In Vitro and In Vivo Mab Production

Kristie Brown

Nov 21, 2019

Science 1


This paper
is mainly based on the publication of the National Research Council (US)
Committee on Methods of Producing Monoclonal Antibodies. Monoclonal Antibody
Production. Washington (DC): National Academies Press (US); 1999, referred to
in the text as (NCR 1999) a few times.

0-309-51904-7, 74 pages, 6 x 9


Large parts
of the content of this paper has been ignored and outdated arguments have been
taken out and replaced by contents from later publications.


are important tools used by many investigators in their biomedical research and
they have led to many medical advances. Although for short term use polyclonal
antibodies may suffice, monoclonal antibodies (mAbs) are commonly preferred when
persistent use of the same antibody over time is required. The traditional way
of producing mAbs requires immunizing an animal, usually a mouse, a rat or a rabbit.
Antibody-producing cells from the animal’s spleen (B-cells) are fused with a
cancer cell (a myeloma) to make them grow and divide indefinitely while they
continue producing antibodies (1). A tumour of the fused cells is called a
hybridoma, but in practice the fused cells initially grown in vitro and
selected for its secreted antibodies are called hybridomas as well. The
hybridoma cells will secrete the antibodies into the cell culture medium, which
can be tested for the right specificity.  The created hybridoma cell lines that are
producing the desired antibody are isolated and cloned to obtain the maximal yield
of antibodies.

Once the
desired hybridoma cell lines have been cloned and selected, production of the
antibodies is the next stage. This can be done either in vivo or in vitro: injecting
hybridoma cells into the peritoneal cavity of a mouse (in vivo) or using in
vitro cell-culture techniques. When injected into a mouse, the hybridoma cells
multiply and produce fluid (ascites) in its abdomen, which contains a high
concentration of antibody. The mouse ascites method is inexpensive, easy to
use, and quick.

if too much fluid accumulates or if the hybridoma is an aggressive cancer, the
mouse will likely experience pain or distress. If the in vivo method produces pain
or distress in animals, regulations call for a search for alternatives. The alternative
is to grow hybridoma cells in a tissue-culture medium. This technique requires
some expertise, requires special media, and can be expensive and time
consuming. There has been considerable research on in vitro methods for growing
hybridomas and these newer methods are less expensive, are faster, and produce antibodies
in higher concentration than has been the case in the past (see below).

It is recommended
to discourage mAb production by the mouse ascites method, but it should be
permitted when it is scientifically justified and approved by the relevant authorities.
Any discomfort, stress and pain should be minimized when this method is being
used, and when there are such signs, prompt euthanasia is recommended. In many
European countries, the ascites method is banned and advanced in vitro
production methods are the only option.

introduction of molecular biology techniques into the world of antibody
production has shifted the traditional dilemmas over producing either in vivo
or in vitro. Transiently expressed DNA-clones of recombinant monoclonal
antibodies (recmAbs) in existing cell lines like CHO or HEK293 are now widely
used for in vitro production and such cell lines are not at all used for
ascites production. This takes out the factor of instability of hybridoma cell
lines and the risk of losing a hybridoma clone. Since the CDR sequences of the mAb
are known, the molecular characteristics of the mAb are forever preserved. Yet,
even when producing recmAbs, most considerations outlined below remain relevant
unless stated otherwise.

General considerations

The mouse
ascites method is generally familiar, well understood, and widely available in
many laboratories across the world; but the mice require careful watching to
minimize the pain or distress induced by excessive accumulation of fluid in the
abdomen or by invasion of the viscera. The expansion of hybridomas in animals
has been the topic of much discussion in the scientific community (NRC 1999),
and it is becoming less acceptable worldwide. Hence, advanced in vitro
production methods have been tried and optimized and are currently used for
industrial-scale production. But in vitro methods can be expensive and
time-consuming compared to the costs and time required by the mouse ascites method,
especially when low amounts of antibodies are required. And in vitro production
may fail due to properties of the hybridoma cell line, even with skilled

anticipated use of the mAb will determine the amount required (2): Very small
amounts of mAb (less than 0.1 g) are required for most research projects and
many analytic purposes. Small-scale quantities (0.1-1 g) are used for
production of diagnostic kits and reagents and for efficacy testing of new mAb
in animals. Medium-scale and large-scale production of mAb are defined, in this
context, as over 1 gram and over 100 grams respectively. These larger
quantities are used for routine diagnostic procedures and for therapeutic
purposes. Commercial-scale production is generally performed to produce mAb for
diagnosis, therapy, and research on and development of new therapeutic agents. Such
production requires more than just the culturing of large batches of cells. It
requires considerable pre-production effort to ensure that the cell line is
stable, can produce commercially appropriate quantities of a stable antibody,
and can produce an uncontaminated product. Commercial production also involves
building a high-quality facility for in vitro production and for processing of
the antibody. There is a need for quality control and quality assurance
departments to meet the requirements of good manufacturing practices that are
required for commercial products. Batch-to-batch testing is necessary to ensure
product reproducibility. Production-process verification and documentation are
necessary to protect the consumer and are required by regulatory authorities
such as FDA in their regulatory guidelines.

mAb production facilities do no longer use the mouse ascites method. In
large-scale production runs, in vitro systems are economically competitive and are
usually selected because they reduce animal use and decrease the presence of contaminating
foreign antigens because serum-free media can be used. Only when the speed of
mAb production is critical and small amounts are required, in vivo production may
be selected. Time requirements for in vitro systems vary considerably with the
type of culture vessel and the required yield of antibodies. Commercial-scale
in vitro production from hybridoma cell lines required more time than in vivo
production because of the lengthy optimization process and the increased time
for producing a given quantity of mAb (33, 34, 35, 36). However, this argument
is becoming outdated since the production of recmAbs make the use of hybridoma
cell lines obsolete (51).

therapeutic industry uses primarily serum-free in vitro technology because of a
concern for treatment-related allergic responses caused by repeated foreign-antigen
exposure. Immune responses are of concern here because mice are the source of
the cell lines used in most mAb production methods. The human immune system
tends to reject mouse-derived antibodies, which can lead to allergies or
decreased effectiveness of injected mAb. Therefore, techniques that replace
most of the mouse’s antibody genes with human DNA have been developed and humanized
antibodies have alleviated this problem (37, 38).

In the
diagnostic industry, keen competition leads to overriding cost considerations, whereas
the presence of foreign antigens is less important. As a result, in
vivo-derived products have been commonly used. In vivo procedures are optimized
to increase productivity by reducing hybridoma invasiveness and increasing mAb secretion
(39). This optimization can result in a reduction in animal use by a factor of
2-10 that greatly reduces production costs. Ascites production costs are
important because ascites production has a high variable cost component.
However, the research industry—that is, industry concerned with research on and
development of new therapeutic agents—is most concerned with production time
and binding affinity of the mAb. Therefore, whether in vivo or in vitro methods
are used depends on the purpose of the project and on the quality of mAb
produced by the cell line in that system. For very-small-scale production,
ascites production is often preferred (done in countries where this is still
allowed) because it is a much more accessible and quicker procedure than in
vitro production and can be done without optimizing cell lines in an in vitro

In vitro production

commercial in vitro systems meet the different needs and requirements of users.
The many systems can be split in two types: single-compartment systems that
allow only low-density cell culture, and double-compartment systems that allow
high-density cell culture, which results in increased mAb concentration. There
is also the distinction between static and agitated suspension cultures.
Agitated cultures allow higher gas exchange and thus permit higher volumes of

cells to
be cultured compared to static suspension cultures.

For small-scale
production (less than 10g), both the low-density cell-culture systems (such as culture
flasks, roller bottles, gas permeable bags) and the high-density cell culture
systems (such as the hollow-fiber bioreactors) are used. For medium-scale
production (10-100 g), double compartment, high-density cell-culture systems (semi-permeable
membrane-based systems) are the best choice (55). High-scale production (over 100
g) is performed in large capital-intensive systems, such as homogeneous suspension
culture in deep-tank stirred fermentors, perfusion-tank systems, airlift reactors,
and continuous-culture systems (51).

antigen-free product can be obtained by adapting the hybridoma cell line to low
serum or serum-free media, with generally minor inhibitory effects on the cells
(40). The benefits of in vitro production are: the absence of live-animal use,
although some products in the culture media come from animals; the possibility
of low-serum or serum-free media production (41); and the absence of
host-contributed immunoglobulin or antigens.

Problems described
in the past with in vitro systems, often associated with hybridoma cell lines:

  • material, labour, and equipment costs are
    higher than for the in vivo method (11, 19, 42, 43)
  • characteristics of the hybridoma are more
    critical than in vivo
  • about 3-5% of all hybridoma clones cannot be
    maintained by in vitro systems (44, 45)
  • the great potential for microbial
    contamination, poor growth, and monitoring and attention every day (46)
  • the design of downstream processing is
    emphasized because large volumes of media are required to obtain large
    quantities of mAb and to ensure product economy and purity (47)
  • residual endotoxin, residual DNA from cell
    death, and bovine IgG contamination with cell lines that require some serum all
    complicate the process.

It is
difficult for a user to choose a particular in vitro system based on manufacturers’
claims because of how costs are calculated and because the amount of antibody
secreted by different hybridoma lines in identical medium and culture
conditions can vary by a factor of as much as 100 (48). Therefore, it is
important to compare the productivity of several systems by using several cell
lines and to include optimization costs of each system in calculating the
overall cost per gram. Numerous commercial-volume systems are available, and
none is inexpensive. However, with the emergence of recombinant antibodies
(recmAbs) expressed in non-hybridoma cell lines, some of these concerns have
been alleviated (see below).

One of the
most common causes of failure of in vitro methods is poor adherence to basic
tissue-culture techniques, such as sterilization of culture-ware, equipment,
and media and humidity and temperature control in the system. In large-scale
and medium-scale production, it is important to have tight procedural and
environmental controls to minimize losses due to system microbial
contamination. To help avoid a major economic effect of such losses in
commercial production, expensive facilities and tightly controlled procedures
are implemented, all of which add to the high fixed cost of in vitro mAb

tissue culture

The simplest approach for producing mAb in vitro is to grow the
hybridoma cultures in batches and purify the mAb from the culture medium. Fetal
bovine serum (FBS) is used in most tissue-culture media and contains bovine
immunoglobulin at about 50 μg/ml. The use of such serum in hybridoma culture
medium can account for a substantial fraction of the immunoglobulins being present
in the culture fluids (3). To avoid contamination with bovine immunoglobulin, several
companies have developed serum-free media specifically formulated to support
the growth of hybridoma cell lines (4, 5, 6). In most cases, hybridomas growing
in 10% FBS can be adapted within four passages (8-12 days) to grow in less than
1% FBS or in FBS-free media. However, this adaptation can take much longer and
in 3-5% of the cases the hybridoma will never adapt to the low FBS media. After
this adaptation, cell cultures are incubated in commonly used tissue-culture
flasks under standard growth conditions for about 10 days; mAb is then
harvested from the medium. The above approach yields mAb at concentrations that
are typically below 20μg/ml. Methods that increase the concentration of
dissolved oxygen in the medium may increase cell viability and the density at
which the cells grow and thus increase mAb concentration (7, 8). Some of those
methods use spinner flasks and roller bottles that keep the culture medium in
constant circulation and thus permit nutrients and gases to distribute more
evenly in large volumes of cell-culture medium (5, 9). The gas-permeable bag
(available through Baxter and Diagnostic Chemicals) increases concentrations of
dissolved gas by allowing gases to pass through the wall of the culture
container. All these methods can increase productivity substantially, but
antibody concentrations remain in the range of micrograms per milliliter (10, 11,

Most research applications require mAb concentration of 0.1-10
mg/ml, much higher than mAb concentrations in batch tissue-culture media (13). If
un-purified antibodies are sufficient for the research application, low
molecular weight cut off filtration devices that rely on centrifugation or gas
pressure can be used to increase mAb concentration. Alternatively, antibodies
from tissue-culture supernatants can be purified by a protein A or protein G
affinity column (11, 14). However, bovine or other immunoglobulin present in
the culture medium will contaminate the monoclonal antibody preparation. In
short, batch tissue-culture methods are technically relatively easy to perform,
have relatively low start-up costs, have a start-to-finish time (about 3 weeks)
that is similar to that of the ascites method, and produce quantities of mAb sufficient
for research purposes. The disadvantages of these methods are that large
volumes of tissue-culture media must be processed, the mAb concentration
achieved will be low (around a few micrograms per milliliter), and some mAb are
denatured during concentration or purification (15). In fact, a random screen
of mAbs revealed that activity was decreased in 42% by one or another of the
standard concentration or purification processes (16).

membrane-based systems

As mentioned above, growth of hybridoma cells to higher densities
in culture results in larger amounts of mAb that can be harvested from the
media. The use of a barrier, either a hollow fiber or a membrane, with a
low-molecular-weight cut-off (10,000-30,000 Dalton), has been implemented in several
devices to permit cells to grow at high densities (17, 18, 19). These devices
are called semipermeable-membrane- based systems. The objective of these
systems is to isolate the cells and mAb produced in a small chamber separated
by a barrier from a larger compartment that contains the culture media. Culture
can be supplemented with numerous factors that help optimize growth of the
hybridoma (20). Nutrient and cell waste products readily diffuse across the
barrier and are at equilibrium with a large volume, but cells and mAb are
retained in a smaller volume (1-15 ml in a typical membrane system or small
hollow-fiber cartridge). Expended medium in the larger reservoir can be replaced
without losing cells or mAb; similarly, cells and mAb can be harvested
independently of the growth medium. This compartmentalization makes it possible
to achieve mAb concentrations comparable with those in mouse ascites. Below
follows a summary of devices that have been assessed by Dewar et al (55):

The CELLine 1000 (Integra Bioscience, Chur, Switzerland) device is
a membrane-based disposable cell culture system that is easy to use. It is composed
of two compartments, a cultivation chamber (20ml) and a nutrient supply compartment
(1000ml) separated by a semipermeable dialysis membrane (10kD molecular weight cut-off),
which allows small nutrients and growth factors to diffuse to the production chamber.
Oxygen supply of the cells and CO2 diffusion occur through a
gas-permeable silicone membrane. Antibodies concentrate in the production medium.
This culture system requires a CO2 incubator.

The miniPERM (Vivascience, Hannover, Germany) is a modified roller
bottle two-compartment bioreactor in which the production module (35ml) is
separated from the nutrient module (450ml) by a semipermeable dialysis
membrane. Nutrients and metabolites diffuse through the membrane, and secreted
antibodies concentrate in the production module. Oxygenation and CO2
supply occur through a gas-permeable silicone membrane at the outer side of the
production module and through a second silicone membrane extended into the nutrition
module. The miniPERM must be placed on a roller base inside a CO2

The Cell-Pharm system 100 (CP100, Bio-Vest, Minneapolis, MN) is a fully
integrated 0.14 m2 hollow-fiber cell culture system. It does not
require a CO2 incubator because air and CO2 bottles are
connected directly to the system while temperature and pH (air/CO2
flow) are set on the control unit. The cell culture unit consists of two
cartridges: one that serves as a cell compartment and the other, as an
oxygenation unit.

The Cell-Pharm system 2500 (CP2500, Bio-Vest) is a hollow-fiber
cell culture production system that can produce high-scale quantities of MAbs.
Unlike CP100, it consists of two fiber cartridges for the cells and hence offers
a larger cell growth surface (3.25 m2). A third cartridge serves for
oxygenation of the medium.

The FiberCell (Fibercell Systems Inc., Frederick, MD) hollow-fiber
cell culture system is composed of a culture medium reservoir (250ml) and a
60ml fiber cartridge (1.2 m2), both connected to a single
microprocessor-controlled pump. It is possible to prolong the media supply
cycles by replacing the original medium reservoir with a 5-litre flask. In
contrast to the Cell-Pharm systems, the FiberCell bioreactor is used inside a
CO2 incubator. Oxygenation occurs by a gas-permeable tubing.

The Tecnomouse (Integra Biosciences, Chur, Switzerland) cell production
unit provides separation of cultivation (12 mL) and nutrient (10 L) chambers
via hollow fibers in combination with two thin gas-permeable silicone membranes
to enable oxygenation. Tecnomouse is the only compartmentalized system in which
five different hybridoma cell lines can be cultured in parallel in separate cell
culture cassettes.

A large set of hybridomas were tested at GSK in the above systems
(55). The concentrations of antibodies obtained were 50 to 200 times greater
than with the static T-flask. Supernatant concentrations were between 100 and
2100 mg/ml reflecting the high variability
of hybridomas in terms of secretion capacity. The consumption of medium was
higher for the hollow-fiber systems than for the suspension systems. The miniPERM
consumed less medium (about 60%) than CELLine1000 per milligram of antibody,
while CELLine1000 was more cost effective in medium than CP100 (270 %), CP2500
(330 %), and Fibercell (240%).

Recombinant antibody production in large scale

development of recombinant technology based on cloning and expression of the
heavy and light chain antibody genes in Chinese Hamster Ovary (CHO) cells enabled
mAb production to take advantage of the common technologies already used for
recombinant products like tissue plasminogen activator, erythropoietin, Factor
VIII, etc. These recombinant cell culture processes for antibody production
initially had low expression levels, with titers typically well below 1 mg/ml
(50). The combination of low titers and large market demands for some of the
first recombinant mAbs like rituximab (Rituxan), trastuzumab (Herceptin),
infliximab (Remicade) and others drove many companies and contract
manufacturing organizations (CMOs) to build large production plants containing multiple
bioreactors with volumes of 10,000 litres or larger. Other products derived
from mammalian cell culture in the mid-90s also required large production
capacity (Enbrel, while not a mAb, is an Fc-fusion protein which is produced
using a similar manufacturing process), driving further expansion. In parallel
with the increase in bioreactor production capacity throughout the
bioprocessing industry, improvements in the production processes resulted in
increased expression levels and higher cell densities, which combined provided
much higher product titers (51).

cells are used for expression of all commercial therapeutic mAbs, and grown in
suspension culture in large bioreactors. The use of mammalian cells for the
expression of the antibodies ascertain appropriate glycosylation, thus
mimicking in vivo production. The majority of commercial mAbs are derived from
just a few cell lines (52) such as CHO, NS0 and Sp2/0, with CHO being the
dominant choice because of its long history of use since the licensure of
tissue plasminogen activator in 1987. CHO cells have attractive process
performance attributes such as rapid growth, high expression, and the ability
to be adapted for growth in chemically-defined media. Typical production
processes will run for 7–14 days with periodic feeds when nutrients are added to
the bioreactor. These fed-batch processes will accumulate mAb titers of 1–5 mg/ml,
with some companies reporting 10–13 mg/ml for extended culture durations.
Production bioreactor volumes range from 5,000 litres to 25,000 litres.

antibody purification process is initiated by harvesting the bioreactor using
industrial continuous disc stack centrifuges followed by clarification using
depth and membrane filters. The mAb is captured and purified by Protein A
chromatography, which includes a low pH elution step that also serves as a
viral inactivation step. Two additional chromatographic polishing steps are
typically required to meet purity specifications, most commonly anion- and
cation-exchange chromatography (53). A virus retentive filtration step provides
additional assurance of viral safety, and a final ultrafiltration step
formulates and concentrates the product – the step order of the virus filter
and two polishing steps is somewhat flexible, and may vary among company
platforms (54). Overall purification yields from cell cultured fluid range from
70–80%, and the concentrated bulk drug substance is stored frozen or as a
liquid, and then shipped to the drug product manufacturing site. While the
purification processes developed in the 1990s using the separations media
(chromatographic resins and membranes) available at the time were not capable
of purifying 2–5 mg/ml feed-streams, improvements in separations media make it
possible today for many facilities to purify up to 5 mg/ml, which would
generate batches of 15–100 kg from 10,000 –25,000 litre bioreactors. Large
manufacturing plants are designed with multiple bioreactors supplying one (or
sometimes two) purification train(s). The individual purification unit
operations can be completed in under two days, and often in just one day, and
therefore several bioreactors can be matched to the output of a single
purification train. The increased capacity of these plants arising from the
elevated titers will decrease the costs of goods (COGs), by the virtue of the economies
of scale afforded by the large bioreactors. These plants produce enormous
quantities of mAbs against very attractive costs (51).

In vivo production

Optimal in
vivo production requires reduction of the invasive nature of a cell line so
that all mice survive completion of a production run. Selecting appropriate
clones and altering hybridoma cell concentration injected into the peritoneal
cavity of the mice are two ways to optimize production. The volume and
concentration of mAb produced depend on the clone selected, and this makes systematic
comparisons difficult. Therefore, the best way to achieve maximal in vivo
yields is to screen clones in mice and to use the clone that provides the best yield.
Cell growth conditions are optimal in vivo, so almost all cell lines will produce
antibody, even when they are not optimized (45). That is why injecting into
mice usually saves cell lines that are difficult to grow in vitro.

production is a simple procedure, once proper technique is learned. Daily
observation of the mice requires skilled observers to determine the optimal time
for tapping the fluid and to determine when the mouse should be euthanized. It
is quicker, is more forgiving, is more economical for small-scale and
medium-scale production, produces a high concentration of mAb, and is easy to
scale up in production (NRC 1999). The major
problems associated with in vivo production are the use of animals, the
possibility that the animal could be harmed if technicians are not properly
trained and procedures are not followed properly, the presence of endogenous
mouse immunoglobulin contamination except when immunodeficient mice are used (49),
and the possibility of contamination with murine pathogens, which requires the
use of high-quality animals and a high-quality program for health assurance.

Mouse Ascites

Although in vitro techniques can be used for more than 90% of mAb
production, it must be recognized that there are situations in which in vitro
methods will be ineffective, especially when (still) dependant on hybridoma
cell lines. Because hybridoma characteristics vary and mAb production needs are
diverse, in vitro techniques are not suitable in all situations, and their use
might impede research, especially when large numbers of mAbs must be screened
for efficacy or specificity in the treatment of disease. In some cases, in
vitro production of mAb has not met the scientific aims of a project (MCR 1999).

There are seven circumstances that may prevent the use of in vitro
mab production:

  1. Some
    hybridomas do not adapt well to in vitro culturing.
    In some
    instances, continued in vitro culture does not support hybridoma growth; in
    these instances, the rising concentration of antibody might adversely affect
    hybridoma growth or secretion. One may expect a 3% failure rate of hybridomas
    grown in vitro (22).
  2. In vivo
    experiments in mice may require mabs produced in murine ascites.
    The need
    for the mouse ascites method arises when small volumes of concentrated antibody
    are needed for a rapid in vivo screening in mice to select hybridomas with the
    desired bioreactivity. In vitro production would be too slow because of slow
    adaptation of the hybridoma to in vitro growth conditions, there are limitations
    to in vitro produced antibodies for in vivo testing several hybridomas at the
    same time, and there will be contamination of inactivated antibodies
    co-purified from the in vitro growth media.
  3. Rat
    hybridomas are often limited to the mouse ascites production method.
    In some
    situations, mAbs to mouse epitopes are required, necessitating the use of
    another species (usually rat) for immunization. Although some rat hybridomas
    adapt to in vitro conditions, this often requires tedious manipulation of the
    culture. When small volumes of concentrated rat mAb are needed and the
    hybridoma does not easily adapt to culture conditions, the mouse ascites method
    using immunocompromised mice is required (23).
  4. Downstream
    purification leads to a subpopulation of denatured and inactivated antibodies
    . There
    are many laboratory situations in which the concentration of antibody
    obtainable by current in vitro methods is not high enough for experimental
    studies and absolute purity of the antibody reagent is not essential. Other
    situations that require the mouse ascites method of producing mAb are related
    to the need for high binding affinities, the presence of complement-fixing
    activities, and mAbs that are naturally glycosylated. Many of the in
    vitro-produced antibodies cannot be readily concentrated from culture
    supernatant, because standard procedures result in losses of antigen binding
    activity or other antigen-antibody features (15, 24), although such a
    concentration step might not be required with semipermeable-membrane-based
    systems. For example, immunoglobulin M (IgM) and immunoglobulin G3 (IgG3)
    antibodies often undergo denaturation during in vitro purification techniques,
    resulting in the loss of complement-binding activity (25). Downstream
    purification is particularly difficult for immunoglobulin A (IgA) mAb, in which
    monomeric IgA (with poor antigen-binding abilities) must be separated from
    dimeric and polymeric IgA (15). The mouse ascites method avoids such problems.
  5. Obtaining
    serum-free antibodies is not always possible through in vitro production
    . Some
    hybridomas can be readily adapted to low serum or no-serum culture media, but
    others cannot, or antibody production may be affected by serum-deprived culture
    media. The mouse ascites method might be
    required when mAb to infectious agents or tumor antigens are being tested for
    toxicity and efficacy in mouse models of human diseases. Such testing is
    usually needed to establish a proof of principle (that is, showing that the mAb
    in fact is effective therapeutically) or for the preclinical studies required
    by federal agencies. In those situations, large numbers of mAb of different
    isotypes and specificities often must be tested in dose escalation studies
    before a candidate is chosen for more detailed analysis, and this requires
    initial production of large amounts of mAb so that enough subjects can be
    challenged to establish a statistically significant result. Unexpected
    toxicities or questions of efficacy sometimes require additional batches; in
    these cases, the presence of non-mouse contaminating proteins and the immune
    responses to them can distort the results.
  6. Differences
    in IgG glycosylation between in vitro and in vivo production methods.

    the expression of glycosylation enzymes to achieve the correct in vitro
    placement of sugars, sialic acids, and so on, on the IgG molecule is a
    formidable task, extremely expensive, and often not attainable with present
    technology (26, 27, 28, 29) when dependant on hybridoma culturing. In vitro
    glycosylation patterns might yield mAb with preferred pharmacokinetic
    characteristics for in vivo applications (30, 31, 32). This argument is dealt
    with when producing recmAbs expressed by a mammalian cell line.
  7. Contaminated
    hybridomas can be cleared through mouse ascites method.
    fungal, or mycoplasma contamination of in vitro cultures of hybridoma can be
    removed by passing cells from the culture through mice.

Summary of advantages and disadvantages of in vitro and in vivo production

Advantages of in vitro production:

  • Reduction of sacrificing animal lives
  • Economic reasons (for easy to generate
    antibodies): to obtain high yields against low cost
  • Avoidance of animal welfare issues and
    submission of animal protocols to authorities
  • Avoidance of animal facilities and expertise
    of animal handling
  • Use of semipermeable membrane-based systems
    allow as high yields as ascites and without contaminating mouse proteins
  • Expression of recombinant antibodies in
    mammalian cell lines ascertain correct glycosylation

Disadvantages of in vitro production:

  • Some hybridomas do not grow well or are
    unstable in tissue culture
  • Presence of serum limits antibody applications
  • Loss of proper glycosylation (partly solved by
    recmabs expressed in proper cell line)
  • Limited yields, or complicated/slow
    purifications required
  • Antibody dilution in conditioned media too
  • Affinity might be lower compared to in vivo
  • Too expensive for small-scale or medium-scale
  • Limitation to numbers of clones to propagate
  • Performance of antibody may vary from one
    production to the next (batch-to-batch)

Advantages of mouse ascites method:

  • Extreme high concentrations of antibody
  • Relatively low levels of contaminants
  • Correct glycosylation
  • Avoidance of equipment and expertise on tissue
  • Easy purification of antibody from ascites

Disadvantages of mouse ascites method:

  • Requirement of continued use and monitoring of
    animals and their welfare
  • Contaminants may prevent certain applications,
    such as clinical use.
  • When immunodeficient mice are required, costs
    become a factor


  1. Köhler, G., C. Milstein. 1975. Continuous
    cultures of fused cells secreting antibody of predefined specificity. Nature
  2. Marx, U. et al. 1997. Monoclonal antibody
    production: The report and recommendations of ECVAM Workshop 23. ATLA 25:121-
  3. Darby, C., K. Hamano, K. Wood. 1993.
    Purification of monoclonal antibodies from tissue culture medium depleted of
    IgG. J Immunol 159: 125-129.
  4. Federspiel, G., K.C. McCullough, U. Kihm.
    1991. Hybridoma antibody production in vitro in type II serum-free medium using
    Nutridoma-SP supplements. Comparison with in vivo methods. J Immunol
  5. Tarleton, R., A. Beyer. 1991. Medium-scale
    production and purification of monoclonal antibodies in protein-free medium.
    BioTechniques 11:590-593.
  6. Velez, D. et al. 1986. Kinetics of monoclonal
    antibody production in low serum medium. J Immunol Methods 86:45-52.
  7. Boraston, R. et al. 1984. Growth and oxygen
    requirements of antibody producing mouse hybridoma cells in suspension culture.
    Develop Biol Standard 55:103-111.
  8. Miller, W., C. Wilke, H. Blanch. 1987. Effects
    of dissolved oxygen concentration on hybridoma growth and metabolism in
    continuous culture. J Cell Physiol 132:524-30.
  9. Reuveny, S. et al. 1986. Factors affecting
    cell growth and monoclonal antibody production in stirred reactors. J Immunol
    Methods 86:53-59.
  10. Heidel, J. 1997. Monoclonal antibody
    production in gas-permeable tissue culture bags using serum-free media. Center
    for Alternatives to Animal Testing: Alternatives in Monoclonal Antibody Production
  11. Peterson, N., J. Peavey 1998. Comparison of in
    vitro monoclonal antibody production methods with an in vivo ascites production
    technique. Contemp Top Lab Anim Sci 37(5):61-66.
  12. Vachula, M. et al. 1995. Growth and phenotype
    of a pheresis product mononuclear cells cultured in life cell flasks in
    serum-free media with combinations of IL-2, OKT3, and anti-CD28. Exp Hematol
  13. Coligan, J.E., A.M. Kruisbeek, D.H. Margulies,
    E.M. Shevach, W. Strober, eds. 1998. Current protocols in immunology. R. Coico,
    series ed. New York: Wiley & Sons.
  14. Akerstrom, B., T. Brodin, K. Reis, L. Bjorck.
    1985. Protein G: A powerful tool for binding and detection of monoclonal and
    polyclonal antibodies. J Immunol 135:2589-2592.
  15. Lullau, E. et al. 1996. Antigen binding
    properties of purified immunoglobulin A and reconstituted secretory
    immunoglobulin A antibodies. J Biol Chem 271:16300-16309.
  16. Underwood, P.A., P.A. Bean. 1985. The
    influence of methods of production, purification and storage of monoclonal
    antibodies upon their observed specificities. J Immunol Methods 80:189-197.
  17. Evans, T., R. Miller. 1988. Large-scale
    production of murine monoclonal antibodies using hollow fiber bioreactors.
    BioTechniques 6:762-767.
  18. Falkenberg, F., H. Weichert, M. Krane. 1995.
    In vitro production of monoclonal antibodies in high concentration in a new and
    easy to handle modular minifermentor. J Immunol 179:1329.
  19. Jackson, L. et al. 1996. Evaluation of hollow
    fiber bioreactors as an alternative to murine ascites production for small
    scale monoclonal antibody production. J Immunol Methods 189:217-231.
  20. Jaspert, R., T. Geske, A. Teichmann, Y.M
    Kabner, K. Kretzschman, J. L’age-Stehr. 1995. Laboratory scale production of
    monoclonal antibodies in a tumbling chamber. J Immunol Methods 178:77-87.
  21. Knazek, R., P. Gullino, P. Kohler, R. Dedrick.
    1972. Cell culture on artificial capillaries. Science 178:65-67.
  22. Hendriksen, C. et al. 1996. The production of
    monoclonal antibodies: Are animals still needed? ATLA 24:109-110.
  23. Wolf, M. 1998. CL6-well experimental screening
    device application: Murine and rat hybridoma. CELLine Technical Report IV.
  24. Underwood, P.A., P.A. Bean. 1985. The
    influence of methods of production, purification and storage of monoclonal
    antibodies upon their observed specificities. J Immunol Methods 80:189-197.
  25. Roggenbuck, D. et al. 1994. Purification and
    immunochemical characterization of a natural human polyreactive monoclonal IgM
    antibody. J Immunol Methods 167:207-218.
  26. Wright, A., S.L. Morrison. 1994. Effect of altered
    CH2-associated carbohydrate structure on the functional properties and in vivo
    fate of chimeric mouse-human immunoglobulin G1. J Exp Med 180:1087-1096.
  27. Wright, A, S.L. Morrison. 1998. Effect of
    C2-associated structure on Ig effector function: Studies with chimeric
    mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary
    cells. J Immunol 160:3393-3402.
  28. Wright, A, S.L. Morrison. 1997. Effect of
    glycosylation on antibody function: Implications for genetic engineering.
    Trends Biotechnol 15:26-32.
  29. Matsuuchi, L., L.A. Wims, S.L. Morrison. 1981.
    A variant of the dextran-binding mouse plasmacytoma J558 with altered
    glycosylation of its heavy chain and decreased reactivity with polymeric
    dextran. Biochemistry 20:4827-4835.
  30. Maiorella B.L. et al. 1993. Effect of culture
    conditions on IgM antibody structure, pharmacokinetics, and activity.
    Biotechnology 11:387-392.
  31. Monica, T.J., C.F. Goochee, B.L. Maiorella.
    1993. Comparative biochemical characterization of a human IgM produced in both
    ascites and in vitro cell culture. Biotechnology 11:512-515.
  32. Patel, S.S., R.B. Parekh, B.J. Moellering,
    C.P. Prior. 1992. Different culture methods lead to differences in
    glycosylation of a murine IgG monoclonal antibody. J Biochem 285:839-845.
  33. Butler, M., N. Huzel. 1995. The effect of
    fatty acids on hybridoma cell growth and antibody productivity in serum-free
    cultures. J Biotech 39:165-173.
  34. Moro, A.M., M.T. Rodrigues, M.N. Gouvea, M.L.
    Silvestri, J.E. Kalil, I. Raw. 1994. Multiparametric analyses of hybridoma growth
    on glass cylinders in a packed-bed bioreactor system with internal aeration.
    Serum-supplemented and serum-free media comparison for mAb production. J
    Immunol Methods Nov. 10; 176(1):67-77.
  35. Stoll T.S., P.A. Ruffieux, E. Lullau, U. von
    Stockar, I.W. Marison. 1996a. Characterization of monoclonal IgA production and
    activity in hollow-fiber and fluidized-bed reactors. Pp. 608-614 in Immobilized
    Cells: Basics and Applications, R.H. Wijffels, R.M. Buitolarr, C. Bucke, J.
    Tramper, eds. The Netherlands: Elsevier Science B.V.
  36. Stoll, T.S., K. Muhlethaler, U. von Stockar,
    I.W. Marison. 1996b. Systematic improvement of a chemically-defined
    protein-free medium for hybridoma growth and monoclonal antibody production. J
    Biotechnol. Feb. 28;45(2):111-123.
  37. Boyd, J.E., K. James. 1989. Human monoclonal
    antibodies: Their potential, problems, and prospects. Pp. 1-43 in Monoclonal
    Antibodies: Production and Application. A. Mizrahi, ed. New York: Alan R. Liss,
  38. Reuveny, S., A. Lazar. 1989. Equipment and
    procedures for production of monoclonal antibodies in culture. Adv Biotechnol
    Processes 11:45-80.
  39. Harlow, E., D. Lane. 1988. Antibodies: A
    laboratory manual, p 274-275. New York: Cold Spring Harbor Laboratory Press.
  40. Kurkela, R., E. Fraune, P. Vihko. 1993. Pilot-scale
    production of murine monoclonal antibodies in agitated, ceramic-matrix, or
    hollow-fiber cell culture systems. BioTechniques 15:674-693.
  41. Klerx, J., C. Jansen Verplanke, C. Blonk, L.
    Twaalfhoven. 1988. In vitro production of monoclonal antibodies under
    serum-free conditions using a compact and inexpensive hollow fibre cell culture
    unit. J Immunol Methods 111:179-188.
  42. Brodeur, B., P. Tsang. 1986. High yield
    monoclonal antibody production in ascites. J Immunol Methods 86:239-241.
  43. Lipman, N. 1997. Hollow fibre bioreactors: An
    alternative to the use of mice for monoclonal antibody production. Pp.10-15. in
    Alternatives in Monoclonal Antibody Production. Johns Hopkins Center for
    Alternatives to Animal Testing Technical Report #8.
  44. deGeus, B., C. Hendriksen, eds. 1998. 74th
    Forum in Immunology: In vivo and in vitro production of monoclonal antibodies:
    Current possibilities and future perspectives. Res Immunol 149:529-620.
  45. Hendriksen, C., W. de Leeuw. 1998. Production
    of monoclonal antibodies by the ascites method in laboratory animals. Res
    Immunol 149:535-542
  46. Lebherz, W. 1987. Batch production of
    monoclonal antibody by large-scale suspension culture. Pp.93-118 in Commercial
    Production of Monoclonal Antibodies. S. Seaver, ed. New York: Marcel Dekker.
  47. Stang, B.V., P.A. Wood, J.J. Reddington, G.M.
    Reddington, J.R. Heidel. 1998. Monoclonal antibody production in gas-permeable
    flexible flasks, using serum-free media. Contemp Top Lab Anim Sci 37(6):55-60.
  48. Seaver, S. 1987. Culture method affects
    antibody secretion of hybridoma cells. Pp. 49-71 in Commercial Production of
    Monoclonal Antibodies. S. Seaver, Ed. New York:Marcel Dekker, Inc.
  49. Ware, C., N. Donato, K. Dorshkind. 1985.
    Human, rat or mouse hybridomas secrete high levels of monoclonal antibodies
    following transplantation into mice with severe combined immunodeficiency
    disease (SCID). J Immunol Methods 85:353-361.
  50. Birch JR, Onakunle Y. Biopharmaceutical
    Proteins: Opportunities and Challenges. In: Smales CM, James DC. Methods in
    Molecular Biology, vol. 308: Therapeutic Proteins: Methods and Protocols.
    Totowa, NJ: Humana Press Inc., 2005; 1-16.
  51. Kelly B. 2009. Industrialization of mAb
    production technology: the bioprocessing industry at a crossroads. MAbs
  52. Wurm FM. Production of recombinant protein
    therapeutics in cultivated mammalian cells. Nature 2004; 22:1393-8.
  53. Fahrner RL, et al. Industrial purification of
    pharmaceutical antibodies: development, operation and validation of
    chromatography processes. Biotechnol Genet Eng Rev 2001; 18:301-27.
  54. Shukla AA, Hubbard B, Tressel T, Gunhan S, Low
    D. Downstream processing of monoclonal antibodiesapplication of platform
    approaches. J Chromatogr B 2007; 848:29-39.
  55. Dewar V, Voet P, Denamur F, Smal J. Industrial
    implementation of in vitro production of monoclonal antibodies. ILAR J.