Purpose: We have developed an in-house generic PK assay using ELISA to measure human IgG1 therapeutic antibodies in non-clinical samples. The colorimetric ELISA format of the assay uses anti-human antibodies as capture and detection. Standard calibration curves are generated using a 5-parameter logistical (5PL) regression model and the concentrations of samples are interpolated from their respective standard curves in SoftmaxR.
We also developed a prototype version of this generic PK assay on the ELLA platform from Protein Simple. ELLA is an automated immunoassay system utilizing a microfluidic Simple Plex cartridge where samples and buffer manually loaded into the cartridge are placed in the ELLA device. The analyte of interest binds to the antibody coated Glass Nano Reactors (GNR). Triplicate results from each GNR are generated from factory-calibrated standard curve encoded in each cartridge reducing hands-on time and yielding results under 2 hours.
To compare the PK profiles of human IgG1 therapeutic in cynomolgus serum and cerebrospinal fluid (CSF), using generic PK ELLA prototype cartridge with the generic PK ELISA.
Methods: The in-house developed generic PK ELISA was used to evaluate standards and QCs prepared in assay buffer, cynomolgus serum and CSF, together with matched serum and CSF samples from cynomolgus monkeys dosed with a human IgG1 drug. To compare platforms, the same samples mentioned above, QCs and cynomolgus monkey samples were tested on the ELLA assay. Data for the ELISA was generated in 8 hours and that for ELLA in 2 hours.
Results: For the standard curve prepared in pooled cyno serum, data reduction of a user generated calibration curve using Softmax demonstrated similar performance to that generated using the cartridge provided calibration curve. Results from study samples analyzed using the generic PK ELISA format and the ELLA format met the criteria for incurred sample reproducibility (ISR).
For the standard prepared in CSF, the calibration curve generated from ELISA overlapped with theoretical concentration, while the one generated from the ELLA cartridge built-in curve over-recovered compared to theoretical concentration and the user generated curve using Softmax. Data from CSF samples thereby did not meet the ISR criteria.
Conclusion: The generic PK using ELLA platform offers a simple, user friendly, and high throughput measurement of serum PK profile for NHP studies. The performance of ELLA cartridge is comparable to ELISA for serum sample testing. While the prototype ELLA cartridge works for serum, more development and evaluation should be done for other metrices, such as, NHP plasma and CSF.