Critical reagent characterization and re-evaluation to ensure long-term stability: two case studies.

Characterization of critical reagents can mitigate adverse impact to ligand-binding assay performance. We investigated the conjugation conditions of a bispecific protein to SULFO-TAG NHS-Ester™ ruthenium to resolve a steady increase in ligand-binding assay background signal. Functional and biophysical attributes in stability samples revealed low pH (4.0) conjugation and formulation buffers were key to decrease aggregate formation. We also identified pH-specific (3.0) purification conditions to reduce aggregate levels from 37% to <5% of a mouse IgG3 reagent antibody. These case studies support the utility of biophysical and functional characterization of critical reagents as a proactive approach to maintain long-term stability and provide the basis for our recommendations a risk-based approach to establish re-evaluation intervals for traditional and novel reagents.

Feasibility of Singlet Analysis for Ligand Binding Assays: a Retrospective Examination of Data Generated Using the Gyrolab Platform.

There are many sources of analytical variability in ligand binding assays (LBA). One strategy to reduce variability has been duplicate analyses. With recent advances in LBA technologies, it is conceivable that singlet analysis is possible. We retrospectively evaluated singlet analysis using Gyrolab data. Relative precision of duplicates compared to singlets was evaluated using 60 datasets from toxicokinetic (TK) or pharmacokinetic (PK) studies which contained over 23,000 replicate pairs composed of standards, quality control (QC), and animal samples measured with 23 different bioanalytical assays. The comparison was first done with standard curve and QCs followed by PK parameters (i.e., Cmax and AUC). Statistical analyses were performed on combined duplicate versus singlets using a concordance correlation coefficient (CCC), a measurement used to assess agreement. Variance component analyses were conducted on PK estimates to assess the relative analytical and biological variability. Overall, 97.5% of replicate pairs had a %CV of <11% and 50% of the results had a %CV of ≤1.38%. There was no observable bias in concentration comparing the first replicate with the second (CCC of 0.99746 and accuracy value of 1). The comparison of AUC and Cmax showed no observable difference between singlet and duplicate (CCC for AUC and Cmax >0.99999). Analysis of variance indicated an AUC inter-subject variability 35.3-fold greater than replicate variability and 8.5-fold greater for Cmax. Running replicates from the same sample will not significantly reduce variation or change PK parameters. These analyses indicated the majority of variance was inter-subject and supported the use of a singlet strategy.

Challenges in selectivity, specificity and quantitation range of ligand-binding assays: case studies using a microfluidics platform.

BACKGROUND: Method developers of plate-based ligand-binding assays (LBAs) often face challenges establishing selectivity, specificity and range of quantitation to meet the needs of a particular study. Case studies are presented to compare different ligand-binding immunoassay platforms (plate-based vs microfluidic system) in method development to support pharmacokinetic and pharmacodynamic studies. RESULTS: Studies highlight the challenges of plate-based LBAs to establish selectivity, specificity and range of quantitation as a result of nonspecific background signal, matrix interference, lack of linearity and drug interference. The fast assay kinetics of a microfluidic immunoassay system was shown to generally reduce nonspecific background and matrix effects, while increasing assay linear range and drug tolerance. CONCLUSION: The short incubation times with microfluidics can be beneficial for LBAs burdened by matrix effects and in these cases had superior assay performance compared with widely used immunoassay platforms in bioanalysis, for example, Meso Scale Discovery(®) and enzyme-linked immunosorbent assay.

One mouse, one pharmacokinetic profile: quantitative whole blood serial sampling for biotherapeutics.

PURPOSE: The purpose of this study was to validate the approach of serial sampling from one mouse through ligand binding assay (LBA) quantification of dosed biotherapeutic in diluted whole blood to derive a pharmacokinetic (PK) profile. METHODS: This investigation compared PK parameters obtained using serial and composite sampling methods following administration of human IgG monoclonal antibody. The serial sampling technique was established by collecting 10 µL of blood via tail vein at each time point following drug administration. Blood was immediately diluted into buffer followed by analyte quantitation using Gyrolab to derive plasma concentrations. Additional studies were conducted to understand matrix and sampling site effects on drug concentrations. RESULTS: The drug concentration profiles, irrespective of biological matrix, and PK parameters using both sampling methods were not significantly different. There were no sampling site effects on drug concentration measurements except that concentrations were slightly lower in sodium citrated plasma than other matrices. CONCLUSIONS: We recommend the application of mouse serial sampling, particularly with limiting drug supply or specialized animal models. Overall the efficiencies gained by serial sampling were 40-80% savings in study cost, animal usage, study length and drug conservation while inter-subject variability across PK parameters was less than 30%.

Bioanalytical platform comparison using a generic human IgG PK assay format.

A comparison of four different ligand-binding assay technology platforms (ELISA, Meso Scale Discovery®, Gyros® and AlphaLISA®) was conducted using quantitative assays for the measurement of a human IgG1 monoclonal antibody (MAb) in rat serum. The assays used common reagents for Fc-specific measurement to determine total levels of a human IgG MAb drug analyte, and all were fully optimized for use on each platform. Mock MAb study samples were prepared and analyzed using all platforms to assess assay performance. Assay parameters such as sensitivity, dynamic range, minimum required dilution and sample volume as well as other considerations such as per-run cost, technology availability, requisite equipment and necessary reagent modifications were evaluated toward the determination of a default go-to assay platform for monoclonal antibody biotherapeutics in this laboratory. Based primarily on superior assay performance, Meso Scale Discovery and Gyros were selected from the four technologies evaluated as our default platforms for non-regulated (discovery) study support. As an adjunct, immunoaffinity LC-MS/MS was explored as an alternate platform for generic Fc quantitation and was found to perform similarly to the ligand-binding assays.

Use of response surface methods and path of steepest ascent to optimize ligand-binding assay sensitivity.

Response surface methods (RSM) combined with a steepest ascent approach is a powerful technique to optimize assay performance. In this case, a ligand-binding assay (LBA) to quantitate a peptide biotherapeutic was optimized for improved sensitivity using this technique. Conditions were elucidated to enable pg/mL quantitation of the peptide in human plasma using steepest ascent to efficiently optimize assay factors. Instead of relying solely on assay development experience and intuition to improve assay sensitivity, this systematic approach takes advantage of a predictive mathematical model generated through response surface methods that defines a specific path towards greater predicted assay sensitivity. The actual response observed along the steepest ascent path was in good agreement with the model for several steps, until reagent concentrations moved beyond the physical limits of the system, and model breakdown occurred. RSM combined with steepest ascent method proved a useful tool for sensitivity optimization in three ways: (1) The required LBA sensitivity performance (approximately 200 pg/mL), measured as a signal-to-noise ratio (SNR) at the targeted lower limit of quantitation (LLOQ), was efficiently achieved in only two optimization experiments; (2) Steepest ascent confirmed that the desired sensitivity was found within the initial RSM design space, and no further gain in sensitivity was found venturing beyond this design space along the steepest ascent path; (3) The desired assay sensitivity was maintained over a reasonable range of reagent concentrations along the steepest ascent path, indicating assay robustness for this parameter.

Pharmacokinetic, biodistribution, and biophysical profiles of TNF nanobodies conjugated to linear or branched poly(ethylene glycol).

Covalent attachment of poly(ethylene glycol) (PEG) to therapeutic proteins has been used to prolong in vivo exposure of therapeutic proteins. We have examined pharmacokinetic, biodistribution, and biophysical profiles of three different tumor necrosis factor alpha (TNF) Nanobody-40 kDa PEG conjugates: linear 1 × 40 KDa, branched 2 × 20 kDa, and 4 × 10 kDa conjugates. In accord with earlier reports, the superior PK profile was observed for the branched versus linear PEG conjugates, while all three conjugates had similar potency in a cell-based assay. Our results also indicate that (i) a superior PK profile of branched versus linear PEGs is likely to hold across species, (ii) for a given PEG size, the extent of PEG branching affects the PK profile, and (iii) tissue penetration may differ between linear and branched PEG conjugates in a tissue-specific manner. Biophysical analysis (R(g)/R(h) ratio) demonstrated that among the three protein-PEG conjugates the linear PEG conjugate had the most extended time-average conformation and the most exposed surface charges. We hypothesized that these biophysical characteristics of the linear PEG conjugate accounts for relatively less optimal masking of sites involved in elimination of the PEGylated Nanobodies (e.g., intracellular uptake and proteolysis), leading to lower in vivo exposure compared to the branched PEG conjugates. However, additional studies are needed to test this hypothesis.