The effects of viral inactivation agents on the activities of monoclonal antibodies

The aim of this thesis was to determine whether or not two viral inactivation methods which had been developed for use with blood products, affected either antigen binding or secondary biological functions of two selected model antibody molecules. It was postulated that at least one of the viral inactivants, BPL, would affect the functional integrity of antibody molecules since it is reactive with proteins and has been used to block the complement-fixing properties of gamma-globulins. As one of the most important functions of antibodies is opsonization, which occurs through binding of the F region to the Fe receptors in the cell membrane and binds complement, an in vitro model for this process such as hemolysis, serves ac a useful assay. Because haemagglutination and hemolysis serve as useful models for determining the functional integrity of antibodies in situ, these were the initial assays chosen to detect whether either of the two chemical viral inactivants, PL and TNBP, had a detrimental effect on the variable (antigen-binding) region or Fc region (effector function) of the two model monoclonal antibodies. However, the results from these assays were not clear cut and difficult to interpret due to both the inherent difficulty in reading the assays, i.e. the absence of an electronic component to provide sensitivity in measurement and the sensitivity of the antibody reaction to its physicochemical environment. The ELISA provides a useful comparison to haemagglutination and haemolysis assays as it overcomes these limitations. ELISAs can be used as a comparison but not a substitute for haemagglutination and haemolysis since the proteins are fixed onto a solid substrate so their mobility is restricted and the Fe is not assessed for its effector functions. It is therefore proposed that a series of assays is required to determine the functionality of the variable and F regions of antibodies. In the future NMR will have a large part to play in the analysis of antibody purity but so far its use is limited to observing the conformation of variable domains. For the present, the assays chosen for this thesis were seen as the appropriate option to obtain data of greatest significance. By ELISA it has been determined that the viral inactivation treatment using BPL with an IgM monoclonal antibody preparation resulted in a 25 % reduction in antigen-binding activity of the variable region, i.e. a 25 % reduction of antigen-antibody complex was detected. The results obtained from haemagglutination and haemolysis assays were less clear cut for reasons discussed previously. Initially, a reduction in titre for both haemagglutination and haemolysis was observed so that, as with the ELISA, a reduction in antigen binding activity followed treatment with BPL. Because an antigen-antibody complex has a greater affinity for an Fc receptor than an unbound antibody, the reduction in haemolytic activity does necessarily indicate a chemical modification of the Fc region. Subsequently, when the assays were repeated in the presence of 5 % BSA, a reduction in neither the haemagglutination nor the haemolytic titres were observed. It is conceivable that while the affinity constant and hence detectability was increased using BSA, the precision of the measurement was reduced to such a degree that the differences in avidity of the antibody molecules could not be detected. No significant alteration in overall biological activity, as measured by ELISA, haemagglutination or haemolysis resulted when the same IgM monoclonal antibody preparation was subjected to the viral inactivation procedure using TNBP. No significant alteration in the antigen-binding activity, measured by haemagglutination resulted when a model human IgG1 monoclonal antibody preparation was subjected to the viral inactivation procedures using either TNBP or BPL; however, this assay was carried out in the presence of 5 % BSA and, as been concluded from the previous experiments, further assays may be required to detect an alteration. It may be concluded that although the use of EPL as an viral inactivant is the only chemical treatment method available to inactivate all viruses for which it has been tested, it significantly reduces the antigen-binding ability of a mouse IgM molecule; also, it is likely that /LPL modifies the Fc region to reduce its functional integrity (115). Procedures such as filtration or lyophilisation have advantages in that they have been demonstrated to yield a safe product; however, filters available cannot remove very small viruses and the use of lyophilisation for inactivation has not been substantially investigated