Immunoaffinity proteomics for kidney injury biomarkers in male beagle dogs.

Drug-induced kidney injury (DIKI) is a cause of drug development failure. Dogs represent a common non-rodent animal model in pre-clinical safety studies; however, biomarker assays for detecting nephrotoxicity in dogs are limited. To identify novel proteins and gain insight into the molecular mechanisms involved in DIKI, we developed an assay to evaluate proteomic changes associated with DIKI in male beagle dogs that received nephrotoxic doses of tobramycin for 10 consecutive days. Label-free quantitative discovery proteomics analysis on representative kidney cortex tissues collected on Day 11 showed that the tobramycin-induced kidney injury led to a significant differential regulation of 94 proteins mostly associated with mechanisms of nephrotoxicity such as oxidative stress and proteasome degradation. For verification of the proteomic results, we developed a multiplex peptide-centric immunoaffinity liquid chromatography tandem mass spectrometry assay (IA LC-MS/MS) to evaluate the association of eight DIKI protein biomarker candidates using kidney cortices collected on Day 11 and urine samples collected on Days -4, 1, 3, 7 and 10. The results showed that most biomarkers evaluated were detected in the kidney cortices and their expression profile in tissue aligned with the label-free data. Cystatin C was the most consistent marker regardless of the magnitude of the renal injury while fatty acid-binding protein-4 (FABP4) and kidney injury molecule-1 (KIM-1) were the most affected biomarkers in response to moderate proximal tubular injury in absence of changes in serum-based concentrations of blood urea nitrogen or creatinine. In the urine, clusterin is considered the most consistent biomarker regardless of the magnitude and time of the renal injury. To our knowledge, this is the most comprehensive multiplex assay for the quantitative analysis of mechanism-based proximal tubular injury biomarkers in dogs.

Multi-laboratory evaluation of immunoaffinity LC-MS-based glucagon-like peptide-1 assay.

Background: Although the fit-for-purpose approach has been proposed for biomarker assay validation, practical data should be compiled to facilitate the predetermination of acceptance criteria. Methods: Immunoaffinity LC-MS was used to analyze glucagon-like peptide-1 as a model biomarker in six laboratories. Calibration curve, carryover, parallelism, precision, relative accuracy and processed sample stability were evaluated, and their robustness among laboratories was assessed. The rat glucagon-like peptide-1 concentrations in four blinded samples were also compared. Results: The obtained results and determined concentrations in the blinded samples at all laboratories were similar, with a few exceptions, and robust, despite the difference in optimization techniques among laboratories. Conclusion: The results provide insights into the predefinition of the acceptance criteria of immunoaffinity LC-MS-based biomarker assays.

Plasma biomarkers TAP, CPA1, and CPA2 for the detection of pancreatic injury in rat: the development of a novel multiplex IA-LC-MS/MS assay and biomarker performance evaluation.

Drug-induced pancreatic injury (DIPI) is an issue seen in drug development both in nonclinical and clinical contexts. DIPI is typically monitored by measurement of lipase and/or amylase, however, both enzymes lack sensitivity and specificity. Although candidate protein biomarkers specific to pancreas exist, antibody-based assay development is difficult due to their small size or the rapid cleavage by proteolytic enzymes released during pancreatic injury. Here we report the development of a novel multiplexed immunoaffinity-based liquid chromatography mass spectrometric assay (IA-LC-MS/MS) for trypsinogen activation peptide (TAP) and carboxypeptidases A1 and A2 (CPA1, CPA2). This method is based on the enzymatic digestion of the target proteins, immunoprecipitation of the peptides with specific antibodies and LC-MS/MS analysis. This assay was used to detect TAP, CPA1, and CPA2 in 470 plasma samples collected from 9 in-vivo rat studies with pancreatic injury and 8 specificity studies with injury in other organs to assess their performance in monitoring exocrine pancreas injury. The TAP, CPA1, and CPA2 response was compared to histopathology, lipase, amylase and microRNA217. In summary, TAP, CPA1, and CPA2 proteins measured in rat plasma were sensitive and specific biomarkers for monitoring drug-induced pancreatic injury; outperforming lipase and amylase both by higher sensitivity of detection and by sustained increases in plasma observed over a longer time period. These protein-based assays and potentially others under development, are valuable tools for use in nonclinical drug development and as future translatable biomarkers for assessment in clinical settings to further improve patient safety.

Immunoaffinity LC-MS/MS is more suitable than ELISA to quantify a PEGylated molecule in cynomolgus monkey serum.

Background: Polyethylene glycolylation (PEGylation) technology is a long-acting delivery platform used to increase the half-life of protein therapeutics. Quantitation of PEGylated anti-Factor D Fab (PEG-aFD) poses bioanalytical challenges. Results: An ELISA was developed to determine total Fab concentration in cynomolgus monkey serum following intravitreal administration of PEG-aFD. However, assay characterization showed a low recovery of about 25% for free unconjugated Fab whereas recovery for PEG-conjugated Fab was within 80-120%. To overcome this challenge, an immunoaffinity liquid chromatography tandem mass spectrometry (IA LC-MS/MS) assay was developed, achieving recovery within 80-120% for both free and conjugated Fab. Conclusion: Immunoaffinity LC-MS/MS is more suitable than ELISA to accurately quantify the total protein concentration of PEG-aFD in cynomolgus monkey serum.