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Lily Chu and David K Robinson: Industrial Choices For Protein Production by Large-Scale Cell Culture
Lily Chu and David K Robinson: Industrial Choices For Protein Production by Large-Scale Cell Culture
First, recombinant therapeutics, which are mainly recombi- Suspension cell-culture processes
nant proteins or antibodies, typically require more significant The ability to adapt many cell types to suspension culture
production quantities; therefore, this category is the best and the use of polymeric additives to reduce shear damage
representation of large-scale protein production. In this cate- have enabled the widespread application of suspension
gory, at least 70% of the licensed processes use stirred-tank cell culture [3]. As seen in our review of license applica-
bioreactors, and at least 50% use serum-free medium. tions (Table 1), suspension cell-culture processes are the
system of choice for large-scale applications, owing to the
Second, vaccines and tissue-replacement products are well-understood principles of scaling parameters and the
produced in many different cell lines that require special- ease of process control in homogeneous systems.
ized bioreactor systems and medium.
As shown by Moran et al. [4••] and Schenerman et al. [5••],
Third, although diagnostic products, which are mainly mAbs, very similar process performance and product characteris-
are similar to recombinant therapeutics, the required tics are achieved for suspension cultures conducted at
quantities are significantly lower. This allows manufacturers scales ranging from 3 to 10,000 L. A common theme in
to consider more specialized cell lines and bioreactor systems. recent publications is that certain cell-culture conditions
can affect product quality. Andersen et al. [6•] demonstrat-
Therefore, current industrial processes for large-scale ed that tissue plasminogen activator (t-PA) glycosylation
production using mammalian cell culture appear to be corresponds to CHO cell growth rate and may even be
consolidating on a common platform of suspension related to cell state.
cultures in stirred-tank reactors. The ability to use a
common set of cell lines (murine myelomas or CHOs) Schenerman et al. [5••] described the manufacture of a
enhances the ability of using a platform system. Other humanized mAb (Synagis) using NS0 cells in a stirred-
common features in these processes include a trend tank bioreactor. They found that cell-culture harvest time
towards serum-free medium, and more sophisticated affects the product profile of their antibody, whereas an
feeding or perfusion processes. increase in scale, the number of cell generations, and glu-
cose conditions did not affect product quality. Their cells
Recently published processes were cultured under serum-free, fed-batch conditions at
Recently approved biological products represent accept- the 20 to 10,000 L scale. Although there were few process-
ed industrial processes that were established early in the ing details in the publication, Schenerman et al. described
clinical development of the product. As such, these tech- a useful approach to biological process validation by testing
nologies may not represent current trends in cell-culture three critical parameters outside the proposed control
development. These industrial processes have addressed specifications and by evaluating parameter effects with
many of the barriers to large-scale processing. biochemical and functional comparisons.
Specifically, adherent cell lines can be a challenge to Moran et al. [4••] demonstrated another approach to biolog-
scale-up; therefore, many products are produced in sus- ical process validation. They produced a mAb (IgG1) using
pension cell lines including suspension-adapted CHO or NS0 cells in stirred-tank suspension cultures. The serum-
murine myelomas. Shear damage from agitation and aera- free, fed-batch process could be cultured at the 3 to 8000 L
tion can be minimized by appropriate reactor design and scale with similar process and product characteristics. The
control, and by the addition of shear-protecting agents authors identified five critical parameters and created a
such as Pluronic F68 to the media. Accumulation of waste half-fractional factorial experimental design, which tested
products can be controlled by more sophisticated feeding 16 of the 32 possible combinations of parameters. Both of
schemes involving perfusion systems, which continually these publications show that well-designed validation pro-
remove waste products, or by using the glutamine syn- tocols, in combination with accurate analytical methods,
thetase expression system developed by Lonza (formerly can provide significant insight into both the manufacturing
known as Celltech), which reduces the formation of waste process and product quality.
products. Process control systems have been either adapt-
ed from fermentation systems or newly developed for Much of the published literature continues to focus on the
cell-culture bioreactors. use of nutrient feeding to increase volumetric productivi-
ties either by increasing cell density or by increasing
Recently published literature continues to address many specific productivity [7]. Basic batch suspension processes
of these issues with more sophisticated solutions, and in a stirred-tank bioreactor have become increasingly less
also considers newly arising challenges such as how to common, because nutrient feeding schemes can reduce
maximize productivity while maintaining product quali- waste accumulation and nutrient fluctuations, which
ty, how to remove all animal derived components from allows higher cell densities and productivities to be
cell-culture medium, how to minimize contamination reached. Recently designed suspension processes tend to
during large-scale production, and how to control carbon fall into three categories: batch-refeed, fed-batch or perfu-
dioxide removal. sion processes. Some processes need to be designed with
182 Biochemical engineering
Table 1
Specific details for each BLA product generated in mammalian cell-culture systems, 1996–2000*.
BLA name Manufacturer Approval Type of product Cell line Method Medium
date
Recombinant therapeutics
Tenecteplase/TNKase
Reduction of mortality Genentech 6/2/00 Recombinant CHO Not revealed Not revealed
associated with acute glycoprotein
myocardial infarction
Antihemophilic factor
(recombinant)/ReFacto
Control of hemorrhagic Genetics 3/6/00 Recombinant CHO Continuous Serum-free, containing
episodes Institute glycoprotein perfusion human serum albumin
and recombinant insulin
Interferon α-n1,
lymphoblastoid/Wellferon
Treatment of chronic Wellcome Foundation 3/25/99 Endogenous Human lympho- Not revealed Not revealed
hepatitis C Limited, Wellcome glycoprotein blastoid cell line
Research Laboratories
Coagulation factor VIIa
(recombinant)/NovoSeven
Treatment of bleeding in Novo Nordisk A/S 3/25/99 Recombinant BHK Not revealed Serum (NCS)-
hemophilia A or B patients glycoprotein containing
Etanercept/Enbrel
Treatment of rheumatoid Immunex 11/2/98 IgG1 fusion with CHO Stirred-tank Not revealed
arthritis TNF receptor bioreactor,
suspension
Trastuzumab/Herceptin
Treatment of metastatic Genentech 9/25/98 mAb, humanized CHO Stirred-tank Serum-free,
breast cancer IgG1-κ with bioreactor, containing gentamicin
murine CDRs suspension
Infliximab/Remicade
Treatment of Crohn’s disease Centocor 8/24/98 mAb, mouse/human Murine myeloma Stirred-tank Serum-free, containing
chimeric IgG1-κ cell line, bioreactor, protein hydrolysates,
recombinant (NS0 suspension, bovine transferrin,
based on media perfusion, insulin, serum albumin,
components) culture and Excyte
(spin filter)
Palivizumab/Synagis
Prophylaxis of respiratory MedImmune 6/19/98 mAb NS0, Stirred-tank Serum-free
syncytial virus recombinant bioreactors,
suspension,
fed-batch
Basiliximab/Simulect
Prophylaxis of acute organ Novartis 5/12/98 mAb, mouse/human Murine myeloma Stirred-tank Serum-free
rejection Pharmaceuticals chimeric IgG1-κ cell line, bioreactor,
recombinant suspension,
perfusion
culture
Daclizumab/Zenapax
Prophylaxis of acute organ Hoffman-LaRoche 12/10/97 mAb, mouse/human SP2/0 Not revealed Not revealed
rejection chimeric IgG1
Rituximab/Rituxan
Treatment of B-cell IDEC Pharmaceutical/ 11/26/97 mAb, mouse/human CHO Stirred-tank Containing
non-Hodgkin’s lymphoma Genentech chimeric IgG1-κ bioreactors, gentamicin
suspension
development timelines as the constraint; therefore, a Cacciuttolo et al. [8] focused on removing toxic by-product
batch-refeed process can be the most efficient route to a build-up, specifically ammonium, by using medium exchanges
highly productive process [8,9]. and medium optimizations; this allowed an increase in cell
Industrial choices for protein production Chu and Robinson 183
Table 1 (continued)
BLA name Manufacturer Approval Type of product Cell line Method Medium
date
Coagulation factor IX
(recombinant)/Benefix
Control of hemorrhagic Genetics Institute 2/11/97 Recombinant CHO Stirred-tank Serum-free, containing
episodes in patients with glycoprotein bioreactors, recombinant insulin
hemophilia B suspension,
batch-refeed
Interferon β-1a/Avonex
Treatment of multiple Biogen 5/17/96 Recombinant CHO Stirred-tank Not revealed
sclerosis glycoprotein bioreactors,
suspension
Vaccines
Rotavirus vaccine, live, oral,
tetravalent/RotaShield
Immunization for rotavirus Wyeth Laboratories 8/31/98 Live viral vaccine FRhL-2 Not revealed Serum (FBS)-
containing cell growth
medium with neomycin
sulfate and
amphotericin B
Rabies vaccine/RabAvert
Immunization for rabies Chiron Behring 10/20/97 Vaccine Not revealed Not revealed Not revealed
Hepatitis A vaccine,
inactivated/VAQTA
Immunization for hepatitis A Merck & Co 3/29/96 Inactivated virus Human MRC-5 Adherent, NCF Serum-containing
diploid fibroblasts to Costar Cubes,
continuous fed-
batch culture
Tissue-culture products
Autologous cultured
chondrocytes/Carticel
SM Service
Repair of cartilaginous Genzyme Tissue 8/22/97 Autologous cells Chondrocytes Tissue-culture Serum-containing
defects Repair flasks
Diagnostic products
Human T-lymphotropic virus
types I and II/Abbott HTLV-I/
HTLV-II EIA
For use in enzyme Abbott Laboratories 8/15/97 mAb Chronically Not revealed Not revealed
immunoassay infected human
T and B lymphocytes
Capromab pendetide/
ProstaScint
Cancer imaging agent Cytogen 10/28/96 mAb, murine IgG1-κ Murine hybridoma Hollow fiber Serum-free, containing
cell line bovine serum albumin
and transferrin
Nofetumomab/Verluma
Cancer imaging agent Dr Karl Thomae 8/20/96 mAb, murine IgG2b Mammalian cells Not revealed Serum-free
FAb fragment
Imciromab pentetate/
Myoscint
Cardiac imaging agent Centocor 7/3/96 mAb, murine IgG2-κ Murine hybridoma Not revealed Serum-containing,
FAb fragment cell line containing gentamicin
*Source: http://www.fda.gov. BHK, baby hamster kidney; CDR, complementarity-determining region; FAb, fragment of antigen binding; FBS, fetal
bovine serum; FRhL-2, fetal rhesus diploid cell line; NCS, newborn calf serum; TNF, tumor necrosis factor.
density to greater than 5 × 106 cells/mL and an extension of 6 oxygen limitations, decreased cell growth, and increased lactic
days in culture longevity. This process was developed at scales acid production. These issues were addressed by increasing the
of 500 mL to 20 L, and was demonstrated in a 200 L stirred- dissolved oxygen from 20 to 50% (relative to air saturation).
tank reactor. All parameters were easily scaled from 3 to 200 L
with the exception of dissolved oxygen control. The authors Another approach to decreasing process development time
proposed that non-ideal mixing at the 200 L scale resulted in requirements is to develop a platform cell-culture process
184 Biochemical engineering
Table 2
13 3 4 1
Stirred- Speciality Unknown Stirred- Speciality Unknown Stirred- Speciality Unknown Stirred- Speciality Unknown
tank tank tank tank
9 0 4 0 1 2 0 1 3 0 1 0
Batch/ Perfusion Unknown Batch/ Perfusion Unknown Batch/ Perfusion Unknown Batch Perfusion Unknown
fed-batch fed-batch fed-batch fed-batch
6 3 4 1 0 2 0 1 3 0 0 1
Serum- Serum- Unknown Serum- Serum- Unknown Serum- Serum- Unknown Serum- Serum- Unknown
free containing free containing free containing free containing
6 1 6 0 2 1 2 1 1 0 1 0
*BLA products are categorized on the basis of product application, and subcategories for each product application include reactor type, process
feeding design and medium type. Source: www.fda.gov.
that can be used to produce many different recombinant a suspension stirred-tank process. The authors examined
proteins or mAbs. Sauer et al. [10••] developed a generic batch, fed-batch and perfusion culture at the 3 L develop-
serum-free, fed-batch culture in stirred-tank bioreactors ment scale, and implemented the perfusion process with
using Sp2/0 and NS0 cell lines to produce human or an external hollow fiber module at the 20 L scale, because
humanized IgG isotypes. The process was optimized at this gave the highest cell density (1.3 × 107 cells/mL) and
the 3 L scale, and scaled-up directly to the 15 and 750 L the highest mAb concentration (150 U/L). By adding a
scales with similar cell densities, specific mAb productivi- controlled feed of glucose to the perfusion culture, they
ty rates, and product quality. The same fed-batch process were able to increase both cell density and mAb concen-
was used in seven additional Sp2/0 cell lines and one NS0 trations to 1.8 × 107 cells/mL and 275 U/L, respectively.
cell line. The authors demonstrated that choosing a Yang et al. [16••] have proposed that controlled-fed perfu-
platform system can significantly decrease process devel- sion significantly increases the daily mAb productivity
opment timelines. Sauer et al. [10••] also suggested that through two combined effects: increased cell density and
the same process can be applied to other cell lines as a minimized product dilution resulting from the glucose
generic fed-batch process, but they published only data for feed. They noticed a difference in glycosylation distribu-
one additional cell line (NS0), which is related to the SP2/0 tion between roller bottle cultures and the final optimized
line. Additional processes have been developed using controlled-fed perfusion cultures. Therefore, as noted in
serum-free, fed-batch suspension cultures in stirred-tank other papers examining product quality, cell-culture differ-
bioreactors including CHO/humanized mAb and 293/ade- ences can affect product characteristics.
noviral green fluorescent protein systems [11,12].
Adherent cell-culture processes
Perfusion suspension cultures seem to be the most com- Adherent cell-culture processes present unique problems to
mon processing format for protein production. When industrial scale-ups. Because adherent cell lines have sur-
designing a stirred-tank perfusion process, cell retention face-attachment requirements, the surface to volume ratio
can be achieved through many methods: centrifugation in the bioreactor needs to be maximized for maximal cell
[P1], internal and external spin filters [13,14,15•], and hol- densities. Traditional approaches to adherent cell culture
low fiber modules [16••,17]. Iding et al. [15•] performed a include roller bottle cultures [P2,21•], multilayer stacked
detailed analysis on the effects of perfusion and culture plate systems such as Costar cellcubes and Nunc cell facto-
conditions on the performance of an internal spin filter ries [P3], and microcarriers suspended in stirred-tanks
using a CHO/prothrombin system at the 200 L scale [15•]. [22,23,24•,25]. The current use of roller bottle cultures and
Some researchers have used hollow fiber bioreactors for multilayer stacked plate systems is limited to more special-
more specialized perfusion systems, but the productivity ized fields including erythropoetin production, gene
of these tends to be much lower [18–20]. therapy, and vaccine production. Unger et al. [21•] modeled
the flow and fluid mixing in roller bottle bioreactors and
Yang et al. [16••] merged the concepts of controlled feeding confirmed theoretical results by particle imaging velocime-
for fed-batch cultures into perfusion culture processing, try in experimental systems. They could improve the speed
which they described as controlled-fed perfusion cultures. and completeness of mixing in roller bottle systems by
They grew Sp2/0-Ag14 cells producing hLL2 (humanized adding a vertical rocking motion. Their work has shown that
B-cell specific IgG2a) in serum-free medium. The process even well-established bioreactor processes can still benefit
was originally a roller bottle process that was adapted into from design improvements based on engineering principles.
Industrial choices for protein production Chu and Robinson 185
Non-traditional bioreactors for adherent cell culture include The authors demonstrated that carbon dioxide accumula-
a bubble column bioreactor, a membrane dialysis reactor tion can be problematic for microcarrier suspensions at the
with integrated radial-flow fixed bed, and a perfusion 30 to 2000 L scale in stirred-tank reactors where the total
packed-bed reactor [26–29]. aeration has been minimized by microaeration with pure
oxygen to avoid damage from excessive sparging. Nitrogen
The most predominant bioreactor format for adherent cul- sparging was used, together with the on-line analysis of
tures remains microcarriers suspended in stirred-tanks. dissolved carbon dioxide, to control the level of accumu-
The use of microcarriers in stirred-tank bioreactors allows lated dissolved carbon dioxide to acceptable levels.
operation at scales of up to 10,000 L, and provides for more
efficient process optimization and flexibility in feeding Contamination of cell-culture processes remains a signifi-
process designs. This is further evidence that industry cant issue for large-scale industrial processes. Some
favors platform technologies with similar technical con- contamination issues are due to regulatory concerns of
cepts. The same principles and themes emerge when viruses or prions that might be transferred potentially from
developing feeding schemes for suspension and adherent animal components in the culture medium [3,32]. Other
culture processes — the goal is to reduce waste accumula- contamination issues result from imperfect processing con-
tion and nutrient variability, and establish a high cell ditions, which result in the need to include antibiotics in
density culture. the large-scale culture, as judged by the number of com-
mercial products where antibiotics are used in the
The predominant process design for adherent cultures in cell-culture process (Table 1).
recent literature reports are perfusion cultures with micro-
carrier beads. Kong et al. [22] implemented perfusion The regulatory push for serum-free and protein-free medi-
microcarrier culture at 3 and 15 L scales in stirred-tank bio- um will require significant changes in process design, in
reactors to produce monocyte-colony inhibiting factor with particular for adherent cell cultures. Almost all the suspen-
CHO cells. Internal spin filters were installed in the bioreac- sion cell-culture processes reviewed above have been
tors to retain cells/microcarriers over a period of 2 months. A designed using serum-free medium and many are even
unique feature of this process was the ability to inoculate protein-free, but the opposite is true for adherent cell-cul-
microcarrier cultures directly without a cell-removal step. ture processes. Therefore, industry may find it easier to
Most parameters scaled-up easily from the 3 to the 15 L adapt processes to suspension cultures with well-estab-
scale, but Kong et al. [22] found it necessary to re-optimize lished cell lines in order to design a serum-free and
both the microcarrier concentration to increase surface area protein-free process, rather than try to adapt adherent cell
and the impeller design for high microcarrier loads. lines to serum-free processes. Other sources of contamina-
tion are currently controlled with the use of antibiotics in
The direct transfer of cells between microcarrier beads the culture medium, but promising research in the field of
without a cell removal step has also been reported in other robot automation for stirred-tank bioreactors is on going.
studies [23,24•]. Zhang et al. [25] used a 3 L stirred-tank Lutkemeyer et al. [33•] have shown that a robot can be
bioreactor equipped with an internal spin filter for perfu- implemented at 20 and 100 L scales to perform sterile
sion culture to produce recombinant human stanniocalcin sampling procedures and deliver the samples to the
(hSTC) in CHO cells. In parallel, hSTC was also produced appropriate analytical machines.
in Sf9 cells at the 60 L scale. Although both methods pro-
duced similar amounts of product, hSTC produced in For more specialized applications where flexibility, dispos-
CHO cells had a different glycosylation pattern to that of ability and additional process control are preferred, an
the Sf9-derived products. These glycosylation differences additional disposable culture system has been introduced
show that mammalian cells must be used if glycosylation recently. The Wave bioreactor (Wave Biotech/Panacea
patterns are important. Solutions, Inc, Bedminster, NJ) takes advantage of the
development of sterile bags, more commonly used for media
Common themes for suspension and adherent cell- storage, which can be adapted for the growth of adherent and
culture processes suspension cell lines [34•,35•]. The system has been imple-
Many of these papers have addressed issues of oxygen lim- mented for different cell/protein systems including
itation as bench-scale processes are transferred to the NS0/mAb, 293/adenovirus and CHO/recombinant protein.
manufacturing scale, and improvements continue in this
area [30]. Another cell-culture process control issue — the Conclusions
measurement and control of dissolved carbon dioxide — Reviewing the current processes used in the large-scale
has emerged partly because of the success of oxygen addi- cell-culture production of recombinant proteins and anti-
tion with minimal aeration. Until recently, reliable bodies indicates that industry has converged on using
measurement of dissolved carbon dioxide was only avail- suspension cultures in stirred-tank bioreactors with some
able by off-line analysis. However, Pattison et al. [31••] form of controlled feeding (e.g. fed-batch or perfusion).
report the use of an on-line carbon dioxide analyzer (YSI Both recently approved FDA BLAs and the published lit-
8500) in both microbial and mammalian culture processes. erature suggest that companies may be considering the use
186 Biochemical engineering
of platform technologies to speed up process development 10. Sauer PW, Burky JE, Wesson MC, Sternard HD, Qu L: A high-
•• yielding, generic fed-batch cell culture process for production of
timelines. For example, more than 50% of approved BLAs recombinant antibodies. Biotechnol Bioeng 2000, 67:585-597.
using mammalian cell culture chose to use CHO or murine Development of a generic fed-batch process that can be applied to seven
Sp2/0 and one NS0 cell lines from the 15 to the 750 L scale. This is a good
myeloma cells, and the same trend was apparent in the indication of the industry trend towards platform or generic technologies that
review of published literature. reduce process development timelines.
11. Schoenherr I, Stapp T, Ryll T: A comparison of different methods to
There are exceptions in niche applications for which spe- determine the end of exponential growth in CHO cell cultures for
optimization of scale-up. Biotechnol Prog 2000, 16:815-821.
cific cell lines are required and disposables allow
adaptation to a variety of cell lines (e.g. vaccine produc- 12. Nadeau I, Jacob D, Perrier M, Kamen A: 293SF metabolic flux
analysis during cell growth and infection with an adenoviral
tion). Large-scale cell-culture production is still faced with vector. Biotechnol Prog 2000, 16:872-884.
significant challenges — maximizing productivity while 13. Monica TJ, Montgomery T, Ayala JL, Schoofs GM, Whiteley EM,
maintaining product quality, removing all animal derived Roth G, Garbutt JJ, Harvey S, Castillo FJ: Monitoring adenovirus
infections with on-line and off-line methods. Biotechnol Prog
components from cell-culture medium, minimizing conta- 2000, 16:866-871.
mination issues at large-scale production, and controlling
14. Stiens LR, Buntemeyer H, Lutkemeyer D, Lehmann J, Bergmann A,
carbon dioxide removal. Weglohner W: Development of serum free bioreactor production
of recombinant human thyroid stimulating hormone receptor.
Biotechnol Prog 2000, 16:703-709.
References and recommended reading
Papers of particular interest, published within the annual period of review, 15. Iding K, Lutkemeyer D, Fraune E, Gerlach K, Lehmann J: Influence of
have been highlighted as: • alterations in culture conditions and changes in perfusion
parameters on the retention performance of a 20 micron spinfilter
• of special interest during a perfusion cultivation of a recombinant CHO cell line in
•• of outstanding interest pilot scale. Cytotechnology 2000, 34:141-150.
1. Henry CM: The next pharmaceutical century. Chem Eng News Studies the effect of cell-culture conditions on the effectiveness of a commer-
2000, 85-100. cially available 20 micron internal spin filter. Determines that cell retention is not
affected by spin filter rotation velocity nor perfusion rate. Cell retention is
2. Glacken MW, Fleischaker RJ, Sinskey AJ: Large-scale production increased by increased operation time, cell diameter and culture aggregation.
of mammalian cells and their products: engineering principles
and barriers to scale-up. Ann New York Acad Sci 1983, 16. Yang J-D, Angelillo Y, Chaudhry M, Goldenberg C, Goldenberg DM:
413:355-372. •• Achievement of high cell density and high antibody productivity
by a controlled-fed perfusion bioreactor process. Biotechnol
3. van der Pol L, Tramper J: Shear sensitivity of animal cells from a Bioeng 2000, 69:74-82.
culture-medium perspective. Trends Biotechnol 1998, 16:323-328. Integrates the concepts of fed-batch and perfusion processing into a single
process, which increases overall productivity twofold relative to the original
4. Moran EB, McGowan ST, McGuire JM, Frankland JE, Oyebade IA, perfusion process.
•• Waller W, Archer LC, Morris LO, Pandya J, Nathan SR et al.:
A systematic approach to the validation of process control 17. Furey J: Continuous cell culture using the ATF system. Genet Eng
parameters for monoclonal antibody production in fed-batch News 2000, 20:52.
culture of a murine myeloma. Biotechnol Bioeng 2000,
69:242-255. 18. Prilliman KR, Lindsey M, Jackson KW, Cole J, Bonner R,
Well-designed validation experiments that address parameter range specifi- Hildebrand WH: Complexity among constituents of the
cations in a systematic approach. Shows that statistical design experiments HLA-B·1501 peptide motif. Immunogenetics 1998, 48:89-97.
can be a powerful tool when considering the effects of a large group of
process parameters. May be an indication of future requirements for regula- 19. Lamers CHJ, Gratama JW, Luider-Vrieling B, Bolhuis RLH,
tory authority approval of biological process validation. Bast EJEG: Large-scale production of natural cytokines during
activation and expansion of human T lymphocytes in hollow fiber
5. Schenerman MA, Hope JN, Kletke C, Singh JK, Kimura R, Tsao E, bioreactor cultures. J Immunother 1999, 22:299-307.
•• Folena-Wasserman G: Comparability testing of a humanized
monoclonal antibody (Synagis) to support cell line stability, 20. Gramer MJ, Poeschl DM: Comparison of cell growth in T-flasks, in
process validation, and scale-up for manufacturing. Biologicals micro hollow fiber bioreactors, and in an industrial scale hollow
1999, 27:203-215. fiber bioreactor system. Cytotechnology 2000, 34:111-119.
A targeted approach to validating three process parameters — population 21. Unger DR, Muzzio FJ, Aunins JG, Singhvi R: Computational and
doubling, glucose concentration, and culture harvest time. The authors • experimental investigation of flow and fluid mixing in the roller
demonstrate that, with the correct selection of analytical methods, ‘compa- bottle bioreactor. Biotechnol Bioeng 2000, 70:117-130.
rability’ can be shown for different processing conditions, and they validate Examines the velocity profile in the traditional roller bottle bioreactor using
the process specification ranges. theoretical modeling and experimental imaging techniques. These results
6. Andersen DC, Bridges T, Gawlitzek M, Hoy C: Multiple cell culture indicate that the mixing was incomplete, particularly in the axial direction,
• factors can affect the glycosylation of Asn-184 in CHO-produced under typical operating conditions. The authors redesign the operating con-
tissue-type plasminogen activator. Biotechnol Bioeng 2000, ditions to include an additional direction of rocking, which improves the
70:25-31. speed and completeness of mixing.
This paper examines the effect of specific cell growth parameters on t-PA site
22. Kong D, Gentz R, Zhang J: Long-term stable production of
occupancy. The authors demonstrate that feed composition, feeding process,
monocyte-colony inhibition factor (M-CIF) from CHO microcarrier
and temperature result in different glycosylation patterns. Further investigation
suggests that these parameters affect cell growth rate and cell state. A cor- perfusion cultures. Cytotechnology 1998, 26:131-138.
relation is established between cell growth state and site occupancy. 23. Kumar A, Goel AS, Payne JK, Evans C, Mikolajczyk SD,
7. Kundu PK, Prasad NS, Datta D: Monoclonal antibody: high density Kuus-Reichel K, Saedi MS: Large-scale propagation of
culture of hybridoma cells and downstream processing for IgG recombinant adherent cells that secrete a stable form of human
recovery. Indian J Exp Biol 1998, 36:125-135. glandular kallikrein, hK2. Protein Expres Purif 1999, 15:62-68.
8. Cacciuttolo MA, Patchan M, Lamey K, Allikmets E, Tsao E: Large- 24. Hu X, Xiao C, Huang Z, Guo Z, Zhang Z, Li Z: Pilot production of
scale production of a monoclonal IgM in a hybridoma suspension • u-PA with porous microcarrier cell culture. Cytotechnology 2000,
culture. Pharm Technol 1998, 22:44-58. 33:13-19.
Demonstrates direct inoculation from the 1 or 5 L scale to 30 L bioreactors
9. Hammill L, Welles J, Carson GR: The gel microdrop secretion using CHO cells grown on microcarrier beads. The cell/microcarrier system
assay: identification of a low productivity subpopulation arising can also migrate between microcarrier beads without a trypsinization step.
during the production of human antibody in CHO cells. These two papers [26,27•] show that direct bead to bead transfer is feasi-
Cytotechnology 2000, 34:27-37. ble and can greatly simplify microcarrier processes.
Industrial choices for protein production Chu and Robinson 187
25. Zhang J, Alfonso P, Tahotakura NR, Su J, Buergin M, Parmelee D, of animal origin contaminants in animal cell culture.
Collins AW, Oelkuct M, Gaffney S, Gentz S et al.: Expression, Cytotechnology 2000, 33:27-36.
purification, and bioassay of human stanniocalcin from
baculovirus-infected insect cells and recombinant CHO cells. 33. Lutkemeyer D, Poggendorf I, Scherer T, Zhang J, Knoll A, Lehmann J:
Protein Expres Purif 1998, 12:390-398. • First steps in robot automation of sampling and sample
management during cultivation of mammalian cells in pilot scale.
26. Rodrigues MTA, Vilaca PR, Garbuio A, Takagi M, Barbosa S Jr, Leo P, Biotechnol Prog 2000, 16:822-828.
Laignier NS, Silva AAP, Moro AM: Glucose uptake rate as a tool to Demonstrates that robot automation may be feasible for large-scale mam-
estimate hybridoma growth in a packed bed bioreactor. malian culture. This provides an alternative to antibiotic addition for reducing
Bioprocess Eng 1999, 21:543-546. large-scale contamination issues in mammalian cultures.
27. Portner R, Ludemann I, Reher K, Neumaier M, Markl H: Fixed-bed 34. Singh V: Disposable bioreactor for cell culture using wave-
dialysis culture of a transfectoma cell line producing chimeric • induced agitation. Cytotechnology 1999, 30:149-158.
Fab-fragments with ‘nutrient-split’-feeding strategy. Biotechnol Many cell/product systems are produced at the 2 to 200 L scale using the
Tech 1998, 12:501-505. Wave bioreactor. This paper demonstrates that these bioreactors are quite
adaptable to different processes including suspension and microcarrier
28. Kaufman JB, Wang G, Zhang W, Valle MA, Shiloach J: Continuous processes. Direct comparison with conventional stirred-tank systems shows
production and recovery of recombinant Ca2+ binding receptor that they have similar growth rates and productivities.
from HEK 293 cells using perfusion through a packed bed
bioreactor. Cytotechnology 2000, 33:3-11. 35. Namdev PK, Lio P: Assessing a disposable bioreactor for
• attachment-dependent cell cultures. Biopharm 2000, 13:44-50.
29. Hu Y-C, Kaufman J, Cho MW, Golding H, Shiloach J: Production of The authors grow 293 and CHO cells attached to microcarriers to
HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 show that fed-batch and perfusion cultures can be performed in 2 L
expression system. Biotechnol Prog 2000, 16:744-750. Wave bioreactors.
30. Dutton G: Improved tools for large-scale bioprocessing. Genet
Eng News 2000, 20:11, 57, 63.
Patents
31. Pattison RN, Swamy J, Mendenhall B, Hwang C, Frohlich BT: P1. Bonarius H: Method for producing heterologous proteins in
•• Measurement and control of dissolved carbon dioxide in eukaryotic cells on an industrial scale using nucleotide-
mammalian cell culture processes using an in situ fiber optic manipulating agents. US Patent 1999, WO 200032754.
chemical sensor. Biotechnol Prog 2000, 16:769-774.
This paper addresses the problem of controlling dissolved carbon dioxide in P2. Carcagno C, Criscuolo M, Melo C, Vidal J: Method for the massive
large-cell mammalian cultures (30 to 2000 L scale). The authors success- culture of cells producing recombinant human erythropoietin.
fully use an on-line dCO2 probe (YSI 8500) to control carbon dioxide levels US Patent 2000, WP 200027997.
with N2 sparging at all bioreactor scales.
P3. Zhang S, Thwin C, Wu Z, Cho T, Gallagher S: An improved method
32. Jayme DW, Smith SR: Media formulation options and for the production and purification of adenoviral vectors.
manufacturing process controls to safeguard against introduction US Patent 2000, WP 200032754.