Generic method approaches for monoclonal antibody therapeutics analysis using both ligand binding and LC-MS/MS techniques.

Monoclonal antibody (mAb) and mAb-derived biotherapeutics are being developed to interact with specific target molecule(s) to intervene disease formation or progression. LC-MS/MS methods have emerged to compensate for the limitations of conventional ligand-binding assays. Application of a generic LC-MS/MS method to multiple mAb candidates can save method development time as most mAb biotherapeutics are IgG 1, 2 and 4 isotypes. Three common components are essential to a generic LC-MS/MS method: a common workflow, a common surrogate peptide and the corresponding stable isotope-labeled internal standard. The generic LC-MS/MS method is translatable from a single- into a multiple-analyte method, and from a generic into a specific method. Strategies and caveats on method applications are discussed in this chapter.

Recommendations for validation of LC-MS/MS bioanalytical methods for protein biotherapeutics.

This paper represents the consensus views of a cross-section of companies and organizations from the USA and Canada regarding the validation and application of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for bioanalysis of protein biotherapeutics in regulated studies. It was prepared under the auspices of the AAPS Bioanalytical Focus Group's Protein LC-MS Bioanalysis Subteam and is intended to serve as a guide to drive harmonization of best practices within the bioanalytical community and provide regulators with an overview of current industry thinking on applying LC-MS/MS technology for protein bioanalysis. For simplicity, the scope was limited to the most common current approach in which the protein is indirectly quantified using LC-MS/MS measurement of one or more of its surrogate peptide(s) produced by proteolytic digestion. Within this context, we considered a range of sample preparation approaches from simple in-matrix protein denaturation and digestion to complex procedures involving affinity capture enrichment. Consideration was given to the method validation experiments normally associated with traditional LC-MS/MS and ligand-binding assays. Our collective experience, thus far, is that LC-MS/MS methods for protein bioanalysis require different development and validation considerations than those used for small molecules. The method development and validation plans need to be tailored to the particular assay format being established, taking into account a number of important factors: the intended use of the assay, the test species or study population, the characteristics of the protein biotherapeutic and its similarity to endogenous proteins, potential interferences, as well as the nature, quality, and availability of reference and internal standard materials.

ADME of monoclonal antibody biotherapeutics: knowledge gaps and emerging tools.

Absorption, distribution, metabolism and excretion (ADME) data are pivotal for small-molecule drug development, with well-developed in vitro and in vivo correlation tools and guidances from regulatory agencies. In the past two decades, monoclonal antibody (mAb) biotherapeutics have been successfully approved, including derived novel conjugates of active molecules (toxins or bioactive peptides) for specific target delivery or half-life extension. However, ADME information of mAb therapeutics lags behind that of small molecules due to the complex nature of the molecules and lack of appropriate tools to study drug exposure, biotransformation, and target engagement in the vascular and tissue spaces. In this perspective, the current knowledge gaps on ADME of mAb-related therapeutics are reviewed with potential solutions from emerging analytical technologies.

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.

Simultaneous analysis of multiple monoclonal antibody biotherapeutics by LC-MS/MS method in rat plasma following cassette-dosing.

We have recently developed a general liquid chromatography-tandem mass spectrometric (LC-MS/MS) method using a stable isotope-labeled (SIL) monoclonal antibody (mAb) as an internal standard (IS) for single-analyte quantification of mAb (Li et al. Anal Chem 84(3):1267-1273, 2012). The method offers an advantage over ligand binding assay in reducing the time and resources needed for bioanalytical support in preclinical stages of drug development. In this paper, we report another marked increase in assay efficiency for multi-analyte bioanalysis using unique surrogate peptides for each analyte and the strategic choice of the SIL-IS peptide. The method was qualified for the simultaneous determinations of four mAbs in rat plasma and applied to samples from discrete- and cassette-dosed rats. The pharmacokinetic parameters of the four mAbs of cassette dosing were comparable to those of discrete dosing and of enzyme-linked immunosorbent assay results. Although there may be limitations and special considerations for cassette-dosing of biologics, these results demonstrate the robust performance of the multi-analyte LC-MS/MS method allowing cassette-dosing that would ultimately reduce animal use and improve efficiency.

Bioanalysis of target biomarker and PK/PD relevancy during the development of biotherapeutics.

The majority of biotherapeutic drugs act on specific targets, which may serve as biomarkers to be evaluated for target engagement and validation. Together with subsequent pathway biomarkers, these data can provide proof-of-mechanism and understanding of the biological drug affect. A major task during early development is to predict, for the first first time in human clinical trials, the starting dose and simulate the PK/PD relationship. However, determinations of the biotherapeutic drug and target concentrations are not straightforward due to temporal changes of drug-target binding and challenges in developing reliable methods to measure the free and total drug and target. Herein, the bioanalysis of the target biomarker and the biotherapeutics in the context of PK/PD relevancy during drug development is reviewed. Binding of the target to the biotherapeutic will affect target clearance and drug disposition, resulting in nonlinear PK. Reliable and specific methods are crucial for the correct PK/PD modeling and interpretation.

Polycystin-1 regulates the stability and ubiquitination of transcription factor Jade-1.

Autosomal-dominant polycystic kidney disease (ADPKD) and von Hippel-Lindau (VHL) disease lead to large kidney cysts that share pathogenetic features. The polycystin-1 (PC1) and pVHL proteins may therefore participate in the same key signaling pathways. Jade-1 is a pro-apoptotic and growth suppressive ubiquitin ligase for beta-catenin and transcriptional coactivator associated with histone acetyltransferase activity that is stabilized by pVHL in a manner that correlates with risk of VHL renal disease. Thus, a relationship between Jade-1 and PC1 was sought. Full-length PC1 bound, stabilized and colocalized with Jade-1 and inhibited Jade-1 ubiquitination. In contrast, the cytoplasmic tail or the naturally occurring C-terminal fragment of PC1 (PC1-CTF) promoted Jade-1 ubiquitination and degradation, suggesting a dominant-negative mechanism. ADPKD-associated PC1 mutants failed to regulate Jade-1, indicating a potential disease link. Jade-1 ubiquitination was mediated by Siah-1, an E3 ligase that binds PC1. By controlling Jade-1 abundance, PC1 and the PC1-CTF differentially regulate Jade-1-mediated transcriptional activity. A key target of PC1, the cyclin-dependent kinase inhibitor p21, is also up-regulated by Jade-1. Through Jade-1, PC1 and PC1 cleaved forms may exert fine control of beta-catenin and canonical Wnt signaling, a critical pathway in cystic renal disease. Thus, Jade-1 is a transcription factor and ubiquitin ligase whose activity is regulated by PC1 in a manner that is physiologic and may correlate with disease. Jade-1 may be an important therapeutic target in renal cystogenesis.

General LC-MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies.

Ligand binding assays (LBAs) are widely used for therapeutic monoclonal antibody (mAb) quantification in biological samples. Major limitations are long method development times, reagent procurement, and matrix effects. LC-MS/MS methods using signature peptides are emerging as an alternative approach, which typically use a stable isotope labeled signature peptide as the internal standard (IS). However, a new IS has to be generated for every candidate, and the IS may not correct for variations at all processing steps. We have developed a general LC-MS/MS method approach employing a uniformly heavy-isotope labeled common whole mAb IS and a common immunocapture for sample processing. The method was streamlined with automation for consistency and throughput. Method qualification of four IgG(2) and four IgG(1) mAbs showed sensitivity of 0.1 µg/mL and linearity of 0.1-15 µg/mL. Quality control (QC) data of these eight mAbs were accurate and precise. The QC performance of the whole molecule labeled IS was better than those of synthetic labeled IS peptides tested. The pharmacokinetic results of two mAbs (an IgG(2) and IgG(1) candidate) dosed in rats were comparable to those of LBA. The general LC-MS/MS method approach overcomes the limitations of current methods to reduce time and resources required for preclinical studies.

Bioanalytical considerations in the comparability assessment of biotherapeutics.

Comparison of biotherapeutic products before and after manufacturing changes is required to show that the products are highly similar. Besides in vitro assessment on the critical quality attributes and potency, biocomparability studies are sometimes required to demonstrate similarities in pharmacokinetic and pharmacodynamic characteristics. The complex and diverse nature of biotherapeutics requires multifaceted considerations in the biocomparability study design, bioanalytical measurements of drug concentrations and/or pharmacodynamic responses, immunogenicity analysis, data interpretation and decision making. A major perspective is to understand the structure and biological functions of the biotherapeutics in relation to the indication. Issues of a common standard and the importance of the use of ligand-binding assays that are sensitive to structural changes are discussed. It would not be possible to use the same process and one-size-fit-all criteria for biocomparability studies of all biologics. Previous examples from industry and our experience of the bioanalytical considerations for fit-for-purpose pharmacokinetic support and immunogenicity assessments are presented.

Bioanalytical approaches to quantify "total" and "free" therapeutic antibodies and their targets: technical challenges and PK/PD applications over the course of drug development.

The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono- and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.

Quality assessment of bioanalytical quantification of monoclonal antibody drugs.

Monoclonal antibody biotherapeutics are developed to bind to a specific target to affect the target's biological effect. Reliable measurements of monoclonal antibodies in biological fluids using ligand-binding assays are vital for understanding the pharmacokinetic and pharmacodynamic relationships for efficacy/safety evaluations and dose-regimen selection. The method should be properly characterized and demonstrate adequate assay performance to generate credible data for the right decision making at each specific stage, with considerations on the constraints of timeline, reagent availability and assay caveats. Quality assessment of the assay performance should be based on whether the method is 'fit-for-use' to meet the objectives of the study. The basic industrial requirements and recent trends in method and data quality of ligand-binding assays for drug exposure studies at various development stages are discussed.

Ligand-binding mass spectrometry to study biotransformation of fusion protein drugs and guide immunoassay development: strategic approach and application to peptibodies targeting the thrombopoietin receptor.

The knowledge of in vivo biotransformation (e.g., proteolysis) of protein therapeutic candidates reveals structural liabilities that impact stability. This information aids the development and confirmation of ligand-binding assays with the required specificity for bioactive moieties (including intact molecule and metabolites) for appropriate PK profiling. Furthermore, the information can be used for re-engineering of constructs to remove in vivo liabilities in order to design the most stable candidates. We have developed a strategic approach of ligand-binding mass spectrometry (LBMS) to study biotransformation of fusion proteins of peptides fused to human Fc ("peptibodies") using anti-human Fc immunoaffinity capture followed by tiered mass spectrometric interrogation. LBMS offers the combined power of selectivity of ligand capture with the specificity and detailed molecular-level information of mass spectrometry. In this paper, we demonstrate the preclinical application of LBMS to three peptibodies, AMG531 (romiplostim), AMG195(linear), and AMG195(loop), that target the thrombopoietin receptor. The data show that ligand capture offers excellent sample cleanup and concentration of intact peptibodies and metabolites for subsequent query by matrix-assisted laser desorption ionization time-of-flight mass spectrometry for identification of in vivo proteolytic points. Additional higher-resolution analysis by nanoscale liquid chromatography interfaced with electrospray ionization mass spectrometry is required for identification of heterogeneous metabolites. Five proteolytic points are accurately identified for AMG531 and two for AMG195(linear), while AMG195(loop) is the most stable construct in rats. We recommend the use of LBMS to assess biotransformation and in vivo stability during early preclinical phase development for all novel fusion proteins.

Strategies to minimize variability and bias associated with manual pipetting in ligand binding assays to assure data quality of protein therapeutic quantification.

Bioanalytical laboratories require accurate and precise pipetting to assure reproducible and accurate results for reliable data. Two areas where pipetting differences among analysts lead to poor reproducibility are long term stability testing and sample dilution. The purpose of this paper is to illustrate the problems with manual pipetting, describe an automation strategy to mitigate risks associated with manual pipetting, and provide recommendations on a control strategy that properly monitors samples requiring dilutions. We determined differences among various manual pipetting techniques by analysts within a laboratory. To reduce variability in pipetting, a flexible modular liquid handling script was created on the Hamilton Microlab Star (HMS) to perform sample dilution, pre-treatment and plate loading. The script is capable of handling variable dilution factors. Additionally, two dilution controls were prepared and tested at concentrations of high and mid quality controls (QC). These same dilution controls were incorporated into both pre-study validation and in-study QCs to monitor dilution processing and assay performance. Variability of manual pipetting among 11 analysts was more negatively biased with increasing dilution. Forward and reverse pipetting delivering different volumes contributed to the discordance. The dilutional bias with manual pipetting was eliminated using the liquid handler. Total error of dilution controls was less than 20%. The in-study pass rate was 100%. Application of liquid handlers minimizes the variability and bias due to manual pipetting differences among analysts. The incorporation of dilution QCs serves a dual purpose to monitor the dilution process of the samples as well as the binding assay performance.

Tartrate-resistant acid phosphatase (TRACP 5b): a biomarker of bone resorption rate in support of drug development: modification, validation and application of the BoneTRAP kit assay.

A commercial kit assay of tartrate-resistant acid phosphatase (TRACP 5b) used for the diagnosis of bone resorption was modified with a 'Fit-For-Purpose' approach for drug development of anti-resorptive therapeutics. The modifications included changing the standard matrix from buffer to serum, using a consistent bulk reference material to prepare standards and quality controls (QC), and adding sample controls (SC) prepared from authentic sample pools. Method validation experiments were conducted for: inter- and intra-assay accuracy and precision, establishment of SC, range finding of different population groups, selectivity tests, parallelism and stability. The analytical range was 1.00-10.0 U/L and the total errors of lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) validation samples were 8% and 21%, respectively. Data of range finding experiment showed that serum samples should be collected in tubes instead of bags. Selectivity results showed accurate spike recovery among the majority of test samples from target populations. Samples were demonstrated to be stable for up to four freeze/thaw cycles and for 24 months at -70+/-10 degrees C. Our results show that the modified TRACP 5b method is reliable for the quantification of TRACP 5b in human serum samples to support clinical trials of bone resorptive effect reflected by TRACP 5b activities. The method was robust with similar assay performance characteristics shown in three bioanalytical laboratories.

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.

Method validation of protein biomarkers in support of drug development or clinical diagnosis/prognosis.

Protein biomarkers are used for various purposes in drug development and clinical diagnosis and prognosis. In this review, a fit-for-purpose method validation approach is discussed that fulfills the needs of exploratory and advanced applications in both the pharmaceutical and diagnostic arenas. Method validation for protein biomarkers is typically applied to ligand binding assays (LBA) although hyphenated mass spectrometric methods can be used as adjunct methodologies to confirm LBA specificity or provide valuable information during early discovery or demonstrative phases of a novel biomarker. Pre-analytic variables of protein biomarkers, such as the purpose of the intended application, analyte(s), biological matrix, availability of reference standard, calibrator matrix, assay platform, and sample collection/handling, must be considered in any method development and validation plan. Method validation for exploratory applications involves basic experiments for assay range finding, accuracy and precision, selectivity, specificity, and minimal stability. For advanced method validation, more rigorous tests with a wider scope are performed. These tests include additional patient population ranges, more runs on accuracy and precision from multiple analysts/reagent lots/instruments, selectivity and specificity tests using patient samples, and stability tests subjected to conceivable conditions over long-term use. Differences in biomarker method validation for drug development vs. clinical diagnosis and issues of using developmental commercial kits are discussed. The co-development of biomarkers for drug development and diagnostics presents collaborative opportunities between the pharmaceutical and diagnostic sectors.

Method validation and application of protein biomarkers: basic similarities and differences from biotherapeutics.

Protein drug development and biomarkers share common bioanalytical technologies that are applied for different purposes. A fit-for-purpose approach should be used for biomarker assays at various stages of novel biomarker development and their application to drug development. Biomarker quantifications can be absolute or relative, depending upon the characteristics of the standard curve, which include the reference standard, substituted matrix and parallelism. Appropriate method-validation experiments should be carried out on sample collection, relative accuracy and precision, range finding, parallelism, selectivity, specificity and stability in order to meet the need for exploratory or advanced application that is specified for a study. The interaction of a biotherapeutic with the target ligand or inter-related biomarkers should be taken into consideration for method platform choice and validation. Direct adoption of commercial diagnostic kits can produce confounding data. Therefore, kit comparison, modification and appropriate validation experiments are often carried out to meet the specific purpose for drug development. Multiplex assays and physicochemical methods can complement the single-analyte ligand-binding assay for protein drugs and biomarkers.

Chylomicron-bound LPS selectively inhibits the hepatocellular response to proinflammatory cytokines.

BACKGROUND/AIMS: Pretreatment of rodent hepatocytes with chylomicron-bound lipopolysaccharide (CM-LPS) renders these cells unresponsive to subsequent stimulation by proinflammatory cytokines. We sought to test the selectivity of this response. METHODS: Cellular responses to hypoxia, oxidative stress, apoptosis, and heat-shock response, and thermotolerance induced in CM-LPS pretreated hepatocytes were compared with responses in non-pretreated cells. RESULTS: CM-LPS inhibited the hepatocellular response to proinflammatory cytokines without affecting the response to the other cellular stressors. It did not affect the response to oxidative stress, as measured by mitochondrial activity after hydrogen peroxide was added, or protein induction before or after stimulation with cobalt chloride. Also, induction of heat shock proteins did not differ between the CM-LPS pretreated cells and non-pretreated cells. CM-LPS did not interfere with the adoption of the thermotolerant phenotype, as shown by similar mitochondrial activity between pretreated and non-pretreated cells. Although stimulation with tumor necrosis factor-alpha and actinomycin D increased activity of the apoptotic enzymes, there were no differences between cells pretreated with CM-LPS and non-pretreated hepatocytes. CONCLUSION: When the response to proinflammatory cytokines is inhibited, hepatocellular responses to hypoxia, oxidative stress, heat shock, and apoptosis remain intact after pretreatment with CM-LPS. CM-LPS may have a specific anti-inflammatory effect on hepatocytes.