Study protocol
Two commercially available PCSK9 monoclonal antibodies, including alirocumab 75 mg/ml (Praluent®; Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA) and evolocumab 140 mg/ml (Repatha®; Amgen, Inc., Thousand Oaks, CA, USA), were used to test the effect of various temperatures on the stability of these drugs under 3 different temperature conditions, including room temperature (RT), cooler device with cold pack, and freeze-thaw. The same batches of PCSK9 monoclonal antibody from each company were used to minimize production-related variation (batch number 1091418 for evolocumab, and batch number 9 W0512 for alirocumab).
Drugs were dispensed from pre-filled auto-injector pens, with 160 μl of drug from each pen aliquoted into each of 6 Eppendorf tubes (Fig. 1a). For RT condition, the tested drugs were placed in a closed room without direct sunlight exposure for 9 or 18 h. Temperature recorders (Lascar Electronics Ltd., Wiltshire, UK) were placed together with the drugs to monitor temperature and humidity hourly during the test date. For cooler device with cold pack condition, the study drugs were placed together with a temperature recorder in a standard cooler device (size 14x21x14 cm) with one cold pack (size 8 × 12 cm) for 9 or 18 h. For freeze-thaw condition, the drugs were frozen to -20 °C for 9 or 18 h and then thawed by 27 °C water bath for 2 min (Fig. 1b).
PCSK-9 quantification
The stability of PCSK9 monoclonal antibody was analyzed using a quantitative enzyme-linked immunosorbent assay (ELISA) method. Serum samples were purified by protein G Agarose (Abcam193258; Abcam, Cambridge, UK) and washing buffer (PBST 20X) before an experiment to separate other IgGs from serum and drug complex. Both study drugs were diluted to half concentration (37.5 mg/mL for alirocumab, and 70 mg/mL for evolocumab). One hundred microliters of each drug was combined with 10 μl of the subject’s plasma and incubated in 37 °C for 20 min. After the binding of free PCSK9 and PCSK9 monoclonal antibody, PCSK9 ELISA kits (Human PCSK9 Simple Step ELISA Kit, ABCAM model ab209884) was used to determine unbound PCSK9 concentration in plasma. The ELISA kit used employed an affinity tag labeled capture antibody, and a reporter conjugated detector antibody that immunocaptured the sample analysis in solution. This entire complex (capture antibody/analyte/detector antibody) was in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. The signal intensity was measured at 450 nm by ELISA reader (Ultra reader: EL808; Bio-Tek Instruments, Winooski, VT, USA). Plasma PCSK9 concentration was calculated by comparing sample luminescence to the standard luminescence curve. The PCSK9 concentration in plasma was measured before and after incubation with PCSK9 mAb. The percent inhibition of plasma PCSK9 was calculated using the following formula: (measured PCSK9 level after incubation with drugs – pre-incubation plasma PCSK9 level) / pre-incubation plasma PCSK9 level × 100). Percent inhibition represents the ability of the drug to decrease plasma PCSK9 levels. The stability of the study drugs was inversely related with unbound PCSK9 concentration. High unbound PCSK9 concentration reflects poor drug efficacy. Percent plasma PCSK9 inhibition with proper storage drug was used as baseline. Comparison between percent plasma PCSK9 inhibitions for drugs maintained in each of the 3 study conditions and baseline was calculated and reported as change in percent PCSK9 inhibition from baseline.
This study was conducted in cooperation with the Department of Endocrinology and Metabolism and the Department of Immunology, Faculty of Medicine Siriraj Hospital Mahidol University, Bangkok, Thailand. The study protocol was approved by the Siriraj Institutional Review Board and informed consent was obtained from each patient.
Sample size calculation
A previous study reported that PCSK9 monoclonal antibody has an ability to reduce plasma LDL-C level by 57.0% [4], and 99.5% of subjects were good responders [12]. The sample size was calculated by using an estimating proportion of one group formula. The relative potency of the study drug to inhibit plasma PCSK9 was 70.0% with an acceptable error of 3.0 and 95% confidence interval. Our calculation showed that plasma samples from 9 subjects were required to test the efficacy of these drugs in the 3 different study conditions.
Inclusion and exclusion criteria
Patients with dyslipidemia aged over 18 years who visited the endocrine or diabetic clinic at Siriraj Hospital or Phyathai 2 Hospital during September to December 2019 were recruited. The exclusion criteria were patients who were currently being treated with PCSK9 mAb, and patients with history of PCSK9 mAb or immunoglobulin allergy.
Statistical analysis
The data included demographics (age, gender, hometown), underlying diseases, treatment regimen, and plasma lipid levels. Categorical variables were expressed as percentages, while continuous variables were expressed as mean plus/minus standard deviation (SD) or median (range), as appropriate. Paired t-test was used to compare change in percent inhibition of PCSK9 levels at each timepoint of the 3 study conditions. A two-tailed P-value of < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS Statistics version 18.0 (SPSS, Inc., Chicago, IL, USA).