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Alternatives to Monoclonal Antibody Production (Proceedings)

Hollow Fiber Bioreactors: An Alternative To The Use Of Mice For Monoclonal Antibody Production

Neil S. Lipman1 and Lynn R. Jackson2
1Memorial Sloan-Kettering Cancer Center and the
 Cornell University Medical College, New York, NY
 2Massachusetts Institute of Technology, Cambridge, MA

Laboratory scale quantities of monoclonal antibodies from hybridomas have classically been produced in ascites in histocompatible or immunodeficient mice. Although a useful technique, it has been criticized on the basis of humane concerns and the potential for contamination of the monoclonal antibody with adventitious murine viruses, endogenous murine immunoglobulin, other proteins, nucleic acids and residual priming agent. Additionally, gross morphological and conformational changes in DNA as well as changes in DNA content have been observed in hybridomas passaged as ascitic tumors. Alternatively, hybridomas can be grown in cell culture. A variety of culture techniques are available, however many require the use of large quantities of media and extensive post-production processing to attain concentrations of monoclonal antibody comparable to that obtained in ascites.

We have critically compared monoclonal antibody production in hollow fiber bioreactors to production in murine ascites. Hollow fiber bioreactors permit growth of hybridomas to extremely high densities and can be maintained for several months. As a result of the fibers' permeability characteristics, antibody can be harvested at concentrations that meet or exceed that attainable in ascites. Three different hybridoma lines were grown in each of three commercially available laboratory scale hollow fiber bioreactor systems and in 20 mice. Monoclonal antibody production was compared based on the technical expertise required, the equipment needed, and the concentration, quantity, and cost of producing antibody.

The total quantity of antibody produced during the nine 65-day bioreactor runs was equivalent to production in 4 to 48 mice, dependent upon cell line and hollow fiber bioreactor system used. While mice on average produced 31 mg of antibody at a concentration of 2.5 to 15 mg/ml, bioreactors produced as little as 91 to as much as 1287 mg of monoclonal antibody at concentrations of 0.71 to 11 mg/ml. Production of monoclonal antibody in ascites was, dependent on cell line, accompanied by minimal to moderate morbidity and some mortality and required at least daily observation of animals to ensure they were tapped before ascites accumulation became extreme. Material and time logs were carefully maintained for production in both hollow fiber bioreactors and mice. Cost for producing a mg of antibody ranged from $ 0.72 to $ 9.73 in hollow fiber bioreactors dependent on cell line and hollow fiber bioreactor system used, excluding costs for capital equipment. Production in mice ranged from $ 1.10 to $ 1.18 per mg antibody produced. Although it was generally more expensive to produce monoclonal antibody in hollow fiber bioreactors, the downstream processing costs required to purify monoclonal antibody in ascites was not evaluated.

In consideration of the humane aspects of monoclonal antibody production in mice, we consider production in hollow fiber bioreactors to be more humane. Our studies suggest hollow fiber bioreactors are one of several in vitro alternatives that merit close consideration when producing monoclonal antibody. Clearly, additional research is needed using a large number of cell lines with a variety of cell culture techniques, before one can determine if in vitro techniques can be consistently applied to produce monoclonal antibodies.

The Nobel Prize winning discovery by Köhler and Milstein in 1975, in which fusion of an antibody secreting lymphocyte and a plasmacytoma produced antibodies of predetermined specificity, has had and continues to have a tremendous impact on numerous scientific disciplines (1). Monoclonal antibodies (MAbs) are extremely valuable tools which are routinely utilized in both research and clinical settings. MAbs are used in vitro as components of immunodiagnostic assays, as biosensors, in affinity purification, fluorescent activated cell sorting (FACS), and immunohistochemistry, for tissue or cell typing, and as enzymatic antibodies (2). In vivo use continues to expand as many new therapeutic strategies employ MAbs for imaging, immunotherapy, and as cell specific cytotoxins (3,4).

Mice have been used extensively to produce antibody rich ascitic fluid following intraperitoneal inoculation of hybridoma cells (5-7). Although a useful technique, it has been criticized on the basis of human concerns (8-10). In addition to humane issues, in vivo production has been criticized for other reasons, particularly in relation to products for human clinical use. Antibodies produced in vivo may potentially be contaminated by adventitious murine viruses and endogenous murine immunoglobulin, other unwanted and frequently antigenic proteins, nucleic acids, and residual pristane, which is frequently used to prime the peritoneum prior to hybridoma cell inoculation (11,12). These issues pose additional difficulties in purification of MAbs.

Although monoclonal antibodies of murine origin can generally be produced in high concentrations by growth of hybridomas as ascitic tumors in histocompatible mice, similar attempts to grow heterohybridomas have been less successful and require immunodeficient mice (13-15). Additional methods including whole-body irradiation, treatment with immunosuppressive drugs, and prior subcutaneous adaptation of hybridoma cells to in vivo growth have been employed to improve production (15-19). These methods are labor intensive, expensive, and require special facilities for maintaining and handling immunodeficient rodents (11).

Gross morphological changes and alteration of DNA content of human hybridoma cells have been observed in ascites passaged cells from nude mice in comparison to cells grown in culture (15). Conformational changes have been detected in the DNA form hybridoma cells and correlated with the presence of cell-associated pristane (20). These findings suggest that the potential for genetic alteration of hybridoma cells grown in vivo exists.

Although the development of hybridomas still largely depends on the use of mice, the production of monoclonal antibody (MAb) from established hybridomas offers options, which for many hybridomas may not require the use of animals. Hybridomas are anchorage independent cell lines which can be grown in cell culture. In fact, hybridoma development utilizes cell culture for growth and selection of antibody secreting clones with desirable specificity (2). Therefore the question is not whether hybridomas can be grown in vitro, but rather, whether they can be grown in culture at densities and for sufficient periods of time to yield the needed quantity and concentration of antibody for its desired purpose. A variety of in vitro cell cultures techniques have been employed to produce monoclonal antibodies from hybridomas on a laboratory scale. The methods commonly utilized include stationary cultures in T-flasks and suspension cultures, including roller bottles and spinner flasks, all of which are handled as batch or fed-batch cultures (10,11,18). Laboratory scale stirred tank reactors and air-lift fermentors have also been used (21-23). However, shear forces exerted on cells grown in these systems may adversely influence cell growth and antibody production. Large volumes of culture media and extensive post-production concentration techniques are required as the concentrations of antibody achieved using these methods is 1/100th to 1/1000th that obtained in murine ascites (10). Other techniques including growth of hybridomas in dialysis tubing, hollow fiber bioreactors, modular fermentors, oscillating bubble dialysis chambers, and entrapment of cells in agarose beads have also been recently described (24-28). Antibody produced by some of these techniques begin to approach concentrations attained in and offer viable alternatives to murine ascites.

Hollow fiber bioreactor (HFB) systems have, within the past decade, been integrated into commercially available systems suitable for laboratory scale production. These systems offer significant advantages over many other in vitro culture techniques. Hollow fiber technology, introduced by Knazek et al in 1972, was developed as an artificial capillary system for in vitro growth of cells (29). The system provided a more physiologic environment for cell growth with respect to nutrient supply, waste removal, and pH while maintaining a stable pericellular environment without shear (30). With technological advances in hollow fiber membrane construction, bioreactors have been fabricated which provide an extracapillary space (ECS) for hybridoma cell growth which is both compartmentally and physiologically separate from the intracapillary space (ICS). Hybridoma cells are provided nutrients from culture media continuously circulating through the ICS; small molecular weight metabolic waste products are removed from the ECS, while immunoglobulin secreted (molecular weight - >/= 150 KD) can be retained in the ECS for periodic collection.

Hollow fiber bioreactors (HFBs) consistently produce concentrated antibody, when comparing different cell culture techniques, ranging from 0.7 to 2.3 mg/ml, and concentrations exceeding 17 mg/ml have been reported (31-33). The hollow fivers are packaged in parallel as a bundle and are potted at both ends within a plastic cartridge. Media is pumped into one end of the cartridge, through the lumen of the fibers and out the opposite end. The ECS which surrounds the fibers is accessed directly from the harvest ports. The fibers' walls are constructed of a material which acts as a semi-permeable ultrafiltration membrane. The size of the membrane's pores, characterized by its molecular weight cut-off, can be small enough to retain cells and immunoglobulin in the ECS while permitting nutrients and waste products to move freely down their concentration gradients. Hollow fibers used for monoclonal antibody production are generally manufactured from cellulose and are selected to have a molecular weight cutoff (>/=45-50 KD at 95% retention) well below that of immunoglobulin.

HFBs have a number of advantages in comparison to other in vitro and in vivo production methods. They can support the growth of a large number of both anchorage-dependent and independent cell lines from a variety of species, including primary and continuous, normal and malignant, and fibroblast and epithelial cells lines (30). The continuous perfusion of the bioreactor with media simulates the in vivo environment and provides an uninterrupted supply of nutrients and removal of metabolic waste products necessary for optimal production while protecting the cells from shear (31,34). Cells can be grown to very high cell densities (> 108 cells/ml) and viability and production can be maintained for relatively long periods of time, generally months (11,30,34,35). Antibody can be harvested from the bioreactor repeatedly during production, decreasing the potential for antibody degradation resulting from prolonged exposure to cellular proteases (11). Purity of antibody has been facilitated without inhibiting production by gradual reduction of serum in the media or by using serum-free media (30,31,33). Growth of hybridomas in HFBs takes maximum advantage of the type II antibody production kinetics of most murine and human hybridomas (11,36).

Experimental results obtained in our laboratory support the use of HFBs as an alternative to murine ascites for monoclonal antibody production. We have critically compared MAb production in three commercially available laboratory scale HFB systems to production in murine ascites based on the technical expertise required, the equipment needed, and the concentrations, quantity, and cost of producing antibody (37). We utilized three different hybridomas which differed with respect to the plasmacytoma fusion partner, the isotype of antibody secreted, and antibody specificity. Two hybridomas were mouse X mouse and one was a rat X mouse heterohybridoma. Each hybridoma was inoculated into each of 3 HFB systems and also into 20 mice following pristane priming. Mice were tapped a maximum of three times for collection of ascites. Ascites volumes and daily clinical observations were recorded and necropsies were performed on all animals at the end of the study. Bioreactors were harvested three times weekly for 65 days and were monitored by cell counts, cell viability and media glucose consumption. Detailed time and material logs were maintained to ascertain the cost of producing antibody by these methods.

The total quantity of MAb produced in 20 mice versus the mean production for the three different bioreactors in 65 days was as follows: cell line 2B11, 45 mg vs. 168 mg; cell line 3C9, 446 mg vs. 565 mg; and cell line RMK, 997 mg vs. 1023. When examined as mouse equivalents based on actual yields from mice and HFBs in our study, HFB runs were equivalent to producing MAb in a minimum of 4 to a maximum of 48 mice per production run dependent upon specific cell line and HFB system utilized.

Immunocompetent mice (CAF1/J) produced 22 mg antibody per mouse on average for cell lines 2B11 and 3C9. Cell line RMK, the rat X heterohybridoma, was grown in immunocompromised (C.B.-17/IcrTac-scidDF) scid mice which produced 48 mg antibody per mouse, considerably more than produced in the immunocompetent F1 hybrids. The mean harvest ranged form 1.2 to 3.2 ml per mouse per tap. The percentage of antibody contributed by each tap to the total produced from all three taps, differed by cell line. The first tap for cell line 2B11, the second tap for cell line 3C9 and the third tap for cell line RMK yielded the greatest amount of antibody. In fact, more than 66% of all antibody produced by mice inoculated with RMK was collected during the third tap. In addition, all the SCID mice used for RMK production remained clinically healthy for the entire production run, whereas 7 out of 40 CAF1 mice inoculated with cell lines 2B11 or 3C9 died or required sacrifice. The increase in mortality associated with these cell lines correlated with the harvest of blood contaminated serosanguinous ascites and more aggressive pathologic lesions.

Material costs for producing antibody were carefully evaluated. Material costs for HFB production runs, exclusive of capitol costs for the purchase of HFB systems, were generally higher than mice at $469.00, $341.00, and $554.00 for the three systems respectively. Capitol costs for the purchase of HFB systems ranged from $4,000.00 to $7,235.00. In comparison, production in mice inclusive of animal purchase and per diem expenses, but exclusive of labor, were approximately $300.00 for production in CAF1 mice and $900.00 in SCID mice. Time spent in labor for production of a single cell line in HFBs for the 65 day production run ranged from 16 h 42 min to 23 h 22min. Technician time required to set up and maintain the HFB systems was approximately twice the 7 h 49 min to 9 h needed to produce MAb in 20 mice. Assuming a technician's salary at $25.00 per h inclusive of employee benefits, total costs for producing MAb in HFBs, exclusive of costs for capital equipment, ranged form $0.72 to $9.73 per mg which was more expensive for producing 2 of the 3 cell lines evaluated on a per mg basis. Total cost for production in mice ranged for $1.10 to $1.18 per mg antibody produced.

Although our study indicates that is was more expensive to produce MAb for 2 of the 3 hybridoma lines in HFBs, we did not consider the downstream processing costs that may be required to purify ascites nor did we consider the subsidy that many institutions provide to the operation of their animal care and use program in artificially reducing the cost of in vivo production. This subsidy may exceed 50% at some institutions.

When comparing antibody production in mice and HFBs, each method has advantages and disadvantages. Production can be initiated more quickly and can be sustained over longer periods in HFBs; however, larger total quantities of antibody were frequently produced in a shorter time period in mice. The antibody harvested from a HFB is considerably more pure, especially when utilizing serum-free media or when serum supplementation is reduced (30,31,33,38). Antibody produced in ascites is contaminated with murine proteins including albumin and endogenous murine immunoglobulin. Growth of hybridomas in cell culture is subject to microbial contamination and mechanical failure is possible when producing antibody in a HFB. Some hybridomas will not secret or may produce minimal antibody when grown in vitro necessitating the use of mice (39). Growth in cell culture, in general, requires greater technical expertise. Importantly, HFBs can be operated on a 3 day per week schedule; however, mice used for ascites should be observed 7 days per week, often more than once daily, independent of weekends and holidays, to ensure that animals are tapped before ascites accumulation becomes extreme. Daily examination also ensures that animals in distress are humanely sacrificed in order to minimize morbidity and mortality.

Importantly, the biologic activity of a MAb may differ dependent upon whether the antibody was produced in vivo or in vitro, a result of differences in antibody glycosylation (40-42). Although these differences may be insignificant in many applications, when used clinically they may alter the antibody's pharmacokinetics (41). Neither in vivo or in vitro production are necessarily better, this depends solely on the specific antibody.

In consideration of the humane aspects, we consider production of MAb in HFBs to be more humane than production in mice. Our studies suggest HFBs are one of several in vitro alternatives that merit close consideration when producing MAbs. Additional research is needed to explore potential factors which contribute both positively and negatively to MAb production in vitro. Ultimately it may be possible to reduce the cost of in vitro production by refining culture technique to increase antibody yield or to obtain reasonable harvests from lines which previously were not productive when grown in vitro. Although preliminary, our findings suggest that ascites production in immunodeficient SCID mice may yield greater quantity per mouse while reducing adverse clinical effects and contaminating endogenous murine immunoglobulin.


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