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
been addressed, with the standard stirred-tank reactor
emerging as industry’s technology of choice. The issues of
adapting cells to suspension culture, shear sensitivity and
oxygen supply have been largely resolved. But for many low-
volume and speciality applications, such as the production of
viral vaccines and gene therapies, reactor technology remains
diversified, with reactor types ranging from roller bottles to
stacked plates and hollow fibers.
Addresses
Bioprocess R&D, Merck Research Laboratories, Merck & Co, Inc,
PO Box 2000, Rahway, NJ 07065, USA
*e-mail: lily_chu@merck.com
Current Opinion in Biotechnology 2001, 12:180–187
0958-1669/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
BLA biological license approval
CHO Chinese hamster ovary
FDA Food and Drug Administration
hSTC human stanniocalcin
mAb monoclonal antibody
Introduction
The biotechnology industry has grown significantly in the
past decade and continues to grow at a rapid rate. Experts
predict that protein therapeutics are just beginning to enter
the marketplace, with a surge of protein, antibody and pep-
tide drugs expected in the next 10–20 years [1]. This rapid
growth in protein therapeutics has been led by a diverse
range of products produced in mammalian cell culture that
have been licensed in the past five years, including, among
others, a monoclonal antibody (mAb) for treating breast can-
cer, Herceptin, an immunoglobulin-TNF (tumor necrosis
factor) receptor fusion protein for treating rheumatoid arthri-
tis, Enbrel, and an inactivated hepatitis A vaccine, Vaqta.
As indicated by the successful licensure in the USA of 21 dif-
ferent therapeutic and diagnostic products produced in
mammalian cell culture since 1996, cell-culture technology
has progressed to a status allowing the reliable development,
scale-up, and subsequent operation on a commercial level. In
the 1980s, as nicely summarized by Glacken et al. [2] in 1983,
many reactor types were considered for the development of
cell-culture processes. Key barriers to large-scale mammalian
culture were considered to include oxygen supply limita-
tions, waste product accumulation, the need for more
sophisticated process control, shear sensitivity of animal
cells, and the challenges of growing adherent cell lines [2].
As highlighted in this review, the reactor technology for much
large-scale cell culture has settled on the standard stirred-tank
reactor as the technology of choice, with the issues of
adapting cells to suspension culture, shear sensitivity and oxy-
gen supply mostly resolved. The remaining challenges in
these cases are the control of product quality while maximiz-
ing productivity, the control of carbon dioxide concentrations
on scale-up, and the minimization of the risk of contamina-
tion by adventitious agents through raw material and
manufacturing controls. For many low-volume and speciality
applications, however, the reactor technology remains diversi-
fied with reactor types ranging from roller bottles to stacked
plates and hollow fibers in use for the production of products
such as viral vaccines, gene therapies and diagnostics. The
use of transgenic plants and animals as production vehicles,
although not covered in this review, remains an area of active
development that may play a role in applications requiring
exceptional product volumes (e.g. in excess of 100 kg per year
for monoclonal antibodies [mAbs]), but substantial regulatory
hurdles remain to be addressed and products have yet to be
licensed using transgenics as the production vehicles.
Here we review some of the products recently approved by
the USA Food and Drug Administration (FDA), together
with recently published methods that have been used in
large-scale cell-culture processes.
Current Food and Drug Administration
(FDA)-approved products
We have reviewed recent FDA biological license approvals
(BLAs) from January 1996 to November 2000. More than half
of these approved biotechnology products (21 out of 33) are
manufactured using mammalian cell systems (see FDA web-
site http://www.fda.gov). To determine patterns in the
technologies applied, we have categorized these products on
the basis of their applications into therapeutic recombinants,
vaccines, tissue-culture products or diagnostic products.
Generated from information available in the public domain,
Table 1 lists the following data for each approved product: the
production mammalian cell line; the type of bioreactor and
process; and the type of production medium.
There are clearly three principal mammalian cell lines of
choice: Chinese hamster ovary (CHO) cells; and the
murine myeloma lines SP2/0 and NS0. These three cell
lines have been used to produce 11 out of 21 approved
products, and nearly all recombinant therapeutics.
We have summarized the data in Table 1 into more relevant
categories in Table 2. Again, only products generated in
mammalian cell systems are considered in Table 2, and the
four subcategories are based on product application. For
each of these product applications, the reactor type, process
feeding design, and medium type are summarized.
Biological licensing approval statistics show the
following trends.

First, recombinant therapeutics, which are mainly recombi-
nant proteins or antibodies, typically require more significant
production quantities; therefore, this category is the best
representation of large-scale protein production. In this cate-
gory, at least 70% of the licensed processes use stirred-tank
bioreactors, and at least 50% use serum-free medium.
Second, vaccines and tissue-replacement products are
produced in many different cell lines that require special-
ized bioreactor systems and medium.
Third, although diagnostic products, which are mainly mAbs,
are similar to recombinant therapeutics, the required
quantities are significantly lower. This allows manufacturers
to consider more specialized cell lines and bioreactor systems.
Therefore, current industrial processes for large-scale
production using mammalian cell culture appear to be
consolidating on a common platform of suspension
cultures in stirred-tank reactors. The ability to use a
common set of cell lines (murine myelomas or CHOs)
enhances the ability of using a platform system. Other
common features in these processes include a trend
towards serum-free medium, and more sophisticated
feeding or perfusion processes.
Recently published processes
Recently approved biological products represent accept-
ed industrial processes that were established early in the
clinical development of the product. As such, these tech-
nologies may not represent current trends in cell-culture
development. These industrial processes have addressed
many of the barriers to large-scale processing.
Specifically, adherent cell lines can be a challenge to
scale-up; therefore, many products are produced in sus-
pension cell lines including suspension-adapted CHO or
murine myelomas. Shear damage from agitation and aera-
tion can be minimized by appropriate reactor design and
control, and by the addition of shear-protecting agents
such as Pluronic F68 to the media. Accumulation of waste
products can be controlled by more sophisticated feeding
schemes involving perfusion systems, which continually
remove waste products, or by using the glutamine syn-
thetase expression system developed by Lonza (formerly
known as Celltech), which reduces the formation of waste
products. Process control systems have been either adapt-
ed from fermentation systems or newly developed for
cell-culture bioreactors.
Recently published literature continues to address many
of these issues with more sophisticated solutions, and
also considers newly arising challenges such as how to
maximize productivity while maintaining product quali-
ty, how to remove all animal derived components from
cell-culture medium, how to minimize contamination
during large-scale production, and how to control carbon
dioxide removal.
Industrial choices for protein production Chu and Robinson 181
Suspension cell-culture processes
The ability to adapt many cell types to suspension culture
and the use of polymeric additives to reduce shear damage
have enabled the widespread application of suspension
cell culture [3]. As seen in our review of license applica-
tions (Table 1), suspension cell-culture processes are the
system of choice for large-scale applications, owing to the
well-understood principles of scaling parameters and the
ease of process control in homogeneous systems.
As shown by Moran et al. [4••] and Schenerman et al. [5••],
very similar process performance and product characteris-
tics are achieved for suspension cultures conducted at
scales ranging from 3 to 10,000 L. A common theme in
recent publications is that certain cell-culture conditions
can affect product quality. Andersen et al. [6•] demonstrat-
ed that tissue plasminogen activator (t-PA) glycosylation
corresponds to CHO cell growth rate and may even be
related to cell state.
Schenerman et al. [5••] described the manufacture of a
humanized mAb (Synagis) using NS0 cells in a stirred-
tank bioreactor. They found that cell-culture harvest time
affects the product profile of their antibody, whereas an
increase in scale, the number of cell generations, and glu-
cose conditions did not affect product quality. Their cells
were cultured under serum-free, fed-batch conditions at
the 20 to 10,000 L scale. Although there were few process-
ing details in the publication, Schenerman et al. described
a useful approach to biological process validation by testing
three critical parameters outside the proposed control
specifications and by evaluating parameter effects with
biochemical and functional comparisons.
Moran et al. [4••] demonstrated another approach to biolog-
ical process validation. They produced a mAb (IgG1) using
NS0 cells in stirred-tank suspension cultures. The serum-
free, fed-batch process could be cultured at the 3 to 8000 L
scale with similar process and product characteristics. The
authors identified five critical parameters and created a
half-fractional factorial experimental design, which tested
16 of the 32 possible combinations of parameters. Both of
these publications show that well-designed validation pro-
tocols, in combination with accurate analytical methods,
can provide significant insight into both the manufacturing
process and product quality.
Much of the published literature continues to focus on the
use of nutrient feeding to increase volumetric productivi-
ties either by increasing cell density or by increasing
specific productivity [7]. Basic batch suspension processes
in a stirred-tank bioreactor have become increasingly less
common, because nutrient feeding schemes can reduce
waste accumulation and nutrient fluctuations, which
allows higher cell densities and productivities to be
reached. Recently designed suspension processes tend to
fall into three categories: batch-refeed, fed-batch or perfu-
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
days in culture longevity. This process was developed at scales
of 500 mL to 20 L, and was demonstrated in a 200 L stirred-
tank reactor. All parameters were easily scaled from 3 to 200 L
with the exception of dissolved oxygen control. The authors
proposed that non-ideal mixing at the 200 L scale resulted in
oxygen limitations, decreased cell growth, and increased lactic
acid production. These issues were addressed by increasing the
dissolved oxygen from 20 to 50% (relative to air saturation).
Another approach to decreasing process development time
requirements is to develop a platform cell-culture process
184 Biochemical engineering

Table 2

Numerical summary of BLA products generated in mammalian cell-culture systems, 1996–2000*.

Recombinant therapeutics Vaccines Diagnostics Tissue culture

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
proteins or mAbs. Sauer et al. [10••] developed a generic
serum-free, fed-batch culture in stirred-tank bioreactors
using Sp2/0 and NS0 cell lines to produce human or
humanized IgG isotypes. The process was optimized at
the 3 L scale, and scaled-up directly to the 15 and 750 L
scales with similar cell densities, specific mAb productivi-
ty rates, and product quality. The same fed-batch process
was used in seven additional Sp2/0 cell lines and one NS0
cell line. The authors demonstrated that choosing a
platform system can significantly decrease process devel-
opment timelines. Sauer et al. [10••] also suggested that
the same process can be applied to other cell lines as a
generic fed-batch process, but they published only data for
one additional cell line (NS0), which is related to the SP2/0
line. Additional processes have been developed using
serum-free, fed-batch suspension cultures in stirred-tank
bioreactors including CHO/humanized mAb and 293/ade-
noviral green fluorescent protein systems [11,12].
Perfusion suspension cultures seem to be the most com-
mon processing format for protein production. When
designing a stirred-tank perfusion process, cell retention
can be achieved through many methods: centrifugation
[P1], internal and external spin filters [13,14,15•], and hol-
low fiber modules [16••,17]. Iding et al. [15•] performed a
detailed analysis on the effects of perfusion and culture
conditions on the performance of an internal spin filter
using a CHO/prothrombin system at the 200 L scale [15•].
Some researchers have used hollow fiber bioreactors for
more specialized perfusion systems, but the productivity
of these tends to be much lower [18–20].
Yang et al. [16••] merged the concepts of controlled feeding
for fed-batch cultures into perfusion culture processing,
which they described as controlled-fed perfusion cultures.
They grew Sp2/0-Ag14 cells producing hLL2 (humanized
B-cell specific IgG2a) in serum-free medium. The process
was originally a roller bottle process that was adapted into
a suspension stirred-tank process. The authors examined
batch, fed-batch and perfusion culture at the 3 L develop-
ment scale, and implemented the perfusion process with
an external hollow fiber module at the 20 L scale, because
this gave the highest cell density (1.3 × 107 cells/mL) and
the highest mAb concentration (150 U/L). By adding a
controlled feed of glucose to the perfusion culture, they
were able to increase both cell density and mAb concen-
trations to 1.8 × 107 cells/mL and 275 U/L, respectively.
Yang et al. [16••] have proposed that controlled-fed perfu-
sion significantly increases the daily mAb productivity
through two combined effects: increased cell density and
minimized product dilution resulting from the glucose
feed. They noticed a difference in glycosylation distribu-
tion between roller bottle cultures and the final optimized
controlled-fed perfusion cultures. Therefore, as noted in
other papers examining product quality, cell-culture differ-
ences can affect product characteristics.
Adherent cell-culture processes
Adherent cell-culture processes present unique problems to
industrial scale-ups. Because adherent cell lines have sur-
face-attachment requirements, the surface to volume ratio
in the bioreactor needs to be maximized for maximal cell
densities. Traditional approaches to adherent cell culture
include roller bottle cultures [P2,21•], multilayer stacked
plate systems such as Costar cellcubes and Nunc cell facto-
ries [P3], and microcarriers suspended in stirred-tanks
[22,23,24•,25]. The current use of roller bottle cultures and
multilayer stacked plate systems is limited to more special-
ized fields including erythropoetin production, gene
therapy, and vaccine production. Unger et al. [21•] modeled
the flow and fluid mixing in roller bottle bioreactors and
confirmed theoretical results by particle imaging velocime-
try in experimental systems. They could improve the speed
and completeness of mixing in roller bottle systems by
adding a vertical rocking motion. Their work has shown that
even well-established bioreactor processes can still benefit
from design improvements based on engineering principles.

Non-traditional bioreactors for adherent cell culture include
a bubble column bioreactor, a membrane dialysis reactor
with integrated radial-flow fixed bed, and a perfusion
packed-bed reactor [26–29].
The most predominant bioreactor format for adherent cul-
tures remains microcarriers suspended in stirred-tanks.
The use of microcarriers in stirred-tank bioreactors allows
operation at scales of up to 10,000 L, and provides for more
efficient process optimization and flexibility in feeding
process designs. This is further evidence that industry
favors platform technologies with similar technical con-
cepts. The same principles and themes emerge when
developing feeding schemes for suspension and adherent
culture processes — the goal is to reduce waste accumula-
tion and nutrient variability, and establish a high cell
density culture.
The predominant process design for adherent cultures in
recent literature reports are perfusion cultures with micro-
carrier beads. Kong et al. [22] implemented perfusion
microcarrier culture at 3 and 15 L scales in stirred-tank bio-
reactors to produce monocyte-colony inhibiting factor with
CHO cells. Internal spin filters were installed in the bioreac-
tors to retain cells/microcarriers over a period of 2 months. A
unique feature of this process was the ability to inoculate
microcarrier cultures directly without a cell-removal step.
Most parameters scaled-up easily from the 3 to the 15 L
scale, but Kong et al. [22] found it necessary to re-optimize
both the microcarrier concentration to increase surface area
and the impeller design for high microcarrier loads.
The direct transfer of cells between microcarrier beads
without a cell removal step has also been reported in other
studies [23,24•]. Zhang et al. [25] used a 3 L stirred-tank
bioreactor equipped with an internal spin filter for perfu-
sion culture to produce recombinant human stanniocalcin
(hSTC) in CHO cells. In parallel, hSTC was also produced
in Sf9 cells at the 60 L scale. Although both methods pro-
duced similar amounts of product, hSTC produced in
CHO cells had a different glycosylation pattern to that of
the Sf9-derived products. These glycosylation differences
show that mammalian cells must be used if glycosylation
patterns are important.
Common themes for suspension and adherent cell-
culture processes
Many of these papers have addressed issues of oxygen lim-
itation as bench-scale processes are transferred to the
manufacturing scale, and improvements continue in this
area [30]. Another cell-culture process control issue — the
measurement and control of dissolved carbon dioxide —
has emerged partly because of the success of oxygen addi-
tion with minimal aeration. Until recently, reliable
measurement of dissolved carbon dioxide was only avail-
able by off-line analysis. However, Pattison et al. [31••]
report the use of an on-line carbon dioxide analyzer (YSI
8500) in both microbial and mammalian culture processes.
Industrial choices for protein production Chu and Robinson 185
The authors demonstrated that carbon dioxide accumula-
tion can be problematic for microcarrier suspensions at the
30 to 2000 L scale in stirred-tank reactors where the total
aeration has been minimized by microaeration with pure
oxygen to avoid damage from excessive sparging. Nitrogen
sparging was used, together with the on-line analysis of
dissolved carbon dioxide, to control the level of accumu-
lated dissolved carbon dioxide to acceptable levels.
Contamination of cell-culture processes remains a signifi-
cant issue for large-scale industrial processes. Some
contamination issues are due to regulatory concerns of
viruses or prions that might be transferred potentially from
animal components in the culture medium [3,32]. Other
contamination issues result from imperfect processing con-
ditions, which result in the need to include antibiotics in
the large-scale culture, as judged by the number of com-
mercial products where antibiotics are used in the
cell-culture process (Table 1).
The regulatory push for serum-free and protein-free medi-
um will require significant changes in process design, in
particular for adherent cell cultures. Almost all the suspen-
sion cell-culture processes reviewed above have been
designed using serum-free medium and many are even
protein-free, but the opposite is true for adherent cell-cul-
ture processes. Therefore, industry may find it easier to
adapt processes to suspension cultures with well-estab-
lished cell lines in order to design a serum-free and
protein-free process, rather than try to adapt adherent cell
lines to serum-free processes. Other sources of contamina-
tion are currently controlled with the use of antibiotics in
the culture medium, but promising research in the field of
robot automation for stirred-tank bioreactors is on going.
Lutkemeyer et al. [33•] have shown that a robot can be
implemented at 20 and 100 L scales to perform sterile
sampling procedures and deliver the samples to the
appropriate analytical machines.
For more specialized applications where flexibility, dispos-
ability and additional process control are preferred, an
additional disposable culture system has been introduced
recently. The Wave bioreactor (Wave Biotech/Panacea
Solutions, Inc, Bedminster, NJ) takes advantage of the
development of sterile bags, more commonly used for media
storage, which can be adapted for the growth of adherent and
suspension cell lines [34•,35•]. The system has been imple-
mented for different cell/protein systems including
NS0/mAb, 293/adenovirus and CHO/recombinant protein.
Conclusions
Reviewing the current processes used in the large-scale
cell-culture production of recombinant proteins and anti-
bodies indicates that industry has converged on using
suspension cultures in stirred-tank bioreactors with some
form of controlled feeding (e.g. fed-batch or perfusion).
Both recently approved FDA BLAs and the published lit-
erature suggest that companies may be considering the use
186 Biochemical engineering
of platform technologies to speed up process development
timelines. For example, more than 50% of approved BLAs
using mammalian cell culture chose to use CHO or murine
myeloma cells, and the same trend was apparent in the
review of published literature.
There are exceptions in niche applications for which spe-
cific cell lines are required and disposables allow
adaptation to a variety of cell lines (e.g. vaccine produc-
tion). Large-scale cell-culture production is still faced with
significant challenges — maximizing productivity while
maintaining product quality, removing all animal derived
components from cell-culture medium, minimizing conta-
mination issues at large-scale production, and controlling
carbon dioxide removal.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1. Henry CM: The next pharmaceutical century. Chem Eng News
2000, 85-100.
2. Glacken MW, Fleischaker RJ, Sinskey AJ: Large-scale production
of mammalian cells and their products: engineering principles
and barriers to scale-up. Ann New York Acad Sci 1983,
413:355-372.
3. van der Pol L, Tramper J: Shear sensitivity of animal cells from a
culture-medium perspective. Trends Biotechnol 1998, 16:323-328.
4. Moran EB, McGowan ST, McGuire JM, Frankland JE, Oyebade IA,
•• Waller W, Archer LC, Morris LO, Pandya J, Nathan SR et al.:
A systematic approach to the validation of process control
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culture of a murine myeloma. Biotechnol Bioeng 2000,
69:242-255.
Well-designed validation experiments that address parameter range specifi-
cations in a systematic approach. Shows that statistical design experiments
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10. Sauer PW, Burky JE, Wesson MC, Sternard HD, Qu L: A high-
•• yielding, generic fed-batch cell culture process for production of
recombinant antibodies. Biotechnol Bioeng 2000, 67:585-597.
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
indication of the industry trend towards platform or generic technologies that
reduce process development timelines.
11. Schoenherr I, Stapp T, Ryll T: A comparison of different methods to
determine the end of exponential growth in CHO cell cultures for
optimization of scale-up. Biotechnol Prog 2000, 16:815-821.
12. Nadeau I, Jacob D, Perrier M, Kamen A: 293SF metabolic flux
analysis during cell growth and infection with an adenoviral
vector. Biotechnol Prog 2000, 16:872-884.
13. Monica TJ, Montgomery T, Ayala JL, Schoofs GM, Whiteley EM,
Roth G, Garbutt JJ, Harvey S, Castillo FJ: Monitoring adenovirus
infections with on-line and off-line methods. Biotechnol Prog
2000, 16:866-871.
14. Stiens LR, Buntemeyer H, Lutkemeyer D, Lehmann J, Bergmann A,
Weglohner W: Development of serum free bioreactor production
of recombinant human thyroid stimulating hormone receptor.
Biotechnol Prog 2000, 16:703-709.
15. Iding K, Lutkemeyer D, Fraune E, Gerlach K, Lehmann J: Influence of
• alterations in culture conditions and changes in perfusion
parameters on the retention performance of a 20 micron spinfilter
during a perfusion cultivation of a recombinant CHO cell line in
pilot scale. Cytotechnology 2000, 34:141-150.
Studies the effect of cell-culture conditions on the effectiveness of a commer-
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
increased by increased operation time, cell diameter and culture aggregation.
16. Yang J-D, Angelillo Y, Chaudhry M, Goldenberg C, Goldenberg DM:
•• Achievement of high cell density and high antibody productivity
by a controlled-fed perfusion bioreactor process. Biotechnol
Bioeng 2000, 69:74-82.
Integrates the concepts of fed-batch and perfusion processing into a single
process, which increases overall productivity twofold relative to the original
perfusion process.
17. Furey J: Continuous cell culture using the ATF system. Genet Eng
News 2000, 20:52.
18. Prilliman KR, Lindsey M, Jackson KW, Cole J, Bonner R,
Hildebrand WH: Complexity among constituents of the
HLA-B·1501 peptide motif. Immunogenetics 1998, 48:89-97.
19. Lamers CHJ, Gratama JW, Luider-Vrieling B, Bolhuis RLH,
Bast EJEG: Large-scale production of natural cytokines during
activation and expansion of human T lymphocytes in hollow fiber
bioreactor cultures. J Immunother 1999, 22:299-307.
20. Gramer MJ, Poeschl DM: Comparison of cell growth in T-flasks, in
micro hollow fiber bioreactors, and in an industrial scale hollow
fiber bioreactor system. Cytotechnology 2000, 34:111-119.
21. Unger DR, Muzzio FJ, Aunins JG, Singhvi R: Computational and
• experimental investigation of flow and fluid mixing in the roller
bottle bioreactor. Biotechnol Bioeng 2000, 70:117-130.
Examines the velocity profile in the traditional roller bottle bioreactor using
theoretical modeling and experimental imaging techniques. These results
indicate that the mixing was incomplete, particularly in the axial direction,
under typical operating conditions. The authors redesign the operating con-
ditions to include an additional direction of rocking, which improves the
speed and completeness of mixing.
22. Kong D, Gentz R, Zhang J: Long-term stable production of
monocyte-colony inhibition factor (M-CIF) from CHO microcarrier
perfusion cultures. Cytotechnology 1998, 26:131-138.
23. Kumar A, Goel AS, Payne JK, Evans C, Mikolajczyk SD,
Kuus-Reichel K, Saedi MS: Large-scale propagation of
recombinant adherent cells that secrete a stable form of human
glandular kallikrein, hK2. Protein Expres Purif 1999, 15:62-68.
24. Hu X, Xiao C, Huang Z, Guo Z, Zhang Z, Li Z: Pilot production of
• u-PA with porous microcarrier cell culture. Cytotechnology 2000,
33:13-19.
Demonstrates direct inoculation from the 1 or 5 L scale to 30 L bioreactors
using CHO cells grown on microcarrier beads. The cell/microcarrier system
can also migrate between microcarrier beads without a trypsinization step.
These two papers [26,27•] show that direct bead to bead transfer is feasi-
ble and can greatly simplify microcarrier processes.

25. Zhang J, Alfonso P, Tahotakura NR, Su J, Buergin M, Parmelee D,
Collins AW, Oelkuct M, Gaffney S, Gentz S et al.: Expression,
purification, and bioassay of human stanniocalcin from
baculovirus-infected insect cells and recombinant CHO cells.
Protein Expres Purif 1998, 12:390-398.
26. Rodrigues MTA, Vilaca PR, Garbuio A, Takagi M, Barbosa S Jr, Leo P,
Laignier NS, Silva AAP, Moro AM: Glucose uptake rate as a tool to
estimate hybridoma growth in a packed bed bioreactor.
Bioprocess Eng 1999, 21:543-546.
27. Portner R, Ludemann I, Reher K, Neumaier M, Markl H: Fixed-bed
dialysis culture of a transfectoma cell line producing chimeric
Fab-fragments with ‘nutrient-split’-feeding strategy. Biotechnol
Tech 1998, 12:501-505.
28. Kaufman JB, Wang G, Zhang W, Valle MA, Shiloach J: Continuous
production and recovery of recombinant Ca2+ binding receptor
from HEK 293 cells using perfusion through a packed bed
bioreactor. Cytotechnology 2000, 33:3-11.
29. Hu Y-C, Kaufman J, Cho MW, Golding H, Shiloach J: Production of
HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7
expression system. Biotechnol Prog 2000, 16:744-750.
30. Dutton G: Improved tools for large-scale bioprocessing. Genet
Eng News 2000, 20:11, 57, 63.
31. Pattison RN, Swamy J, Mendenhall B, Hwang C, Frohlich BT:
•• Measurement and control of dissolved carbon dioxide in
mammalian cell culture processes using an in situ fiber optic
chemical sensor. Biotechnol Prog 2000, 16:769-774.
This paper addresses the problem of controlling dissolved carbon dioxide in
large-cell mammalian cultures (30 to 2000 L scale). The authors success-
fully use an on-line dCO2 probe (YSI 8500) to control carbon dioxide levels
with N2 sparging at all bioreactor scales.
32. Jayme DW, Smith SR: Media formulation options and
manufacturing process controls to safeguard against introduction
Industrial choices for protein production Chu and Robinson 187
of animal origin contaminants in animal cell culture.
Cytotechnology 2000, 33:27-36.
33. Lutkemeyer D, Poggendorf I, Scherer T, Zhang J, Knoll A, Lehmann J:
• First steps in robot automation of sampling and sample
management during cultivation of mammalian cells in pilot scale.
Biotechnol Prog 2000, 16:822-828.
Demonstrates that robot automation may be feasible for large-scale mam-
malian culture. This provides an alternative to antibiotic addition for reducing
large-scale contamination issues in mammalian cultures.
34. Singh V: Disposable bioreactor for cell culture using wave-
• induced agitation. Cytotechnology 1999, 30:149-158.
Many cell/product systems are produced at the 2 to 200 L scale using the
Wave bioreactor. This paper demonstrates that these bioreactors are quite
adaptable to different processes including suspension and microcarrier
processes. Direct comparison with conventional stirred-tank systems shows
that they have similar growth rates and productivities.
35. Namdev PK, Lio P: Assessing a disposable bioreactor for
• attachment-dependent cell cultures. Biopharm 2000, 13:44-50.
The authors grow 293 and CHO cells attached to microcarriers to
show that fed-batch and perfusion cultures can be performed in 2 L
Wave bioreactors.
Patents
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eukaryotic cells on an industrial scale using nucleotide-
manipulating agents. US Patent 1999, WO 200032754.
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culture of cells producing recombinant human erythropoietin.
US Patent 2000, WP 200027997.
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for the production and purification of adenoviral vectors.
US Patent 2000, WP 200032754.