Although the original research was principally an in vitro technique, it was also apparent that monoclonal antibodies could be produced by injecting the hybridoma cells into the abdominal cavities of different species of rodents. This was the initial use of the ascites method. Since these MAbs were easily made in any laboratory and the ascites process, widely viewed as both simple and inexpensive, was introduced early, its use rapidly expanded. Unfortunately, the original possibility of MAb production replacing uses of laboratory animals was and often continues to be overlooked or ignored. In the decades that followed the original discovery, tens of millions of animals suffered and died despite the availability of more humane alternatives.
During this same period of time the appropriateness of using the ascites method was increasingly being questioned in Europe. Milstein noted that "in later years, both on practical and humane grounds, I became concerned with the use of ascitic fluids" (personal communication, 1997). As new in vitro alternative techniques were developed and validated, it became more difficult to justify the suffering associated with the use of ascites. It simply was not possible to humanely produce MAbs using animal-based procedures.
In 1989, The Netherlands government introduced a Code of Practice for the Production of Monoclonal Antibodies (1), which provided detailed descriptions of the veterinary problems and pathophysiology associated with the ascites process and placed restrictions on its use. The resulting increased humane awareness among Dutch researchers provided further encouragement for adoption of in vitro approaches to MAb production. In 1995, a symposium held in Bilthoven, The Netherlands, concluded that progress in development of such alternatives (both in efficacy and cost) was sufficient that the use of ascites could no longer be justified. The resulting prohibition of animal-based MAb production caused no serious difficulties within the Dutch biomedical research community. Despite initial academic resistance, bans on ascites in Germany and Switzerland experienced similar results, as did the restrictions placed on ascites use in Sweden and the United Kingdom.
By 1996 in vitro production of MAbs was the method of choice in Europe for commercial concerns and others needing large quantities and on the increase for the smaller-scale needs of individual researchers. This latter group made up about 60 percent of European MAb users, primarily for basic research and some diagnostic procedures. Their MAb needs were often met using the ascites technique. A similar situation exists in the United States.
Scientist representatives from several member states of the European Union met in October 1996 at the European Center for the Validation of Alternative Methods to discuss the current status of in vitro and in vivo methods of monoclonal antibody production. After careful consideration of the different types of research and commercial needs for MAbs and the available production options, they concluded that "for all levels of MAb production; there are one or more in vitro methods which are not only scientifically acceptable, but are also reasonably and practically available; and as a consequence, in vivo production can no longer be justified and should cease." (15) The group further called for a Europe-wide prohibition on the routine use of ascites methods of MAb production. In Europe the trend is toward adoption of in vitro alternatives at all stages of the MAb process.
There are three principal steps in production of monoclonal antibodies: 1) immunization, 2) hybridoma formation and 3) MAb production. Each has its own potential for adoption of alternative methods. Although currently done primarily as an in vivo procedure using a few animals, it is possible, especially with human MAbs, to conduct the immunization process entirely in cell cultures. Some technical difficulties remain to be solved before this becomes a routine non-animal-based procedure.
Formation of the hybridoma cells has always been an in vitro technique. However, final production of the monoclonal antibodies involves use of either the ascites or in vitro alternative approaches. The present review briefly focuses on this last step, with an emphasis on the availability of multiple alternative MAb production techniques, suitable for the small-to-medium-scale research and commercial laboratories.
Experience in European laboratories strongly suggests that "these arguments are increasingly being challenged by documents showing that, depending on the amount and concentration of MAbs needed, in vitro production methods are equally well suited"as ascites (Hendriksen, personal communication). With continually increasing expenses associated with the care of laboratory animals and decreasing prices associated with either the newer in vitro methods or decreased production costs for existing in vitro equipment, the cost differential between animal- and non-animal-based methods is rapidly disappearing.
In contrast with in vivo methods, in vitro approaches to monoclonal antibody production have many positive attributes. In general, MAbs derived from in vitro alternatives express immunoreactivity in ranges of 90 to 95 percent regardless of the method used. This is significantly higher than that with MAbs produced by ascites. Similarly, in vitro cultures rarely fail (3 percent or less), while a much higher percentage of ascitic mice do not produce antibodies. Further, the quality of in vitro MAbs is equal to or better than that derived from in vivo methods.
Glycosylation has been raised as an issue with in vitro methods for producing Mabs. (Ed. note: Glycosylation can influence the antigen-binding capacity, the resistance of an antibody to proteolysis, and other biologically important processes.) However, glycosylation patterns are more easily regulated in vitro, since with ascites, they can vary between each individual animal. The ECVAM committee concluded that "there are no reasonable arguments based on antibody glycosylation which support the use of in vivo methods." (15).
Ascites is also subject to criticism on both technical and humane criteria. The major disadvantages of animal-based methods include: (1) association with severe cruelty and suffering to the animals; (2) ascitic fluids may be contaminated with rodent plasma proteins, immunoglobulins (reducing its immunoreactivity), infectious agents and bioreactive cytokines; (3) the need for extensive animal facilities, associated support services with each of the individual animals requiring daily monitoring 7 days a week; (4) some hybridomas (for example, human) are difficult to grow in rodents; (5) rodents only produce MAbs for a few days; (6) from 60 to 80 percent of mice may not produce ascites due to premature death, development of solid tumors, or failure to establish in vivo hybridoma growth (14); and (7) individual batches of ascites may vary significantly in quality and quantity.
The most compelling arguments against in vivo production of monoclonal antibodies are based on the suffering associated with the induction of ascites in the animals. It is known from human clinical experience that the growth of abdominal tumors is very painful (fig. 1). Further, ascites fluid accumulation in human patients is associated with abdominal distension, anorexia, nausea, vomiting, respiratory distress, edema, decreased mobility and fatigue. Such individuals are treated for discomfort and pain while never being allowed to progress to advanced stages routinely seen during ascites production in animals.
Animals used for ascites accumulation of MAbs frequently exhibit a spectrum of clinical symptoms including: (1) roughened haircoat, hunched posture, abdominal distention, anorexia, cachexia, anemia; (2) decreased activity and body mass, dehydration, shrunken eyes; (3) difficulty walking, respiratory distress due to an elevated diaphragm; (4) circulatory shock due to excessive fluid removal; (5) decreased venous, arterial and renal blood flow; (6) classical peritonitis; (7) immunosuppression associated with adjuvant use; and (8) up to 20 percent mortality after removal of ascitic fluid. It is not uncommon for fluid to be withdrawn in amounts greater than the entire blood volume of the animal. These symptoms become increasingly severe the longer the animals are allowed to survive (1, 9).
Pathological changes associated with ascites production of MAbs are known for each step in the process. Use of adjuvants produces mild to severe peritonitis and inflammation. Fluid removal may cause hemorrhage, edema, and death. As expected, growth of the ascitic tumors creates a variety of responses including: (1) adhesions of the abdominal wall, bladder, diaphragm, kidneys, liver, seminal vesicles, testicles and ureters; (2) linear lesions in diaphragm muscles; (3) enlarged thoracic lymph nodes and lymphatic obstruction; (4) tumors with extensive hemorrhagic and necrotic areas; (5) disseminated tumors in mesenteric, lumbar, kidney, and testicular regions; (6) centro-lobular liver necrosis; and (7) solid tumors throughout the abdomen. (1, 9)
The significance of the list of abnormalities associated with ascites production is further emphasized by observations that the animals may experience severe pathologic changes in their abdomens and chests, but appear to be clinically normal. By themselves, frequent abdominal injections are known to be a major stressful factor. Further, although abdominal distention is the most obvious consequence of ascites production, severe pain from infiltrated growth of tumors and peritonitis is a significantly more serious problem for the animals.
From all of the above, it is apparent that animals used for ascites production of monoclonal antibodies are routinely subjected to chronic pain and distress. Use of adjuvants further complicates this situation by injuring the animals before the process begins.
Only a few of the available in vitro methods meet all of these criteria, but there are a wide variety of options available to investigators, ranging from simple, individual cell culture containers to giant, commercial bioreactors.
For individuals needing only small quantities of MAbs, simple, inexpensive stationary cell cultures may be suitable. These involve a variety of flasks and bottles and are simple and easy to use with existing equipment and skills. Batches can be run for days, weeks, or months, depending on the amount of nutrient supplementation and waste removal. These latter processes can be automated. Regardless of the configuration selected, the MAbs must be concentrated after production.
Modifications of the stationary cell cultures primarily involve improved nutrient supply and waste removal, continuous MAb harvesting, and increased hybridoma cell densities. One of the simplest of these is the use of gas permeable tissue culture bags (fig. 2) which provide a practical, economical, and flexible system, using readily available laboratory equipment. In addition to simple convenience, the system is more efficient than rigid tissue culture flasks on a per cell basis and is significantly less expensive than rodent ascites on a cost per milligram basis and total yield . Because the bags are a closed system with no mechanical parts, they are relatively free of contamination and require little or no monitoring. MAbs are produced in quantities sufficient for small, medium, and large-scale needs (17, 23).
Stationary cultures can also be upgraded by a variety of mechanisms to keep the cells in suspension. These include improvements in stirring and overflow replacement of media (for example, the cytostat (20)) and agitation of cell cultures in roller bottles, spinner flasks, and stirred tanks (2, 14, 19). These alternatives require specific, relatively inexpensive equipment and are simple, fast, and effective.
Roller bottles produce low to medium monoclonal antibody yields at a price similar to that of ascites. Comparative studies of this system that examined 39 different types of hybridomas found that all grew well and produced MAbs (14). Oscillating bubble systems produce higher yields of MAbs, with quantities in each tube exceeding that produced by one ascitic mouse, as well as using equipment that is reusable (19). Due to shear forces in the culture media, spinner flasks may damage the hybridoma cells, and thus are a less effective alternative.
To protect fragile hybridoma cells in suspension cultures and some types of bioreactors, it is possible to encapsulate them in polymers. This provides high yields of MAbs (comparable to ascites methods), with ten-fold increases in cell density over simple suspension systems. Because the cells are isolated from the culture media, it is possible to establish a long-term, continuous operation (up to 40 days in an expanded bed bioreactor). Although available for smaller-scale needs, this technology is primarily used for commercial production of large quantities of MAbs (21).
Dialysis tubing or chambers are used in several different configurations to achieve high hybridoma cell densities with MAb concentrations and purity similar to that found in ascites. The cells may be placed in dialysis bags or other containers within a nutrient chamber to produce up to a gram of MAb within a few weeks. This represents a very inexpensive and simple system which allows several hybridomas to be grown simultaneously (22).
When dialysis tubing is combined with a simple roller-bottle-like mechanism, up to four hybridomas may be grown at once at 10 to 30 fold increase in concentration over stationary cultures. Such compound devices produce MAbs in quantities and purity often equivalent to that of ascites (4).
Combination with a tumbling chamber system provides passive gas exchange with simple, inexpensive, reusable equipment. This approach is capable of producing up to 300 mg of MAb in 21 days and is "universally adaptable in any research laboratory" (10).
One of the most promising in vitro methods for producing high yields of MAbs at a concentration, purity, and cost comparable to mice are the new modular minifermentors (5). These systems meet all the requirements for the perfect in vitro alternative. The hybridoma cells are grown in disposable culture chambers, separated from a reusable media chamber by a gas-permeable dialysis membrane. From 9 to 159 milligrams of MAb can be inexpensively produced in 1 to 4 weeks without the use of serum. Based on current European monoclonal antibody guidelines, these devices have yields equivalent to that of three ascitic mice.
Airlift (packed-bed) bioreactors provide long-term, serum-free production of MAbs in gram amounts. They are particularly cost-effective, since all of the components are reusable (18).
Hollow fiber bioreactors are designed to provide a more physiologically stable environment for the hybridomas and come in both small and large-scale units that produce high cell densities and MAb yields under low or serum-free conditions. Although somewhat more expensive to initially establish than other types of in vitro alternatives, their ability to produce concentrated, relatively pure MAbs at up to half the cost of ascites and in amounts that can be equivalent to 200 mice, 60 large spinner flasks, or a 60 liter fermentor, makes them an attractive option for basic biomedical research and smaller-scale commercial needs. They also require technical expertise to operate without contamination and equipment failures (7, 8).
As noted by the participants in the 1996 ECVAM meeting, there are a variety of in vitro approaches to producing MAbs, some of which are briefly described above. For all levels of consumption, replacements for the use of ascites methods are available. Some of the principal characteristics of these alternative options are summarized in tables 1 and 2.
Table 1. Antibody production in different bioreactor systems (Adapted from Stoll et al., 1995 (24)).
Table 2. Comparisons of different monoclonal production methods. (Adapted from Hendriksen et al., 1996 (6); Kamp amd de Leeu 1996 (11)).
As mentioned above, the initial immunization procedures are most commonly done in vivo, although in vitro techniques are available in many cases. There is a need to further develop these in vitro options so they can be applied to the broad range of MAb needs, not just the production of human-specific antibodies.
Because of serious humane concerns related to its production, the use of fetal calf serum and similar products with in vitro alternatives (either for MAbs or in general) represents another candidate for development of appropriate replacements. Whenever possible, hybridoma cells should be conditioned to grow in serum-free media. This has the additional advantages of reducing the overall expense and post-production processing of the monoclonal antibodies. More than a decade of experience in Europe suggests this type of switch can be easily accomplished with little or no decrease in the production of MAbs. All of the high yield alternatives described above are designed to work effectively without the use of animal serum in their culture media.
Finally, there is a need in the production of hybridomas to replace the use of animal cells with those derived from humans. This is already a critical concern for MAbs produced for therapeutic applications, since murine-derived antibodies are severely limited for effective use in human recipients. For this reason, clinical uses of such antibodies focus on those derived from human cells. To completely replace the use of animals in all steps of MAb production, such concerns need to become more generalized among all producers and consumers of monoclonal antibodies.
These entirely in vitro, high technology approaches mimic and, in some cases, improve upon the natural immune system mechanisms. They accomplished this by creating large libraries of all possible antibodies, including those that would be difficult or impossible to prepare using standard immunization and MAb hybridoma techniques. This is particularly important for the production and use of human MAbs, which are not readily produced using ascites, but are easily derived from the use of recombinant techniques (3, 12, 25, 26).
At present these biotechnology approaches to antibody differentiation and production are not equivalent to or less expensive than ascites or in vitro hybridoma methods. They also require specialized knowledge and skills but will become more competitive and eventually the system of choice as the methodology is refined and developed into commercial kits suitable for use in small to large-scale laboratory applications.
For the last decade, researchers in Europe and the United States have systematically developed, validated, and adopted multiple in vitro replacements for the use of rodent ascites. This process of technique development has progressed to the point that it is now possible to prohibit use of ascites in all but the most unusual and rare circumstances.
Replacement of animals (either intact, cells, or serum) in production of MAbs will finally allow for implementation of the humane possibilities of Kohler and Milstein's original discovery, replace a method widely acknowledged to be cruel with more humane options, and promote a greater understanding and acceptance of the alternatives approach to planning and conducting biomedical research (16).
For additional information on alternatives in biomedical research, testing and education, or to subscribe to our complimentary newsletter, please contact: John McArdle, Ph.D., Director, Alternatives Research & Development Foundation, 14280 Golf View Drive, Eden Prairie, MN 55346-3000, USA, phone: 612-949-2409, fax: 612-949-2619.
|Table 1. Antibody production in different bioreactor systems (Adapted from Stoll et al., 1995 (24)).|
|Productivity||Required for the |
production of 1 g
|In vivo||2200||2||180 mice|
|Stirred-tank bioreactor, batch||47||180a||39 days|
|Stirred-tank bioreactor, fed-batch||120||250a||28 days|
|Hollow fiber reactor||1600||1400||26; 5 daysb|
|aThe total cycle time (innoculation, culture and cleaning) was taken into account.|
bFirst number is for first gram; second number is for second gram.
|Table 2. Comparisons of different monoclonal production methods. (Adapted from Hendriksen et al., 1996 (6); Kamp amd de Leeu 1996 (11)).|
|Production System||Scale||Volume (ml)||Concentration (mg/ml)||Production Time|
|Estimated Cost (per gram)||Quality|
|Ascites (in vivo)||20-250 mg||5-10||< 20||2-3||1000-6000||Low|
|Dialysis membrane||< 50 mg||10-25||0.1-1.5||2-5||High|
|Roller bottles||< 2 g||100-2000||0.01-0.2||2-6||2000-6000||High|
|Fermentor||2-100g||< 2000 liters||0.05-0.5||2-12||1000-6000||High|
|aExcluding immunization, including up-scaling cell cultures.|
Contents, Animal Welfare Information Center Newsletter
Top of Document | Introduction - History | Pros and Cons: Ascites vs. In Vitro | In Vitro Alternatives | Related Uses Of Alternatives | The Future Of MAb Production | Conclusion | References
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