Scale monoclonal antibodies

Figure 10.3. Schematic representation of monoclonal antibody production using immortalized hybrid cells that secrete antibodies selective for the target antigen. The mortal, immune B cells Isolated from mice immunized with a target antigen are fused with myeloma, immortal B cells that express defective antibodies. The selecting of antigen-specific, immortal hybrid cells (hybridomas) results in identification of unique clones of cells that express antibodies with high specificity and affinity (monoclonal antibodies). These cells are cloned and expanded for large-scale monoclonal antibody preparations.

Projected Intensified Large-Scale Monoclonal Antibody Manufacturing Process... 

Lihme, A., and Bendix Hansen, M. (1997). Protein A mimetic for large scale monoclonal antibody purification. Biotechnol. Lab. 15, 30-31. 

Kelley, B. (2007) Very large scale monoclonal antibody purification the... 

The main disadvantage of all these systems is the Hmitation of scale-up. Monoclonal antibodies are produced by multiplying the hollow fiber systems and stirred tank reactors with membrane aeration are known up to 100 liter. Small quantities of product can be produced by these systems but they are not suitable for real industrial scale-up. 

Fig 2 Immunogold negative staining, with a monoclonal antibody (JIM 5 (13)) that recognises a relatively unesterified pectic epitope, of rhamnogalacturonans extracted from onion cell walls. Arrows indicate 5 nm colloidal gold particles. Scale bar represents 200nm. 

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

Monoclonal Antibodies. HA-1 A is a human monoclonal IgM antibody that binds to LPS and lipid A. The initial phase 3 trial demonstrated no overall benifit of HA-1 A compared with placebo (Z5). However, HA-1A appeared to afford significant protection to a subgroup of patients with gram-negative bacteremia (Z5). A second large-scale trial documented a lack of overall clinical benefit of HA-1A (W11). 

The 1980 s and the early 1990 s have seen the blossoming development of the biotechnology field. Three-phase fluidized bed bioreactors have become an essential element in the commercialization of processes to yield products and treat wastewater via biological mechanisms. Fluidized bed bioreactors have been applied in the areas of wastewater treatment, discussed previously, fermentation, and cell culture. The large scale application of three-phase fluidized bed or slurry bubble column fermen-tors are represented by ethanol production in a 10,000 liter fermentor (Samejima et al., 1984), penicillin production in a 200 liter fermentor (Endo et al., 1986), and the production of monoclonal antibodies in a 1,000 liter slurry bubble column bioreactor (Birch et al., 1985). Fan (1989) provides a complete review of biological applications of three-phase fluidized beds up to 1989. Part II of this chapter covers the recent developments in three-phase fluidized bed bioreactor technology. 

Ghetie, V., Ghetie, M.-A., Uhr, J.W., and Vitetta, E.S. (1988) Large scale preparation of immunotoxins constructed with the Fab fragment of IgGl murine monoclonal antibodies and chemically deglyco-sylated ricin A chain./. Immunol. Meth. 112, 267-277. 

Mammalian cell suspension cultures are the preferred choice for large-scale recombinant protein production in stirred-tank bioreactors. The most widely used systems are Chinese hamster ovary (CHO) cells and the murine myeloma fines NSO and SP2/0. In half of the biological license approvals from 1996-2000, CHO cells were used for the production of monoclonal antibodies and other recombinant glycosylated proteins, including tPA (tissue plasminogen activator) and an IgGl fusion with the tumor necrosis factor (TNF) receptor, the latter marketed as Enbrel [7]. 

In addition to the above extracellular parameters, cell concentration and cell activity are two important cell-associated parameters that determine how well a fermentation process is performing. The manufacturing of biological products (antibiotics, amino acids, monoclonal antibodies, and other protein products) at large scales requires that cells be cultured at high cell densities and stay metabolically active. Consequently, much effort has been expended to develop techniques that can allow the estimation of cell concentration and cell activity in real time during a fermentation. 

Fig. 1. Hydra oligactis whole mount labeled with monoclonal antibody JDl. (A) Isolated ganglionic neuron in the body column, scale bar = 50 om (B) hypostomal nerve net with sensory neurons of the mouth at the left and ganglionic neurons of the perihypostomal ring to the right, scale bar = 50 pm (C) cell bodies of hypostomal sensory neurons extending from the mesoglea (processes) to the surface of the ectoderm, scale bar = 25 pm.

Fig. 2. Hypostome (mouth) of a Hydra oligactis whole mount labeled with monoclonal antibody DB5. (A) Nonconfocal image of sensory neurons and processes (B) confocal optical section of the same field as (A) illustrating details of neuronal cell bodies and processes. Scale bar = 25 pm.

Fig. 3. Head of a Hydra oligactis labeled with monoclonal antibody DBS. (A) Neurons of hypostome and perihypostomal ring (left) and base of tentacle (right) at high magnification (x 20 objective) note considerable out-of-focus labeling of the tentacle (B) same specimen as in (A) but at lower magnification (x 10 objective) although the hypostome is out of focus, the neuronal net of the tentacles is much clearer. Scale bars = 50 pm.

Mammalian cells are commonly employed for the production of therapeutic and diagnostic proteins, since they are able to correctly synthetize the large and complex structures that the human body requires as medicine [1]. Nowadays, they are employed for the large-scale production of recombinant therapeutic proteins, monoclonal antibodies (MAbs) and viruses used in the preparation of vaccines (e.g. against rabies, hepathytis B, polio, etc) [2]. An overview of some licensed/approved products derived from mammalian cell culture is given in Table 1. 

Over half of all biopharmacuticals thus far approved are produced in recombinant E. coli or S. cerevisiae. Industrial-scale bacterial and yeast fermentation systems share many common features, an overview of which is provided below. Most remaining biopharmaceuticals are produced using animal cell culture, mainly by recombinant BFIK or CFiO cells (or hybridoma cells in the case of some monoclonal antibodies Appendix 1). While industrial-scale animal cell culture shares many common principles with microbial fermentation systems, it also differs in several respects, as subsequently described. Microbial fermentation/animal cell culture is a vast speciality area in its own right. As such, only a summary overview can be provided below and the interested reader is referred to the Further Reading section. 

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