Proceedings of the Production of Monoclonal Antibodies Workshop

August 29, 1999 | Bologna, Italy

This document has been adapted with permission from a publication created by the Alternatives Research & Development Foundation (ARDF)

A Comprehensive Cost Comparison of In Vitro and Ascites Approaches to MAb Production

Norman C. Peterson, DVM, PhD
Department of Clinical Studies, School of Veterinary Medicine
University of Pennsylvania
Philadelphia, PA 19003-6010

The need to re-assess current popular practices for the production of monoclonal antibodies (MAb) has come about because of a growing international concern for the animals involved in the MAb ascites production protocols and the availability of alternative methods1,2,3. Adoption of in vitro methodologies to produce MAbs has been met with some resistance, largely due to the perception that the alternative technology is much more costly than the ascites method. This report will discuss some of the factors which need to be considered when comparing the in vitro methods with the ascites method for producing MAbs in order that more justifiable decisions can be made. As seen in Table 1, many of the factors are complex and proper evaluation must include direct and indirect monetary considerations as well as personal and societal values.

Table 1: Cost considerations when comparing tissue culture techniques to the ascites method for monoclonal antibody production

Time

Start to Finish (days)

Actual Technical Working Hours

Labor

Technical Difficulty

Space

Materials

Convenience

Endproduct Quality

Personal Values

Completion of Animal Use Forms and Review

Animal Well Being

Laws and published guidelines in several countries require that alternatives be considered whenever animals are used in research4. In the United States, each institution which uses animals in research is required to establish a committee (Institutional Animal Use and Care Committee, IACUC) which reviews proposed research protocols to ensure that the stipulations of the Animal Welfare Act are being followed5. Similar types of centralized committees in many other countries also serve to review animal use protocols6,7. The time and expense of these committees to review all animal use protocols is often overlooked when considering the costs of animal use. This may be because the researcher is never directly billed for these costs and the institution usually absorbs these expenses. In addition to the committee's costs, the researcher should also include the time necessary to complete his/her institution's animal use forms in these expenditures.

If the ascites method is approved by the IACUC, national policy requires that the animals do not experience unnecessary pain or distress5. In this regard, IACUCs at most institutions limit the number of taps and amount of weight that a mouse is permitted to gain8. Dependent upon these restrictions and the aggressiveness of the hybridoma cell line used, the quantity of MAbs harvested may be limited. Comparison of in vivo and in vitro methods of MAb should be made under the terms of these IACUC stipulations. Additionally, the health of the animals which are developing the ascites needs to be monitored at least daily. Because most animal facilities are at either remote or centralized locations and protective clothing (cap, gown, gloves, and/or shoe covers) is commonly required, adequate time (20 minutes or more) for daily travel, "gowning up", and inspection, should be included in the cost estimate for the use of these animals. In vitro techniques can be performed in the research laboratory, thus minimizing the time required to check the cell culture conditions.

Facilities

Another area which is largely subsidized by the institution and under-estimated in the cost analysis of ascites produced MAbs is the animal facilities and husbandry. The amount of subsidization varies among institutions and this makes comparisons difficult. Although the percentage of space utilized for ascites protocols may be relatively small, it does contribute to the demand upon which facilities are built. This may be of greater significance when space is limited or special facilities, such as those for immunocompromised mice, are needed. Dependent upon the method used, the antibody concentration, and amount of product needed, incubator space limitations may also be an important factor for in vitro produced MAbs.

Materials

Material expenditures are one of the most obvious factors included in any cost analysis. Mice are relatively inexpensive to purchase, however, if a mouse which is syngeneic to the hybridoma cell line is unavailable, additional expense will be incurred for the purchase of immunocompromised mice. Occasionally, ascites does not develop in some animals. This may be a result of inadvertent injection of cells into the lumen of an organ (intestines or bladder) or other sites not conducive to hybridoma cell growth9. Non-ascites producing mice should also be included in the MAb yield per mouse estimates and overall cost comparisons.

A major cost of the in vitro systems is the culture media required to keep a large number of cells viable and producing MAb. Cell lines differ in their efficiency at producing MAbs in vitro and this contributes to the large disparity in cost comparisons of multiple cell lines. Those hybridomas which grow rapidly in culture and expend less energy at MAb production will require more media per milligram of MAb, and, hence, require greater financial and labor investments10. Equipment costs correlate well with the scale of MAb they are designed to produce. Relatively inexpensive standard T-flask or gas permeable bag may suffice for the production of small quantities (1-25 mg) by the batch method, whereas membrane-based systems such as the Celline (Integra Bioscience, Ijamsville, MD) and the mini-Perm (Unisyn Technologies, Hopkinton, MA) which range in price from $300-$800 are more suitable for intermediate levels (10-500 mg) of production. Hollow fiber bioreactors (HFB) ($1,200 and up) are typically used for larger scale preparations. In order to accurately determine the production cost per milligram of MAb, the equipment cost must be included with respect to its total capacity. For example, the HFB (excluding the cartridge) may be used for several applications and, therefore, it is most appropriate to divide the production cost among the products of the bioreactor's lifetime use.

Time & Labor

Labor costs for in vitro monoclonal antibody production parallel those of the material costs10. This is largely because the culture media volume is a common determinant of both expenses. Time is required to either process and concentrate the large volumes of culture supernatant produced by the batch method or to monitor and supply the media reservoir of HFB system. The intensity of the labor required in the production protocol is also of concern. Although the labor involved in producing MAbs in our laboratory was similar for the batch method and the HFB, we found that the time spent with the HFB was more intensive. This was especially true during the early phases of production when cell viability and media acidity had to be monitored closely.

The desirability of a particular technique is also influenced by its aesthetic quality or how it effects one's emotional disposition. For this reason, some technicians may be reluctant in harvesting (tapping) mice for ascites. The in vitro methods may be a practicable alternative for these individuals, and it is likely that technicians would welcome the challenge of trying some of these newer alternatives to animals.

The start to finish time should not be of major concern, as the amount of time to produce the same quantity of MAbs was estimated to be similar (3-4 weeks) when four different approaches were considered (ascites, T-flask, gas permeable bags, and HFB)10. The HFB has the advantage that its production can be continually monitored and maintained until a desired amount of MAbs has been produced (provided the cartridge does not become clogged with cellular debris).

Some knowledge and skills are required to use either the in vitro approaches or the ascites method. Creation of a hybridoma cell line requires both the handling of mice and familiarity with tissue culture techniques, therefore, if a laboratory has made the cell line, additional skills needed for either approach should be minimal. If the hybridoma cell-line was not produced in-house or is of unknown origin, the institution may require that the cell-line be Mouse Antibody Protection (MAP) tested to avoid the introduction of rodent pathogens into the animal colony. MAP testing can add considerable expense and delays up to one month to any research protocol can be expected.

Quality

In order for the in vitro production methods to serve as a suitable replacement for the ascites method, the MAbs produced by the tissue culture methods must also retain the same level of activity as those produced by the ascites method. In some instances the glycosylation patterns of in vitro and in vivo produced MAbs were shown to be different and this may alter the effector function of each of the MAbs11. This does not appear to be a significant factor given that several core facilities use primarily in vitro methods to supply different researchers with their MAb needs and HFB are commonly used to produce the large amounts of antibodies that are used in clinical trials12,13. In vitro produced MAbs in my laboratory were equally effective in binding their targeted proto-oncogenic receptors which resulted in previously observed biological effects10. There may also be some concern as to the impurities such as cellular debris (in vitro) or mouse proteins or endogenous antibodies (ascites) which may contaminate the MAb preparation8. However, in a recent study by Lipman, et al, the purity of MAbs produced by a two-chambered minifermentor was purer than that produced by the ascites method14.

Cost Comparisons

Several cost comparisons between the in vitro and ascites methods have been reported by biotechnology companies and in independent studies. It may be difficult to form conclusions from these studies because different hybridomas were used and details necessary to standardize results are often not provided. Additionally, as stated above, different levels of institutional subsidizations for animal housing and care are not provided or too complex to be included in these reports. However, analyses of these estimates may provide a perception of the current state of technology.

The HFB has been the method most commonly used to produce MAbs at concentrations similar to that achieved in the mouse, and, therefore, it is the method most commonly compared to the in vivo technique. Jackson, et al, compared the ascites method to small scale MAb production in HFB using three different cell lines15. Rough estimates of their data indicate that the cost per milligram of MAb was approximately two to ten times greater by the in vitro method than by the ascites method. In a comparison by Peterson and Peavey, the estimated costs for the production of 100 mg of HFB MAb was similar to that of the ascites generated product10. Using a hybridoma cell line, which was found to be an average producer at a contract laboratory, Chandler compared the cost to produce different quantities of MAbs by using HFB and mice16. It was about three times more expensive to produce the 10 mg of MAbs by the HFB than by ascites method ($12/mg MAb, ascites vs. $35/mg HFB); however, as the quantity of MAb produced increased to 10 g this difference significantly decreased ($2.53/mg MAb, ascites vs. $3.58/mg MAb, HFB).

As other in vitro systems have become available, they have also been compared to the standard ascites method for producing Mabs. Hendriksen compiled cost estimates from ten different sources17. In his study, he found that the US dollar cost per milligram of MAb produced by the ascites method ranged from $0.5-$12, whereas the cost for the in vitro methods was $0.75-$40 per milligram dependent upon the method used. Peterson and Peavey produced two different MAbs by three different methods (T-flask, gas permeable bag, and HFB) and found that the in vitro methods averaged similar to and five times greater in cost than the in vivo method10. Data provided by Integra Bioscience and Diagnostic Chemicals Limited indicate that their in vitro systems (Celline and i-Mab, respectively) were slightly less expensive than the mouse for the production of MAbs18,19.

In summarizing the information to date, the overall impression is that in the majority of cases the in vitro techniques will tend to be financially more expensive than the ascites method for MAb production. Exceptions include large-scale production with HFBs, use of special immuno-compromised mice, or the need for MAP testing. In these cases the ascites method may be similar to, if not more expensive than the in vitro methods. Costs for the in vitro systems rarely exceed ten times that of the ascites method, and conceptually this may be the point at which the in vitro systems are considered unsuccessful. One should also consider that the quality of the hybridomas placed into the system will greatly influence its success rate. Petrie, et al, found that by sub-cloning the cell cultures to select for higher level producers, they could obtain sufficient MAb yields from HFBs in two of three cell-lines that previously failed to produce significant yields20.

Although the in vitro systems may average about 2-3 times more expensive than the ascites method, the added expense of producing MAbs by the in vitro methods should not place additional financial burdens upon the researcher. This is because the MAb needs for most protocols are relatively small (less than 10 mg) and thus the additional expenditure that may be needed is insignificant. Additionally, due to the public concern for reducing animal use in research, most funding agencies will have no reservations at increasing allowances for alternative means of producing MAbs. Lastly, the value of using fewer animals in a research protocol and the acceptance of the technicians performing the work needs to be considered in the total cost analysis.

References

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