A study was made of the stability of the murine bispecific trinoma OC/TR with respect to secretion of both types of parental heavy and light chains. OC/TR is a cell line producing bispecific antibody that reacts with the CD3 antigen on T cells and the folate-binding receptor--frequently found to be overexpressed on ovarian carcinoma cells. Of the 10 different IgG combinations theoretically possible with 2 heavy and 2 light chains, 6 combinations were secreted. Subclones varied considerably in relative production of the two parental heavy and light chains. A detailed analysis was made of the binding characteristics and retargeting activity of each of the IgGs produced. From a clone producing a relatively high quantity of bispecific IgG, a large-scale production was initiated. The purification of clinical grade bispecific F(ab')2 from harvest fluids is described. The yield from this purification process was found to be comparable to the yield of bispecific F(ab')2 after chemical cross-linking of two different Fab'.

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Measurement of adsorption breakthrough curves in packed beds has shown that the amounts and rates of uptake of immunoglobulin M (IgM) onto the commonly used anionic ion-exchanger Q Sepharose Fast Flow (based on 6% agarose) are severely limited as a result of the large molecular size of this adsorbate (RMM 950,000). A similar ion-exchanger based on a more porous 4% agarose, Q Sepharose 4 Fast Flow was evaluated as an alternative adsorbent for the purification of IgM. Equilibrium adsorption isotherms and the effective diffusivities of IgM within these two adsorbents were measured. Q-Sepharose 4 Fast Flow was found to have a maximum capacity for IgM 2.5 times greater than that of Q Sepharose 6 Fast Flow and the effective diffusivity of IgM was found to be between 6 and 7 times greater than with the latter material. Comparison of the breakthrough curves obtained for these adsorbents at a variety of flow velocities confirm that Q Sepharose 4 Fast Flow is a superior adsorbent for the capture and purification of large proteins.

HA-1A, a human IgM mAb, has been shown to significantly reduce mortality in septic patients with Gram-negative bacteremia, especially those with septic shock, in a controlled clinical trial. To confirm the reported specificity of this antibody for the lipid A domain of endotoxin, several assay systems were developed. These assay systems included an ELISA, which measured the binding of HA-1A to lipid A adsorbed to a solid phase; a rate nephelometry assay, which measured the ability of HA-1A to bind and aggregate lipid A in solution; and a dot-blot immunoassay, which measured the ability of HA-1A to interact with lipid A adsorbed to Immobilon-P. In all three assay systems, HA-1A bound in a dose-dependent manner to lipid A prepared from Salmonella minnesota R595 LPS, whereas negative control human IgM mAb or polyclonal antibodies did not. Several experimental approaches were employed to demonstrate the specificity of HA-1A in these assay systems. Both polymyxin B and murine IgG mAb (8A1) with a specificity for lipid A were able to competitively inhibit HA-1A reactivity with lipid A in a dose-dependent manner. Furthermore, a murine IgG anti-Id mAb (9B5.5) developed against HA-1A was also able to block the binding of HA-1A to lipid A in these assay formats. HA-1A reactivity with synthetic lipid A confirmed that HA-1A binding to the natural lipid A was not the result of contaminants in the latter. Finally, the reactivity of HA-1A against a variety of glucosamine-containing and fatty acid-containing compounds was assessed. Some weak interaction was seen with cardiolipin and chitin, but not with serum proteins, lipoteichoic acid, or DNA. Collectively, these results conclusively establish that HA-1A binds to the lipid A region of LPS by an interaction with the V region of the antibody.