180

Industrial choices for protein production by large-scale cell culture
Lily Chu* and David K Robinson
Traditional barriers to large-scale mammalian culture have now adapting cells to suspension culture, shear sensitivity and oxy-
been addressed, with the standard stirred-tank reactor gen supply mostly resolved. The remaining challenges in
emerging as industry’s technology of choice. The issues of these cases are the control of product quality while maximiz-
adapting cells to suspension culture, shear sensitivity and ing productivity, the control of carbon dioxide concentrations
oxygen supply have been largely resolved. But for many low- on scale-up, and the minimization of the risk of contamina-
volume and speciality applications, such as the production of tion by adventitious agents through raw material and
viral vaccines and gene therapies, reactor technology remains manufacturing controls. For many low-volume and speciality
diversified, with reactor types ranging from roller bottles to applications, however, the reactor technology remains diversi-
stacked plates and hollow fibers. fied with reactor types ranging from roller bottles to stacked
plates and hollow fibers in use for the production of products
Addresses such as viral vaccines, gene therapies and diagnostics. The
Bioprocess R&D, Merck Research Laboratories, Merck & Co, Inc, use of transgenic plants and animals as production vehicles,
PO Box 2000, Rahway, NJ 07065, USA although not covered in this review, remains an area of active
*e-mail: lily_chu@merck.com
development that may play a role in applications requiring
Current Opinion in Biotechnology 2001, 12:180–187 exceptional product volumes (e.g. in excess of 100 kg per year
for monoclonal antibodies [mAbs]), but substantial regulatory
0958-1669/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved. hurdles remain to be addressed and products have yet to be
licensed using transgenics as the production vehicles.
Abbreviations
BLA biological license approval
CHO Chinese hamster ovary
Here we review some of the products recently approved by
FDA Food and Drug Administration the USA Food and Drug Administration (FDA), together
hSTC human stanniocalcin with recently published methods that have been used in
mAb monoclonal antibody large-scale cell-culture processes.

Introduction Current Food and Drug Administration
The biotechnology industry has grown significantly in the (FDA)-approved products
past decade and continues to grow at a rapid rate. Experts We have reviewed recent FDA biological license approvals
predict that protein therapeutics are just beginning to enter (BLAs) from January 1996 to November 2000. More than half
the marketplace, with a surge of protein, antibody and pep- of these approved biotechnology products (21 out of 33) are
tide drugs expected in the next 10–20 years [1]. This rapid manufactured using mammalian cell systems (see FDA web-
growth in protein therapeutics has been led by a diverse site http://www.fda.gov). To determine patterns in the
range of products produced in mammalian cell culture that technologies applied, we have categorized these products on
have been licensed in the past five years, including, among the basis of their applications into therapeutic recombinants,
others, a monoclonal antibody (mAb) for treating breast can- vaccines, tissue-culture products or diagnostic products.
cer, Herceptin, an immunoglobulin-TNF (tumor necrosis Generated from information available in the public domain,
factor) receptor fusion protein for treating rheumatoid arthri- Table 1 lists the following data for each approved product: the
tis, Enbrel, and an inactivated hepatitis A vaccine, Vaqta. production mammalian cell line; the type of bioreactor and
process; and the type of production medium.
As indicated by the successful licensure in the USA of 21 dif-
ferent therapeutic and diagnostic products produced in There are clearly three principal mammalian cell lines of
mammalian cell culture since 1996, cell-culture technology choice: Chinese hamster ovary (CHO) cells; and the
has progressed to a status allowing the reliable development, murine myeloma lines SP2/0 and NS0. These three cell
scale-up, and subsequent operation on a commercial level. In lines have been used to produce 11 out of 21 approved
the 1980s, as nicely summarized by Glacken et al. [2] in 1983, products, and nearly all recombinant therapeutics.
many reactor types were considered for the development of
cell-culture processes. Key barriers to large-scale mammalian We have summarized the data in Table 1 into more relevant
culture were considered to include oxygen supply limita- categories in Table 2. Again, only products generated in
tions, waste product accumulation, the need for more mammalian cell systems are considered in Table 2, and the
sophisticated process control, shear sensitivity of animal four subcategories are based on product application. For
cells, and the challenges of growing adherent cell lines [2]. each of these product applications, the reactor type, process
feeding design, and medium type are summarized.
As highlighted in this review, the reactor technology for much
large-scale cell culture has settled on the standard stirred-tank Biological licensing approval statistics show the
reactor as the technology of choice, with the issues of following trends.

which ty. fed-batch or perfu- dioxide removal. the required tics are achieved for suspension cultures conducted at quantities are significantly lower. this category is the best and the use of polymeric additives to reduce shear damage representation of large-scale protein production. The serum- murine myelomas. A common theme in to consider more specialized cell lines and bioreactor systems. which reduces the formation of waste process and product quality. very similar process performance and product characteris- are similar to recombinant therapeutics. recombinant therapeutics. Industrial choices for protein production Chu and Robinson 181 First. As shown by Moran et al. which tested products can be controlled by more sophisticated feeding 16 of the 32 possible combinations of parameters. because nutrient feeding schemes can reduce maximize productivity while maintaining product quali. tocols. Their cells Recently published processes were cultured under serum-free. ease of process control in homogeneous systems. Specifically. Although there were few process- ed industrial processes that were established early in the ing details in the publication. tions (Table 1). Accumulation of waste half-fractional factorial experimental design. and at least 50% use serum-free medium. 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]. [5••]. Some processes need to be designed with . and more sophisticated affects the product profile of their antibody. Shear damage from agitation and aera. sion processes. products. Suspension cell-culture processes nant proteins or antibodies. recent publications is that certain cell-culture conditions can affect product quality. described clinical development of the product. [5••] described the manufacture of a enhances the ability of using a platform system.000 L. in combination with accurate analytical methods. therefore. The control. Schenerman et al. and in a stirred-tank bioreactor have become increasingly less also considers newly arising challenges such as how to common. This allows manufacturers scales ranging from 3 to 10. They found that cell-culture harvest time towards serum-free medium. [4••] and Schenerman et al. Recently designed suspension processes tend to during large-scale production. the 20 to 10. adherent cell lines can be a challenge to Moran et al. although diagnostic products. waste accumulation and nutrient fluctuations. ized bioreactor systems and medium. which are mainly mAbs. the number of cell generations. 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. Both of schemes involving perfusion systems. In this cate. suspension cell-culture processes are the system of choice for large-scale applications. which are mainly recombi. 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. whereas an feeding or perfusion processes.000 L scale. Andersen et al. or by using the glutamine syn. thetase expression system developed by Lonza (formerly can provide significant insight into both the manufacturing known as Celltech). As such. ical process validation. Third. and glu- cose conditions did not affect product quality. The ability to use a common set of cell lines (murine myelomas or CHOs) Schenerman et al. As seen in our review of license applica- bioreactors. typically require more significant The ability to adapt many cell types to suspension culture production quantities. which continually these publications show that well-designed validation pro- remove waste 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. vaccines and tissue-replacement products are well-understood principles of scaling parameters and the produced in many different cell lines that require special. these tech. owing to the Second. [6•] demonstrat- Therefore. have enabled the widespread application of suspension gory. cultures in stirred-tank reactors. 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. how to remove all animal derived components from allows higher cell densities and productivities to be cell-culture medium. at least 70% of the licensed processes use stirred-tank cell culture [3]. Basic batch suspension processes of these issues with more sophisticated solutions. increase in scale. free. many products are produced in sus. They produced a mAb (IgG1) using pension cell lines including suspension-adapted CHO or NS0 cells in stirred-tank suspension cultures. Other humanized mAb (Synagis) using NS0 cells in a stirred- common features in these processes include a trend tank bioreactor. therefore. how to minimize contamination reached. These industrial processes have addressed specifications and by evaluating parameter effects with many of the barriers to large-scale processing. and how to control carbon fall into three categories: batch-refeed. [4••] demonstrated another approach to biolog- scale-up. fed-batch conditions at Recently approved biological products represent accept. biochemical and functional comparisons. and by the addition of shear-protecting agents authors identified five critical parameters and created a such as Pluronic F68 to the media.

bioreactor. Not revealed Not revealed hepatitis C Limited. lymphoblastoid/Wellferon Treatment of chronic Wellcome Foundation 3/25/99 Endogenous Human lympho. bovine transferrin. perfusion culture Daclizumab/Zenapax Prophylaxis of acute organ Hoffman-LaRoche 12/10/97 mAb. protein hydrolysates. mouse/human Murine myeloma Stirred-tank Serum-free. [8] focused on removing toxic by-product batch-refeed process can be the most efficient route to a build-up. insulin. containing chimeric IgG1-κ cell line. based on media perfusion. suspension Trastuzumab/Herceptin Treatment of metastatic Genentech 9/25/98 mAb. 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 gentamicin murine CDRs suspension Infliximab/Remicade Treatment of Crohn’s disease Centocor 8/24/98 mAb. gentamicin suspension development timelines as the constraint.182 Biochemical engineering Table 1 Specific details for each BLA product generated in mammalian cell-culture systems. serum albumin. specifically ammonium. suspension.9]. Stirred-tank Serum-free syncytial virus recombinant bioreactors. therefore. recombinant (NS0 suspension. mouse/human SP2/0 Not revealed Not revealed rejection chimeric IgG1 Rituximab/Rituxan Treatment of B-cell IDEC Pharmaceutical/ 11/26/97 mAb. this allowed an increase in cell . by using medium exchanges highly productive process [8. humanized CHO Stirred-tank Serum-free. and medium optimizations. 1996–2000*. a Cacciuttolo et al. fed-batch Basiliximab/Simulect Prophylaxis of acute organ Novartis 5/12/98 mAb. mouse/human CHO Stirred-tank Containing non-Hodgkin’s lymphoma Genentech chimeric IgG1-κ bioreactors. bioreactor. breast cancer IgG1-κ with bioreactor. containing episodes Institute glycoprotein perfusion human serum albumin and recombinant insulin Interferon α-n1. recombinant suspension. 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. mouse/human Murine myeloma Stirred-tank Serum-free rejection Pharmaceuticals chimeric IgG1-κ cell line. components) culture and Excyte (spin filter) Palivizumab/Synagis Prophylaxis of respiratory MedImmune 6/19/98 mAb NS0.

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. 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. oral. These issues were addressed by increasing the of 500 mL to 20 L. This process was developed at scales acid production. live. newborn calf serum. density to greater than 5 × 106 cells/mL and an extension of 6 oxygen limitations. FRhL-2. recombinant insulin hemophilia B suspension.fda. murine IgG1-κ Murine hybridoma Hollow fiber Serum-free. tank reactor. NCS. fragment of antigen binding. murine IgG2-κ Murine hybridoma Not revealed Serum-containing. FAb fragment cell line containing gentamicin *Source: http://www. 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. TNF. batch-refeed Interferon β-1a/Avonex Treatment of multiple Biogen 5/17/96 Recombinant CHO Stirred-tank Not revealed sclerosis glycoprotein bioreactors. All parameters were easily scaled from 3 to 200 L with the exception of dissolved oxygen control. containing episodes in patients with glycoprotein bioreactors. fetal rhesus diploid cell line. suspension Vaccines Rotavirus vaccine. inactivated/VAQTA Immunization for hepatitis A Merck & Co 3/29/96 Inactivated virus Human MRC-5 Adherent. murine IgG2b Mammalian cells Not revealed Serum-free FAb fragment Imciromab pentetate/ Myoscint Cardiac imaging agent Centocor 7/3/96 mAb. baby hamster kidney. complementarity-determining region. CDR. dissolved oxygen from 20 to 50% (relative to air saturation). containing cell line bovine serum albumin and transferrin Nofetumomab/Verluma Cancer imaging agent Dr Karl Thomae 8/20/96 mAb.gov. and was demonstrated in a 200 L stirred. fetal bovine serum. and increased lactic days in culture longevity. BHK. decreased cell growth. NCF Serum-containing diploid fibroblasts to Costar Cubes. FAb. 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 . tumor necrosis factor. FBS.

they ty rates.25]. [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. Unknown Serum. 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. and subcategories for each product application include reactor type. By adding a scales with similar cell densities. specific mAb productivi. Unknown 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. controlled feed of glucose to the perfusion culture. The authors examined proteins or mAbs. Speciality Unknown Stirred.17]. Speciality Unknown Stirred. [16••] merged the concepts of controlled feeding confirmed theoretical results by particle imaging velocime- for fed-batch cultures into perfusion culture processing. Source: www. Recombinant therapeutics Vaccines Diagnostics Tissue culture 13 3 4 1 Stirred. and hol.12]. 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]. . Traditional approaches to adherent cell culture low fiber modules [16••. 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. because humanized IgG isotypes. internal and external spin filters [13.21•]. Sauer et al. Sauer et al. respectively. and product quality. When industrial scale-ups. Serum. and scaled-up directly to the 15 and 750 L the highest mAb concentration (150 U/L). cell retention face-attachment requirements. that can be used to produce many different recombinant a suspension stirred-tank process. process feeding design and medium type. Therefore. and microcarriers suspended in stirred-tanks using a CHO/prothrombin system at the 200 L scale [15•]. the surface to volume ratio can be achieved through many methods: centrifugation in the bioreactor needs to be maximized for maximal cell [P1]. therapy.24•. and completeness of mixing in roller bottle systems by They grew Sp2/0-Ag14 cells producing hLL2 (humanized adding a vertical rocking motion. Adherent cell-culture processes present unique problems to mon processing format for protein production. Speciality Unknown Stirred. The authors demonstrated that choosing a Yang et al. but the productivity ized fields including erythropoetin production. 1996–2000*. Iding et al. ences can affect product characteristics. Serum. [22. cell-culture differ- bioreactors including CHO/humanized mAb and 293/ade.184 Biochemical engineering Table 2 Numerical summary of BLA products generated in mammalian cell-culture systems. fed-batch and perfusion culture at the 3 L develop- serum-free. They noticed a difference in glycosylation distribu- one additional cell line (NS0). [21•] modeled the flow and fluid mixing in roller bottle bioreactors and Yang et al.8 × 107 cells/mL and 275 U/L. The process was optimized at this gave the highest cell density (1. Adherent cell-culture processes Perfusion suspension cultures seem to be the most com.fda. Additional processes have been developed using controlled-fed perfusion cultures. fed-batch suspension cultures in stirred-tank other papers examining product quality. but they published only data for feed. They could improve the speed which they described as controlled-fed perfusion cultures. sion significantly increases the daily mAb productivity opment timelines. [15•] performed a include roller bottle cultures [P2. [16••] have proposed that controlled-fed perfu- platform system can significantly decrease process devel. Serum. Serum. noviral green fluorescent protein systems [11. cell line. [10••] developed a generic batch. Unknown Serum. as noted in serum-free.gov.3 × 107 cells/mL) and the 3 L scale.23. fed-batch culture in stirred-tank bioreactors ment scale. and vaccine production. 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. Because adherent cell lines have sur- designing a stirred-tank perfusion process.14. 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. Unger et al. Their work has shown that B-cell specific IgG2a) in serum-free medium. gene of these tends to be much lower [18–20]. 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. densities. which is related to the SP2/0 tion between roller bottle cultures and the final optimized line. try in experimental systems.15•].

However. 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.32]. 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. dissolved carbon dioxide. [33•] have shown that a robot can be bioreactor equipped with an internal spin filter for perfu. ture processes. which can be adapted for the growth of adherent and culture processes suspension cell lines [34•. 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. [25] used a 3 L stirred-tank Lutkemeyer et al. to control the level of accumu- The use of microcarriers in stirred-tank bioreactors allows lated dissolved carbon dioxide to acceptable levels.35•]. The system has been imple- Many of these papers have addressed issues of oxygen lim. the large-scale culture.24•]. A designed using serum-free medium and many are even unique feature of this process was the ability to inoculate protein-free. studies [23. Some favors platform technologies with similar technical con. together with the on-line analysis of tures remains microcarriers suspended in stirred-tanks. and improvements continue in this area [30]. fed-batch or perfusion). reliable bodies indicates that industry has converged on using measurement of dissolved carbon dioxide was only avail. These glycosylation differences additional disposable culture system has been introduced show that mammalian cells must be used if glycosylation recently.g. Therefore. NJ) takes advantage of the development of sterile bags. Nitrogen The most predominant bioreactor format for adherent cul. This is further evidence that industry cant issue for large-scale industrial processes. Although both methods pro- duced similar amounts of product. and establish a high cell ditions. in Sf9 cells at the 60 L scale. Zhang et al. [31••] form of controlled feeding (e. Solutions. [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. Almost all the suspen- CHO cells. more commonly used for media Common themes for suspension and adherent cell. 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). manufacturing scale. 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. Pattison et al. dispos- CHO cells had a different glycosylation pattern to that of ability and additional process control are preferred. In parallel. suspension cultures in stirred-tank bioreactors with some able by off-line analysis. an the Sf9-derived products. recent literature reports are perfusion cultures with micro- carrier beads. but Kong et al. contamination issues are due to regulatory concerns of cepts. 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. Inc. erature suggest that companies may be considering the use . operation at scales of up to 10. sion cell-culture processes reviewed above have been tors to retain cells/microcarriers over a period of 2 months. 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. storage. hSTC was also produced appropriate analytical machines. in reactors to produce monocyte-colony inhibiting factor with particular for adherent cell cultures. The Wave bioreactor (Wave Biotech/Panacea patterns are important. aeration has been minimized by microaeration with pure oxygen to avoid damage from excessive sparging. um will require significant changes in process design. sparging was used. and provides for more efficient process optimization and flexibility in feeding Contamination of cell-culture processes remains a signifi- process designs. lines to serum-free processes. Internal spin filters were installed in the bioreac. contamination issues result from imperfect processing con- tion and nutrient variability. cell-culture production of recombinant proteins and anti- tion with minimal aeration. which result in the need to include antibiotics in density culture. 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. mented for different cell/protein systems including itation as bench-scale processes are transferred to the NS0/mAb. Until recently. Bedminster. rather than try to adapt adherent cell and the impeller design for high microcarrier loads. but the opposite is true for adherent cell-cul- microcarrier cultures directly without a cell-removal step. a membrane dialysis reactor tion can be problematic for microcarrier suspensions at the with integrated radial-flow fixed bed. 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. 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. Other culture processes — the goal is to reduce waste accumula. hSTC produced in For more specialized applications where flexibility.000 L. 293/adenovirus and CHO/recombinant protein. and a perfusion 30 to 2000 L scale in stirred-tank reactors where the total packed-bed reactor [26–29].

Biotechnol Prog components from cell-culture medium. Welles J. Payne JK. Biotechnol 3. These two papers [26. feeding process. Morris LO. Kamen A: 293SF metabolic flux analysis during cell growth and infection with an adenoviral tion). 16. Goldenberg DM: 413:355-372. Singhvi R: Computational and doubling. Tsao E. 34:111-119. May be an indication of future requirements for regula. The authors • experimental investigation of flow and fluid mixing in the roller demonstrate that. mination issues at large-scale production. which increases overall productivity twofold relative to the original 4.: A systematic approach to the validation of process control 17. Integrates the concepts of fed-batch and perfusion processing into a single process. Schenerman MA. Ayala JL. Kimura R. Moran EB. Stiens LR. determine the end of exponential growth in CHO cell cultures for optimization of scale-up. Schoenherr I. and culture harvest time. bioreactor cultures. 11. 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. cific cell lines are required and disposables allow adaptation to a variety of cell lines (e. 85-100. Tramper J: Shear sensitivity of animal cells from a Bioeng 2000. of mammalian cells and their products: engineering principles and barriers to scale-up. Castillo FJ: Monitoring adenovirus infections with on-line and off-line methods. •• Achievement of high cell density and high antibody productivity by a controlled-fed perfusion bioreactor process. Kundu PK. For example. Cole J. carbon dioxide removal. Hildebrand WH: Complexity among constituents of the cations in a systematic approach. during the production of human antibody in CHO cells. and the same trend was apparent in the indication of the industry trend towards platform or generic technologies that review of published literature. •• Waller W. Cell retention is 2. cell diameter and culture aggregation. Oyebade IA. Pharm Technol 1998. Prasad NS. Shows that statistical design experiments HLA-B·1501 peptide motif. Bergmann A. Indian J Exp Biol 1998. minimizing conta. in process validation. 34:141-150. monocyte-colony inhibition factor (M-CIF) from CHO microcarrier and temperature result in different glycosylation patterns. Huang Z. Determines that cell retention is not affected by spin filter rotation velocity nor perfusion rate. Garbutt JJ. Gratama JW. 22:44-58. Patchan M. Burky JE. Glacken MW. . Lamey K. culture-medium perspective. 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. Henry CM: The next pharmaceutical century. Iding K. generic fed-batch cell culture process for production of timelines. ‘compa. speed and completeness of mixing. and controlling 14. Gawlitzek M. 18.27•] show that direct bead to bead transfer is feasi- Cytotechnology 2000. Angelillo Y. 70:117-130. Xiao C. Biotechnol Bioeng 2000. 12. Tsao E: Large. perfusion process. The authors demonstrate that feed composition. 16:323-328. Montgomery T. Biotechnol Prog 2000. Cytotechnology 2000. Well-designed validation experiments that address parameter range specifi. 2000. Biotechnol Prog 2000. relation is established between cell growth state and site occupancy. 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. Hoy C: Multiple cell culture indicate that the mixing was incomplete. Ryll T: A comparison of different methods to There are exceptions in niche applications for which spe. Chem Eng News Studies the effect of cell-culture conditions on the effectiveness of a commer- 2000. 69:74-82. Weglohner W: Development of serum free bioreactor production of recombinant human thyroid stimulating hormone receptor. A targeted approach to validating three process parameters — population 21. Biotechnol Prog 2000. Kumar A. Gerlach K. Demonstrates direct inoculation from the 1 or 5 L scale to 30 L bioreactors 9. Further investigation suggests that these parameters affect cell growth rate and cell state. Archer LC. Lutkemeyer D. Trends Biotechnol 1998. van der Pol L. This is a good myeloma cells. fiber bioreactor system. 1. Biotechnol Bioeng 2000. Furey J: Continuous cell culture using the ATF system. Mikolajczyk SD. Wesson MC. Protein Expres Purif 1999. Lehmann J. 16:703-709. Harvey S. Large-scale cell-culture production is still faced with vector. 23. vaccine produc. Poeschl DM: Comparison of cell growth in T-flasks. ditions to include an additional direction of rocking. Luider-Vrieling B. Sternard HD. These results 6. Hu X. 19. Carson GR: The gel microdrop secretion using CHO cells grown on microcarrier beads. published within the annual period of review. perfusion cultures. cially available 20 micron internal spin filter. Sinskey AJ: Large-scale production increased by increased operation time. References and recommended reading Papers of particular interest. Aunins JG. Biotechnol Bioeng 2000. reduce process development timelines. Goldenberg C. Cytotechnology 1998. significant challenges — maximizing productivity while 13. 48:89-97. •• Folena-Wasserman G: Comparability testing of a humanized monoclonal antibody (Synagis) to support cell line stability. tory authority approval of biological process validation. Perrier M. • factors can affect the glycosylation of Asn-184 in CHO-produced under typical operating conditions. particularly in the axial direction. 16:866-871. 15. with the correct selection of analytical methods. Chaudhry M. Yang J-D. This paper examines the effect of specific cell growth parameters on t-PA site 22. Lindsey M. Biologicals micro hollow fiber bioreactors. Gramer MJ. Lamers CHJ. Bonner R. Goel AS. and they validate Examines the velocity profile in the traditional roller bottle bioreactor using the process specification ranges. Cytotechnology 2000. Guo Z. Andersen DC. ble and can greatly simplify microcarrier processes. Whiteley EM. Evans C. Bolhuis RLH. 7. 24. Jackson KW. 36:125-135. 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