Application of multi-factorial design of experiments to successfully optimize immunoassays for robust measurements of therapeutic proteins.

Developing a process that generates robust immunoassays that can be used to support studies with tight timelines is a common challenge for bioanalytical laboratories. Design of experiments (DOEs) is a tool that has been used by many industries for the purpose of optimizing processes. The approach is capable of identifying critical factors and their interactions with a minimal number of experiments. The challenge for implementing this tool in the bioanalytical laboratory is to develop a user-friendly approach that scientists can understand and apply. We have successfully addressed these challenges by eliminating the screening design, introducing automation, and applying a simple mathematical approach for the output parameter. A modified central composite design (CCD) was applied to three ligand binding assays. The intra-plate factors selected were coating, detection antibody concentration, and streptavidin-HRP concentrations. The inter-plate factors included incubation times for each step. The objective was to maximize the logS/B (S/B) of the low standard to the blank. The maximum desirable conditions were determined using JMP 7.0. To verify the validity of the predictions, the logS/B prediction was compared against the observed logS/B during pre-study validation experiments. The three assays were optimized using the multi-factorial DOE. The total error for all three methods was less than 20% which indicated method robustness. DOE identified interactions in one of the methods. The model predictions for logS/B were within 25% of the observed pre-study validation values for all methods tested. The comparison between the CCD and hybrid screening design yielded comparable parameter estimates. The user-friendly design enables effective application of multi-factorial DOE to optimize ligand binding assays for therapeutic proteins. The approach allows for identification of interactions between factors, consistency in optimal parameter determination, and reduced method development time.

Serum sample stability in ligand-binding assays: challenges in assessments of long-term, bench-top and multiple freeze-thaw.

Chris Macaraeg has been a lead scientist for method development, validation, and study support intended for regulated pre-clinical/clinical studies within the Pharmacokinetics and Drug Metabolism department at Amgen Inc, Thousand Oaks, CA. He joined Amgen in 2006. His expertise also includes automation and method transfer to CROs. Chris received his BS degree in Physiological Science and Neuroscience from the University of California, Los Angeles, CA and MS in Forensic Science from Pace University, New York, NY. Stability of therapeutic proteins in biological matrix is an important parameter to evaluate in bioanalytical support of regulated nonclinical or clinical studies. Despite industry guidance publications, many questions still arise as to how these practices are implemented to establish therapeutic protein stability in bioanalytical method validations. This article presents findings from long-term, bench-top and freeze-thaw stability assessments for three therapeutic monoclonal antibodies using either ELISA or electrochemiluminescent technology. Studies illustrate the principles and challenges in stability tests which represent scenarios that samples will likely encounter during sample analysis. Thoughtful consideration of each study requirements and a fit-for-purpose approach is essential in successful establishment of the sample stability parameters in method validation.

In Silico Evaluation of the Potential Impact of Bioanalytical Bias Difference between Two Therapeutic Protein Formulations for Pharmacokinetic Assessment in a Biocomparability Study.

Formulation changes at later stages of biotherapeutics development require biocomparability (BC) assessment. Using simulation, this study aims to determine the potential effect of bias difference observed between the two formulations after spiking into serum in passing or failing of a critical BC study. An ELISA method with 20% total error was used to assess any bias differences between a reference (RF) and test formulations (TF) in serum. During bioanalytical comparison of these formulations, a 9% difference in bias was observed between the two formulations in sera. To determine acceptable level of bias difference between the RF and TF bioanalytically, two in silico simulations were performed. The in silico analysis showed that the likelihood of the study meeting the BC criteria was >90% when the bias difference between RF and TF in serum was 9% and the number of subjects was ≥20 per treatment arm. An additional simulation showed that when the bias difference was increased to 13% and the number of subjects was <40, the likelihood of meeting the BC criteria decreased to 80%. The result from in silico analysis allowed the bioanalytical laboratory to proceed with sample analysis using a single calibrator and quality controls made from the reference formulation. This modeling approach can be applied to other BC studies with similar situations.

Validation of a microfluidic platform to measure total therapeutic antibodies and incurred sample reanalysis performance.

BACKGROUND: A microfluidic platform-based assay was validated to measure a humanized or fully human IgG in rat serum samples. MATERIALS & METHODS: The cumulative assessment for accuracy and precision was performed with three accuracy and precision runs. RESULTS: The inter-assay accuracy (mean %bias) ranged from -4.3 to 3.8%, and inter-batch %CV ranged from 5.0 to 9.2%. The method acceptance criterion was determined as 15% total error. The assay dynamic range was 50 to 10000 ng/ml. Incurred sample reanalysis passed with 95% of samples meeting incurred sample reanalysis acceptance criteria. Potential carryover effect was not observed. CONCLUSION: This study demonstrated the need for evaluating additional platform-specific processes when new technologies are employed to ensure the reproducibility of a successfully validated microfluidic platform method.

Specific method validation and sample analysis approaches for biocomparability studies of denosumab addressing method and manufacture site changes.

Manufacturing changes during a biological drug product life cycle occur often; one common change is that of the manufacturing site. Comparability studies may be required to ensure that the changes will not affect the pharmacokinetic properties of the drug. In addition, the bioanalytical method for sample analysis may evolve during the course of drug development. This paper illustrates the scenario of both manufacturing and bioanalytical method changes encountered during the development of denosumab, a fully human monoclonal antibody which inhibits bone resorption by targeting RANK Ligand. Here, we present a rational approach to address the bioanalytical method changes and provide considerations for method validation and sample analysis in support of biocomparability studies. An updated and improved ELISA method was validated, and its performance was compared to the existing method. The analytical performances, i.e., the accuracy and precision of standards and validation samples prepared from both manufacturing formulation lots, were evaluated and found to be equivalent. One of the lots was used as the reference standard for sample analysis of the biocomparability study. This study was sufficiently powered using a parallel design. The bioequivalence acceptance criteria for small molecule drugs were adopted. The pharmacokinetic parameters of the subjects dosed with both formulation lots were found to be comparable.

Assessment of incurred sample reanalysis for macromolecules to evaluate bioanalytical method robustness: effects from imprecision.

Incurred sample reanalysis (ISR) is recommended by regulatory agencies to demonstrate reproducibility of validated methods and provide confidence that methods used in pharmacokinetic and toxicokinetic assessments give reproducible results. For macromolecules to pass ISR, regulatory recommendations require that two thirds of ISR samples be within 30% of the average of original and reanalyzed values. A modified Bland-Altman (mBA) analysis was used to evaluate whether total error (TE), the sum of precision and accuracy, was predictive of a method's passing ISR and to identify potential contributing parameters for ISR success. Simulated studies determined minimum precision requirements for methods to have successful ISR and evaluated the relationship between precision and the probability of a method's passing ISR acceptance criteria. The present analysis evaluated ISRs conducted for 37 studies involving ligand-binding assays (LBAs), with TEs ranging from 15% to 30%. An mBA approach was used to assess accuracy and precision of ISR, each with a threshold of 30%. All ISR studies met current regulatory criteria; using mBA, all studies met the accuracy threshold of 30% or less, but two studies (5%) failed to meet the 30% precision threshold. Simulation results showed that when an LBA has ≤15% imprecision, the ISR criteria for both the regulatory recommendation and mBA would be met in 99.9% of studies. Approximately 71% of samples are expected to be within 1.5 times the method imprecision. Therefore, precision appears to be a critical parameter in LBA reproducibility and may also be useful in identifying methods that have difficulty passing ISR.

Applications of a planar electrochemiluminescence platform to support regulated studies of macromolecules: benefits and limitations in assay range.

Development and validation of ligand binding methods that can measure therapeutic antibodies (TA) accurately and precisely are essential for bioanalysis that supports regulated pharmacokinetic (PK) and toxicokinetic (TK) studies. Non-bead (planar) electrochemiluminescence (ECL) methods are known to have high sensitivity and a wide assay range and are therefore potentially useful in supporting research studies in the early phases of development as well as for diagnostic fields and multiplex biomarker applications. Here, we demonstrate the applications for using ECL for regulated studies associated with protein drug development. Three planar ECL methods were developed, validated, and implemented to quantify three different TAs to support PK/TK studies. An automated liquid handler was used for the preparation of standards, quality controls, and validation samples to minimize assay variability. Robustness and ruggedness were tested during pre-study validations. During method optimization, the potential assay ranges were 3 log orders. To improve assay accuracy and precision, assay ranges in all 3 methods were truncated by at least 50% at the upper end before proceeding to pre-study validations. All 3 methods had assay ranges of about 2 logs during pre-study validations. The inter-assay accuracy and precision during pre-study validations were <6% and 8%, respectively. The total error of the assays was <15% for both in-study and pre-study validations in all 3 methods. With the incorporation of a robotic workstation we concluded that performance in all 3 planar ECL methods was extremely precise and accurate during pre-study and in-study validations, enabling >90% assay success during sample analyses. Although there were limitations in the assay ranges, the strength of this technology in assay accuracy, precision, and reproducibility can be beneficial for macromolecule analyses in support of PK and TK studies in a regulated environment.

Bioanalytical method requirements and statistical considerations in incurred sample reanalysis for macromolecules.

BACKGROUND: Incurred sample reanalysis (ISR) is the most recent in-study validation parameter that regulatory agencies have mandated to ensure reproducibility of bioanalytical methods supporting pharmacokinetic/toxicokinetic and clinical studies. The present analysis describes five representative case studies for macromolecule therapeutics. METHOD: Single ISR acceptance criteria (within 30% of the averaged or original concentration) and a modified Bland-Altman (BA) approach were used to assess accuracy and precision of ISR results. General concordance between the two criteria was examined using simulation studies. RESULTS: All five methods met the ISR criteria. The results indicated that thorough method development and prestudy validation were prerequisites for a successful ISR. The overall agreement between the original and reanalyzed results as determined by BA was within 20%. Simulation studies indicated that concordance between the ISR criteria and BA was observed in 95% of the cases. Dilution factors had no significant impact on the ISR, even for C(max) samples where 1:100 or higher dilutions were used. CONCLUSION: The current ISR acceptance criteria for macromolecules was scientifically and statistically meaningful for methods with a total error of 25% or less.

Technical advances: measurement of collateral flow in the lung with a dedicated endobronchial catheter system.

Interest in assessment of collateral flow measurement has resurfaced with the advent of endoscopic lung volume reduction studies, namely valve technologies (eg Emphasys EBV), that achieve volume reduction through lobar isolation. It is currently thought that collateral ventilation could be a contributing factor for endoscopic lung volume reduction failures, as isolated lobes can "backfill" through these channels. In an attempt to quantify collateral ventilation, the Chartis System has been developed by Pulmonx, Inc. to measure pressure and flow, and calculate resistance to airflow through collateral channels in isolated lung compartments.