Minimizing mischaracterization of cell products


Research and pre-clinical studies in cellular therapy make extensive use of antibodies to accurately characterize the cells which are intended for transplantations. Antibodies are not only used for product characterization and post-transplant testing (e.g. to screen for residual disease), but are often also used for cell selection processes and cell activation. Likewise, antibodies are essential reagents in commercial cellular therapy.

It is important to recognize that antibodies used for product characterization and diagnostics are regulated different from antibodies used for cell activation and selection. In the context of safety, antibodies used in these last applications may be categorized under therapeutic antibodies as they come in touch with the cells that are injected into patients. In contrast to most therapeutic antibodies and antibodies used for cell activation, antibodies for cell selection should be inert and should not affect the functionality of the cell product. Because the cells used for product characterization and diagnostics are not injected into patients, it is generally not relevant whether or not an antibody affects cell functionality when used in these applications.

Regardless of the applications mentioned above, specificity and selectivity are critical properties to define accurately. While a monoclonal antibody is quickly named after the biomarker of interest, it binds to just a small part (the epitope it was specifically raised against) of the overall structure of the biomarker. Hence, it is not uncommon to find a number of monocloncal antibodies named after the same biomarker, yet, raised against different epitopes of that biomarker. Because the binding characteristics of each antibody-epitope complex are unique, optimized sample preparation methods for reliable identification can be different with different antibodies despite they target the same biomarker.

Optimizing sample preparation methods is even more complicated in analytics that require fluorochrome-conjugated antibodies (e.g. in fluorescence microscopy and flow cytometry). Here, two identical monocloncal antibodies may acquire very different binding characteristics after being conjugated to different fluorochromes. For instance, the Phycoerythrin-conjugated mouse IgG anti-human CD34 monocloncal antibody raised against the so called Class II epitope of the CD34 protein structure, can give markedly different results from the Fluorescein-Isothiocyanate-conjugated version. A generally accepted explanation for this is that the overall negative charge of FITC limits access to the predominant negative charge of the cave-like structure in which the Class II epitope is located (Lanza F et al. 2001).

Aside from the obvious practice to document ‘everything there is to know’ about the antibodies [such as: Name (approved, alternative names), Clone, Isotype, Workshop number (if applicable), Conjugate, Reactivity / Cross reactivity, Fluorochrome/Protein (F/P) ratio, or Degree of Labeling (DOL), Percentage of free dye, Formulation, Formulated Concentration, Qualified Applications (live, fixed, fixed-permeabilized), Recommended Storage, Brief description of target antigen, references], analytical assays like this should undergo extensive qualification studies to establish the boundaries of the conditions under which the assay provides reliable results.