Monoclonal antibodies play a critical role in research, diagnostics and product development and provide numerous benefits to patients in diverse therapeutic areas.
mAbs are capable of recognizing only one epitope found on an antigen. Therefore they have greater specificity than polyclonal antibodies (pAbs), which makes them ideal for diagnostic and therapeutic uses. Custom monoclonal antibody production requires a different skillset than polyclonal antibody production, and often results in additional costs and time.
The specificity of mAbs has made them ideal for medical applications such as diagnostic and therapeutic use. So what are the key considerations in the production of monoclonal antibodies?
Hybridoma generation
Hybridoma cells have the ability to reproduce and secrete the antibody of interest while continuing to proliferate indefinitely. The unlimited replicative potential of hybridoma cells produces significant levels of mAbs. The generation of hybridomas and production of mAbs generally takes nine to ten months.
Traditional methods such as in vivo ascites production in animals are less common today; ascites production is banned outright in Europe. Large quantities of highly concentrated antibodies can be produced using various approaches such as tissue culture flasks, spinners, roller bottles, stirred tank fermenters and hollow fiber bioreactors.
Antibody phage display (APD)
Scientists use APD to generate fully human antibodies against a selected antigen. APD has been utilized to generate research antibodies and antibody-based therapeutics (Nixon, 2014). Phage display technology was originally described by George Smith in 1985 (Smith, 1985). Following this discovery, scientists demonstrated that antibody fragments could be displayed on the surface of phage particles (McCafferty, 1990; Barbas, 1991). This breakthrough allows human antibodies to be manufactured using in vitro processes.
In general, APD involves three major steps. First, a vector is constructed to allow a large number of different light chain and heavy chain genes to be incorporated (Lerner, 2016). Next, the vector is expressed in a host type that allows the coupling of the genotype (i.e., antibody gene) with the phenotype (i.e., antibody molecule expressed outside the host) (Lee, 2007; Lerner, 2016). Once a large collection of phage-bearing antibody molecules has been generated, researchers apply a selection process to isolate phages that bind to a given antigen. The third step, known as “panning,” occurs when phages are exposed to the antigen and only the antigen-bound phages are replicated via infection of E. coli to amplify the monoclonal antibody construct (Hoogenboom; 2005; Lerner, 2016). The antibody genes in these phage particles are used in expression systems to generate purified antibodies (Shukra, 2014; Lerner, 2016).
Humanized mice
In the 1990’s, scientists disclosed the first genetically engineered mice that expressed fully human antibody repertoires (Lonberg, 1994; Green, 1994). These human immunoglobulin transgenic mice express B cell receptors that are hybrids of mouse and human components, and their B cells develop and mature into seemingly normal B cell subtypes (Lonberg, 2008). IgM (Green, 1994) and IgB (Lonberg, 1994) mAbs, which specifically recognize the antigens of interest, are isolated.
In 2006, the FDA approved the first human mAb generated in a transgenic mouse for epidermal growth factor receptor-expressing colorectal cancers. (Jakobovits, 2007). Recently, Regeneron Pharmaceuticals developed the VelocImmune® mouse, which enabled researchers to rapidly progress ten different fully human antibodies into human clinical trials. (Murphy, 2014).
Another approach is the use of humanized immune system models to generate mAbs. For instance, inoculating newborn immunodeficient mice with human fetal or umbilical cord hematopoietic stem cells, which can result in a robust engraftment of a number of immune cells. (Becker, 2010). However, results generally have been underwhelming. (e.g., Villaudy, 2014).
Process for optimal antigen specific mouse mAb production
Mice are most commonly used for the production of mAbs, in contrast with pAb production which generally uses rabbits and other larger animals. Researchers must use great care when fusing B-cells with myeloma cells. Hybridoma cells are very fragile immediately after the fusion process. Fetal bovine serum (FBS) is commonly used to provide cellular factors necessary for growth. Use of FBS has drawbacks, however, such as the potential for contamination with infectious agents. As a result, many commercial serum-free media (SFM) products have been developed to support the growth and maintenance of cells.
When researchers determine that particular hybridoma clones are acceptable for production, they are frozen in cryopreservation media and stored in the vapor phase of liquid nitrogen.
Scaling up production of mAbs
Scientists can escalate production of mAbs in a variety of ways, such as the hollow fiber reactor method. These are vessels used to collect tissue culture supernatants. Thousands of semi-permeable hollow fibers are arranged in parallel within a cartridge that provides a 3D environment for cellular growth and has inlet and outlet ports on both sides of the fibers (Dewar et al.,2005). One of the benefits of this method is the continuous removal of waste products.
Careful planning and preparation are critical when producing high quality hybridomas and mAbs. By working with an experienced partner, researchers can eliminate many of the common pitfalls while also saving time and money.
Looking toward the future of antibody optimized production
The production of a custom antigen specific mouse mAb is a time-consuming and technically demanding process. However, once stable hybridomas have been established, they can be expanded and frozen to create a reliable source of the mAb. Scaling up the production of mAbs can be done using a wide range of various methodologies.
Careful planning and preparation are required to produce high-quality hybridomas and mAbs. Working with a partner with deep practical experience can both ensure the optimization of each step along the way and save time and money by eliminating the most common pitfalls.
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