Development of an Automated Complement C5 Functional Assay for Personalized Monitoring of Patients on C5 Inhibitors

Abstract

Abstract Background Humanized anti-C5 monoclonal antibody therapeutics has been heralded as a breakthrough treatment for Paroxysmal Nocturnal Hemoglobinuria and atypical Hemolytic Uremic Syndrome. Therapeutic drug monitoring is currently not widely available but assessing complement blockage maybe useful to monitor and optimize Eculizumab or Ravlizumab treatment efficacy. Recent studies have suggested using C5 functional assays as a specific target to assess complement blockage. The aim of this study was to assess the analytical performance of an automated C5 functional assay we developed by modifying the complement CH50 assay used in our laboratory. Methods Patient sera mixed in different ratios (20-90%) with commercially available C5-deficient (C5d) serum (Quidel corp. SD) were tested for lytic activity using the CH50 liposome assay (Optilite, Binding site, Birmingham, UK). Normal lysis ensures intact C5 functional activity exclusively from the patient serum. A ratio that led to normal lysis in healthy sera and in patient sera defective in complement factors other than C5 was considered optimal. This ratio was adopted for preliminary reference intervals (n=34 normal donors) using bi-weight quantile method estimation at a 90% confidence interval. Accuracy was assessed by recovery studies. Specimens (n=2) were created with varying levels of C5 functional activity using Eculizumab (Biovision, CA). Recovery was calculated (measured/expected) by adding back 100 ug/mL of purified C5 (Quidel corp. SD) and comparing the CH50 values to the original results. To evaluate specificity, patient specimens (n=22) defective in complement components other than C5 (C2, C3, C4) were mixed with C5d and lytic activity measured. Results A ratio of 75% C5d to 25% patient serum was used to establish a reference interval of 32.3 – 46.7 U/mL. The two accuracy samples had initial CH50 values of 37.9 and 41.7 U/mL and 14.2 and 20.6 U/mL after Eculizumab depletion, respectively. Adding C5 back to the Eculizumab-depleted samples yielded values of 36.5 and 39.9 U/mL and recoveries of 96%. The non-C5 complement-deficient specificity samples had low CH50 values ranging from 11.8 to 28.8 U/mL. After mixing with C5d, 21/22 samples demonstrated low-normal or normal lytic activity (range: 28.4 -41.6 U/mL). Conclusion A ratio of 75% C5d to 25% patient serum was optimal for maintaining CH50 activity in normal specimens and recovering activity in specimens with complement deficiencies other than C5. The C5-depleted specimens did not regain normal CH50 activity after mixing with C5d serum. Adding C5 to these specimens regained normal CH50 activity, confirming that C5 was non-functional and the C5 inhibitor was not interfering with the assay. Specimens’ defective in complement components other than C5 recovered lytic activity when mixed with C5d serum in 95.5% of the specimens, confirming the analytical specificity of the assay. These data demonstrate an acceptable analytical performance for the C5 functional assay developed.