Monoclonal antibodies standardization

At the Pharmaceutical Production Research Facility of the University of Calgary experimental data have been collected (Linardos, 1991) to investigate the effect of glucose to glutamine ratio on monoclonal antibody (anti-Lewish IgM) productivity in a chemostat and they are reproduced here in Tables 17.8, 17.9 and 17.10. Data are provided for a 5 1 (standard for cell culture media), 5 2 and 5 3 glucose to glutamine ratio in the feed. The dilution rate was kept constant at 0.45 d . 

M. L. Roy, M. J. Pikal, E. C. Rickard, and A. Maloney, The effects of formulation and moisture on the stability of a freeze-dried monoclonal antibody-vinca conjugate A test of the WLF glass transition theory Dev. Biol. Standards, 74, 323 (1991). 

With the advent of monoclonal antibodies, the search for tumour-specific antigens became the biggest cottage industry since unemployment. It rapidly became apparent that a 90 kD disulfide-bridged transmembrane protein was present in many tumour cells - it was the transferrin receptor, and as they say, the rest is history. It has become a standard procedure to determine the in vivo growth potential of tumours by measuring transferrin receptor expression. 

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

Fig. 2.2 Stability of IgCi monoclonal antibody added to sterile plant and animal cell culture media. ( ) Murashige and Skoog (MS) medium (A) Dulbecco s minimal essential medium (DMEM) with 10% serum and (A) serum-free Ex-cell 302 medium. The error bars indicate standard errors from triplicate flasks. (Reproduced with permission, from B. M. -Y. Tsoi and P. M. Doran, Biotechnol. Appi. Bio-chem. 2002, 35, 171-180. Portland Press on behalf of the IUBMB.)...

For other plant-derived antibodies, stability was shown to be similar to mammalian counterparts. For instance, a humanized anti-herpes simplex virus monoclonal antibody (IgGl) was expressed in soybean and showed stability in human semen and cervical mucus over 24 h similar to the antibody obtained from mammalian cell culture. In addition, the plant-derived and mammalian antibodies were tested in a standard neutralization assay with no apparent differences in their ability to neutralize HSV-2. As glycans may play a role in immune exclusion mechanisms in mucus, the diffusion of these monoclonal antibodies in human cerival mucus was tested. No differences were found in terms of the prevention of vaginal HSV-2 transmission in a mouse model, i.e. the plant-derived antibody provided efficient protection against a vaginal inoculum of HSV-2 [58]. This shows that glycosylation differences do not necessarily affect efficacy. 

Antibodies have and likely will find additional use in transplantation-related medicine. In general, cell-mediated immunological mechanisms are responsible for mediating rejection of transplanted organs. In many instances, transplant patients must be maintained on immunosuppressive drugs (e.g. some steroids and, often, the fungal metabolite cyclosporine). However, complications may arise if a rejection episode is encountered that proves unresponsive to standard immunosuppressive therapy. Orthoclone OKT-3 was the first monoclonal antibody-based product to find application in this regard. 

The most commonly used format for quantitation assays is the sandwich assay format. Typically, a monoclonal antibody (MAb) is used to capture the product. It is then detected by another antibody, usually enzyme-labeled. A reference standard is used from which to compare the response of an unknown test sample. There is a relative increase in measured response (optical density) with increasing analyte concentration. Figure 11.2 is an example of a typical ELISA standard curve. 

CE is applied to two major categories of quality release testing identity and impurity testing. Identification assays are intended to ensure the unique identity of an analyte in the sample. This is normally achieved by comparison of a property of the sample (e.g., spectrum, chromatographic behavior, chemical reactivity, etc.) to that of a reference standard. As shown in Figure 9, CZE can be used to determine identity for monoclonal antibodies and proteins based on their unique electrophoretic profile. 

A further advance in antibody technology is the development of transgenic mouse human strains. XenoMouse animals have been engineered in such a way that they now produce exclusively human antibodies rather than murine antibodies when immunized. The use of XenoMouse animals to produce MAbs avoids the need for any engineering of the antibody genes, since the products are already 100% human protein. XenoMouse animals are fully compatible with standard hybridoma technology and can be readily adopted by laboratories experienced in monoclonal antibody production [56]. 

In reality, these forays represent miniaturization of the standard sandwich ELISA to attain higher throughput assays by multiplexing a limited number (<50) of analytes, e.g., cytokine panels. Even at these low densities, quantification problems arise in part due to a lack of robustness in the printing process and also in the selection and stability of monoclonal antibodies that must be highly specific and of high binding affinity to be useful for microarrays. 

Fig. 10 Formation of complexes between human rhinovirus HRV2 and neutralizing monoclonal antibody mAb 8F5 analyzed by CE. A fixed concentration of F1RV2 (15 nM) was incubated with an increasing concentration (indicated in the figure) of mAb 8F5 prior to the CE analysis at room temperature. O-phthalic acid was used as internal standard (IS). (Reprinted with permission from Ref. 34. Copyright 2000 American Chemical Society.)...

Ascites production, however, suffers from a number of drawbacks. It is costly, and the product is contaminated by significant levels of various mouse proteins, rendering subsequent downstream processing more complex. As a result, monoclonal antibody production by standard animal cell culture techniques has become the method of choice for the production of pharmaceutical-grade monoclonal antibody preparations. 

---