Considerations for in vitro monoclonal antibody production

IN VIVO

Considerations

AND IN VITRO

PRODUCTION

for irt vitro monoclonal

OF mAbs

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antibody production

N.C. Peterson Department of Pathology & Laboratory Medicine University of Pennsylvania, Philadelphia, PA 19104 (USA)

The ascites method has been one of the most popular means for producing large quantities of highly concentrated monoclonal antibodies (mAbs) since its inception in 1972 [l]. Although hybridoma cells secrete mAbs into the culture media and these immunoglobulins can be harvested for use, researchers have been reluctant to use in vitro methods for mAb production protocols. This can be largely attributed to a common belief that in vitro production techniques are costly, technically difficult to produce, and result in poor yields of dilute antibodies. An increasing public concern for the welfare of animals has led to a renewed interest and re-evaluation of alternatives to the use of animals for the production of mAbs and this topic has been the focus of several seminars, workshops and this Forum [2, 31. In order to investigate the practicality of using in vitro techniques to generate large quantities of mAbs, three different culturing techniques and the mouse ascites method are compared in this report. Mice were initially used to produce anti-net&erbB2 mAb 7.16.4 and anti-epidermal growth factor receptor mAb 225. Both hybridoma cell lines produced abdominal swelling in the mice at 10 to 15 days after inoculation. Ascites harvested from 37 mice inoculated with mAb 7.16.4 producing hybridomas yielded an average of 2.38 mg of protein G purified immunoglobulin per mouse. Included in this average yield were 4 inoculated mice (11%) which did not develop ascites. By tapping the mice twice and euthanasia being performed before the final tap, an average 1.65 ml of ascites containing 1.44 mg/ml of mAb 7.16.4 was collected from each mouse. Of the five mice which were inoculated with mAb 225, one (20%) did not produce ascites. The average amount of mAb 225 obtained from each mouse in this group of 5 was 4.53 mg, with an average concentration of 1.19 mg/ml. These values may appear somewhat lower than previously reported yields and this may be attributed to several factors [4, 5, 61. Firstly, hybridomas vary greatly in their production capabilities. Hybridoma

7.16.4 is a rapidly growing cell line and this may have limited its productivity. Secondly, these mAb were purified and then quantified by UV light absorbance, while others typically determine yields of unpurified samples by immunoassays [4, 5, 61. Mice also vary in their acceptance of the inoculated cells and non-producers should be included in calculations in order to accurately reflect yield and animal use. Lastly, Institutional Animal Care and Use Committee (IACUC) guidelines for mAb production were followed in that only two ascites harvests were taken. Some earlier studies report as many as seven taps and mice remaining in ascites production protocols until death [4]. Aggressive approaches such as these, although less humane, may result in higher mAb yields. The use of animals for the production of mAbs can be avoided by harvesting the immunoglobulins from the culture media of hybridoma cells. In order to avoid contamination of bovine antibodies present in the serum of the culture media, the hybridomas grown in either a tissue flask or gas-permeable bag must first be adapted to grow in serum-free media. A variety of serum-free media are commercially available and the growth and production response of a specific hybridoma cell line to each culture medium will tend to be variable. If hybridomas do not thrive in the culture media which contain reduced serum levels, the hollow fibre cartridge (HFC) bioreactor may be more suitable for in vitro production. The molecular weight cut-off (lo35 kDa) of the fibres which separate the cells from the circulating culture media do not allow cross-contamination of antibodies. However, I found that production levels for mAb 7.16.4 in the HFC could be improved if the serum levels were lowered to 2.5 % in protein-free hybridoma growth media compared to growth conditions of 10% foetal calf serum in DMEM. The highest mean concentration of in vitro-produced mAb 7.16.4 was achieved from harvests of the HFC (fig. 1). This was slightly less than l/3 of

Received July 10, 1998. Present and corresponding ladelphia, PA 19104-6010.

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mAb 7.16.4 mAb 225

Fig. 1. Comparisonof mAb concentrationby in vivo and in vitro methods. mAbs producedby the methodsindicatedwerepurified

by proteinG affinity chromatography,andultraviolet absorbance(280 nm) was used to determine the total amount of mAb purified.This valuewasdivided by the volumeof the supematants purified to derive the ascitesandtissueculture mAb concentrations.Concentrationvalues are indicated aboveeachbar (reprinted with permission from Contemporary Topics in Laboratory

Animal Science).

the concentration of mAb 7.16.4 in ascites. mAb concentrations in the media from 10 days of 7.16.4 hybridoma cultured in rigid plastic flasks had the lowest mean concentration (9.4 pg/ml) of mAb which was approximately 150-fold lower than the ascites concentration (fig. 1). A 70% gain in concentration of mAb 7.16.4 in tissue culture media was achieved when hybridomas were grown in gaspermeable bags instead of plastic tissue culture flasks (fig. 1). Hybridoma cells which secrete mAb 225 did not grow as well in culture as mAb 7.16.4producing cells. This was reflected by a lower mean concentration (2.85 pg/ml) of mAbs in the culture supematant (fig. 1). The concenrrarion ot mAb 225 could be slightly increased to 3.53 pg/ml (24% increase) by incubation of the hybridomas in gaspermeable bags. Production of mAb 225 was not analysed by the HFC bioreactor method becausethe large quantities typically generated by this method would exceed our requirements and this would add unnecessary labour and expense Although the concentrations of the in vitro-produced mAbs were significantly lower than that

obtained by the ascites method, protein G-sepharose affinity purification of the expended hybridoma culture media and the ascites resulted in products of similar concentration and quality. However, because the in vitro methods produce mAbs at lower concentrations, large volumes of media must be purchased and processed. In order to determine if this would make the in vitro methods cost-prohibitive, the time and costs necessary to produce 100 mg of each of the mAbs by each method was estimated for comparison. The material costs for the production of mAb 7.16.4 ranged from $624 per 100 mg for the gaspermeable bags to $791 for ascites (fig. 2). Although the material expenses to produce mAb 7.16.4 in vitro were slightly less than the those of the in vivo approach, mAb 225 was about five-fold more expensive to produce in tissue culture flasks than by ascites ($2,063/mg versus $418/100 mg, respectively) (fig. 2). tnAb 7.16.4 required 42 mice to produce the 100 mg, whereas only 22 mice could generate the same amount of mAb 225. The purchase of the mice accounted for 77% of the material costs with the remaining expense largely attributed to per diem costs. Tissue culture media tended to be a significant expense for the in vitro techniques and accounted for 5 l-68 % of the mAb production costs. The ability to achieve higher concentrations of mAb 7.16.4 than mAb 225 in the supernatant of tissue culture flasks resulted in less expenditure for culture media. An estimation of 10.8 1 was required to generate 100 mg of mAb 7.16.4, whereas 35 1 was needed to produce the same amount of mAb 225. Growth of hybridoma cells was less expensive for mAb 7.16.4 in the gas-permeable bags than in the standard tissue culture flasks (fig. 2). Although the price of a 225-cm2 tissue culture flask is less than that of a gas-permeable bag, we found that if the volume of the 225-cm* tissue culture flask exceeded 300 ml, the hybridoma cells did not grow well, presumably due to limited gas diffusion. As a result, a l-l gas-permeable bag could contain the equivalent culture media of over three large tissue culture flasks and occupy significantly less room in the tissue culture incubator. Additional reduction in cost of the gas-permeable bag method for the production of mAb 7.16.4 could also be attributed to higher mAb concentrations that could be achieved in the supernatants and thus allowing the purchase of less media. However, the slight gains that were achieved by growing mAb 225-producing hybridomas in the gas-permeable flasks were not sufficient to compensate for the slightly more expensive purchase price of the gas-permeable bags when compared to tissue culture flasks. A disadvantage of the HCF bioreactor is the initial investment in the apparatus. This system is designed primarily for high production. In order to

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mAb 7.16.4

Fig. 2. Cost analysis of mAb producedby in vitro andin vivo techniques. Material and labour costsrequiredto produce 100 mg of m4bs. 7.16.4 and 225 were calculated as described in the text. Labour costs were derived by multiplying the estimated time required to produce the mAbs by an hourly wage of $l.Vbour and is represented by the open bars (reprinted with permission from Contemporary Topics in Laboratory Animal Science).

fairly compare the HCF bioreactor to the other methods, we assumed a potential for production of one gram of mAb 7.16.4 ; thus the apparatus cost included in our estimate for 100 mg was l/10 of the original cost ($1,500). Eight harvests of the HFC were required to produce the 100 mg of mAb 7.16.4. During this period, 14 1 of culture media were expended. The other in vitro methods described required less media per milligram of mAb 7.16.4 becausecultures were maintained in the samemedia until cells had expired, at which point the supernatant was harvested. The medium in the HFC bioreactor was monitored for cellular metabolic byproducts and changed before growth inhibitory levels were reached. An additional expense ($130$160 per 100 mg) that was incurred with the tissue culture flask and gas-permeablebag method was the need for several 500-ml volume filtration units (one per 500-ml of supernatant). The smaller volumes from ascites and the HFC contained less debris and could be filtered through the less expensive syringe filter units. In order to estimate the tota! time necessary to produce 100 mg of mAbs 7.16.4 and 225, the protocols were reviewed and the time necessary to processthe calculated amount of material for each step of the procedures was itemized and summed (for a more detailed description of the time/cost analysis see reference 7). The gas-permeable bags which required 12.5 h of technical work time represented the most efficient means of producing mAb 7.16.4. Eight

harvests,

daily

monitoring

of lactic

acid pro-

duction, and four changes in the media reservoir, contributed in part to the 15 h required to produce the same amount of antibody in the HFC bioreactor. Tissue culture flasks and ascites required similar processing times (19 and 22 h, respectively). Because fewer mice were needed for mAb 225 production and the in vitro methods required larger volumes of media, the differences in time commitments were greater. Ascites production of mAb 225 required 11.5 h, whereas, the tissue culture flask method required 59.5 h and the gas-permeable bag approach demanded51 h. The estimated time commitments were factored by an intermediate level technician hourly rate of $15 an hour to derive the labour costs and these results are presented with the material costs to reflect the total costs of production (fig. 2). These results show that the time commitment and labour cost profiles paralleled those of the material costs. This was not surprising, as both the material and labour costs of the in vitro methods were highly influenced by the amount of media which was FMchased and processed. Factors wnich were not included as a direct expense in the cost estimates, but should be emphasized in these considerations, include: cost of building the animal care facility, institutional subsidization of animal care, investigator time involved in completing the appropriate animal use regulation form.;, and time invested to review these forms. Additional cOst: wl,icl~ Lallnot be quantitated, but should greatly influence choices in using alternatives, involve the pain and suffering

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associated with the ascites production method [8, 91 and the psychological discomfort of the technician performing this procedure. Although our results showed little difference in the time commitment necessary to produce mAb 7.16.4 by the in vitro methods, routine monitoring of the HFC culture media was necessary to prevent buildup of toxic metabolites. This was especially true during the first two weeks before a regular pattern of cell harvesting and media reservoir changes could be established. Because of the initial financial investment and development of technical skill with the HFC bioreactor, greater financial benefits may be achieved if repeated use of this system is anticipated. Alternatively, many institutional core facilities [lo, 1 l] and some commercial vendors [12] offer in vitro mAb production services. In the United States, the National Institutes of Health subsidize a national cell culture centre which provides these services at minimal cost [13]. As demonstrated by the results presented in this report and others, hybridoma cell lines differ in their response to different growth environments. This makes defining the best method for mAb production which will be applicable to every situation difficult. General characteristics of each of the methods discussed here are summarized in table I. When the costs and production levels of in vitro and in vivo made mAbs are similar, such as with mAb 7.16.4, the in vitro methods are an attractive alternative to the use of animals in mAb production protocols. Other situations may occur in which the ascites

Table I. Summary Method

IN IMMUNOLOGY method may be more efficient in terms of time and cost than the tissue culture techniques. Although mAb 22.5 was shown to be much more expensive to produce in vitro than in vivo, a 500-ml tissue culture harvest was sufficient to generate milligram quantities of purified mAb 225. Production at this scale was sufficient to provide reagents necessary to perform multiple experimental procedures, such as immunohistochemistry, immunoprecipitations and flow cytometric analyses. Over 60% of the research mAb needs falls below 100 mg2. At this lower end of the production scale, the net costs of generating mAbs in vitro, as opposed to in vivo, would not reflect significant budgetary differences. In accordance with standards established in several countries to reduce the use of animals in research [2, 141, the in vitro techniques discussed above and others described in this Forum offer a comparable alternative. Those considering using in vitro techniques for the first time are advised to first try the simplest method of harvesting mAb from serum-free culture media from hybridomas grown in plastic tissue culture flasks. The method requires that cells be slowly adapted to the serum-free media and then allowed to incubate until the media turns acidic and viability drops to about 5-10%. The supematant can then be purified and concentrated by protein A (or G) affinity chromatography. Once satisfactory results have been obtained, the other methods can be investigated to improve yields. As the research community begins to accept the in vitro techniques as the standard means of producing mAbs, additional improvements in this methodology are likely to follow.

of mAb production

techniques.

Disadvantages

Advantages

Ascites

Causes pain and distress in mice [8, 91 Some cells form solid tumours with no production Animal facilities are usually remote from lab

Large yield in a small volume

Tissue culture flask (TCF)

Cells must be adapted to serum-free media Occupies the most incubator space Large volumes of media must be processed

Technically

Gas-permeable bags

Cells must be adapted to serum-free media Large volumes of media must be processed

Technically easy Occupies less space than TCF May require less media than TCF

Hollow fiber cartridge

Large initial investment Technically more challenging Mechanical disruption or contamination -3 start over Large amounts of media expended

Large yield in a small volume Culture media may contain high concentration of foetal calf serum

easy

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23. Alternative

[3]

[4] [5]

[6]

to Laboratory

Animals.

ATL.A, 25,

121-137. Alternatives in Monoclonal Antibody Production.A Workshopof The John HopkinsCenter for Altematives to Animal Testing and the Office of Research Risks,NIH. 1997. Brodeur,B. & Tsang,P. (1986), High yield monoclonal antibody productionin ascites.J Immunol. Methods,86(2), 239-241. Mueller, U., Hawes,C. & Jones,W. (1986), Monoclonal antibody production by hybridomagrowth in Freund’sadjuvant primed mice. J. immunol. Methods,87(2), 193-196. Noeman,S., Misra, D., Yankes,R., Kunz, H. & Gill,

557

T. (1982), Growth of rat-mousehybridomasin nude mice and nude rats. J. Immunol. Methods, 55(3), 319-26. [7] Peterson,N. & Peavey, J. (1998), Practical applications of in vitro monoclonal antibody production.

References [l] Potter, M., Pumphrey, J. & Walters, J. (1972), Growth of primary plasmacytomas in the mineraloilconditionedperitonealenvironment.J. Nat. Cancer Instit., 49( 1). 305-308. [2] Marx, U., Embleton,M. J., Fischer,R., Gruber, F. P. et al. (1997). Monoclonal antibody production: the report and recommendationsof ECVAM workshop

OF mAbs

Contemp. Top. Lab. Anim. Sci. (in press).

[S] Amyx, H. L. (1987), Control of animal pain and distressin antibody production and infectious diseasestudies.J. Am. Vet. Med. Assoc., 191, 128712890. [9] McGill, M. W. & Rowan,A. N. (1989), Refinement of monoclonalantibody productionand animalwellbeing.ILAR News, 31, 7-l 1. [IO] Penno, M. Director: Johns Hopkins Cell Culture CoreFacility. [ 111Kazazian, H. Director: University of Pennsylvania, Cell CenterServices. [ 121Cellco, Inc Germantown,MD 20874. [ 131Cell Culture Center,Minneapolis,Mn 55433. [141 “Guide for the CareandUseof LaboratoryAnimals.” National ResearchCouncil, Institute of Laboratory Animal Resources.(1996) National Academy Press. Washington,DC.

Membrane-based cell culture technologies : a scientifically and economically satisfactory alternative to malignant ascites production for monoclonal antibodies U. Marx University of Leipzig, Institute for Clinical Immunology Department of Medical Biotechnology, Delitzscher Strasse

Based on the principles of perfusion bioreactor systemsdeveloped in the late 197Os,a new powerful generation of membrane-based cell culture devices especially designed for small-scale production of monoclonal antibodies entered the market over the last few years. These culture devices enable scientifically satisfactory in vitro production methods which are of moderate cost and either better than or equal to ascites production method in terms of antibody quality.

ReceivedJuly 10, 1998.

and Transfusion Medicine, 141, 04129 Leipzig (Germany)

Introduction Following the discovery of hybridoma technology by Kijhler and Milstein in 1975, monoclonal antibodies have had profound implications not only in terms of medical research, diagnosis and therapy, but also for biology in general. In the early days of hybridoma technology, the hybridomas developed in vitro were injected into the peritoneal cavity of an animal so that useful amounts of the desired mono-