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In Vitro and In Vivo monoclonal antibody Production

Kristina Whitfield

Nov 21, 2019

Antibody Engineering Recombinant Antibodies

Antibodies are important tools used by many researcher in their research. The use of antibodies has led to many medical advances and monoclonal antibodies (mAbs) are commonly preferred when persistent use of the same antibody over time is required. Learn more about the different methods of producing monoclonal antibodies and the advantages and disadvantages of each production method.

Author: Kristina Whitfield Undertakes marketing activities for Pivotal Scientific and their clients.

Kristina Whitfield


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.

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

PMID: 22934324,

Large parts of the content of this paper has been ignored and outdated arguments have been taken out and replaced by contents from later publications.


Antibodies are important tools used by many investigators in their biomedical research and they have led to many medical advances. Although, 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.

However, 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.

The 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 manipulation.

The 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.

Commercial 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).

The 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 culture.

In vitro production

Different 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).

An 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 production.

Batch 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. This 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 μg/ml (10, 11, 12).

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).

Semi-permeable 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 incubator.

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 bioreactors

The 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).

Mammalian 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, 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 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 (nutrients are added to the bioreactor). These fed-batch processes will accumulate mAb titers of 1–5 mg/ml, some companies reporting 10–13 mg/ml for extended culture durations. Production bioreactor volumes range from 5,000 litres to 25,000 litres.

The 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.

Ascites 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 Production

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. Manipulating 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. Yeast, 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 methods

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 high
  • Affinity might be lower compared to in vivo production
  • Too expensive for small-scale or medium-scale production
  • 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 culturing
  • 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


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