Advances in the Development of Therapeutic Monoclonal Antibodies

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, costeffective
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.
Susan Dana Jones, PhD, is a senior
consultant, Francisco J. Castillo, PhD,
is a senior consultant, and
Howard L. Levine, PhD, is a
principal consultant, all at
BioProcess Technology Consultants, Inc.,
Acton, MA, 978.266.9159,
sjones@bioprocessconsultants.com.
Listen to a podcast interview with
Howard Levine at
biopharminternational.com/biopharmnow
The first therapeutic monoclonal
antibody (MAb) product
entered the market in
1986, but it took another
decade before the potential of this new
class of biologic products began to be
realized. From the mid 1990s until
today, almost 30 therapeutic monoclonal
antibodies (MAbs) have been
approved throughout the world along
with several antibody-related products
(e.g., Fc-fusion proteins) making MAbs
and related products a dominant component
of the biopharmaceutical market,
generating revenues of several
billion dollars. The first approved MAb
was a murine antibody. This was followed
by several chimeric MAbs containing
a mix of murine and human
regions. These early antibody products
posed a moderate risk of immunogenicity
to patients from their residual
murine components, somewhat limiting
the development of MAb products.
To address this issue, new technologies
for creating MAbs that were predominately
or entirely of human origin were
developed. Today, almost all antibody
products currently in development are
humanized or fully human.
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 biologics,
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 specific antibody productivity
from a given cell line and improve
overall productivity in bioreactors. These
advances include the use of new expression
vectors and transfection technology
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Monoclonal Antibodies
Figure 1. Annual approval of recombinant biologic products and
monoclonal antibody products. 2,3 The total number of biologics, including
MAb products, approved by FDA for market each year since 1982 is
shown in green. MAb product approvals only are shown in black.
Antibody-related products such as Fc fusions, engineered antibody
fragments, or other products derived from antibodies but not containing
an antibody binding region are not included in the MAb figures. However,
those products are included in the total product figures.
14
12
10
8
6
4
2
0
82 84 86 88 90 92 94 96 98 0 2 4 6
Total biologics
including MAbs
MAbs
to improve cell line generation; novel parental
cell lines that have been selected or designed
to grow to maximum density and productivity
under standard bioreactor conditions; and
high-throughput, robust screening technologies
to select the highest producing clones rapidly
and more effectively. As a result, the
production of cell lines expressing multigram
quantities of antibody per liter of culture
medium is now routine.
These advances, coupled with improvements
in cell culture media and greatly optimized
bioreactor processes, have made the
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. These challenges
include the need to streamline downstream
processing to enable the processing of
increased product quantities; the implementation
of Quality by Design (QbD) and other
new regulatory concepts to reduce the cost
and development timelines for MAb products
without adversely affecting their quality;
the need for high-concentration product
formulations with sufficient stability to
address the increasing doses of antibody
products; and the development of alternative
delivery systems.
DISCOVERY OF ANTIBODY THERAPEUTICS
In 1984, Kohler and Milstein received the
Nobel Prize in Medicine for their pioneering
work on the production of MAbs. 1 One
of the most significant advantages of this
new technology over traditional techniques
for producing antibodies was the
development of an immortalized cell line
creating a continuous source of the same
antibody with a single antigen specificity.
This enabled the development of highly
specific antibodies directed toward a single
epitope on the target antigen. Initially,
MAbs were used as laboratory reagents, but
they were quickly adopted as clinical diagnostic
reagents, and eventually as therapeutic
agents. The development of
therapeutic MAbs commenced in the early
1980s and by 1986 the first monoclonal
antibody for human use—Orthoclone
OKT3 (Ortho Pharmaceuticals)—was
approved for the prevention of kidney transplant
rejection. Following the approval of
OKT3, the enthusiasm for MAbs as therapeutic
products grew with the next wave of antibody
products generally being developed as
anticancer agents. Several of these products
were approved in the US and Europe in the
mid to late 1990s, a trend that continues to
grow today. Since the commercialization of
the first therapeutic MAbs, these products
have become a dominant component of the
biopharmaceutical market, representing
approximately 20% of all biologic products,
with combined revenues of over $20 billion
in 2006. 4 The growth of MAb products over
the past 25 years, as shown in Figure 1, confirms
the importance of these products and
also shows that MAbs represent a significant
subset of all biopharmaceuticals on the market
and in development. With over 300 antibody
products currently in development,
this unique and effective category of therapeutic
compounds is poised to grow significantly
in the coming years.
MOLECULAR STRUCTURES OF
ANTIBODIES:THEN AND NOW
Murine Antibodies
The initial technology for producing MAbs
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Monoclonal Antibodies
involved fusing individual antibody-secreting
spleen cells from immunized mice with a
murine myeloma cell line to generate
immortalized cell lines that secreted individual,
or monoclonal, antibodies. Hence, the
first MAbs developed for use as potential
human therapeutics were murine antibodies.
While initial interest in these murine MAbs
was high and several companies began
developing products based on this technology,
OKT3 was the only murine monoclonal
antibody that was approved for human therapeutic
use. Despite the fact that OKT3 has
been moderately successful in the market,
the use of murine MAbs as therapeutic
agents quickly ran into many roadblocks.
One of the potential advantages of MAbs as
therapeutic agents is their long circulating
half-life, allowing them to provide a therapeutic
effect in patients over several days.
However, when murine MAbs were repeatedly
administered to humans during clinical
trials, it was observed that the half-life
decreased and the products became less
effective with each injection. This was
because of the immunogenicity of murine
proteins in humans and the rapid development
of a human antimurine antibody
(HAMA) response in the patients. This
HAMA response neutralized the effectiveness
of the murine antibodies and resulted in
their rapid clearance from the body. For
example, it has been reported that OKT3 can
elicit a HAMA response in up to 86% of
patients treated, leading to some limitations
in its efficacy. 5
Chimeric Antibodies
To overcome the HAMA responses occuring
from the usage of murine MAbs as therapeutics,
several approaches were developed in an
attempt to make MAbs more human-like and
less immunogenic. In the early 1990s,
molecular biology techniques enabled the
creation of “chimeric” antibodies by linking
the murine genes encoding the antigenbinding
portion of the antibody (the variable
region) to the genes encoding the constant
region of human immunoglobulin light and
heavy chains. Because over 75% of the protein
sequence of the resulting chimeric antibodies
was of human origin, these chimeric
MAbs elicited much lower HAMA responses
in patients. Moreover, because the antibody
Future therapeutic monoclonal antibody
products will be predominantly
humanized or fully human.
constant region in these chimeric antibodies
is human, it is capable of activating other
components of the human immune system
to potentially create more effective therapeutic
agents. Many of the MAbs approved for
commercialization in the 1990s and early
2000s were chimeric antibodies, including
the highly successful anticancer antibodies
Rituxan (approved in 1997) and Erbitux
(approved in 2004), as well as the antiinflammatory
product Remicade (approved
in 1998). Chimeric antibody products are
superior to murine antibody products but
they still pose a moderate risk of immunogenicity
to patients from their residual
murine components. Therefore, antibody
engineering approaches that further reduce
the murine component or that remove
immunogenic portions of the chimeric antibody,
have been developed and used to generate
fully “humanized” antibody products.
Humanized Antibodies
In 1991, Protein Design Labs (PDL) developed
and patented the first technology for successfully
humanizing MAbs. 6 The antigen binding
specificity of any antibody is determined by
the amino acids present in three distinct
highly variable regions per antibody chain,
referred to as complementarity determining
regions (CDRs), and located in a more conserved
framework sequence in the variable
regions. Therefore, PDL scientists developed
methods for engineering an antibody gene in
which the CDRs of a human antibody gene
were replaced by those from the CDR of a
murine MAb gene. The resulting humanized
antibody has the same antigen binding properties
as the original murine antibody but
contains minimal murine sequences and,
therefore, elicits a lower HAMA response in
patients. The CDR-grafted human antibody
can be used as is or, in cases where affinity of
the chimeric antibody is slightly reduced
from the original murine antibody, additional
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Monoclonal Antibodies
Table 1. Comparison of sales for antibody-based anti-inflammatory products
Product
Company
Year
approved
2006 sales worldwide
($ million) Market share
Humira Abbott 2002 2,000 15.8%
Remicade Johnson & Johnson 1998 4,253 33.7%
Enbrel Amgen 1998 4,379 34.7%
changes can be made in the antibody
sequence to regain or enhance its binding
properties. Like chimeric antibodies, humanized
antibodies can activate other parts of the
immune system to create a more effective
product. Several humanized antibody products
are currently on the market, including
Synagis (approved in 1998), Herceptin
(approved in 1998), Mylotarg (approved in
2000), Xolair (approved in 2003), and Avastin
(approved in 2004).
In addition to the production of chimeric
and humanized antibodies, other technologies
have been developed to help minimize
the HAMA response in patients. These
include human engineering or deimmunization,
in which amino acids on the surface of
the murine variable region that are known to
be effective immunogenic sequences are
changed to their non-immunogenic human
counterpart, leaving the other non-immunogenic
murine sequences unchanged. 7 The
advantage of this approach is that the structural
integrity of the variable region is better
maintained and reduction of affinity for the
target is minimized.
Fully Human Antibodies
The latest advancement in creating less
immunogenic therapeutic antibody products
is the ability to generate fully human MAbs.
Several technologies exist to develop fully
human antibodies, each falling into one of
the two general classes—in vivo approaches
using a murine system in which the
immunoglobulin genes have been replaced by
their human counterparts or in vitro
approaches using libraries containing millions
of variations of antibody sequences coupled
with a mechanism to express and screen these
antibodies in vitro. Humira (approved in 2004)
is the first fully human antibody to be
approved. This anti-TNF-α antibody was first
identified by scientists at Cambridge
Antibody Technology (CAT, now part of
AstraZeneca) using an in vitro
molecular engineering technology
known as phage display. In the marketplace,
this human MAb competes
with Enbrel, an Fc fusion
protein, and Remicade, a chimeric
antibody. The power of the fully
human antibody platform can be
seen in the sales figures for these
three products. Although Humira was
approved four years later than the other products,
it has successfully taken a significant
market share from them, garnering almost
16% market share in 2006. Worldwide sales in
2006 for all three products are shown in
Table 1.
Many antibody products currently in early
clinical development are fully human,
because the technologies that enable the
generation of human antibodies are now
accessible through partnerships or licensing
from the companies that have developed
these approaches. Moreover, the expectation
in the medical and regulatory community is
that companies will use the best approach
for their product to achieve humanization.
There will be exceptions to this generalization,
for example when a short half life is
desired or when a toxic or radioactive payload
is linked to the antibody, but for
unmodified therapeutic antibody products
the industry standard has changed; most
future antibody products will be humanized
or fully human antibodies.
Most MAb products are naked antibodies,
which rely on either blocking an important
biological function or on activating the
immune system, to elicit a therapeutic effect.
However, antibodies are also well suited as
targeting agents to deliver potent chemo- or
radioactive agents specifically to target cells.
For example, Mylotarg contains a cytotoxic
compound conjugated to a monoclonal antibody.
This immunoconjugate product is
designed to deliver the potent cytotoxic
compound selectively to cancer cells. The
radio-immunoconjugate products Zevalin
and Bexxar (both anti-CD20 MAbs), deliver
radioisotopes for the treatment of lymphoma.
Both these products are murine antibodies
because the human or humanized
forms of these products would bind to and
target not only the CD20 positive target cells
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 components
to the site of a foreign antigen. By
inadvertently targeting these cells, human
antibody-based radio-immunoconjugates
could do more harm to nontarget cell types
than to the targeted cancer cell.
All of the above technologies now allow
the generation of better designed antibody
products with fully human sequences and
optimized function. Combining in vivo and in
vitro discovery and molecular engineering
technologies allows exquisite control of the
antibody sequences and properties that was
not possible 20 years ago. New approaches for
the rapid production of cell lines suitable for
large-scale commercial production have
enabled the development of MAb therapies to
treat myriad diseases and made these products
available to an increasing number of patients.
In addition to enabling more efficient and
economic production of MAbs, the above
antibody engineering technologies, coupled
with advances in cell culture production discussed
below, have greatly increased our ability
to control or alter the properties of the
resulting antibodies. For example, the extent
of glycosylation, which can increase effector
function and thereby increase product efficacy,
can be controlled by both cell line engineering
and cell culture technologies.
In the future, human cell lines may
replace CHO and other mammalian
cell lines for the production of MAbs.
MARKET DEMANDS AND
CELL LINE PRODUCTIVITY
One challenging feature of most therapeutic
antibody products is that the doses required
for these products are much higher than for
other biologic products. To meet the large
annual production requirements for these
products, companies have made substantial
progress in developing more efficient and costeffective
methods for manufacturing antibody
products. When antibody products were first
developed and approved, expression levels of
MAbs were typically on the order of 100–500
milligrams per liter. Even as recently as five
years ago, antibody titers in excess of 1 g/L
were not common and many MAb products
were launched using production cell lines and
manufacturing processes that produced
approximately 0.5–1.0 g/L antibody. 8 As MAb
products became successful in the marketplace
and as the demands for new products
increased, newer methods of generating highexpressing
antibody production cell lines and
of culturing these cell lines for maximum productivity
have been developed. Today’s technologies
are enabling antibody production in
the bioreactor of 5 g/L or more. 9 Advances in
cell line generation over the past decade
include new expression vectors and transfection
technology to introduce the genes into
cells; novel parental cell lines that have been
selected or designed to grow to maximum
density; and robust screening technologies
that in combination can enable rapid generation
of production cell lines.
ADVANCES IN THE GENERATION
OF PRODUCTION CELL LINES
Today’s MAbs must be manufactured using
reliable production cell lines capable of producing
sufficient quantities of product to
meet the market demand. For most products,
this means that antibody titers in the bioreactor
must be greater than 1 g/L in a fedbatch
process initially and 3–5 g/L following
process optimization. To achieve these levels
of productivity, it is necessary to quickly
develop a cell line expressing reasonably
high quantities of antibody for early preclinical,
formulation, and analytical validation
studies that can be further optimized to
achieve the desired productivity levels. If the
productivity of the initial cell line is high
enough, it can even be used to support initial
clinical development of the product.
Once the initial cell line is established, a production
cell line exhibiting the highest possible
level of production of functional
antibody and capable of supporting commercial
production at a reasonable cost can be
developed. In today’s highly competitive
market, it is important to complete the initial
stages of cell line development as quickly
and efficiently as possible to enable early
entry into human clinical trials but equally
important is to devote sufficient time and
resources to the full development and optimization
of the commercial cell line so that
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Monoclonal Antibodies
The use of parental cell lines adapted to
grow in suspension and serum-free
media can reduce development times.
a suitable cell line is available for commercial
production as soon as possible.
High-Expressing Cell Lines
To create a production cell line for a specific
antibody, expression vectors containing the
heavy- and light-chain genes under control
of strong mammalian promoters are introduced
into the parental cell line. Usually, a
selectable marker is also included so that
cells containing the gene can be easily
selected by adding a drug or substance to the
culture that causes the cell to require the
activity of the selectable marker. The driving
factors behind the selection of a particular
cell clone during cell line generation is the
expression level of the recombinant protein,
which is measured independently of the
selection, and the time that it takes to obtain
a cell line that expresses enough product to
enable nonclinical and clinical development.
Technologies that increase the percentage of
transfectants with high expression levels will
reduce the time needed to identify a production
cell line because the high-expressing
clones will be easier to select without having
to screen thousands of individual clones.
Recent advances in cell line generation
include technologies that increase this percentage,
as well as sophisticated and automated
approaches to screening that enable
more individual transfectants to be screened
for expression levels. 10,12,15,16
Production levels in the bioreactor are a
function of specific productivity—the density
to which the cells can grow and the
longevity of the culture. Before actual testing
in the bioreactor, expression levels are determined
in small culture vessels, from multiwell
plates to shake flasks. Levels of 15–20
picograms of antibody/cell/day (pcd) are
considered appropriate for initial transfectants,
with greater productivity arising from
optimized cell culture conditions, secondary
transfections, or amplification of the transfected
antibody genes using selective pressure.
Using parental cell lines adapted to
grow in suspension and serum-free media
reduces development times and increases the
likelihood of reaching high cell densities
during manufacturing and high product
yields in the grams-per-liter level.
Selection Systems
One of the earliest effective methods for
transfection, selection, and amplification of
foreign genes in mammalian cells was developed
in 1981 by scientists at Columbia
University using dihydrofolate reductase
(DHFR) selection. In this method, a parental
mammalian cell line deficient in the enzyme
DHFR is transfected with an expression vector
containing the DHFR gene under control
of a relatively weak promoter and the antibody
(or other protein) genes under control
of a strong promoter. 11 By performing multiple
rounds of amplification and selection of
cells in the presence of the folate analog
methotrexate (MTX), a potent inhibitor of
DHFR, production cell lines with relatively
high levels of expression of the foreign genes
can be obtained. The original patents for this
technology have now expired but it is still
widely used to generate antibody production
cell lines. However, because each amplification
cycle requires 12 weeks to complete and
up to five cycles or more, about one year
total may be necessary to obtain a clone
with acceptably high expression levels.
Nevertheless, the DHFR system is effective
and has been used in conjunction with other
aspects of cell line development to achieve
multigram-per-liter expression levels of MAb.
Also, alternative systems requiring less time
to reach maximal expression have been
developed. For example, the glutamine synthetase
selection system, developed by scientists
at Celltech (now Lonza), can achieve
production clones with higher levels of antibody
or protein expression in 4–6 months. 12
Glutamine synthetase (GS) is the enzyme
responsible for the biosynthesis of glutamine
from glutamate and ammonia. This
enzymatic reaction provides the only pathway
for glutamine formation in a mammalian
cell. Therefore, in the absence of
glutamine in the growth medium, the GS
enzyme is essential for the survival of the
mammalian cells in culture. Some mammalian
cell lines, such as the murine cell
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Monoclonal Antibodies
Figure 2. In matrix attachment region (MAR) technology, MAR elements are
inserted into expression vectors surrounding the desired transgene and impose
an open chromatin configuration on the nearby chromatin. This open structure
allows RNA polymerase and other transcription factors to access the
transcriptional promoters and enhancers found within the expression vector and
thereby enables greater levels of transcription. This leads to increased productspecific
translation and a higher yield in a greater percentage of transfected
cells. Figure provided courtesy of Selexis SA.
‘Closed’
chromatin
Promoters/
enhancers
‘Open’
chromatin
chromatin
‘Closed’
MAR
lines NSO or SP2/0 widely used for antibody
production, do not express sufficient
GS to survive without added glutamine.
With these cell lines, a transfected GS gene
can function as a selectable marker by permitting
growth in a glutamine-free
medium. Chinese hamster ovary (CHO)
cells, also widely used for antibody and
other recombinant protein production,
contain sufficient active GS to survive
without exogenous glutamine. 13 In these
cases the specific GS inhibitor, methionine
sulphoximine (MSX), can be used to
inhibit endogenous GS activity such that
only transfectants with additional GS activity
can survive. GS selection can be used to
select high-expressing cell lines without
amplification, which reduces the time compared
to the DHFR selection approach. The
GS system has enabled the rapid identification
and selection of production cell lines
that express up to 20–50 pcd and multiple
grams per liter of product as part of an
overall cell culture process development
effort. According to Lonza, more than 85
global pharmaceutical companies are currently
using this technology to create production
cell lines and five products using
the GS system have been approved for
commercial sale, including Synagis and
Zenapax. The GS technology is available
for licensing from Lonza for the use in
research and commercial applications,
making it widely available for the
development of MAb products. 14
Improving Gene Expression
Another recent approach to improve
expression of antibody genes in the
initially transfected cells is to ensure
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.
The transfection of a mammalian cell
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
active, there is a high likelihood that
integration will occur in regions that
are not able to transcribe high levels
of the antibody genes. Targeting the
expression plasmid to locations on the chromatin
that are known to be transcriptionally
active and accessible to the necessary
enzymes would increase the expression of
all genes integrated at these sites. Although
this is an excellent concept in theory,
homologous recombination or targeted integration
has not been widely adapted in practice
because of the lack of information
about which sites are good locations for
integration and the need to have unique
plasmids and cell lines that are able to perform
the recombination.
Rather than targeting a specific site in the
chromatin for integration, an alternative
approach is to include elements on the
expression plasmid. This will cause the random
integration site to become transcriptionally
active and available to the enzymes
that transcribe the genes. There have been
several reports of such genetic elements that
enable the integrated plasmid to create a
transcriptionally active region at any integration
location on the chromosome and to
enable higher transcription levels in a higher
percentage of transfectants. Two types of elements
that function to create a region of
transcriptionally active chromatin are the
ubiquitous chromatin opening elements
(UCOE) and the matrix attachment regions
(MAR) elements. 15,16 These genetic elements
have different mechanisms of action but
both work to increase the expression levels
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of linked genes that are transfected on the
same plasmid as the MAR or UCOE.
The use of MAR elements for improving
expression has been commercialized by
Selexis. The company has developed a set of
expression vectors and transfection technologies
(the “MARtech” technology) that use
these elements to increase the percentage of
cells expressing the desired gene. As shown
schematically in Figure 2, the MAR elements
are inserted into an expression vector such
that the gene for the desired product is surrounded
by these elements to impose an
open chromatin configuration, thereby
allowing RNA polymerase and other transcription
factors to access the transcriptional
promoters and enhancers found in the
expression vector. For this reason, MARtech
increases the number of independently transformed
cells that express the desired protein
and enables expression levels in the initial
transfectants of as much as 50–70 pcd. Selexis
claims that MARtech allows for generation of
clonal mammalian production cell lines in
about 10 weeks. Many companies have
begun exploring the use of MARtech to
enable rapid generation of high producing
cell lines for their antibody products. Later
this year the first product using this technology
will enter clinical trials. 17
UCOE technology, now available through
Millipore Corporation, provides an
approach to increasing gene expression similar
to that of the MARtech technology. The
UCOE elements are functionally similar to
MAR elements although their composition
and structure are different. 16 UCOE consists
of regions that are rich in the sequence CpG,
and that increase the accessibility of the surrounding
chromatin. Therefore, a single
UCOE element can be included on an
expression vector and can increase the
expression levels of linked genes. There is
less commercial experience with UCOE elements
than with MAR elements, but the
intent is to offer the technology to companies
for use in research and in commercial
production cell line generation.
ADVANCES IN CELL CULTURE TECHNOLOGY
Host cell lines currently used to produce
commercial MAb products include murine
hybridoma and myeloma cell lines, CHO cell
lines, and one human cell line (Table 2).
Table 2. Host cell types used in the manufacture of commercial MAbs
Cell line Species Number of products
Hybridoma Murine 5
SP2/0 myeloma Murine 5
NS0 myeloma Murine 3
Other myeloma Murine 1
Chinese hamster ovary (CHO) Hamster 10
EBV-transformed B cell Human 1
E. coli Microbial 1
Those antibody products produced in
hybridoma cell lines generally have lower
dose requirements than others and are also
older than those produced using highly engineered
systems such as CHO, NSO, or SP2/0.
The single product produced in a human cell
line may represent a trend in coming years
as others develop human cell lines capable of
producing antibody products at high levels.
While the use of murine cell lines still prevails
in commercial processes, the use of
CHO cells for producing commercial products
is growing and most antibody products
currently in development are produced from
CHO or human cell lines.
Hybridoma Technology
MAbs were first produced from hybridomas
consisting of a murine B cell producing a
specific antibody fused to an immortal
murine lymphoid cell line. Initially, MAbs
were produced by injecting a hybridoma cell
line into the abdomen of pristane-primed
mice, in which the cells could grow to a significant
level. As the hybridoma cells grow
in the abdomen, MAb-rich ascites fluid accumulates.
The ascites fluid can then be collected
by withdrawing it with needles at
several day intervals. The collected ascites
fluid is very complex in composition and
highly contaminated, but frequently
contains antibody concentrations approaching
1 g/L or greater. This process is widely
used for the production of small to moderate
amounts of antibodies for multiple applications
and one commercial antibody product
is produced today using this technology.
The limitations of large-scale production
in the abdomens of mice were quickly realized
and scientists turned their efforts to use
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Monoclonal Antibodies
Fed-batch processes are
readily scaled-up to commercial
volumes and represent the primary
method in use today.
in vitro culture as an alternative to replace in
vivo production in ascites. These initial
efforts focused on growing hybridomas in
culture, under conditions enabling the same
high level of antibody expression as seen in
the ascites fluid. Initial studies characterized
and compared the growth of hybridomas
and production of antibodies in either batch
suspension cultures using stirred tanks and
airlift fermentors or in perfusion cultures
using a variety of methods for cell retention.
From simple batch cultures, the use of controlled
feeding, also know as fed-batch,
evolved as extremely successful in increasing
maximum cell concentrations, culture
longevities, and corresponding product
titers. Fed-batch is the primary mode of biopharmaceutical
production used today, both
for antibodies and other recombinant protein
products.
Hybridoma technology enabled the creation
and production of MAbs for research,
analytical use, and as limited-dose therapeutic
products. However, these cell lines are
generally difficult to engineer for high levels
of protein expression and usually grow to
only moderate densities in bioreactors.
Hence, although these cells are designed to
produce antibodies, in many cases they do
so at levels that are too low to be optimal for
manufacturing today’s MAbs.
Using CHO Cells as Production Hosts
To circumvent the limitations of hybridomas
for MAb production, scientists began experimenting
with alternative production hosts
that could be grown to higher densities and
transfected with the antibody genes to
enable higher cellular productivity. The
murine myeloma cell lines NSO and SP2/0
were among the first used to produce recombinant
MAbs. At the same time, others began
examining CHO cell lines. The CHO cell
lines proved to be a suitable production host
for antibodies. Today, the vast majority of
biologic products made in mammalian cells
are produced using a CHO host cell line.
Because of the widespread adoption of this
host cell, the growth characteristics, metabolism,
behavior in bioreactors, virulence factors,
and the likely host-cell related
impurities that might be in a process or
product are well understood. Moreover,
because there is a strong regulatory history
of CHO cells, more and more products in
development are now made using CHO cells.
Human Cell Lines
While the use of CHO cells as production
hosts continues, other cell lines, especially
human cell lines, are being developed as alternative
hosts. For example, the PER.C6 cell line
developed by Crucell, has been shown to produce
antibodies at levels similar to or even
greater than CHO cell lines. 18 One potential
advantage of products produced in these cell
lines is that the glycosylation patterns and
other post-translational modifications of antibodies
produced in them may be more similar
to human antibodies. Therefore, the PER.C6
cell line and other human cell lines may
prove to be reliable, safe, scalable, and economical
alternatives to the CHO cell lines currently
in use for the production of MAbs.
Chemically Defined Media
Current regulatory requirements strongly discourage
or ban the use of any products in the
culture media that are derived from animals,
especially from bovine sources. Therefore, the
use of bovine serum, commonly used earlier
in mammalian cell culture, has been discontinued
and significant efforts have been
directed towards the development of cell culture
media, that is free from animal-derived
products. There is a growing trend toward the
use of chemically defined media. In such
media, recombinant proteins such as IGF-1,
transferrin, insulin, or others may be included
to provide the necessary signals for cell
growth. When used, the recombinant human
versions of these proteins are preferred. To
further minimize the risk associated with the
addition of animal-derived components, CHO
and other production host cell lines used for
antibody production are now selected for
their ability to grow and produce product at
high levels in chemically defined media.
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Monoclonal Antibodies
Significant efforts are being devoted
to the continuous improvement
in the safety and quality of MAbs.
Several different chemically defined media are
now commercially available from a variety of
vendors. However, most companies involved
in the development of MAb products today
have developed proprietary cell culture media
and growth conditions suitable for production
of their particular monoclonal antibody
at high titers.
Along with improvements and refinements
in expression systems and cell lines
for MAb production, there have also been
significant advances in cell culture conditions
over the past 20 years to further optimize
antibody production. 19,20 The
optimization of fed-batch processes has
increased antibody titers in culture orders of
magnitude so that expression levels of
greater than 1 g/L are frequently achieved.
Perfusion Technology
One initial approach to increase the yield of
antibody products from a single bioreactor
was the use of perfusion technology in which
the media is continuously removed from the
bioreactor and replaced with fresh media.
Perfusion technology is based on the rationale
that cells in culture could continue to
produce antibody over several weeks if the
conditioned media, containing the antibody
product along with potentially growth limiting
metabolites, were replaced regularly with
fresh media and growth factors. Years of comparative
work have shown that perfusion cultures
can achieve higher volumetric
productivities than fed-batch cultures at the
expense of lower product titers per liter of
medium consumed. Moreover, the continuously
changing media conditions and long
culture times required for perfusion production
frequently lead to inconsistent processes,
variable glycosylation, and other post-translational
modifications in the product over time
in culture. The risk of contamination also
increases. Perfusion operations tend to be
complex, difficult to scale up, and generally
less robust than fed-batch processes. 21,22
Therefore, fed-batch culture is now the
method of choice for robust, reproducible,
and reliable manufacturing processes. While
the capital investments in a manufacturing
facility using fed-batch culture are higher
than those for a perfusion-based facility, the
overall cost of goods for fed-batch and perfusion
processes are similar. While both culture
technologies are successfully used today by
commercial manufacturers, the biopharmaceutical
industry is converging on the use of
fed-batch suspension cultures in stirred-tank
bioreactors with controlled feeding.
FUTURE CHALLENGES IN
ANTIBODY MANUFACTURING
The advances in cell line generation and cell
culture described above have enabled
companies to produce monoclonal antibodies
at very high expression levels. As a result,
early concerns that the industry would not be
able to meet the growing production
demands of MAbs have subsided. While these
significant improvements in upstream
production have resulted in the ability to
express MAbs at levels approaching 10 g/L,
the capacity and ability of downstream
processes to handle these high quantities of
antibody has been strained. The competing
demands of growing production requirements
and reduced cost to the patient present
challenges to the industry to make
manufacturing processes even more efficient.
Improvements in chromatography media for
antibody purification have resulted in media
with higher capacities, faster throughput, and
improved contaminant clearance. Significant
efforts are currently being devoted to
developing alternative techniques to improve
downstream processing to enable the efficient
processing of high levels of antibody, enhance
process robustness and yields, and reduce
overall manufacturing costs. Companies
today are striving to incorporate Quality by
Design and other new regulatory concepts
into the development of MAb products to
further reduce the cost and development
timelines for these products. The
manufacturers are also striving to develop
final product formulations containing high
concentrations of antibody with sufficient
stability to address the increasing doses of
antibody products without adversely
impacting the quality of these products. ◆
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Monoclonal Antibodies
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genes in somatic mammalian cells. Methods Enzymol.
1987;151:85–104.
12. Bebbington, CR et al. High level expression of a
recombinant antibody from myeloma cells using a
glutamine synthetase gene as an amplifiable
selectable marker. Biotechnol. 1992;10:169–175.
13. Wilson RH. Glutamine synthetase gene amplification
in Chinese hamster ovary cells. Gene amplification in
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How Cell Culture Became King
... and May be Usurped
David Estell, vice president of technology at Genencor International,
650.846.7500, dave.estell@danisco.com
In the early 1980s, most recombinant protein production was carried
out in E. coli. The disadvantage of this method was that the proteins
were produced intracellularly and had to be refolded to obtain active
protein. As a result, at Genentech we were looking for ways to produce
properly folded proteins in other cell systems.
By 1981, Art Levinson’s group had developed techniques to allow
selectable, stable expression in mammalian cells. These methods were
initially applied to our hepatitis B surface antigen and tissue plasminogen
activator (t-PA) expression. The resulting proteins were efficiently
expressed in a properly folded form. Meanwhile, James
Stramondo’s group had developed large-scale cell culture processes
to improve performance.
The biggest concern in using transformed cells was that DNA or
viruses could be carried into the final product. The hepatitis B surface
antigen assembled into 22-nm particles that were similar in size and
shape to some viruses, which made the problem particularly difficult. So
the team proceeded to work toward FDA approval. My group was
Bacterial expression systems
can secrete large amounts of
protein in fermentations that take
only a few days per batch.
responsible for creating
the initial recovery process
and for demonstrating
viral clearance and DNA
removal. Several other
research and development
groups also put in a
tremendous amount of work to develop other aspects of the new mammalian-cell-based
processes. In the end, it paid off. Within a few years,
both the hepatitis B vaccine and the t-PA processes were validated and
approved by the FDA.
This new expression technology rapidly spread through the industry
to become the standard production system for recombinant proteins.
Thus, cell culture became king. The fact that most human
proteins are secreted efficiently in properly folded form by mammalian
cells means that the production of test quantities of a new
pharmaceutical protein is now straightforward, and many production
processes have become highly standardized.
Cell culture may not always keep its crown, however. Mammalian
cell expression is highly efficient on a per cell basis, but creating the
initial working cell banks and production trains requires long lead
times and is expensive, leading to costs of $500–$1,000 per gram of
protein. The system’s effectiveness, however, has made the industry
reluctant to investigate other options, such as bacillus and fungal
expression systems. These alternative systems have been demonstrated
to secrete extremely large amounts of protein in fermentations
that take only a few days per batch, and produce several metric
tons of protein per year. Because these microbial systems can be created
in weeks and produce protein at 1/10,000th of the cost of mammalian
cells, they may replace some of the mammalian cell capacity
for high volume, lower-cost pharmaceutical proteins in the future.
So watch out, cell culture. A microbial coup may be in the making.
114 BioPharm International www.biopharminternational.com October 2007