Background: The 50% hemolytic complement (CH50) assay is a non-specific qualitative assay of complement function. CH50 is sometimes used in patients with complement-mediated thrombotic microangiopathy such as atypical hemolytic uremic syndrome (aHUS) to monitor response to complement inhibition. There are multiple commercial assays as well as research-based CH50 assays that are not directly comparable in outcomes or methods. Ravulizumab is a long-acting anti-C5 monoclonal antibody engineered from eculizumab, currently being investigated for the treatment of aHUS. A fully validated free C5 assay, supporting the primary pharmacodynamic endpoint, was utilized in clinical trials of ravulizumab. We have compared local CH50 results from participating sites with the free C5 assay, performed centrally, in patients with aHUS participating in the clinical trial.
Methods: Adults and children with aHUS enrolled in phase 3 trials of ravulizumab (NCT02949128 and NCT03131219) were included in this analysis. A validated Gyros-based immunoassay with a fluorescence readout was used to quantify free C5 during the trial. The regulatory-accepted threshold for complete terminal complement inhibition is a free C5 value <0.5 µg/mL. Numerous CH50 assays were used, locally run and included hemolytic, CH50 EqEIA, ELISA and liposomal assays. The threshold for complete complement blockade varied in units and with each kit (range from 6-27% or units).
Results: CH50 data from 10 patients with aHUS (5 adults and 5 children) from four countries (US, Spain, France and UK) were available. A scatter plot of serum CH50 and free C5 versus time is shown in the figure. Complete terminal complement inhibition throughout the evaluation period was demonstrated by the free C5 assay. Results of CH50 did not consistently confirm full complement inhibition, demonstrating discordance with the free C5 assay. Relative to the adults, there was qualitatively more variability in the CH50 results in the pediatric patients.
Conclusions: Results from the CH50 assays inconsistently demonstrated full complement inhibition, with discordance with free C5 levels. This discordance requires further investigation. In clinical practice, this has potential to cause different interpretations of complement blockade and highlights the important limitations of CH50 as a technique for assessing complete terminal complement blockade in patients receiving ravulizumab. This study also underscores the need for a commercially available validated measure for determining complement inhibition.
Cataland:Alexion: Consultancy, Research Funding; Ablynx/Sanofi: Consultancy, Research Funding. Ariceta:Alexion: Consultancy, Honoraria. Chen:Alexion: Employment. Dixon:Alexion: Consultancy; Apellis Pharmaceuticals: Consultancy; Horizon Pharmaceuticals: Consultancy. Garlo:Alexion: Employment. Greenbaum:Alexion: Consultancy, Honoraria, Research Funding. Rondeau:Alexion: Consultancy, Honoraria. Scully:Novartis: Consultancy; Shire: Research Funding; Shire/Takeda: Consultancy; Alexion: Consultancy; Ablynx/Sanofi: Consultancy. Ortiz:Alexion: Employment.
Asterisk with author names denotes non-ASH members.