Summary of Advantages and Disadvantages of In Vitro and In Vivo Methods
Advantages and disadvantages of in vitro and mouse ascites methods for producing mAb are highlighted in this section. It should be noted that it is likely that in vitro methods will meet more than 90% of the needs for mAb. Some of the advantages and disadvantages are concerned with animal-welfare issues. Others deal with the economics of producing mAb. As noted below under the section titled "In Vivo and In Vitro Methods for Commercial Production of mAb", in vitro methods can cost 1/2 to 6 times the mouse ascites method. Some of the factors that cause in vitro production to be expensive are labor and equipment costs that are usually due to poor hybridoma production of mAb in vitro. If the investigator must use several types of media or different equipment, as happens occasionally, labor costs rise and research is delayed (Moro and others 1994; Stoll and others 1996b; Butler and Huzel 1995).
Advantages of in Vitro Method
- In vitro methods reduce the use of mice at the antibody-production stage but can use mice as a source of feeder cells when antibody generation is under way).
- In vitro methods are usually the methods of choice for large-scale production by the pharmaceutical industry because of the ease of culture for production, compared with use of animals, and because of economic considerations.
- In vitro methods avoid the need to submit animal protocols to IACUCs.
- In vitro methods avoid or decrease the need for laboratory personnel experienced in animal handling.
- In vitro methods using semipermeable-membrane-based systems produce mAb in concentrations often as high as those found in ascitic fluid and are free of mouse ascitic fluid contaminants.
Disadvantages of in Vitro Methods
- It should be noted that each of the items below pertains to only a fraction (3–5%) of hybridomas, but they indicate some of the difficulties associated with in vitro methods.
- Some hybridomas do not grow well in culture or are lost in culture.
- In vitro methods generally require the use of FBS, which limits some antibody uses. The use of in vitro methods for mAb production generally requires the use of FBS, which is a concern from the animal-welfare perspective.
- The loss of proper glycosylation of the antibody (in contrast with in vivo production) might make the antibody product unsuitable for in vivo experiments because of increased immunogenicity, reduced binding affinity, changes in biologic functions, or accelerated clearance in vivo.
- In general, batch-culture supernatants contain less mAb (typically 0.002-0.01) per milliliter of medium than the mouse ascites method. Note that semipermeable-membrane-based systems have been developed that can produce concentrations of mAb comparable with concentrations observed in mouse ascites fluid.
- In batch tissue-culture methods, mAb concentration tends to be low in the supernatant; this necessitates concentrating steps that can change antibody affinity, denature the antibody, and add time and expense. Adequate concentrations of mAb might be obtained in semi permeable-membrane-based systems.
- Most batches of mAb produced by membrane-based in vitro methods are contaminated with dead hybridoma cells and dead hybridoma-cell products, thus requiring early and expensive purification before study.
- mAb produced in vitro might yield poorer binding affinity than those obtained by the ascites method.
- In vitro culture methods are generally more expensive than the ascites method for small-scale or medium-scale production of mAb (Hendriksen and de Leeuw 1998; Jackson and others 1996; Peterson Peavey 1998; Marx 1998; Lipman 1997).
- The number of mAb produced by in vitro methods is limited by the amount of equipment that it is practical to have available.
- The Food and Drug Administration (FDA) estimates that proving the equivalence of an mAb produced by in vitro methods to an mAb previously produced by the mouse ascites method would cost the sponsor $2–10 million (Stein 1998; Maxim 1998)
Advantages of Mouse Ascites Method
- The mouse ascites method usually produces very high mAb concentrations that often do not require further concentration procedures that can denature antibody and decrease effectiveness.
- The high concentration of the desired mAb in mouse ascites fluid avoids the effects of contaminants in in vitro batch-culture fluid when comparable quantities of mAb are used.
- The mouse ascites method avoids the need to teach the antibody producer tissue-culture methods.
Disadvantages of Mouse Ascites Method
- The mouse ascites method involves the continued use of mice requiring daily observation.
- MAb produced by in vivo methods can contain various mouse proteins and other contaminants that might require purification.
- The mouse ascites method can be expensive if immunodeficient mice in a barrier facility must be used.
- In vivo methods can cause significant pain or distress in mice.