Upstream Processing: Regulatory Considerations Regarding Quality Aspects of Monoclonal Antibodies

An alternative to the hybridoma method is producing engineered antibody molecules and fragments by in vitro methods.
Jul 01, 2007


In the past two decades, more than 20 therapeutic monoclonal antibodies (MAbs) targeting a range of antigens (and working through a variety of mechanisms) have been approved for treatment of serious diseases. First to be approved were murine antibodies, followed by humanized molecules with superior efficacy, safety, and tolerance. Most of the licensed MAbs have been made by the hybridoma method and expressed in cell culture.2, 3 This review describes the next generation of MAbs, taking next generation antibodies into account, and suggests considering international regulatory guidelines for qualifying and characterizing monoclonal-antibody-producing source cells at different phases of development. In addition, in vivo and in vitro manufacture of MAbs is outlined. Adhering to the guidelines ensures a finished product of consistent pharmaceutical quality.

Atibodies (immunoglobulins) are glycoprotein molecules. They are made by foreign immunogen-stimulated B-cells and secreted into the blood to react with antigens present on soluble or cell-surface immunogens. Antibodies induce different immunological effects, such as neutralization of pathogens either by forming antibody-antigen complexes or by involving other immune cells.1 Immortalized cell cultures of antibody-producing B cells can be propagated outside a living organism to maintain infinite supply. Antibodies constructed by this method are directed against single epitopes and are known as monoclonal antibodies (MAbs). Medicinal products containing MAbs as active substances have been developed in major therapeutic areas such as cancer, autoimmune and inflammatory diseases, and for several orphan drug indications.


MAbs recognize and bind to receptors (antigens) of specific targets. These potential targets are proteins, which, unlike other cellular macromolecules (nucleic acids, polysaccharides, or lipids), have immunogenic properties. Drug development attempts to identify antibodies that affect protein–protein interactions by competing with endogenous molecules for binding sites.4 To rationally design potent therapeutic antibodies, the target's function should be well understood in the context of the immunopathologic pathway of the disease. It should also play a key role either as soluble factor (e.g., growth factor or cytokine) or as cell type implicated in tissue damage.

After being isolated and purified, target proteins are subjected to antibody screening procedures: Purified protein is used for the immunization of animals. From the natural antibody repertoire, antibody molecules with high specificity and affinity for the target are selected and their potencies defined in bioassays. The selected antibodies may work via interfering, blocking, or stimulating effects as required.5

Table 1. Description of hybridoma technology7
The standard technology to immortalize activated B cells, which has been of fundamental importance in the monoclonal antibody history, is the hybridoma method. Most currently licensed MAbs were created this way. Developed in the mid 1970s by Köhler and Milstein, it is based on the fusion of rodent antibody-secreting B-cells with rodent tumor (myeloma) cells, followed by segregation steps to isolate single clones.6 The hybridoma method is outlined in Table 1.

Because murine antibodies are immunogenic in humans, researchers have tried to limit the human antimouse immune reaction. One strategy has been to transfer mouse hybridoma technology to human cells. Ethical concerns associated with the immunization of human subjects, along with the lack of a suitable human myeloma cell line, have slowed the development of this approach. Nevertheless, it should be noted that human antibody-producing cells can be immortalized by infection with viruses. The small DNA Epstein-Barr herpes virus has been used to infect and immortalize human B cells. But current methods allow only inefficient immortalization rates and low antibody yields.8 Additionally, since immortalization involves the transfer of viral genes into the parent cell line, there is a risk of generating infectious viruses.

Humanized MAbs can alternatively be constructed using transgenic mice carrying human immunoglobulin genes, thereby minimizing the immunogenicity of resulting antibodies.9,10,11 Although such animals express human antibodies, their production still requires standard hybridoma methods (immunization, B-cell isolation, and hybridoma formation).

Also, in mouse cell lines (used both for production purposes and as fusion partners for the preparation of hybridomas), recombinant DNA technologies facilitate the grafting of murine-antigen-recognizing DNA regions to human immunoglobulin genes integrated with mammalian expression vectors. This approach allows the stable expression of humanized antibodies in a range of cell lines such as Chinese hamster ovary (CHO) cells.12


Once a clone has been identified, manufacturers should establish a seed-lot system to ensure a consistent supply of product with reproducible quality. This is achieved by means of creating a GMP-compliant source cell bank system, consisting of a master cell bank (MCB) and a working cell bank (WCB).13,14,15,16