumulation of inhibi-
`tory lactate in the culture as a result of the reduced specific
`glucose consumption rate (22, 23).
`Fed-batch culture is frequently used for the production of
`cell-culture-based recombinant proteins. The major advan-
`tage of fed-batch culture over other culture operations, such
`as batch, perfusion, and continuous cultures, is that it en-
`ables a higher product concentration. The final recombinant
`protein concentration in fed-batch culture is generally deter-
`mined by the viable cell density, viable culture duration, and
`specific production rate of the recombinant protein. There-
`fore, fed-batch processes have been developed aiming at
`maximizing these three parameters using different approaches
`from various perspectives, such as culture medium and en-
`vironmental parameters (24). The viable cell density and
`viable culture duration have been increased by feeding lim-
`iting nutrients, such as glucose, amino acids, and minerals
`(15, 19, 25). Culture pH is one of the key environmental pa-
`rameters that influence cell metabolism and recombinant
`protein production. Recently, Yoon et al. have reported the
`effect of culture pH in the batch culture of recombinant CHO
`cells (26). The specific glucose consumption rate (qGlc) and
`specific lactate production rate (qLac) increased with increas-
`ing pH in the pH range of 6.85 to 7.8, whereas the maxi-
`mum specific growth rate was observed at pH 7.2. The cul-
`ture pH did not influence the specific production rate of
`
`erythropoietin (qEPO) at 37.0°C but significantly influenced
`qEPO at 32.5°C with the maximum qEPO at pH 7.0. Thus,
`while culture pH affects cell growth, metabolism, and re-
`combinant protein production in CHO cell culture, we are
`not aware of any reports on the effect of pH in the fed-batch
`culture of CHO cells.
`In this work, we investigated effects of culture pH, bal-
`anced amino acid feeding, and an increased glutamate con-
`centration on cell metabolism and rAT production in the
`fed-batch culture of GS-transfected CHO cells. When cells
`were grown at pH values of 6.6, 6.8, 7.0, and 7.2, the max-
`imum cell density and maximum lactate concentration de-
`creased with decreasing pH. The highest rAT production
`level was obtained at culture pH 6.8 due to the extended
`culture lifetime. Balanced amino acid feeding increased the
`viable cell number and amount of rAT activity by 25% and
`30%, respectively, compared with the imbalanced amino
`acid feeding. During the course of development of the fed-
`batch culture process, we observed a decrease in qGlc with
`culture time, while the glucose concentration was main-
`tained above 0.7 g l–1. A parallel consumption of lactate and
`alanine was also observed in the late cultivation stage, sug-
`gesting that the decrease in qGlc caused a limited pyruvate
`availability from the glycolytic pathway that led to the par-
`allel consumption of lactate and alanine to produce pyru-
`vate. In addition, a decrease in specific rAT production rate
`(qrAT) occurred after the depletion of lactate. Taken together,
`these results suggest that the reduced availability of cellular
`energy caused by the decrease in qGlc and depletion of lac-
`tate led to the decrease in qrAT. This decrease in qrAT was par-
`tially prevented by increasing the residual glutamate con-
`centration from 1 mM to 7 mM throughout the cultivation,
`thus resulting in a 30% increase in the amount of rAT activ-
`ity. The relationship among qGlc, lactate metabolism and rAT
`production is discussed.
`
`MATERIALS AND METHODS
`
`Cell line and culture medium Two cloned cell lines, 4D11
`and 2D6, producing rAT were used in this study. The host CHO-
`K1 cells and GS expression vector system were provided by Lonza
`Biologics (Slough, Berkshire, UK). The recombinant CHO cell
`lines were established by the transfection of the rAT expression
`vector that was constructed by the insertion of AT cDNA into the
`GS vector. GS catalyzes the formation of glutamine from gluta-
`mate and ammonia. Stable recombinant cells with GS activity were
`selected in glutamine-free medium containing the GS inhibitor
`MSX at a concentration of 25 µM and adapted to suspension cul-
`ture in shaker flasks.
`The cloned cell lines were cultivated in the serum-free, glu-
`tamine-free medium EX-CELL302 GS-modified (JRH, Kansas
`city, MO, USA) without the addition of MSX. Protein-free chemi-
`cally defined feed media developed in house were used for fed-
`batch culture. These feed media contained a number of compo-
`nents, such as glucose, amino acids, nucleosides, vitamins, and trace
`elements.
`Fed-batch cultures were performed in
`Culture conditions
`four identically configured 3-l bioreactors with a 2-l working vol-
`ume (Bio Master D type; Able, Tokyo). The bioreactor consists of
`a flat bottomed glass vessel and a stainless steel head plate with
`ports for holding the rotating shaft, baffles, temperature sensor, pH
`electrode, dissolved oxygen sensor, acid and base inlet, feed me-
`
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`504
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`KUWAE ET AL.
`
`J. BIOSCI. BIOENG.,
`
`cultivation.
`
`(V
`c)i
`
`= (V
`
`c)i–1
`
`+
`
`i 1–
`
`Vc( )
`
`i∫
`---------------- F
`Vi 1–
`
`t
`
`t
`i 1–
`
`t( ) td
`
`−
`
`E t( ) td
`
`t
`
`i∫
`
`t
`i 1–
`
`(2)
`
`in
`
`= V
`
`i–1
`
`= V(t
`
`0)
`
`when i = 1
`
`(V
`c)i–1
`Here, (V
`c)i is the compensated culture volume obtained immedi-
`ately after i-th withdrawal in liter; V
`i–1 is the culture volume ob-
`tained using Eq. 1 and immediately after (i − 1)-th withdrawal in
`liter. The compensated amount of the consumed substrate, such as
`glucose or glutamate, was obtained using
`
`(3)
`
`(G
`
`c)i
`
`= (G
`
`c)i–1
`
`+ S
`
`i–1(V
`
`c)i–1
`
`
`
`− Si(V
`
`c)i
`
`+ S
`
`feed
`
`Vc( )
`
`i∫
`---------------- F
`Vi 1–
`
`i 1–
`
`t( ) td
`
`(4)
`
`s
`
`t
`
`t
`i 1–
`
`(5)
`
`S
`
`i–1(Vc)i–1
`
`
`
`= S(t0)V(t
`
`0)
`
`when i = 1
`
`
`Here, (G
`c)i is the compensated amount of the consumed substrate
`obtained immediately after i-th withdrawal in gram; S
`i is the con-
`centration of the substrate obtained immediately after i-th with-
`drawal in the culture in g l–1; S
`feed is the concentration of the sub-
`strate in the feed medium in g l–1; Fs(t) is the feed rate of the feed
`medium containing the substrate of interest in l h–1. The experi-
`mental data and the above-mentioned compensated data were ana-
`lyzed using IGOR Pro (Wave-Metrics, Lake Oswego, OR, USA) to
`obtain smoothed values using the spline function and also differen-
`tial coefficients for the calculation of specific rates. The specific
`rates of consumption and production were obtained using
`
`dGc
`-----------
`dt
`
`= q
`
`X
`
`V
`
`v
`
`c
`
`s
`
`(6)
`
`= q
`
`X
`
`P
`
`V
`
`c
`
`v
`
`dPVc
`---------------
`dt
`where G
`c is the compensated amount of the consumed substrate in
`mol; q
`s is the specific consumption rate for the substrate in mol
`cell–1 h–1; X
`V is the viable cell density in cell l–1; V
`C is the compen-
`sated culture volume in liter; P is the concentration of lactate in
`mol l–1 or that of rAT in IU l–1; q
`P is the specific production rate of
`lactate in mol cell–1 h–1 or that of rAT in IU cell–1 h–1.
`
`(7)
`
`RESULTS AND DISCUSSION
`
`Impact of culture pH To investigate the effect of cul-
`ture pH on cell growth, metabolism, and rAT production,
`parallel fed-batch cultures of the cell line 4D11 were carried
`out in a 2-l working volume bioreactor. The same number of
`cells was inoculated into each culture (1.5 × 105 cells ml–1),
`and on-line control of pH to 6.6, 6.8, 7.0, and 7.2 was real-
`ized by the addition of 1 M HCl and 0.5 M NaHCO3 to the
`culture. The feed medium that contained glucose and amino
`acids was continuously fed after 2 d of cultivation on the
`basis of the glucose concentration to maintain the glucose
`concentration at a target level of 2 g l–1. However, the glu-
`cose concentration varied between 0.7 and 3.3 g l–1 due to
`the underfeeding and overfeeding with the medium (Fig. 1A).
`The maximum lactate concentration decreased with decreas-
`ing pH (Fig. 1B). Although the culture was started with an
`initial volume of 1.5 l, the culture volume reached a limit of
`2 l before cell viability decreased to below 90% when cells
`were grown at pH 6.8 or above (Fig. 1C). A greater-than-
`expected increase in culture volume was observed at the
`higher culture pHs due to the increased feeding volume of
`the feed medium and the alkaline solution. To continue the
`
`dium inlet, gas inlet, exhaust gas condenser, and sample pipe. The
`glass vessel was graduated for the off-line measurement of the cul-
`ture volume. The exhaust gas condenser was water-cooled at 10°C
`to minimize the evaporation of the culture medium. Sterile glass
`bottles filled with the feed medium, 1 M HCl, and 0.5 M NaHCO3
`were placed on a balance and aseptically connected to their respec-
`tive bioreactors. The initial culture volume was 1.0 l unless other-
`wise noted. The temperature in the bioreactors was controlled at
`37°C, and the stirrer speed was set to be 60 rpm. A gas mixture
`containing air, CO2, and O2 was provided. The dissolved carbon di-
`oxide concentration (DCO2) was measured off-line once a day and
`manually adjusted to 38 mmHg by changing the partial pressure of
`CO2 in the gas mixture. The dissolved oxygen concentration (DO)
`was controlled on-line at 90 mmHg by varying the oxygen partial
`pressure in the gas mixture. The pH was maintained at 6.8 by the
`addition of 1 M HCl and 0.5 M NaHCO3, unless otherwise noted.
`The feed medium that contained glucose and amino acids was con-
`tinuously fed on the basis of the glucose concentration to maintain
`the glucose concentration at a target level of 2 g l–1. Samples of
`20 ml were taken once or twice a day with a syringe for off-line
`analysis. Immediately after sampling, the culture volume and
`amounts of the feed medium, 1 M HCl, and 0.5 M NaHCO3 were
`measured off-line.
`The pH, DO, and DCO2 were measured using a
`Analyses
`blood-gas analyzer (Rapidlab 248; Bayer HealthCare Diagnostics
`Division, Tarrytown, NY, USA). If the off-line pH measurement
`deviated from the on-line pH measurement by 0.03 pH unit or
`more, the on-line pH measurement was calibrated to be equal to
`the off-line pH measurement. Cells were counted using a Coulter
`particle counter (Coulter Counter Z2; Beckman Coulter, Fullerton,
`CA, USA) and a hemocytometer. Cell viability was quantified by
`the erythrosin B dye exclusion method. After the cells were re-
`moved by centrifugation, the concentrations of glucose, lactate, and
`glutamate in the culture supernatant were measured with Biotech-
`analyzer AS-210 (Sakura Seiki, Tokyo). The ammonia concen-
`tration was enzymatically determined using an ammonia assay kit
`(171-A; Sigma, St. Louis, MO, USA). Free amino acids in the cul-
`ture supernatant were derivatized with phenylisothiocarbamoyl
`(PITC) and analyzed by HPLC using the PICO-TAG method on a
`Waters High-Performance Liquid Chromatography system (Waters,
`Milford, MA, USA).
`The activity of rAT was determined as a heparin cofactor (HC)
`activity with a commercial assay kit that uses the thrombin-specific
`chromogenic substrate S-2238 (TESTZYM ATIII 2 kit; Daiichi
`Pure Chemical, Tokyo). The concentration of rAT was measured
`by reverse-phase (RP) HPLC on a POROS R2/10 column (2.1 mm
`ID × 50 mm; Applied Biosystems, Foster City, CA, USA) at a flow
`rate of 2.0 ml min–1. A segmented gradient elution was used: 0–40%
`solvent B for 0–2 min, and 40–55% solvent B for 2–7.25 min,
`where solvent A was 0.1% trifluoroacetic acid (TFA) aqueous so-
`lution and solvent B was 0.1% TFA in 90% acetonitrile. The AT
`contents were monitored at 220 nm using plasma-derived AT
`(Neuart; Mitsubishi Pharma Corporation, Osaka) as a standard.
`The simulated culture volume
`Equations and calculations
`at time t was obtained using
`
`(1)
`
`E t( ) td
`
`t∫
`
`t 0
`
`F
`
`out
`
`t( ) td
`
`−
`
`t∫
`
`t 0
`
`F
`
`in
`
`t( ) td
`
`−
`
`t∫
`
`t 0
`
`V(t) = V(t
`
`0) +
`
`where V(t) is the culture volume in liter; F
`in(t) and Fout(t) are the
`
`volumetric flow rates into and out of the reactor, respectively, in
`l h–1; E(t) is the evaporation rate in l h–1. E(t) was obtained by the
`curve fitting of V(t) to the experimental culture volume and was
`determined to be 0.000125 l h–1. The compensated culture volume
`was obtained using Eqs. 2 and 3, which is based on the assumption
`that the culture was not withdrawn from the reactor throughout the
`
`

`

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`VOL. 100, 2005
`
`CHO CELL FED-BATCH CULTURE FOR ANTITHROMBIN PRODUCTION
`
`505
`
`FIG. 1. Effects of culture pH on cell metabolism, cell growth and rAT production in fed-batch culture of GS-CHO cell line 4D11. Parallel fed-
`batch cultures were carried out in a 2-l working volume bioreactor using serum-free, glutamine-free medium at pH values of 6.6 (closed circles),
`6.8 (open circles), 7.0 (closed triangles), and 7.2 (open squares). (A) Glucose concentration. (B) Lactate concentration. (C) Measured culture vol-
`ume. (D) Compensated culture volume. (E) Viable cell density. (F) Viable cell number. (G) Amount of glucose consumed. (H) Glucose consump-
`tion rate. (I) Specific glucose consumption rate. (J) Amount of lactate produced. (K) Lactate production rate. (L) Specific lactate production rate.
`(M) Cell viability. (N) Volumetric heparin cofactor activity of rAT.
`
`cultivation until cell viability decreased to below 90%, part
`of the culture was withdrawn so that it should not exceed
`the limit of 2 l. To accurately evaluate the effects of culture
`
`pH on cell growth and metabolism, the substantial change in
`culture volume as a result of dilution and withdrawal should
`be considered; thus, the compensation of the culture volume
`
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`

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`506
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`KUWAE ET AL.
`
`J. BIOSCI. BIOENG.,
`
`by calculation was desired. The compensated culture vol-
`ume was calculated on the basis of the assumption that the
`culture was not withdrawn throughout the cultivation pe-
`riod. The compensated data shows that the final culture vol-
`ume increased with increasing pH (Fig. 1D). The final com-
`pensated culture volume at pH 7.2 increased 3-fold compared
`with the initial volume. The viable cell number (Fig. 1F) was
`obtained by multiplying the viable cell density (Fig. 1E) by
`the compensated culture volume. The maximum viable cell
`number increased with increasing pH, indicating that the
`optimal pH for cell growth is pH 7.2 or higher. The maxi-
`mum cell number obtained at pH 6.8 was half that at pH 7.2,
`while the maximum cell density at pH 6.8 was about 80%
`of that at pH 7.2. The cell growth was severely inhibited at
`pH 6.6. The amount of glucose consumed, which was calcu-
`lated using the compensated culture volume, increased with
`increasing culture pH (Fig. 1G). Differentiating the amount
`of glucose consumed with respect to time produced the glu-
`cose consumption rate (GCR). A significant change in GCR
`with time was observed at the higher pHs (Fig. 1H), which
`made it difficult to estimate the change in GCR and thus to
`maintain the glucose concentration at the target level of 2
`g l–1 (Fig. 1A). In a similar manner, the amount of lactate
`produced and lactate production rate (LPR) were obtained.
`The amount of lactate produced decreased with decreasing
`pH (Fig. 1J). The negative values in LPR mean that lactate
`was consumed in the late cultivation stage (Fig. 1K). The
`metabolic shift from lactate production to lactate consump-
`tion occurred earlier at lower culture pH. The specific glu-
`cose consumption rate (qGlc) was obtained by dividing GCR
`
`by the viable cell number. The maximum qGlc decreased with
`decreasing pH from 7.2 to 6.6 (Fig. 1I). A similar result was
`obtained for the specific rate of lactate production (qLac)
`(Fig. 1L), indicating a strong correlation between qGlc and
`qLac. It is worthy to note that qGlc showed a maximum in the
`early cultivation stage and then decreased with increasing
`time at all pHs tested. Other research groups have also re-
`ported the decrease in qGlc with time in various batch cul-
`tures of mammalian cells, where the decrease in qGlc was
`attributed to a decrease in glucose concentration with time
`(27, 28). In our experiments, however, the decrease in qGlc
`cannot be ascribed to a decrease in glucose concentration,
`because the glucose concentration was maintained above
`0.7 g l–1. For all pHs tested, cell viability was maintained
`above 95% until 192 h but rapidly decreased after 216 h of
`cultivation (Fig. 1M). The culture was harvested when cell
`viability decreased to below 90%. Among the culture pHs
`tested, the longest viable culture duration and highest hep-
`arin cofactor (HC) activity of rAT (3200 IU l–1) were ob-
`tained at pH 6.8 (Fig. 1N). Therefore, pH 6.8 was selected
`for further experiments. Another reason for selecting pH 6.8
`is that the increase in culture volume was reduced at pH 6.8
`compared with the higher pHs due to the reduced amount of
`glucose consumption. The final compensated culture volume
`at pH 6.8 was less than 2-fold the initial volume. A small in-
`crease in culture volume would make the scaling-up of the
`process easier.
`Recently, Yoon et al. have reported the effect of culture
`pH in the batch culture of recombinant CHO cells using se-
`rum-free medium (26). When cells were grown at 37°C in
`
`the pH range between 6.85 and 7.8, the highest specific
`growth rate was observed at pH 7.2, where qGlc and qLac de-
`creased with decreasing pH. The highest erythropoietin con-
`centration was obtained at pH 6.85 mainly due to the pro-
`longed culture longevity. Although these results were ob-
`served for batch culture, they are consistent with our results
`for fed-batch culture. Several other investigators also re-
`ported decreases in qGlc and qLac when culture pH was de-
`creased from 7.2 to 7.0 or lower in cultures of mouse hybri-
`doma cells and Sp2/0 cells (29–31). Surprisingly, we found
`no reports on the effect of pH in the fed-batch culture of
`CHO cells and found only a few studies using other mam-
`malian cells. In fed-batch cultures of six Sp2/0 cell lines,
`Sauer et al. (31) reported a 2.4-fold average increase in the
`final antibody concentration when bioreactor pH was re-
`duced from 7.2 to 7.0. This increase in final antibody con-
`centration was attributed to the increases in both the integral
`of viable cell density and the specific antibody production
`rate. Similar to our observation, a reduced lactate accumula-
`tion was observed at lower pH.
`Several researchers reported a reduced accumulation of
`lactate in the fed-batch culture of mammalian cells by main-
`taining the residual glucose concentration at a low level
`(18–20). However, no general relationship was found be-
`tween the reduced lactate accumulation and the final con-
`centration of protein of interest, suggesting that the effect of
`the reduced lactate accumulation on the final concentration
`of protein of interest depends on cell lines. In addition, a
`high degree of instrumentation is generally required to main-
`tain the glucose concentration at an optimal low level. In
`contrast, decreasing culture pH to 7.0 or lower is a relatively
`simple and easy method of reducing lactate accumulation
`and appears to be one of the effective methods of increasing
`the final concentration of the recombinant protein.
`Effect of balanced amino acid feeding We evaluated
`several cloned cell lines including 4D11 in terms of rAT
`productivity under fed-batch culture at pH 6.8 and selected
`a new cell line, 2D6, for further experiments. Cell line 2D6
`showed a high growth rate and the highest production level
`among the cell lines tested. In the present work, the feed
`medium containing glucose and amino acids was continu-
`ously fed on the basis of the glucose concentration to main-
`tain the glucose concentration at the target level of 2 g l–1.
`Therefore, the concentration of each amino acid in the feed
`medium should be balanced against the concentration of
`glucose on the basis of the specific cell requirements. To
`confirm whether the balance of each amino acid relative to
`glucose was adequate so as to avoid limitation and over-
`feeding during the fed-batch cultivation of cell line 2D6, an
`amino acid analysis of the culture supernatants was carried
`out. As shown in Fig. 2, the concentrations of Asp, Glu,
`Asn, Ser, Ala, and cystine decreased to less than 20% of
`their initial values. The concentrations of the other amino
`acids remained above 30% of their initial values and below
`3 mM (data not shown). Therefore, we hypothesized that the
`availability of these six amino acids is limiting for cell
`growth and that improving the balance of amino acids in the
`feed medium increases the cell number and rAT produc-
`tivity. To test these hypotheses, parallel fed-batch cultures
`were carried out using both the existing feed medium with
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`rAT production rate (qrAT) (Fig. 3D–F). These results show
`that the balanced amino acid feeding increased the amount
`of rAT activity not by increasing the specific rAT produc-
`tion rate but largely by increasing the viable cell number. In
`addition, it became clear that the decrease in qGlc was not
`caused by the limited availability of amino acids. As shown
`in Fig. 3D, qGlc rapidly decreased until 100 h of cultivation
`and then gradually decreased. Lactate was produced until
`100 h, and then consumed, leading to its depletion by 171 h
`of cultivation. Moreover, a rapid decrease in Ala concentra-
`tion was observed after 119 h of cultivation (Fig. 3A), indi-
`cating an increased consumption rate of Ala. The parallel
`consumption of lactate and Ala after 119 h of cultivation
`suggests that the decrease in qGlc caused a limited availabil-
`ity of pyruvate from the glycolytic pathway and then led to
`the parallel consumption of lactate and Ala. Lactate and Ala
`can be metabolized via pyruvate as a source of energy to
`yield ATP. Hence, the metabolic shift from lactate produc-
`tion to consumption was presumably caused by the decrease
`in qGlc. Furthermore, it is interesting to note that the de-
`pletion of lactate at 171 h of cultivation coincided with the
`onset of the decrease in qrAT. This result suggests that the
`reduced availability of energy caused by the depletion of
`lactate led to the decrease in qrAT.
`Effect of increased glutamate concentration We
`considered that if the availability of cellular energy sources
`were increased, the decrease in qrAT could be prevented. To
`test this hypothesis, we selected glutamate as the source of
`energy. Glutamate can be oxidized to 2-oxoglutarate by
`glutamate dehydrogenase, and then metabolized to malate
`through the TCA cycle to yield ATP. Malate can be further
`oxidized to pyruvate by the malic enzyme, and completely
`oxidized in the TCA cycle. Thus, an increase in the avail-
`
`FIG. 2. Concentration profiles of amino acids that were potentially
`limiting for cell growth in fed-batch culture of GS-CHO cell line 2D6
`using feed medium with imbalanced amino acid content. Symbols:
`open circles, aspartate; closed circles, glutamate; open diamonds, as-
`paragine; closed squares, serine; open triangles, alanine; closed dia-
`monds, cystine.
`
`an imbalanced amino acid content and a new feed medium
`with a balanced amino acid content that was reformulated
`on the basis of cell-specific amino acid requirements. The
`culture was started with an initial volume of 1.0 l, and pH
`was controlled at 6.8. The balanced amino acid feeding
`maintained the concentrations of amino acids, except that of
`Ala, at more than 20% of their initial values (Fig. 3A) and
`increased the viable cell number and amount of rAT activity
`by 25% and 30%, respectively, compared with the imbal-
`anced amino acid feeding (Fig. 3B, C). However, the bal-
`anced amino acid feeding did not affect the specific glucose
`consumption rate (qGlc), lactate accumulation, or the specific
`
`FIG. 3. Effect of balanced amino acid feeding in fed-batch culture of GS-CHO cell line 2D6. Parallel fed-batch cultures were carried out using
`the feed media with imbalanced (open squares) and balanced amino acid contents (closed triangles). (A) Concentration profiles for aspartate (open
`circles), glutamate (closed circles), asparagine (open diamonds), serine (closed squares), alanine (open triangles), and cystine (closed diamonds) in
`fed-batch culture using feed medium with a balanced amino acid content. (B) Viable cell number. (C) Total amount of heparin cofactor activity of
`rAT. (D) Specific glucose consumption rate. (E) Lactate concentration. (F) Specific production rate of rAT.
`
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`FIG. 4. Effects of increased glutamate concentration on cell metabolism, cell growth, and rAT production. The concentrations of glutamate of
`1 mM (closed triangles) and 7 mM (open circles) were maintained by continuous feeding of respective media that contained glucose and balanced
`amino acids including glutamate, respectively. (A) Glucose concentration. (B) Glutamate concentration. (C) Specific glutamate consumption rate.
`(D) Specific glucose consumption rate. (E) Lactate concentration. (F) Alanine concentration. (G) Ammonia concentration. (H) Viable cell number.
`(I) Cell viability. (J) Total amount of heparin cofactor activity of rAT. (K) Specific production rate of rAT. (L) Volumetric heparin cofactor activity
`of rAT.
`
`ability of a cellular energy source is expected by increas-
`ing the specific consumption rate of glutamate (qGlu). To in-
`crease qGlu, the effect of the residual glutamate concentra-
`tion was