Development and Validation of an LC–MS/MS Assay for The Quantitation of A Pegylated Anti-Cd28 Domain Antibody in Human Serum: Overcoming Interference from Antidrug Antibodies and Soluble Target

Abstract

The fully validated LC-MS/MS assay demonstrates good accuracy (% deviation ≤6.3) and precision (%CV ≤5.2) with an lower limit of quantitation of 10 ng/ml.

Pharmacokinetic, Pharmacodynamic, and Safety Profile of a Novel Anti-CD28 Domain Antibody Antagonist in Healthy Subjects

“…During the MAD study, samples were taken on day 1 at 0 (predose) and 6 hours after dosing and on days 2, 3, 4, 5, 6, and 7; then at predose on days 8, 15, 22, and 29; then on days 30, 31, 32, 33, 34, 35, and 36; then on days 43 ± 2, 50 ± 2, 57 ± 3, 71 ± 3, and 85 ± 3. The serum samples were analyzed for lulizumab by a fully validated liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method (reasons for the change in method are explained below) . Parameters included C max , T max , AUC τ , T 1/2 , and accumulation index for AUC and C max .…”

“…A standard equivalence test was used to determine if the 2 methods produced similar predicted concentrations: the 90% confidence intervals (CIs) for the ratios of the geometric means (LC‐MS/MS vs LBA) should fall within the interval of 0.8 to 1.25, with 80% power, and the LC‐MS/MS results were found to be statistically equivalent to the LBA results. However, equivalence between the assays cannot be assumed to apply generally, as the findings of the LBA can be subject to interference if levels of antidrug antibodies and soluble target or other matrix‐interfering factors are sufficiently high …”

“…The serum samples were analyzed for lulizumab by a fully validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method (reasons for the change in method are explained below). 15 Parameters included C max , T max , AUC τ , T 1/2 , and accumulation index for AUC and C max .…”

“…However, equivalence between the assays cannot be assumed to apply generally, as the findings of the LBA can be subject to interference if levels of antidrug antibodies and soluble target or other matrix-interfering factors are sufficiently high. 15…”

The clinical utility of mass spectrometry based protein assays

“…In the development of humanized monoclonal antibody therapeutics, the quantitative measurement of bound, free, and total target protein is critical for establishing PK/PD relationships. Developing specific and sensitive paired antibody reagents for traditional immunoassay formats against target protein epitopes that are not masked by the therapeutic antibody is challenging, but in multiple cases, LC-MS has provided a solution [70][71][72]. Neubert and colleagues developed and validated an automated sequential protein and peptide immunoaffinity capture LC-MS assay to quantify total human ß-nerve growth factor (NGF), the endogenous target for tanezumab (anti-NGF humanized IgG2) in a clinical study that included tanezumab dosing.…”