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Alternatives to Monoclonal Antibody Production (Proceedings)

Core Laboratories: What is their cost-effectiveness and what are their needs?

Margaret B. Penno, PhD
Department of Medicine
Johns Hopkins University School of Medicine
Baltimore, MD

What are core laboratories? Core Laboratories at academic institutions are not-for-profit, centralized, facilities that offer specialized laboratory services, equipment, and/or reagents to investigators on a fee-for-service basis. Using a core laboratory is a cost-effective way for investigators to complete more work than they could normally accomplish given their space and personnel restraints. At the Johns Hopkins University School of Medicine there are a number of core laboratories dedicated to various specialties including flow cytometry, confocal microscopy, transgenic mice, NMR, protein sequencing, and genetics resources.

The Genetics Resources Core Facility (GRCF) at Johns Hopkins was established in 1987 as a way to bridge the gap between molecular biology and clinical science, especially with regard to inherited disorders. Divisions of the core include the Bioproducts Facility (media, enzymes and other biologicals), Cell Culture Laboratory, DNA Sequencing and Synthesis, Methods Development, Administration, and Training Labs. Nearly 600 principle investigators use the core facility because of its quality services, proximity, added value in delivery, and competitive prices.

The Cell Culture Laboratory of the core facility was initially designed to establish immortalized cell lines from lymphocytes as a renewable source of patient DNA. Additional services include cell banking, fibroblast establishment, mycoplasma testing, and personnel training. During the past year, services of routine and large-scale cell culture, including hybridoma production, were added in response to anticipated needs at Johns Hopkins.

The number of principle investigators using the services of the Core Cell Culture Lab is just over forty and increases every year. The cost-effectiveness for most investigators is significant when compared to the cost of doing the same work in a modest-sized lab. During a twelve-month period, for example, if an investigator had 200 blood specimens for transformation, and 50 cell lines for growth, he or she would need the assistance of an experienced cell culture technician, plus all reagents and materials. A technician of this description earns an average of $25K per year plus an additional $7K in fringe benefits. Supplies for this project would be approximately $10K for a total of $42K. The actual cost to the investigator at the core lab would be just under $21K, a savings of half. In addition to this tangible financial benefit, the core lab offers multiple skilled personnel, bulk-rate discounts on supplies, sample storage, and 24 hour monitoring of equipment.

The demand for large-scale in vitro hybridoma growth was anticipated based on the number of investigators who were using large amounts of monoclonal antibodies at Johns Hopkins.

Hybridoma production at the core facility was begun using the in vitro artificial capillary cell culture method. In this method, cartridge cells are in contact with the perfusion media through a semi-permeable, 30 kDa MW exclusion barrier. One hundred to two hundred million cells are inoculated into the sterile, equilibrated, capillary bed of the Cell Max. Daily sampling of lactate production guides the technician as to the timing of media replacement. During this time the volume of media is increased five-fold from 100 to 500 ml. Recovery of secreted protein begins at day seven when the lactate production reaches optimal levels. Typically 7-10 ml of "supernatant" is recovered. Although we do not purify antibody as a service, we follow up with the investigator to determine the relative yield. Absolute yield depends, for the most part, on the length of time that the cells are grown in the capillary system. On average, hybridoma cells grown for two weeks produce 50 mg of antibody with variability depending on the cell's own secretion properties.

The cost to set up the core hybridoma service was fairly significant. Once protocols were proven, we purchased a four-cartridge artificial capillary system at a cost of just under $5,000. An additional $3,200 was needed to stock a sufficient number of cartridges and supplies needed for several month's operation. Finally, we purchased a lactate analyzer at a cost of $6,800. Thus the final start-up cost to provide this service was just over $15,000.

The demand for this new hybridoma service in the in vitro system is currently 2-3 lines per month but is steadily increasing in response to advertisement and word-of mouth. There are some investigators at Hopkins, for instance, who have harvested monoclonal antibodies from mouse ascites for more than a decade that are, for a variety of reasons, now using the Cell Culture Laboratory's in vitro hybridoma growth services. Their opinion of the quality, quantity, and timeliness of the antibody-containing supernatant, as documented by a post-service survey, has been uniformly favorable. Even so, the number of inquiries is slightly higher than the actual number of users of this service.

We have conducted an informal survey of 10 academic core facilities across the country to determine the relative demand, methods, costs, and general impressions of hybridoma growth. The core labs surveyed were located at the University of Kansas, University of Virginia, University of California at San Francisco, University of California at San Diego, University of Pennsylvania, Sloan-Kettering Cancer Center, University of Wisconsin, the Pennsylvania State University, University of Arizona, and Georgetown University. The demand for hybridoma growth at these facilities was highly variable ranging from a few requests per year to more than 300. This demand did not necessarily reflect the size of the university. In general, there were more inquiries than actual requests for services and many investigators attempted to produce the antibody themselves. Methods for hybridoma preparation varied among labs and included mouse ascites, hollow fiber capillary systems and other minifermentors, roller bottles, and spinner flasks. Six of the 10 labs performed hybridoma growth using more than one method. This was the first suggestion that all methods had room for improvement. Although the timing of hybridoma growth was dependent on the method used and amount of antibody desired, all labs report that the process was complete between 1 and 6 weeks, with an average time of 4 weeks. Comparison of costs was best done on the basis of dollars per milligram antibody. When normalized in this way, both in vitro and in vivo methods could be included. Again, there was a range of values from $1 - $6 per milligram. The average cost was $4 per milligram. Ascites production was less expensive than hollow fiber technology by about $2 per mg of antibody, although it was difficult to make a direct comparison.

Comments from the lab workers were a useful way to sense the limitations and successes of hybridoma production in the academic core facilities. There was strong support for both in vitro and in vivo methods. Reported limitations to mouse ascites production included the problem of mouse protein contamination and restrictions on the number of times that the mouse could be tapped. For the in vitro methods, limitations included the high cost and time requirements. The following are a few specific comments:

"Hollow fiber technology is not difficult, it just needs to be baby-sat. You need to educate people that there are alternatives."

"I revisited hollow fibers after a negative experience several years ago. Although it works, the technology is not quite at the point of cost-effectiveness. We need to decrease the amount of media used and the time tending the cartridge."

"The cost of roller bottles and spinner flasks would decrease if there were more demand."

"We are given a lot of grief over our animal protocols. One of our committee members recently asked me whether monoclonal antibodies are still needed!"

"Hollow fiber technology is prohibitively expensive."

"We need funds for studies to increase the productivity of the hollow fiber system."

"I was disappointed with hollow fibers with serum-free medium."

"The cells need to be babied in the hollow fiber system. Weekends are a problem. It is expensive even without serum."

"People need to know that monoclonal antibodies from cell culture are cleaner than from ascites and, if the protein is precipitated, the amount of antibody is similar. They need to get away from the idea that ascites is easier than cell culture. Once they get used to it, they will be fine."

The Cell Culture Center in Minneapolis is an NIH-supported facility that is available to most academic labs. Its cost for hybridoma production is $1 - $3 per milligram. This price is below the core lab average yet the facility is not used to its fullest capacity. Two main reasons for this were given. First,investigators are not aware of it. Second, some investigators prefer the "hands on" feeling that a local core facility provides. The majority of the cores agreed that an NIH subsidy would encourage the use of local core laboratories.

In summary, there is a significant demand for large-scale hybridoma growth. The concerns for both in vitro and in vivo methods seems to be quality, yield, and cost-effectiveness. The first two concerns appear to be satisfied, for the most part, by both in vitro and in vivo methods. The latter concern seems to be an issue when choosing between in vitro and in vivo methods.

A number of recommendations can be formulated based on the collective experience of the core laboratories across the country. First, the investigator must be educated about the in vitro choices and cores available for this purpose. Various in vitro options should be provided by the animal care and use committees. Second, an advertising campaign should inform investigators about the services of the Cell Culture Center in Minneapolis and other such core facilities. Finally, the technology of the in vitro systems must be improved. This should be done as soon as possible, it should be NIH-subsidized, and involve key core laboratories across the country as well as leading vendors in the field. The goal should be to bring the cost of production to no more than $1 per milligram of antibody produced. This can be done by reducing the technician time needed to tend the system and by reducing the media requirements. With a few key changes, the cost-effectiveness and availability of the in vitro methods for large-scale hybridoma growth can be improved to the point that they will be preferred over the use of animals. A final balance between quality, yield, and price will result in a match between the number of inquires about hybridoma production and the requests for in vitro services from core laboratories.

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