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)

Small-Scale Monoclonal Antibody Production In Vitro: Methods and Resources

Lynn R. Jackson, DVM, MS
Department of Research Administration, Biogen, Inc.
14 Cambridge Center, Cambridge, MA 02142

Laura J. Trudel, BA
Massachusetts Institute of Technology
Division of Bioengineering and Environmental Health, Cambridge, MA

Neil S. Lipman, VMD
Memorial Sloan-Kettering Cancer Center and
the Weill Medical College of Cornell University, NY, NY

Monoclonal antibodies (MAbs) are secreted from a single clone of B lymphocytes and have identical antigenic specificity, thus binding to the same epitope on an antigen. The Nobel Prize-winning discovery of Köhler and Milstein¹ permitted scientists to harness and exploit MAb design and production for a variety of research, clinical, and industrial applications. In the 1970s, they discovered that they could create a cell that secretes MAb of desired specificity by immunizing a mouse with a specific antigen and then fusing a splenocyte from that mouse with a mouse plasmacytoma cell. The result of this fusion was a hybrid cell containing nucleic acid and characteristics of both parental cells. Desirable hybrids, referred to as "hybridomas," are immortalized like the plasmacytoma, but also retain the antibody-secreting capability of the splenocyte. One can grow hybridomas in cell culture or in the peritoneal cavity of the mouse to produce varying quantities of identical antibody.

MAbs are extremely valuable tools in both research and clinical settings. They are indispensable in the research laboratory; without MAbs, numerous important scientific discoveries would not have been possible. MAbs are used in vitro as components of immunodiagnostic assays; as biosensors; in affinity purification, fluorescent-activated cell sorting (FACS), and immunohistochemistry; and as enzymatic antibodies². MAbs are used routinely in ELISA and IFA tests to detect antibody against adventitious viruses present in rodent serum. Research use is characterized generally by the use of a large number, but small quantities (< 0.5 g), of antibodies³.

A number of clinical therapeutic strategies based on MAbs are either FDA-approved or in the drug development pipeline. For example, the humanized cytotoxic MAb, Herceptin® (anti-HER-2), directed against a transmembrane receptor protein (HER-2/neu) that is over-expressed on some human mammary carcinomas, has recently been approved as a treatment for human metastatic breast carcinoma⁴.

Hybridoma Development and MAb Production

There are two phases of MAb production: hybridoma development and production of MAbs from an established hybridoma. Mice have been integral to the process of generating most hybridomas and producing MAbs. Briefly, hybridoma development involves immunizing mice with the desired antigen, followed by sacrifice and splenic harvest. Splenocytes of B-cell lineage are isolated and fused with plasmacytoma cells using polyethylene glycol (PEG). One selects hybrids from unfused plasmacytoma cells, which remain viable in culture, using selective media; plasmacytomas used as fusion partners have a defect in an essential biosynthetic pathway, allowing one to kill unfused plasmactyomas in selective media while hybridomas remain viable. Subsequently, one dilutes and seeds hybridomas onto multi-well plates, such that each well contains cells derived from a single clone. The supernatant from each well is tested to determine if the resultant antibody has the desired specificity.

Desirable clones are expanded in vivo or in vitro to generate MAbs. Except for immunization, the entire process is based in cell culture. One may employ certain techniques -- principally phage display and in vitro immunization -- to generate hybridomas without immunizing mice^5,6. Although these techniques may decrease or eliminate the need for mice to generate hybridomas in the future, they are currently limited to use in a small number of laboratories.

In addition to murine hybridomas, one may produce mixed species, or "heterohybridomas." Heterohybridomas are created by fusing splenocytes obtained from a species other than a mouse with a murine plasmacytoma. In vivo production of MAbs from these hybridomas requires the use of an immunocompromised host. Recombinant DNA technology has also led to the development of humanized MAbs. The advantage of humanized MAbs is that they can be used clinically in humans, while minimizing development of an anti-antibody immune response.

Mice have been used extensively to produce MAb-rich ascitic fluid (serous fluid that collects in the peritoneal cavity). Mice syngeneic to the hybridoma, or, alternatively, immunocompromised stocks or strains, are inoculated intraperitoneally with a priming agent, typically pristane, 7-21 days prior to intraperitoneal inoculation of hybridoma cells. Use of pristane facilitates development of ascites⁷. The antibody is harvested by paracentesis, a process referred to as "tapping."

Although a useful technique, some have criticized ascites production on the basis of humane concerns. Significant clinicopathologic abnormalities develop in the mice as a result of disseminated or solid tumor growth within the peritoneal cavity, and the associated accumulation of ascitic fluid⁸. The progression of abnormalities is hybridoma-dependent; marked differences may be observed among cell lines, likely a result of the hybridoma's growth characteristics or the secretion of potent bioactive substances by the hybridoma or the mouse. The removal of ascites may, by itself, lead to severe physiologic compromise, including death, potentially a result of circulatory collapse following removal of large volumes of the protein-rich ascites. Multiple taps may lead to significant body weight loss⁸.

Regulatory Issues

As a result of these concerns, a number of European countries have established guidelines or regulations which restrict or prohibit ascites production in rodents^3,9,10. Attention from animal welfare organizations, the scientific community, and regulatory agencies is increasing in the US. Recently, the Office for Protection from Research Risks (OPRR), NIH has advised Institutional Animal Care and Use Committees (IACUCs) in a "Dear Colleague" letter of their responsibility to determine if investigators have considered methods that avoid or minimize pain or distress in animals, and that alternatives to ascites production are used, if suitable¹¹.

OPRR's "Dear Colleague" letter, as well as a petition filed by the American Antivivisection Society requesting that the NIH support a policy precluding routine production of MAb in ascites, has brought the issue of alternatives to murine ascites production to the fore. We interpret OPRR's directive to indicate that MAb production be attempted by first using an in vitro technique. As veterinarians and scientists concerned with the welfare and humane care of animals used in research, having produced MAb both in vivo and in vitro ourselves, we believe this action is prudent. Our reasoning is: most hybridomas will grow and produce a suitable quality and quantity of antibody in vitro; one cannot predict whether a specific hybridoma will fail to produce antibody in vitro; technology is available to most laboratories for this purpose; and in vivo production may lead to pain or distress.

Cell Culture Technology

Although the development of hybridomas depends largely on the use of mice, producing MAbs from established hybridomas does not always require the use of animals. Therefore, the question is not whether hybridomas can be grown in vitro, but whether they can be grown in culture in densities and for sufficient periods of time to yield the necessary quantity of antibody for its desired purpose, at a reasonable cost. Hybridomas are anchorage-independent cells, and thus can be grown and maintained in either stationary or suspension culture. The quantity of antibody produced is dependent on the concentration of cells attained and the duration of time they remain viable and secrete MAb. The viability and productivity of hybridomas depends on meeting their nutritional demands; removing metabolic waste products; and providing a stable pH, temperature, and dissolved oxygen for metabolism.

Culture Media

Specially formulated media meets the nutritional needs of growing cells. There are various basal media formulations suitable for growing hybridomas; commonly used formulations include Dulbecco's modified Eagle's medium (DMEM) and RPMI-1640¹². Basal media is frequently supplemented with glutamine, an amino acid used as an energy supplement, which decomposes spontaneously over time at 37°C. Media is usually buffered with sodium bicarbonate, and cultures must therefore be maintained in a carbon dioxide (~5.0%) atmosphere to support cellular metabolism and maintain physiologic pH of the media. A humidified CO₂ incubator, which maintains stable temperature and CO₂ concentrations, is a key component of the cell culture laboratory. One may use organic buffers, such as HEPES, when a CO₂ incubator is unavailable, or as a supplement to bicarbonate-containing media to enhance buffering capability. In some cell lines, however, organic buffers may cause toxicity or decrease cell growth¹³.

Serum (generally fetal calf) is frequently added to media at concentrations of 5-15% to provide essential growth factors and hormones such as insulin, transferrin, and somatomedin¹⁴. Fetal calf serum, which is extremely expensive, can account for up to 70% of the cost of the overall media formulation. In addition to expense, there are several other disadvantages of using serum supplement: it is undefined; its quality and effectiveness vary by batch; it may be contaminated with microbes; and its addition decreases the purity of MAb produced, especially in low-density cultures in which concentrations of antibody are frequently less than 100 m g/ml. As a result, a variety of serum-free, and, more recently, protein-free, media formulations and supplements are available for hybridoma growth. Not all hybridomas grow well or produce antibody well in these defined media formulations. One must allow hybridomas to adapt gradually to the media while monitoring cell viability.

Aseptic Cell Culture Technique

Bacterial, fungal, and mycoplasmal contamination of cells and media is a significant concern in cell culture. Media, personnel, environment, and contaminated cell lines are potential sources of contamination. Media may be supplemented with antibiotics and antifungals; but many laboratories exclude these additives because they may affect cell viability and growth, and thus antibody production. Adherence to aseptic technique is essential. Tissue culture laboratories should be isolated from general purpose laboratories, adhere to strict sanitization standards, minimize personnel traffic, and contain either a Class-II biological safety cabinet or a HEPA-filtered laminar flow hood for cell and media handling and preparation.

Culture Methods

One may expand and maintain hybridomas in either stationary, suspension, or perfusion cultures. In stationary culture (e.g., T flasks), cells and media are not agitated. In suspension culture (e.g., roller bottles or spinner flasks), the cells and media are agitated continuously to maintain the cells in suspension. Suspension culture provides better gas exchange as the media is constantly agitated. A potential disadvantage of suspension cultures is that some hybridomas are sensitive to shear -- the interaction of moving fluid on the cell -- which develops in an agitated culture. Maximum cell concentrations using these methods generally do not exceed several million cells per ml, so they are considered low-density cultures.

In perfusion culture systems (e.g., hollow fiber bioreactors), cells are maintained in a compartment separated from the media reservoir by a selective membrane. Cells are perfused continuously with media, but are not exposed to shear. As perfusion systems are more effective for supplying nutrients and removing waste, high-density cultures approaching 10⁷-10⁸ cells/ml are obtainable.

The way media is processed characterizes cell culture techniques¹⁵. In batch culture, cells and media are inoculated into culture and are not manipulated until harvest. Culture viability is limited by nutrient depletion or metabolic waste accumulation. One may use batch technique in stationary or suspension cultures. The fed-batch technique involves periodically adding fresh media. As nutrients are replenished, the duration of fed-batch culture is prolonged, and can usually be sustained for several weeks. Culture duration is limited by the accumulation of metabolic waste products. Cells attain a higher concentration than in a batch culture. In continuous culture, fresh media is added continually, while spent media with or without cells is removed. As neither nutrient supply nor waste accumulation is limiting, continuous cultures may remain viable for months. One may maintain continuous culture systems as suspension or perfusion cultures.

Perfusion systems are typically maintained as fed-batch or continuous cultures. Growth of hybridomas in continuous perfusion culture takes maximum advantage of the type-II production kinetics of most hybridomas: although some antibody is produced during log-phase cell growth, quantitatively more antibody is produced when cells are maintained in stationary phase, as they are not expending energy for replication¹⁶.

Culture Monitoring

The required degree of culture monitoring depends on the complexity of the in vitro system. While simple stationary batch cultures (e.g., T flasks and gas-permeable bags) require minimal monitoring, high-density culture methods require frequent monitoring. Culture monitoring may include evaluating pH, nutrient, or waste concentrations, as well as cell concentration and viability. Most media formulations containing bicarbonate buffer contain phenol red as a pH indicator; media changes color from red to yellow as it becomes acidic. Glucose is often measured to determine nutrient adequacy; media is replenished when glucose concentration falls below an established set point or range. One may also measure lactate, the end-product of anaerobic glycolysis. Lactate accumulation serves as an indicator of oxygenation, and may also be used to indicate the need to replenish media. One can calculate glucose use or lactate production rates: inexpensive, commercially available glucometers (One Touch™, Lifescan, Inc., Milpitas, CA) are useful for monitoring media glucose¹⁷, and colorimetric test kits for glucose and lactate are also available commercially (Sigma, St. Louis, MO). Cell concentration and viability are determined using a hemocytometer following staining of a culture aliquot with trypan blue. Monitoring frequencies vary, but most laboratories monitor high-density cultures daily. Some laboratories have successfully maintained small-scale systems without weekend monitoring¹⁷.

Concentration and Purification

Purification of antibody may be necessary, depending on the intended use. Purification is generally necessary if antibodies will be coupled to fluorochromes, biotin, or solid-phase affinity matrices². MAbs can be purified using a number of techniques, including precipitation with ammonium sulfate, affinity or ion-exchange chromatography, or gel filtration¹⁸. Purification is more time-consuming and expensive when antibody is obtained in low concentration, e.g., from stationary and suspension cultures. In these culture systems, the antibody may only represent a small fraction of the protein in the sample, with a considerably lower purity and greater sample volume than MAb obtained from or perfusion culture.

Points to Consider When Selecting an In Vitro Method for MAb Production

Investigators use a variety of in vitro culture methods to produce MAb from hybridomas on a research scale (< 1 g of antibody). One should consider a number of factors when selecting an in vitro method or methods for MAb production; this will help to ensure that the methodology selected will satisfy the needs of the producer and end user.

Number and Quantity of MAbs Needed

The number of different hybridomas that must be grown -- i.e., the number of different MAbs that need to be produced, and the quantity of each MAb that will be needed over a given period of time -- is most critical. Consideration may be given to potential advantages or disadvantages of using techniques that produce a small amount of MAb (i.e., potential to perform many small production runs) vs. techniques that produce a larger amount of MAb (i.e., potential to produce the equivalent amount of antibody in fewer production runs). Recommendations for appropriate in vitro culture systems, based on the quantity of MAb to be produced, are available in the literature³.

MAb Concentration

The desired concentration of the antibody is another consideration. Investigators who are accustomed to using murine ascites may require or desire concentrated antibody in the ranges typically found in murine ascites (1.8-13.4 mg/ml)¹⁹. In vitro MAb production techniques vary significantly in the concentration of the MAb product. While some methods (e.g., hollow fiber bioreactors and modular minifermenters) rival the concentrations obtained in mouse ascites, most in vitro methods produce MAbs which are less concentrated than mouse ascites, so consideration must be given to the labor and materials costs required for concentrating dilute MAb products.

MAb Purity

Depending on the intended use of the final product, it may be necessary to purify the MAb. Purity can be significantly enhanced if the hybridoma cells are adapted to growth in reduced-serum or serum-free media.

MAb Quality

Quality refers to the structural characteristics of the MAb that may affect activity (e.g., glycosylation, fragmentation). While the quality of MAbs produced in vitro have been generally shown to be equivalent to those produced in murine ascites, there are examples in the literature that demonstrate differences in MAb quality, dependent on the production method employed. These include differences in MAb glycosylation, solubility, antigen binding activity, conformational stability, and pharmacokinetics^3,20-22.

Laboratory Space and Equipment

The physical laboratory space and available equipment is a limiting factor. As described previously, a laminar flow hood is required for aseptic cell culture technique. CO₂ incubator space is required for most methodologies, although some bench-top systems are now available (Table 1). The amount of CO₂ incubator space required varies significantly for different in vitro methods.

Labor and Cost

The budget should include capital equipment, consumables, and labor. Availability of laboratory personnel with cell culture expertise, or appropriate training and time necessary to develop expertise, are critical. The in vitro methods available for MAb production vary considerably in the necessary cell culture expertise. Some methods require little or no technical expertise, while others require considerable expertise to facilitate optimal system performance.

One should exercise caution in interpreting labor and cost figures reported by equipment suppliers and in published literature. There is frequently insufficient detail to determine exactly what factors were and were not included in labor and cost calculations-costs for syringes, pipettes, and plasticware; labor and material costs for monitoring procedures; costs of concentrating and purifying the MAb; and methods for amortization of capital costs are frequently not included. One should be careful when making cost comparisons, as there is no standard method for reporting these costs. Cost considerations should include labor, materials, and space use costs per mg of MAb produced.

Core Laboratory vs. Investigator Laboratory

Institutions should determine whether it is more feasible for investigators to take responsibility for their own in vitro MAb production, or to establish a central core laboratory operating as an institutional or regional resource. The principal advantages of establishing a core laboratory include centralization of equipment and technical expertise. The decision to establish a core laboratory is based primarily on the anticipated number and quantity of MAbs that must be produced. To the authors' knowledge, no published data suggest a recommended critical production volume necessary to support a dedicated core laboratory.

Contract vs. In-house MAb Production

Consideration might also be given to using an outside contractor for in vitro MAb production. This may be a feasible alternative for some institutions that cannot dedicate the necessary resources for in-house production. Many contract MAb producers use multiple in vitro culture systems for different scales of production. They may also provide such related services as: hybridoma development, screening, characterization, and cryostorage; testing cell lines for adventitious agents; and MAb purification, conjugation, and labeling. Additional information on contract laboratories is provided in Table 2.

Table 2: Resource reference list

In VitroCell Culture Equipment and Supplies
General Cell Culture Supplies Corning Costar Corporation One Alewife Center, Cambridge, MA 02140 Tel: 800-492-1110, Fax: 617-868-2076 http://www.corning.com/lifesciences/ Becton Dickinson Labware (Falcon® and Biocoat® Products) 1 Becton Drive, Franklin Lakes, NJ 07417-1886 Tel: 800-343-2035, Fax: 800-743-6200 email: mail@bdl.bd.com http://www.bdbiosciences.com/ discovery_labware/ Life Technologies (Gibco BRL Products) Grand Island, NY Tel: 800-828-6686, Fax: 800-331-2286 email: info@lifetech.com http://www.lifetech.com Sigma Chemical Company PO Box 14508, St. Louis, MO 63178 Tel: 800-325-3010, Fax: 800-325-5052 email: custserv@sial.com http://www.sigma.sial.com Gas-Permeable Cell Culture Bags i-MAb™ Gas-Permeable Bags Diagnostic Chemicals Limited 160 Christian St., Oxford, CT 06478 Tel: 800-325-2436, Fax: 203-888-1143 email: sales@dclchem.com http://www.dclchem.com Lifecell® Tissue Culture Flasks Baxter Healthcare Corporation, Fenwal Division Deerfield, IL 60015 Tel: 800-766-1077 email: onebaxter@baxter.com http://www.baxter.com/customers/ cust_svc/index.html	Wave Bioreactors™ Panacea Solutions, Inc. 442 Route 202-206 N, Bedminster, NJ 07921-0753 Tel: 500-673-3030 (US) or 732-469-5411 Fax: 732-469-0118 email: info@panasol.com http://www.panasol.com CELLine Culture Systems INTEGRA Biosciences, Inc. 10097 Tyler Place, Suite 10, Ijamsville, MD 21754 Tel: 800-886-8675, Fax: 301-874-5790 email: CELL@integra-biosciences.com http://www.integra-biosciences.com miniPERM® Bioreactor Unisyn® Technologies, Inc. 25 South St., Hopkinton, MA 01748-2217 Tel: 508-435-2000, Fax: 508-435-8111 email: antibody@unisyntech.com http://www.unisyntech.com Cellmax® Culture System Cellco, a Division of Spectrum Laboratories, Inc. 23022 La Cadena Dr., Laguna Hills, CA 92653-1362 Tel: 800-643-3300, Fax: 800-445-7330 email: customerservice@spectrumlabs.com http://www.spectrumlabs.com Cell-Pharm® System CP100 Unisyn® Technologies, Inc. (see above) TECNOMOUSE INTEGRA Biosciences, Inc. (see above)
*Contract In Vitro* MAb Production and Services**
Listings/links at: http://www.antibodyresource.com http://www.nal.usda.gov/awic/ pubs/antibody/company.htm
Core Laboratories
National Cell Culture Center 8500 Evergreen Blvd., Minneapolis, MN 55433 Tel: 800-325-1112, Fax: 612-786-0915 email: ncccinfo@nccc.com http://www.nccc.com
Commercially Available MAbs and Hybridomas
http://www.antibodyresource.com Linscott's Directory of Immunological and Biological Reagents ISBN: 0-9604920-9-7 4877 Grange Rd., Santa Rosa, CA 95404 Tel: 707-544-9555, Fax: 415-389-6025	Hybridoma Data Bank Tel: 703-365-2700 x 508 email: hdb@atcc.org http://www.atcc.org American Type Culture Collection 10801 University Blvd., Manassas, VA 20110-2209 Tel: 800-638-6597, Fax: 703-365-2750 email: www@atcc.org http://www.atcc.org

Select In Vitro Production Methods

The following information highlights some of the in vitro technologies most commonly used for MAb production. Table 1 provides general information for select commercially available in vitro culture systems. It is important to note that MAb production in all in vitro systems is hybridoma cell line-dependent, and is also dependent on the culture method, type of media, and procedural protocols used. For this reason, there is typically a wide range in MAb production parameters among different hybridoma cell lines cultured in vitro.

Tissue Culture Flasks

One may use either tissue culture flasks (T flasks) or other cell culture dishes for stationary culture. Cell culture flasks and dishes are available commercially from many companies (Table 2). T flasks are available in a variety of sizes, from 12.5 cm² to 300 cm², which hold maximum working volumes of approximately 5-400 ml. 225 cm² flasks with a maximum working volume of 370 ml are commonly used for MAb production. Cells and media are placed in the flask, maintained in a CO2 incubator, and handled as a batch culture. The initial seeding density required for reproducible, rapid proliferation of cells varies among cell lines, and the time between the end of cell proliferation, start of decreased cell viability, and peak antibody level is also hybridoma-dependent23.

Incubation times are typically 7-10 days before harvest. MAb concentration is typically in the range of 10-100 µg/ml. Little or no monitoring is required, and the flask can be harvested when the media turns yellow (becomes acidic) and cell viability drops to approximately 5-10%²⁴.

T flasks are simple to use and require minimal technical expertise. They are relatively inexpensive and are stackable, which minimizes incubator space requirements. The principal disadvantage is that MAb concentrations are very low, necessitating concentration, and only small quantities of MAb are produced. T flasks should be considered for use when only small amounts of MAb are needed; low MAb concentrations are satisfactory; personnel have minimal cell culture expertise; and low-cost production methods are required. T flasks are frequently used to grow cells for subsequent inoculation into other culture systems.

Roller Bottles and Spinner Flasks

Investigators have historically used roller bottles and spinner flasks for production of MAbs. MAb concentrations obtained with these two methods are typically greater than stationary culture techniques, ranging from approximately 10-220 µg/ml^10,25, and the average culture duration is longer -- approximately 12 days¹⁰. Culture volumes are typically <2 L³. Advantages and disadvantages are similar to those described for T flasks, but roller bottles and spinner flasks require more incubator space, and are more expensive than T flasks.

Gas-Permeable Cell Culture Bags

Flexible, gas-permeable pre-sterilized disposable cell-culture bags are commercially available for MAb production. The bags feature attached ports, and tubing with roller clamps, which are used for inoculation of cells and media, sampling during production, and harvest (Fig. 1). The bag is inoculated with cells and media, placed in a CO2 incubator, and generally handled as a batch culture. One harvests the media when MAb concentrations plateau or cell viability drops to approximately 10%^26,27.

Figure 1: The iMAb™ gas-permeable bag

Compared to standard T flasks, bags have more surface area for CO₂ and oxygen diffusion, improving cell oxygenation. While cells do not necessarily grow to higher densities by comparison to T flasks, there appears to be more MAb produced per cell²⁶, and cell viability is maintained for longer periods of time²⁷. Cell culture bags have some other advantages over dishes or flasks: the bags are completely enclosed systems, which reduces the opportunity for microbial contamination, and they may be placed flat on the incubator floor or hung on a stand, potentially reducing required incubator space. In the literature, MAb production in gas-permeable bags is compared to production in T flasks^26-28, hollow fiber bioreactors, and ascites²⁸.

Commercially available i-MAb™ MAb production and isolation kits containing a 1 L bag (which holds up to 500 ml culture volume), inoculation syringe, dialysis tubing, and reagents for concentration of the harvest volume to approximately 30-50 ml are available from Diagnostic Chemicals Limited. Bags can also be purchased separately. (Tables 1 and 2). Gas-permeable bags (Lifecell® Tissue Culture Flasks), in 300 ml (#4R2111), 1 L (#4R2110), and 3 L (#4R2113) sizes (which hold 50-150 ml, 100-500 ml, and 0.5-2 L cell culture volumes, respectively), are available from Baxter Healthcare Corporation, Fenwal Division (Table 2). One can use the Lifecell® septum and 33 mm bottle cap (#3L2100) for media transfer via gravity flow from a media bottle to the bag; a solution transfer pump (#4R4345) and transfer set (#4C2474) are also available for automated filling.

Wave Bioreactors™

Recently introduced and featuring new technology, the wave bioreactor™ is a flexible, plastic pre-sterilized, disposable cell culture bag (Cellbag™). The user partially fills the bag with culture media and cells, inflates it to rigidity, and maintains it on a rocking platform in a CO₂ incubator (Fig. 2). The headspace in the bag is continuously aerated. These bags are not gas-permeable; the wave-induced agitation generated by the rocking mechanism effects oxygen transfer and mixing. The wave action increases the air-liquid surface area for oxygen transfer, mixes the fluid in the bioreactor, and suspends the cells with low shear. Two liter bags (0.1-1 L culture volume) and 20 L bags (1-10 L culture volume) are suitable for small-scale MAb production; scales up to 500 L culture volumes have been demonstrated using this technology.

Figure 2: The Wave Bioreactor™

The bag has an inlet air filter and exhaust air filter, a needle-less syringe fitting for sampling from the bag, and a tubing connector for additions to, and harvests from, the bag. A laminar flow hood is not required for adding to, nor sampling from, the bag. The wave bioreactor™ rocking unit with holder accommodates two, 2 L bags or one, 20 L bag; has adjustable rocking speed with a digital read-out; and an electrical air pump that keeps the bag aerated, and requires 2 bar (30 psig) compressed air. A bench-top model with a heater and controller, as well as a CO₂/air mixing unit, is also available. The bioreactor may be handled as a batch or fed-batch culture system. In fed-batch mode, the volume of media may be increased gradually as cell density increases. Cell densities reach > 7 x 10⁶ cells/ml.

CELLine Culture Systems

The CELLine culture devices are based on membrane compartmentalization technology (Fig. 3). Cells and secreted MAb are retained in a small-volume cell compartment, which is separated by a semi-permeable membrane from the larger volume of basal medium contained in the nutrient medium compartment above. A gas-permeable membrane on the bottom of the cell compartment provides for oxygen and CO₂ exchange. There is a wide mouth port with a screw cap for access to the nutrient medium compartment, and a cell compartment port with a screw cap and septum which is accessed via pipette (Fig. 3). The systems are constructed of clear polystyrene, and are pre-sterilized and disposable.

Figure 3: The CELLine culture system

The manufacturer reports that if serum is used, the costs are reduced considerably because serum is only supplemented in the small-volume cell compartment. Cells reach higher densities, and significantly higher concentrations of MAb are obtained in a smaller volume, as compared to other stationary culture techniques, minimizing downstream concentration steps. The systems are easy to handle and use, and the units are interlocking and stackable to minimize CO₂ incubator space requirements. Use of these systems is described in a recent publication²⁹.

Several sizes and types of CELLine culture devices are available. The CL 6-well consists of six parallel chambers, each with a 0.75 ml cell compartment and a 5-30 ml nutrient medium compartment. This system is especially useful for molecular biology, screening different clones, optimizing media, or pharmacological testing. The CL 350 has a 5 ml cell compartment and a 50-350 ml nutrient media compartment, and is useful for small-scale MAb production. The CL 1000 has a 15 ml cell compartment and a 100-1000 ml nutrient medium compartment for increased MAb production.

Protocols for use of these systems are available from the manufacturer. Briefly, cells are inoculated via a pipette into the cell compartment, and basal medium is poured into the nutrient medium compartment. Approximately seven days later, the basal medium is removed from the nutrient medium compartment and a portion of the cell suspension is harvested from the cell compartment. Fresh medium is added to the cell and nutrient compartments. This process is repeated approximately every three days.

miniPERM® Bioreactor

The miniPERM® bioreactor is a modular minifermenter. The components and operation of this system and MAb production parameters have been described in detail and compared with other culture methods^30,31. This modular system consists of a disposable 40 ml production module and a nutrient reservoir which holds up to 550 ml of media. When assembled, a semi-permeable dialysis membrane separates the two modules. The cells and secreted MAb are retained in the production module. Nutrients, metabolic wastes, and dissolved gases are exchanged across the dialysis membrane between the production and nutrient modules. The production module has a gas-permeable silicone membrane that permits oxygen and CO₂ exchange. Baffles in the membrane facilitate suspension of the cells and increase the surface area for gas exchange. Luer-lock connections on the production module are used for cell inoculation and sample collection.

The miniPERM® bioreactor (Fig. 4) is designed to rotate on a bottle turning device within a CO₂ incubator. A larger bottle turning device that accommodates up to four bioreactors is also available (Fig. 5). The bottle-turning device keeps the cells in the production module in suspension, and agitates the nutrient medium to facilitate passage of nutrients and metabolites across the dialysis membrane. The production module is pre-sterilized and disposable. The nutrient reservoirs may be autoclaved and re-used up to 10 times. Pre-assembled, pre-sterilized miniPERM® bioreactors are also available.

Figure 4: The miniPERM® bioreactor

Figure 5: Four miniPERM® bioreactors on a bottle turning device with remote control unit

The user inoculates cells into the production module and adds medium to the nutrient module. The miniPERM® is maintained as a fed-batch culture system with periodic harvest of product from the production module. Nutrient medium is typically replaced every one to four days, depending on cell density. Reported advantages of this system include: growth of cells at high cell densities (> 107 cells/ml); high MAb concentration; high product purity related to serum reduction or removal from the nutrient compartment; relative ease of use; ability to maintain the cultures for a relatively long period of time; and ability to reuse some components.

Hollow Fiber Bioreactors (General Information)

Originally developed by Knazek, et al.³², hollow fiber bioreactor (HFB) systems were modeled after, and intended to simulate, the in vivo capillary system to provide a more physiologic environment for cultured cells with regard to nutrient supply, metabolic waste removal, and pH, while providing a stable pericellular microenvironment³³. Detailed information regarding the design and operation of HFB systems is presented elsewhere³⁴, and is also available from the respective manufacturers of the commercially available systems.

Small-scale production systems are available commercially from several manufacturers (Tables 1 and 2). The basic design of a bioreactor circuit is illustrated schematically in Fig. 6. Included within the circuit is a media bottle; a variable speed pump to maintain continuous, unidirectional media flow; a HFB cartridge; and a means of providing oxygen and CO₂ exchange. Most systems have media sampling ports where media samples are obtained for monitoring. HFBs and media flow-path are pre-sterilized and disposable. All systems have an instrument control module for regulation of media flow rate and other features unique to each system.

Figure 6: Hollow fiber bioreactor circuit

Schematic diagrams of a HFB in longitudinal and cross-sectional views are presented in Fig. 7. The bioreactors typically consist of bundles of hollow fibers in a plastic cartridge, through which culture media is continuously perfused in the intracapillary space. The cells are grown in the extracapillary space that surrounds the fibers. The walls of the hollow fibers serve as semi-permeable ultrafiltration membranes. The pore size of the membrane, depending on the molecular weight cut-off, is small enough to retain cells and secreted MAb in the relatively small volume of the extracapillary space, while permitting gas, nutrients, and metabolic waste products to diffuse freely across the membrane according to hydrostatic pressure differences and concentration gradients. Harvest ports provide access to the extracapillary space for examination of cells and MAb harvest.

Figure 7: Hollow fiber bioreactor: longitudinal and cross-sectional views

Respective manufacturers provide details on techniques for bioreactor system set-up, operation, and maintenance. Briefly, the user sets up the system, perfuses it with media, and inoculates the cells into the bioreactor. One may harvest periodically via syringe from the harvest ports, typically beginning approximately five to seven days after system set-up. Media bottles are replaced with fresh media as necessary.

Hollow fiber bioreactors consistently produce a concentrated MAb product^17,35-38. The production of a highly concentrated MAb product in a small volume precludes the need for extensive downstream concentrating techniques. Other reported advantages of HFB systems are that the cells are protected from shear^35,39; the cells can be grown to very high densities (10⁷-10⁸ cells/ml); and cell viability and production are maintainable for extended periods of time^15,34,40,41, ranging from weeks to months. Purity of the MAb product in HFB systems has been facilitated by gradual serum reduction in the culture medium without significant inhibitory effect on production^34,35,37,42. Disadvantages include the potential for mechanical failure and the need for technical expertise and familiarity with the system. MAb production in HFB systems has been compared to murine ascites^17,28, T flasks, and gas-permeable bags²⁸, including labor and cost comparisons.

Cellmax® Artificial Capillary Cell Culture System

A single HFB module with a pump station and power supply, or a base unit with a central pump station designed to hold up to 4 HFB modules, each with independent media bottle and flow-path, are available (Cellmax®, Fig. 8; and Cell-max® Quad, respectively). The HFB modules are easily disconnected from the pump station for transport to a laminar flow hood for manipulations. An AC motor cable connects the base unit to an electronic control unit that is placed outside the incubator and connected to a standard electrical outlet. Larger-scale systems are also available. Operation and MAb production in this system have been described⁴³.

Figure 8: Cellmax® artificial capillary cell culture system

Cell-Pharm® System CP100™

The Cell-Pharm® System CP100™ is a bench-top culture system with a media heating block, as well as temperature and CO₂ control, eliminating the need for a CO₂ incubator (Fig. 9). The HFB with media flow-path and oxygenator clip easily onto the unit. Bottled or bagged media may be used. The MAb product can be harvested manually via positive displacement through the harvest port septum or via a Cell-Pharm AutoHarvester™ (Fig. 9). A laminar flow hood is not required for manipulation of the system. Larger-scale production systems are also available⁴⁴.

Figure 9: Cell-Pharm® System CP 100™ with AutoHarvester™

TECNOMOUSE

The TECNOMOUSE system consists of an instrument module on which is mounted a removable rack for placement of one to five bioreactors, each with independent media flow-path, and a pump station which contains individual pump cassettes for each bioreactor (Fig. 10). Unlike conventional HFBs containing a cylindrical bundle of fibers within a hard plastic shell, the fibers in this HFB are arranged in single parallel rows and encased in a silicone membrane, within a flat rectangular plastic cartridge. Via a gas dome with individual gassing ports for each bioreactor, incubator air is pumped to channels outside the gas-permeable silicone membrane, thus providing direct gassing to the culture. An AC motor cable connects the instrument module to an electronic control module that is placed outside the incubator, and a power cord connects the control module to a standard electrical outlet. The module can be programmed to change the media flow rate in fixed or variable increments over a specified time interval. A printer port is available for printer interface. A laminar flow hood is not required for manipulation of the system. A thermohood is available for bench-top operation, which does not require a CO₂ incubator.

Figure 10: The TECNOMOUSE system

Other In Vitro MAb Production Methods

Other in vitro techniques include growing cells in dialysis tubing within a culture bottle⁴⁵, and use of oscillating bubble dialysis chambers⁴⁶ or tumbling chambers⁴⁷. Laboratory-scale stirred tank reactors^39,48, fermenters⁴⁹, ceramic-matrix bioreactors³⁷, and packed-bed bioreactors⁵⁰ are also in use. There are reviews of other methods in the literature^3,25.

Commercially Available MAbs and Hybridomas

Two useful resources for finding and purchasing commercially available MAbs are the "Antibody Resource" web site, which contains links to many commercial MAb suppliers, and "Linscott's Directory of Immunological and Biological Reagents" (Table 2). The production method (in vivo or in vitro) for commercially available MAbs may or may not be indicated in the product information.

An international Hybridoma Data Bank provides a comprehensive directory of information on hybridomas and other cloned cell lines and their immunoreactive products, such as MAbs. The American Type Culture Collection is responsible for collection and dissemination of data contained in this resource. Information on a hybridoma's construction; the reactivity and non-reactivity of its secreted MAb; and the availability of individual hybridomas and their MAb products are included in the database (Table 2).

Other Resources

There are additional resources that provide very useful information about in vitro MAb production. The Johns Hopkins Center for Alternatives to Animal Testing (CAAT) has published the proceedings from a workshop entitled, "Alternatives in Monoclonal Antibody Production," which is available on their web site at http://altweb.jhsph.edu/science/mab/proceedings.htm. The workshop, held September 24-25, 1997, was sponsored by The Johns Hopkins CAAT and OPRR. Topics covered during this workshop included scientific presentations on the uses of MAbs; multiple methods of in vitro MAb production, including advantages, disadvantages, and comparisons among different in vitro techniques; comparisons of ascites and in vitro MAb production methods; and animal welfare concerns. Other topics included the responsibility and needs of IACUCs in advising investigators; core laboratories; regulatory issues in the United States; and the European perspective. Conclusions and recommendations follow the workshop proceedings.

Another useful resource is the Animal Welfare Information Center Resource Series No. 3, Information Resources for Adjuvants and Antibody Production: Comparisons and Alternative Technologies, available on the AWIC web site at http://www.nal.usda.gov/awic/pubs/antibody.htm. This resource includes an antibody production bibliography, a guidelines and policies bibliography, a listing of books and proceedings, a select list of companies and institutional resources providing new technologies, educational web site resources, and a listing of related organizations.

Monoclonal Antibody Production: The Report and Recommendations of ECVAM Workshop 23, by U. Marx, J.M. Embleton, R. Fischer, et al. (ATLA; 25:121-137, 1997), was published by the European Centre for the Validation of Alternative Methods following a workshop evaluating the present status of in vitro methods for MAb production and comparing the advantages and disadvantages of the in vitro methods with those of the in vivo ascites method. The specific conclusions and recommendations made during the workshop are included. This report is also available on the AWIC web site described above.

A feature in a recent issue of the journal Research in Immunology entitled, the "74th Forum in Immunology. In Vitro and In Vivo Production of Monoclonal Antibodies: Current Possibilities and Future Perspectives" (149(6):529-620, 1998), is dedicated to the topic of MAb production. Future Directions Many culture methods are now available for small-scale in vitro production of MAbs. As the cumulative experience with these in vitro culture techniques increases, and larger numbers of hybridoma cell lines are critically evaluated in these systems, optimization of culture system design and operational protocols will likely lead to improved MAb production and reduced costs. These technologies provide viable alternatives to the use of mice for MAb production and should be considered, and used, whenever feasible.

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