Utilizing HPLC to assess immunoreactivity of labeled biologics

Introduction: Labeled biologics are utilized in the medical field for a variety of purposes spanning from diagnostics to a variety of therapies. Although the value of labeled biologic agents is highly recognized, the limitations in the field are dependent on the ability to produce these agents in a safe, quick, and inexpensive way. Multiple quality control tests post-production are required to ensure safety and efficacy for injection into the patient. The current standard for analyzing the immunoreactivity of optically labeled biologics is using an Elisa assay; while the potency of radiolabeled biologics is primarily measured via Lindmo assay.^{1, 2} While these assays effectively quantify the binding of the conjugated biologic, they can be very resource and time intensive. Typically, following the production of these imaging agents, the purity of the labeled antibodies is determined via HPLC. Utilizing size exclusion HPLC for analysis of immunoreactivity in addition to purity could decrease overall workflow, time, and expenses required for injection approval, while maintaining the requirements of GMP.

Methods: This project utilized SE-HPLC (size exclusion high performance liquid chromatography) using a Biozen 1.8 mm dSEC-2, 200 Å column. The mobile phase was a buffer solution containing potassium phosphate and potassium chloride. A stock solution of IRDye800-Panitumumab was used for baseline and buffer condition analysis. The antigen, EGFR, was then mixed with the conjugated antibody and run on the column. Under the same logic and methodology, we have also measured the immunoreactivity of [89Zr]Panitumumab and IRDye800-Panitumumab on a Superdex column.

Results: The SE-HPLC indicated sufficient separation of labeled antibody and bound antigen-antibody complex as well as a resolved small peak accounting for aggregates. Figure 1 displays a run done in excess antibody to ensure there is separation between the species. The figure shows the bound complex at 5.7 minutes while the labeled antibody comes off the column at 7.2 minutes. The immunoreactivity is determined by the presence of unbound labeled-antibody compared to the peak indicating the antigen-antibody complex. It is necessary to have an excess of antigen to assess the immunoreactivity of the antibody. The study indicates that 2-fold antigen is necessary to accurately assess the binding ability of the antibody. We are also applying this method for other labeled antibodies targeting PD-1, and HER2; as well as for antibodies binding B-Amyloid protofibrils.

Conclusions: We can conclude that this method shows good resolution of the antibody from the antigen-antibody complex to identify the immunoreactivity of the labeled antibody. This will also identify stability changes in the labeled biologic over time, which is necessary for long term storage of optical agents and short-term storage of radiolabeled agents.