Advances inthe Development

of Therapeutic
Monoclonal Antibodies
Susan Dana Jones, Francisco J. Castillo, Howard L. Levine

ABSTRACT
Monoclonal antibodies (MAbs) and related products are a dominant component of the
biopharmaceutical market, generating revenues of several billion dollars. While MAbs
have proven to be valuable therapeutic products, the typical doses of these products
required for treatment are significantly higher than those required for most other
biologic products, resulting in the need for large-scale production and efficient, cost-
effective manufacturing processes. In the past few years, improvements have been
made in critical areas, such as cell line generation and large-scale cell culture
production, to maximize productivity. These advances, coupled with improvements in
cell culture media and optimized bioreactor processes, have made large-scale
production of MAbs economically viable. However, the increasing production
requirements and the drive to reduce the cost to develop these expensive medicines
continue to present challenges to the industry to further improve the overall efficiency
of manufacturing processes. This article presents a historical review of the discovery,
development, and production of therapeutic antibodies.

T
he first therapeutic mono- ing the development of MAb products.
clonal antibody (MAb) prod- To address this issue, new technologies
uct entered the market in for creating MAbs that were predomi-
1986, but it took another nately or entirely of human origin were
decade before the potential of this new developed. Today, almost all antibody
class of biologic products began to be products currently in development are
realized. From the mid 1990s until humanized or fully human.
today, almost 30 therapeutic mono- While MAbs have proven to be valu-
clonal antibodies (MAbs) have been able therapeutic products, the typical
approved throughout the world along doses of these products required for
with several antibody-related products treatment are significantly higher than
Susan Dana Jones, PhD,is a senior (e.g., Fc-fusion proteins) making MAbs those required for most other biologics,
consultant,Francisco J.Castillo, PhD, and related products a dominant com- resulting in the need for large-scale pro-
is a senior consultant, and ponent of the biopharmaceutical mar- duction and efficient, cost-effective man-
Howard L.Levine, PhD,is a ket, generating revenues of several ufacturing processes. In the past few
principal consultant,all at billion dollars. The first approved MAb years, improvements have been made in
BioProcess Technology Consultants,Inc., was a murine antibody. This was fol- critical areas, such as cell line generation
Acton,MA,978.266.9159, lowed by several chimeric MAbs con- and large-scale cell culture production,
[email protected]. taining a mix of murine and human to maximize specific antibody productiv-
regions. These early antibody products ity from a given cell line and improve
Listen to a podcast interview with
posed a moderate risk of immuno- overall productivity in bioreactors. These
Howard Levine at
genicity to patients from their residual advances include the use of new expres-
biopharminternational.com/biopharmnow
murine components, somewhat limit- sion vectors and transfection technology

96 BioPharm International www.biopharminternational.com October 2007

 Monoclonal Antibodies

Figure 1. Annual approval of recombinant biologic products and address the increasing doses of antibody
monoclonal antibody products.2,3 The total number of biologics, including products; and the development of alterna-
MAb products, approved by FDA for market each year since 1982 is tive delivery systems.
shown in green. MAb product approvals only are shown in black.
Antibody-related products such as Fc fusions, engineered antibody DISCOVERY OF ANTIBODY THERAPEUTICS
fragments, or other products derived from antibodies but not containing In 1984, Kohler and Milstein received the
an antibody binding region are not included in the MAb figures. However, Nobel Prize in Medicine for their pioneer-
those products are included in the total product figures. ing work on the production of MAbs.1 One
of the most significant advantages of this
Total biologics new technology over traditional tech-
14
including MAbs niques for producing antibodies was the
12 MAbs
development of an immortalized cell line
creating a continuous source of the same
10 antibody with a single antigen specificity.
This enabled the development of highly
8 specific antibodies directed toward a single
epitope on the target antigen. Initially,
6
MAbs were used as laboratory reagents, but
4 they were quickly adopted as clinical diag-
nostic reagents, and eventually as thera-
2 peutic agents. The development of
therapeutic MAbs commenced in the early
0 1980s and by 1986 the first monoclonal
82 84 86 88 90 92 94 96 98 0 2 4 6
antibody for human use—Orthoclone
OKT3 (Ortho Pharmaceuticals)—was
to improve cell line generation; novel parental approved for the prevention of kidney trans-
cell lines that have been selected or designed plant rejection. Following the approval of
to grow to maximum density and productivity OKT3, the enthusiasm for MAbs as therapeu-
under standard bioreactor conditions; and tic products grew with the next wave of anti-
high-throughput, robust screening technolo- body products generally being developed as
gies to select the highest producing clones rap- anticancer agents. Several of these products
idly and more effectively. As a result, the were approved in the US and Europe in the
production of cell lines expressing multigram mid to late 1990s, a trend that continues to
quantities of antibody per liter of culture grow today. Since the commercialization of
medium is now routine. the first therapeutic MAbs, these products
These advances, coupled with improve- have become a dominant component of the
ments in cell culture media and greatly opti- biopharmaceutical market, representing
mized bioreactor processes, have made the approximately 20% of all biologic products,
large-scale production of MAbs economically with combined revenues of over $20 billion
viable. However, the increasing production in 2006.4 The growth of MAb products over
requirements and the drive to reduce the the past 25 years, as shown in Figure 1, con-
cost to develop these expensive medicines firms the importance of these products and
continue to present challenges to the indus- also shows that MAbs represent a significant
try to further improve the overall efficiency subset of all biopharmaceuticals on the mar-
of manufacturing processes. These chal- ket and in development. With over 300 anti-
lenges include the need to streamline down- body products currently in development,
stream processing to enable the processing of this unique and effective category of thera-
increased product quantities; the implemen- peutic compounds is poised to grow signifi-
tation of Quality by Design (QbD) and other cantly in the coming years.
new regulatory concepts to reduce the cost
and development timelines for MAb prod- MOLECULAR STRUCTURES OF
ucts without adversely affecting their qual- ANTIBODIES:THEN AND NOW
ity; the need for high-concentration product Murine Antibodies
formulations with sufficient stability to The initial technology for producing MAbs

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 Monoclonal Antibodies

involved fusing individual antibody-secret-

ing spleen cells from immunized mice with a Future therapeutic monoclonal antibody
murine myeloma cell line to generate
immortalized cell lines that secreted individ-
ual, or monoclonal, antibodies. Hence, the
products will be predominantly
first MAbs developed for use as potential
human therapeutics were murine antibodies.
While initial interest in these murine MAbs
humanized or fully human.
was high and several companies began
developing products based on this technol- constant region in these chimeric antibodies
ogy, OKT3 was the only murine monoclonal is human, it is capable of activating other
antibody that was approved for human ther- components of the human immune system
apeutic use. Despite the fact that OKT3 has to potentially create more effective therapeu-
been moderately successful in the market, tic agents. Many of the MAbs approved for
the use of murine MAbs as therapeutic commercialization in the 1990s and early
agents quickly ran into many roadblocks. 2000s were chimeric antibodies, including
One of the potential advantages of MAbs as the highly successful anticancer antibodies
therapeutic agents is their long circulating Rituxan (approved in 1997) and Erbitux
half-life, allowing them to provide a thera- (approved in 2004), as well as the anti-
peutic effect in patients over several days. inflammatory product Remicade (approved
However, when murine MAbs were repeat- in 1998). Chimeric antibody products are
edly administered to humans during clinical superior to murine antibody products but
trials, it was observed that the half-life they still pose a moderate risk of immuno-
decreased and the products became less genicity to patients from their residual
effective with each injection. This was murine components. Therefore, antibody
because of the immunogenicity of murine engineering approaches that further reduce
proteins in humans and the rapid develop- the murine component or that remove
ment of a human antimurine antibody immunogenic portions of the chimeric anti-
(HAMA) response in the patients. This body, have been developed and used to gen-
HAMA response neutralized the effectiveness erate fully “humanized” antibody products.
of the murine antibodies and resulted in
their rapid clearance from the body. For Humanized Antibodies
example, it has been reported that OKT3 can In 1991, Protein Design Labs (PDL) developed
elicit a HAMA response in up to 86% of and patented the first technology for success-
patients treated, leading to some limitations fully humanizing MAbs.6 The antigen binding
in its efficacy.5 specificity of any antibody is determined by
the amino acids present in three distinct
Chimeric Antibodies highly variable regions per antibody chain,
To overcome the HAMA responses occuring referred to as complementarity determining
from the usage of murine MAbs as therapeu- regions (CDRs), and located in a more con-
tics, several approaches were developed in an served framework sequence in the variable
attempt to make MAbs more human-like and regions. Therefore, PDL scientists developed
less immunogenic. In the early 1990s, methods for engineering an antibody gene in
molecular biology techniques enabled the which the CDRs of a human antibody gene
creation of “chimeric” antibodies by linking were replaced by those from the CDR of a
the murine genes encoding the antigen- murine MAb gene. The resulting humanized
binding portion of the antibody (the variable antibody has the same antigen binding prop-
region) to the genes encoding the constant erties as the original murine antibody but
region of human immunoglobulin light and contains minimal murine sequences and,
heavy chains. Because over 75% of the pro- therefore, elicits a lower HAMA response in
tein sequence of the resulting chimeric anti- patients. The CDR-grafted human antibody
bodies was of human origin, these chimeric can be used as is or, in cases where affinity of
MAbs elicited much lower HAMA responses the chimeric antibody is slightly reduced
in patients. Moreover, because the antibody from the original murine antibody, additional

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 Monoclonal Antibodies

Table 1. Comparison of sales for antibody-based anti-inflammatory products AstraZeneca) using an in vitro
molecular engineering technology
Year 2006 sales worldwide known as phage display. In the mar-
Product Company approved ($ million) Market share ketplace, this human MAb com-
Humira Abbott 2002 2,000 15.8% petes with Enbrel, an Fc fusion
protein, and Remicade, a chimeric
Remicade Johnson & Johnson 1998 4,253 33.7%
antibody. The power of the fully
Enbrel Amgen 1998 4,379 34.7% human antibody platform can be
seen in the sales figures for these
changes can be made in the antibody three products. Although Humira was
sequence to regain or enhance its binding approved four years later than the other prod-
properties. Like chimeric antibodies, human- ucts, it has successfully taken a significant
ized antibodies can activate other parts of the market share from them, garnering almost
immune system to create a more effective 16% market share in 2006. Worldwide sales in
product. Several humanized antibody prod- 2006 for all three products are shown in
ucts are currently on the market, including Table 1.
Synagis (approved in 1998), Herceptin Many antibody products currently in early
(approved in 1998), Mylotarg (approved in clinical development are fully human,
2000), Xolair (approved in 2003), and Avastin because the technologies that enable the
(approved in 2004). generation of human antibodies are now
In addition to the production of chimeric accessible through partnerships or licensing
and humanized antibodies, other technolo- from the companies that have developed
gies have been developed to help minimize these approaches. Moreover, the expectation
the HAMA response in patients. These in the medical and regulatory community is
include human engineering or deimmuniza- that companies will use the best approach
tion, in which amino acids on the surface of for their product to achieve humanization.
the murine variable region that are known to There will be exceptions to this generaliza-
be effective immunogenic sequences are tion, for example when a short half life is
changed to their non-immunogenic human desired or when a toxic or radioactive pay-
counterpart, leaving the other non-immuno- load is linked to the antibody, but for
genic murine sequences unchanged. 7 The unmodified therapeutic antibody products
advantage of this approach is that the struc- the industry standard has changed; most
tural integrity of the variable region is better future antibody products will be humanized
maintained and reduction of affinity for the or fully human antibodies.
target is minimized. Most MAb products are naked antibodies,
which rely on either blocking an important
Fully Human Antibodies biological function or on activating the
The latest advancement in creating less immune system, to elicit a therapeutic effect.
immunogenic therapeutic antibody products However, antibodies are also well suited as
is the ability to generate fully human MAbs. targeting agents to deliver potent chemo- or
Several technologies exist to develop fully radioactive agents specifically to target cells.
human antibodies, each falling into one of For example, Mylotarg contains a cytotoxic
the two general classes—in vivo approaches compound conjugated to a monoclonal anti-
using a murine system in which the body. This immunoconjugate product is
immunoglobulin genes have been replaced by designed to deliver the potent cytotoxic
their human counterparts or in vitro compound selectively to cancer cells. The
approaches using libraries containing millions radio-immunoconjugate products Zevalin
of variations of antibody sequences coupled and Bexxar (both anti-CD20 MAbs), deliver
with a mechanism to express and screen these radioisotopes for the treatment of lym-
antibodies in vitro. Humira (approved in 2004) phoma. Both these products are murine anti-
is the first fully human antibody to be bodies because the human or humanized
approved. This anti-TNF-α antibody was first forms of these products would bind to and
identified by scientists at Cambridge target not only the CD20 positive target cells
Antibody Technology (CAT, now part of but also those cells that contain the IgG

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 Monoclonal Antibodies

receptors that function to enable antibodies

to recruit additional immune system compo- In the future,human cell lines may
nents to the site of a foreign antigen. By
inadvertently targeting these cells, human
antibody-based radio-immunoconjugates
replace CHO and other mammalian
could do more harm to nontarget cell types
than to the targeted cancer cell. cell lines for the production of MAbs.
All of the above technologies now allow
the generation of better designed antibody expressing antibody production cell lines and
products with fully human sequences and of culturing these cell lines for maximum pro-
optimized function. Combining in vivo and in ductivity have been developed. Today’s tech-
vitro discovery and molecular engineering nologies are enabling antibody production in
technologies allows exquisite control of the the bioreactor of 5 g/L or more.9 Advances in
antibody sequences and properties that was cell line generation over the past decade
not possible 20 years ago. New approaches for include new expression vectors and transfec-
the rapid production of cell lines suitable for tion technology to introduce the genes into
large-scale commercial production have cells; novel parental cell lines that have been
enabled the development of MAb therapies to selected or designed to grow to maximum
treat myriad diseases and made these products density; and robust screening technologies
available to an increasing number of patients. that in combination can enable rapid genera-
In addition to enabling more efficient and tion of production cell lines.
economic production of MAbs, the above
antibody engineering technologies, coupled ADVANCES IN THE GENERATION
with advances in cell culture production dis- OF PRODUCTION CELL LINES
cussed below, have greatly increased our abil- Today’s MAbs must be manufactured using
ity to control or alter the properties of the reliable production cell lines capable of pro-
resulting antibodies. For example, the extent ducing sufficient quantities of product to
of glycosylation, which can increase effector meet the market demand. For most products,
function and thereby increase product effi- this means that antibody titers in the biore-
cacy, can be controlled by both cell line engi- actor must be greater than 1 g/L in a fed-
neering and cell culture technologies. batch process initially and 3–5 g/L following
process optimization. To achieve these levels
MARKET DEMANDS AND of productivity, it is necessary to quickly
CELL LINE PRODUCTIVITY develop a cell line expressing reasonably
One challenging feature of most therapeutic high quantities of antibody for early preclin-
antibody products is that the doses required ical, formulation, and analytical validation
for these products are much higher than for studies that can be further optimized to
other biologic products. To meet the large achieve the desired productivity levels. If the
annual production requirements for these productivity of the initial cell line is high
products, companies have made substantial enough, it can even be used to support ini-
progress in developing more efficient and cost- tial clinical development of the product.
effective methods for manufacturing antibody Once the initial cell line is established, a pro-
products. When antibody products were first duction cell line exhibiting the highest pos-
developed and approved, expression levels of sible level of production of functional
MAbs were typically on the order of 100–500 antibody and capable of supporting commer-
milligrams per liter. Even as recently as five cial production at a reasonable cost can be
years ago, antibody titers in excess of 1 g/L developed. In today’s highly competitive
were not common and many MAb products market, it is important to complete the ini-
were launched using production cell lines and tial stages of cell line development as quickly
manufacturing processes that produced and efficiently as possible to enable early
approximately 0.5–1.0 g/L antibody.8 As MAb entry into human clinical trials but equally
products became successful in the marketplace important is to devote sufficient time and
and as the demands for new products resources to the full development and opti-
increased, newer methods of generating high- mization of the commercial cell line so that

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 Monoclonal Antibodies

sure. Using parental cell lines adapted to

The use of parental cell lines adapted to grow in suspension and serum-free media
reduces development times and increases the
grow in suspension and serum-free likelihood of reaching high cell densities
during manufacturing and high product
yields in the grams-per-liter level.
media can reduce development times.
Selection Systems
a suitable cell line is available for commercial One of the earliest effective methods for
production as soon as possible. transfection, selection, and amplification of
foreign genes in mammalian cells was devel-
High-Expressing Cell Lines oped in 1981 by scientists at Columbia
To create a production cell line for a specific University using dihydrofolate reductase
antibody, expression vectors containing the (DHFR) selection. In this method, a parental
heavy- and light-chain genes under control mammalian cell line deficient in the enzyme
of strong mammalian promoters are intro- DHFR is transfected with an expression vec-
duced into the parental cell line. Usually, a tor containing the DHFR gene under control
selectable marker is also included so that of a relatively weak promoter and the anti-
cells containing the gene can be easily body (or other protein) genes under control
selected by adding a drug or substance to the of a strong promoter.11 By performing multi-
culture that causes the cell to require the ple rounds of amplification and selection of
activity of the selectable marker. The driving cells in the presence of the folate analog
factors behind the selection of a particular methotrexate (MTX), a potent inhibitor of
cell clone during cell line generation is the DHFR, production cell lines with relatively
expression level of the recombinant protein, high levels of expression of the foreign genes
which is measured independently of the can be obtained. The original patents for this
selection, and the time that it takes to obtain technology have now expired but it is still
a cell line that expresses enough product to widely used to generate antibody production
enable nonclinical and clinical development. cell lines. However, because each amplifica-
Technologies that increase the percentage of tion cycle requires 12 weeks to complete and
transfectants with high expression levels will up to five cycles or more, about one year
reduce the time needed to identify a produc- total may be necessary to obtain a clone
tion cell line because the high-expressing with acceptably high expression levels.
clones will be easier to select without having Nevertheless, the DHFR system is effective
to screen thousands of individual clones. and has been used in conjunction with other
Recent advances in cell line generation aspects of cell line development to achieve
include technologies that increase this per- multigram-per-liter expression levels of MAb.
centage, as well as sophisticated and auto- Also, alternative systems requiring less time
mated approaches to screening that enable to reach maximal expression have been
more individual transfectants to be screened developed. For example, the glutamine syn-
for expression levels.10,12,15,16 thetase selection system, developed by scien-
Production levels in the bioreactor are a tists at Celltech (now Lonza), can achieve
function of specific productivity—the den- production clones with higher levels of anti-
sity to which the cells can grow and the body or protein expression in 4–6 months.12
longevity of the culture. Before actual testing Glutamine synthetase (GS) is the enzyme
in the bioreactor, expression levels are deter- responsible for the biosynthesis of gluta-
mined in small culture vessels, from multi- mine from glutamate and ammonia. This
well plates to shake flasks. Levels of 15–20 enzymatic reaction provides the only path-
picograms of antibody/cell/day (pcd) are way for glutamine formation in a mam-
considered appropriate for initial transfec- malian cell. Therefore, in the absence of
tants, with greater productivity arising from glutamine in the growth medium, the GS
optimized cell culture conditions, secondary enzyme is essential for the survival of the
transfections, or amplification of the trans- mammalian cells in culture. Some mam-
fected antibody genes using selective pres- malian cell lines, such as the murine cell

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 Monoclonal Antibodies

Figure 2. In matrix attachment region (MAR) technology, MAR elements are making it widely available for the
inserted into expression vectors surrounding the desired transgene and impose development of MAb products.14
an open chromatin configuration on the nearby chromatin. This open structure
allows RNA polymerase and other transcription factors to access the Improving Gene Expression
transcriptional promoters and enhancers found within the expression vector and Another recent approach to improve
thereby enables greater levels of transcription. This leads to increased product- expression of antibody genes in the
specific translation and a higher yield in a greater percentage of transfected initially transfected cells is to ensure
cells. Figure provided courtesy of Selexis SA. that the genes are integrated into
regions of the chromatin, which are
easily available to the enzymes that
transcribe the gene into RNA, thereby
increasing the rate of transcription.
Promoters/ MAR The transfection of a mammalian cell
enhancers generally results in the integration of
the DNA into the chromatin in one or
more random locations. Because most
of the genome is not transcriptionally
‘Closed’ ‘Open’ ‘Closed’
chromatin chromatin active, there is a high likelihood that
chromatin integration will occur in regions that
are not able to transcribe high levels
of the antibody genes. Targeting the
lines NSO or SP2/0 widely used for anti- expression plasmid to locations on the chro-
body production, do not express sufficient matin that are known to be transcriptionally
GS to survive without added glutamine. active and accessible to the necessar y
With these cell lines, a transfected GS gene enzymes would increase the expression of
can function as a selectable marker by per- all genes integrated at these sites. Although
mitting growth in a glutamine-free this is an excellent concept in theory,
medium. Chinese hamster ovary (CHO) homologous recombination or targeted inte-
cells, also widely used for antibody and gration has not been widely adapted in prac-
other recombinant protein production, tice because of the lack of information
contain sufficient active GS to survive about which sites are good locations for
without exogenous glutamine. 13 In these integration and the need to have unique
cases the specific GS inhibitor, methionine plasmids and cell lines that are able to per-
sulphoximine (MSX), can be used to form the recombination.
inhibit endogenous GS activity such that Rather than targeting a specific site in the
only transfectants with additional GS activ- chromatin for integration, an alternative
ity can survive. GS selection can be used to approach is to include elements on the
select high-expressing cell lines without expression plasmid. This will cause the ran-
amplification, which reduces the time com- dom integration site to become transcrip-
pared to the DHFR selection approach. The tionally active and available to the enzymes
GS system has enabled the rapid identifica- that transcribe the genes. There have been
tion and selection of production cell lines several reports of such genetic elements that
that express up to 20–50 pcd and multiple enable the integrated plasmid to create a
grams per liter of product as part of an transcriptionally active region at any integra-
overall cell culture process development tion location on the chromosome and to
effort. According to Lonza, more than 85 enable higher transcription levels in a higher
global pharmaceutical companies are cur- percentage of transfectants. Two types of ele-
rently using this technology to create pro- ments that function to create a region of
duction cell lines and five products using transcriptionally active chromatin are the
the GS system have been approved for ubiquitous chromatin opening elements
commercial sale, including Synagis and (UCOE) and the matrix attachment regions
Zenapax. The GS technology is available (MAR) elements.15,16 These genetic elements
for licensing from Lonza for the use in have different mechanisms of action but
research and commercial applications, both work to increase the expression levels

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 Monoclonal Antibodies

of linked genes that are transfected on the Table 2. Host cell types used in the manufacture of commercial MAbs
same plasmid as the MAR or UCOE.
Cell line Species Number of products
The use of MAR elements for improving
expression has been commercialized by Hybridoma Murine 5
Selexis. The company has developed a set of
SP2/0 myeloma Murine 5
expression vectors and transfection technolo-
gies (the “MARtech” technology) that use NS0 myeloma Murine 3
these elements to increase the percentage of
Other myeloma Murine 1
cells expressing the desired gene. As shown
schematically in Figure 2, the MAR elements Chinese hamster ovary (CHO) Hamster 10
are inserted into an expression vector such
EBV-transformed B cell Human 1
that the gene for the desired product is sur-
rounded by these elements to impose an E. coli Microbial 1
open chromatin configuration, thereby
allowing RNA polymerase and other tran- Those antibody products produced in
scription factors to access the transcriptional hybridoma cell lines generally have lower
promoters and enhancers found in the dose requirements than others and are also
expression vector. For this reason, MARtech older than those produced using highly engi-
increases the number of independently trans- neered systems such as CHO, NSO, or SP2/0.
formed cells that express the desired protein The single product produced in a human cell
and enables expression levels in the initial line may represent a trend in coming years
transfectants of as much as 50–70 pcd. Selexis as others develop human cell lines capable of
claims that MARtech allows for generation of producing antibody products at high levels.
clonal mammalian production cell lines in While the use of murine cell lines still pre-
about 10 weeks. Many companies have vails in commercial processes, the use of
begun exploring the use of MARtech to CHO cells for producing commercial prod-
enable rapid generation of high producing ucts is growing and most antibody products
cell lines for their antibody products. Later currently in development are produced from
this year the first product using this technol- CHO or human cell lines.
ogy will enter clinical trials.17
UCOE technology, now available through Hybridoma Technology
Millipore Corporation, provides an MAbs were first produced from hybridomas
approach to increasing gene expression simi- consisting of a murine B cell producing a
lar to that of the MARtech technology. The specific antibody fused to an immortal
UCOE elements are functionally similar to murine lymphoid cell line. Initially, MAbs
MAR elements although their composition were produced by injecting a hybridoma cell
and structure are different.16 UCOE consists line into the abdomen of pristane-primed
of regions that are rich in the sequence CpG, mice, in which the cells could grow to a sig-
and that increase the accessibility of the sur- nificant level. As the hybridoma cells grow
rounding chromatin. Therefore, a single in the abdomen, MAb-rich ascites fluid accu-
UCOE element can be included on an mulates. The ascites fluid can then be col-
expression vector and can increase the lected by withdrawing it with needles at
expression levels of linked genes. There is several day intervals. The collected ascites
less commercial experience with UCOE ele- fluid is very complex in composition and
ments than with MAR elements, but the highly contaminated, but frequently
intent is to offer the technology to compa- contains antibody concentrations approach-
nies for use in research and in commercial ing 1 g/L or greater. This process is widely
production cell line generation. used for the production of small to moderate
amounts of antibodies for multiple applica-
ADVANCES IN CELL CULTURE TECHNOLOGY tions and one commercial antibody product
Host cell lines currently used to produce is produced today using this technology.
commercial MAb products include murine The limitations of large-scale production
hybridoma and myeloma cell lines, CHO cell in the abdomens of mice were quickly real-
lines, and one human cell line (Table 2). ized and scientists turned their efforts to use

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 Monoclonal Antibodies

for antibodies. Today, the vast majority of

Fed-batch processes are biologic products made in mammalian cells
are produced using a CHO host cell line.
readily scaled-up to commercial Because of the widespread adoption of this
host cell, the growth characteristics, metabo-
lism, behavior in bioreactors, virulence fac-
volumes and represent the primary tors, and the likely host-cell related
impurities that might be in a process or
method in use today. product are well understood. Moreover,
because there is a strong regulatory history
in vitro culture as an alternative to replace in of CHO cells, more and more products in
vivo production in ascites. These initial development are now made using CHO cells.
efforts focused on growing hybridomas in
culture, under conditions enabling the same Human Cell Lines
high level of antibody expression as seen in While the use of CHO cells as production
the ascites fluid. Initial studies characterized hosts continues, other cell lines, especially
and compared the growth of hybridomas human cell lines, are being developed as alter-
and production of antibodies in either batch native hosts. For example, the PER.C6 cell line
suspension cultures using stirred tanks and developed by Crucell, has been shown to pro-
airlift fermentors or in perfusion cultures duce antibodies at levels similar to or even
using a variety of methods for cell retention. greater than CHO cell lines.18 One potential
From simple batch cultures, the use of con- advantage of products produced in these cell
trolled feeding, also know as fed-batch, lines is that the glycosylation patterns and
evolved as extremely successful in increasing other post-translational modifications of anti-
maximum cell concentrations, culture bodies produced in them may be more similar
longevities, and corresponding product to human antibodies. Therefore, the PER.C6
titers. Fed-batch is the primary mode of bio- cell line and other human cell lines may
pharmaceutical production used today, both prove to be reliable, safe, scalable, and eco-
for antibodies and other recombinant pro- nomical alternatives to the CHO cell lines cur-
tein products. rently in use for the production of MAbs.
Hybridoma technology enabled the cre-
ation and production of MAbs for research, Chemically Defined Media
analytical use, and as limited-dose therapeu- Current regulatory requirements strongly dis-
tic products. However, these cell lines are courage or ban the use of any products in the
generally difficult to engineer for high levels culture media that are derived from animals,
of protein expression and usually grow to especially from bovine sources. Therefore, the
only moderate densities in bioreactors. use of bovine serum, commonly used earlier
Hence, although these cells are designed to in mammalian cell culture, has been discon-
produce antibodies, in many cases they do tinued and significant efforts have been
so at levels that are too low to be optimal for directed towards the development of cell cul-
manufacturing today’s MAbs. ture media, that is free from animal-derived
products. There is a growing trend toward the
Using CHO Cells as Production Hosts use of chemically defined media. In such
To circumvent the limitations of hybridomas media, recombinant proteins such as IGF-1,
for MAb production, scientists began experi- transferrin, insulin, or others may be included
menting with alternative production hosts to provide the necessary signals for cell
that could be grown to higher densities and growth. When used, the recombinant human
transfected with the antibody genes to versions of these proteins are preferred. To
enable higher cellular productivity. The further minimize the risk associated with the
murine myeloma cell lines NSO and SP2/0 addition of animal-derived components, CHO
were among the first used to produce recom- and other production host cell lines used for
binant MAbs. At the same time, others began antibody production are now selected for
examining CHO cell lines. The CHO cell their ability to grow and produce product at
lines proved to be a suitable production host high levels in chemically defined media.

110 BioPharm International www.biopharminternational.com October 2007

 Monoclonal Antibodies

Therefore, fed-batch culture is now the

Significant efforts are being devoted method of choice for robust, reproducible,
and reliable manufacturing processes. While
to the continuous improvement the capital investments in a manufacturing
facility using fed-batch culture are higher
than those for a perfusion-based facility, the
in the safety and quality of MAbs. overall cost of goods for fed-batch and perfu-
sion processes are similar. While both culture
Several different chemically defined media are technologies are successfully used today by
now commercially available from a variety of commercial manufacturers, the biopharma-
vendors. However, most companies involved ceutical industry is converging on the use of
in the development of MAb products today fed-batch suspension cultures in stirred-tank
have developed proprietary cell culture media bioreactors with controlled feeding.
and growth conditions suitable for produc-
tion of their particular monoclonal antibody FUTURE CHALLENGES IN
at high titers. ANTIBODY MANUFACTURING
Along with improvements and refine- The advances in cell line generation and cell
ments in expression systems and cell lines culture described above have enabled
for MAb production, there have also been companies to produce monoclonal antibodies
significant advances in cell culture condi- at very high expression levels. As a result,
tions over the past 20 years to further opti- early concerns that the industry would not be
mize antibody production. 19,20 The able to meet the growing production
optimization of fed-batch processes has demands of MAbs have subsided. While these
increased antibody titers in culture orders of significant improvements in upstream
magnitude so that expression levels of production have resulted in the ability to
greater than 1 g/L are frequently achieved. express MAbs at levels approaching 10 g/L,
the capacity and ability of downstream
Perfusion Technology processes to handle these high quantities of
One initial approach to increase the yield of antibody has been strained. The competing
antibody products from a single bioreactor demands of growing production requirements
was the use of perfusion technology in which and reduced cost to the patient present
the media is continuously removed from the challenges to the industry to make
bioreactor and replaced with fresh media. manufacturing processes even more efficient.
Perfusion technology is based on the ration- Improvements in chromatography media for
ale that cells in culture could continue to antibody purification have resulted in media
produce antibody over several weeks if the with higher capacities, faster throughput, and
conditioned media, containing the antibody improved contaminant clearance. Significant
product along with potentially growth limit- efforts are currently being devoted to
ing metabolites, were replaced regularly with developing alternative techniques to improve
fresh media and growth factors. Years of com- downstream processing to enable the efficient
parative work have shown that perfusion cul- processing of high levels of antibody, enhance
tures can achieve higher volumetric process robustness and yields, and reduce
productivities than fed-batch cultures at the overall manufacturing costs. Companies
expense of lower product titers per liter of today are striving to incorporate Quality by
medium consumed. Moreover, the continu- Design and other new regulatory concepts
ously changing media conditions and long into the development of MAb products to
culture times required for perfusion produc- further reduce the cost and development
tion frequently lead to inconsistent processes, timelines for these products. The
variable glycosylation, and other post-transla- manufacturers are also striving to develop
tional modifications in the product over time final product formulations containing high
in culture. The risk of contamination also concentrations of antibody with sufficient
increases. Perfusion operations tend to be stability to address the increasing doses of
complex, difficult to scale up, and generally antibody products without adversely
less robust than fed-batch processes. 21,22 impacting the quality of these products. ◆

112 BioPharm International www.biopharminternational.com October 2007

 Monoclonal Antibodies

How Cell Culture Became King

REFERENCES ... and May be Usurped
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So watch out, cell culture. A microbial coup may be in the making.

114 BioPharm International www.biopharminternational.com October 2007