Calibrator material selection: a key criteria during biomarker assay method development.

Biomarker assay method development is a multistep rigorous process and calibrant material selection is integral to ensuring the quality of such assays. However, the impact of selection of calibrator material may often get overlooked. In this article, we highlight three case studies where biomarker calibrant material selection was deemed an essential criterion for consideration. Through these case studies we highlight challenges faced, steps taken and discuss the impact on assay-related decision-making. We also provide additional perspectives for selection and characterization of calibrant proteins in the setting of an evolving biomarker context of use.

High-Throughput Therapeutic Antibody Interference-Free High-Resolution Mass Spectrometry Assay for Monitoring M-Proteins in Multiple Myeloma.

Measurement of monoclonal antibodies (M-proteins) plays an important role in the diagnosis and treatment monitoring of multiple myeloma. Currently available M-protein assays have several limitations, particularly because of their lack of sensitivity and propensity to therapeutic antibody (t-mAb) interference. A previously described mass spectrometry method termed monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) is more sensitive than current clinical tests and can provide a solution for resolving t-mAb interferences. However, the original miRAMM workflow is too complex for the throughput needed to analyze a large number of samples. Here, we describe a high-throughput liquid chromatography-high-resolution mass spectrometry (HT-LC-HRMS) approach that employs a fully automated immunocapture step, significantly improved immunoglobulin recovery, simplified chromatography, and high throughput (HT) data processing. In this HT-LC-HRMS approach, raw spectra of the peaks eluting from the LC column during the predefined time period are automatically deconvoluted without the need to identify and monitor the retention time of each patient-specific M-protein. The deconvoluted peak heights of M-protein and therapeutic antibody light chain are conveniently used for quantitation. With the total LC-HRMS measurement time being only 11.0 min, this method was able to differentiate between the M-protein and elotuzumab mass signatures in 91 out of 92 (98.9%) multiple myeloma serum samples tested. The single interference case was resolved using the mass signature of a heavy chain. In addition to resolving t-mAb interference, the developed assay has a 25-fold improvement in sensitivity over immunofixation electrophoresis and can potentially provide an objective tracking of M-proteins in patients with complete response.

Antipeptide Immunocapture with In-Sample Calibration Curve Strategy for Sensitive and Robust LC-MS/MS Bioanalysis of Clinical Protein Biomarkers in Formalin-Fixed Paraffin-Embedded Tumor Tissues.

Despite huge promises, bioanalysis of protein biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for clinical applications is still very challenging. Here, we describe a sensitive and robust LC-MS/MS assay to quantify clinical protein biomarkers in FFPE tumor sections using automated antipeptide antibody immunocapture followed by in-sample calibration curve (ISCC) strategy with multiple isotopologue reaction monitoring (MIRM) technique. ISCC approach with MIRM of stable isotopically labeled (SIL) peptides eliminated the need for authentic matrices for external calibration curves, overcame the matrix effects, and validated the quantification range in each individual sample. Specifically, after deparaffinization, rehydration, antigen retrieval, and homogenization, the protein analytes in FFPE tumor tissues were spiked with a known concentration of one SIL peptide for each analyte, followed by trypsin digestion and antipeptide immunocapture enrichment prior to MIRM-ISCC-based LC-MS/MS analysis. This approach has been successfully used for sensitive quantification of programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) in 15 representative FFPE tumor samples from lung, colorectal, and head and neck cancer patients. Except for one sample, PD-L1 and PD-1 in all samples were quantifiable using this assay with concentrations of 27.85-798.43 (amol/µg protein) for PD-L1 and 16.96-129.89 (amol/µg protein) for PD-1. These results were generally in agreement with the immunohistochemistry (IHC) data but with some exceptions. This approach demonstrated the feasibility to quantify low abundant protein biomarkers in FFPE tissues with improved sensitivity, specificity, and robustness and showed great potential as an orthogonal analytical approach to IHC for clinical applications.

A flow-through cell counting assay for point-of-care enumeration of CD4 T-cells.

CD4 T-cell count is a priority for staging HIV disease and guiding clinical management as part of HIV care. Conventional CD4 T-cell enumeration methods based on flow cytometry are expensive, require well-trained personnel, and are challenging to use in rural, resource-scarce areas. A simple CD4 T-cell count test that can be used at point-of care, the Flow-Through cell Counting Assay (FTCA), is described in this article. The FTCA is based on the use of: 1) a special membrane that selectively retains white blood cells (WBCs); 2) a sample delivery system; and 3) optical signal detection. To show the feasibility of the FTCA, a proof-of-concept prototype of the FTCA cassette and digital camera or handheld reflectance meter were used for obtaining quantitative assay results within 30 min. The results show that the FTCA allows for quantitative enumeration of CD4 T-cells in the clinically relevant range of CD4 T-cell concentrations. The advantages of the FTCA technology, including simplicity, short analysis time, and portability, suggest that FTCA has great potential for use in clinical practice and wide applicability for other cell-based diagnostic tests.

Critical reagent screening and characterization: benefits and approaches for protein biomarker assays by hybrid LC-MS.

In recent years, hybrid ligand-binding assays (LBAs)/LC-MS assays have been increasingly used for quantitation of protein biomarkers in biological matrices. However, unlike in LBAs where the importance of critical reagent screening and characterization is well understood and widely reported, benefits of well-characterized hybrid LC-MS assay reagents are frequently underestimated. Two groups of analyte-specific reagents, binding reagents and assay calibrators, are considered the critical reagents for biomarker assays. In this article, we summarize the similarities and differences of critical reagents used in LBAs and hybrid LC-MS assays, overview the benefits and approaches of critical reagent screening, characterization, antibody conjugation and discuss bioanalytical considerations in hybrid LC-MS assay development for robust measurements of protein biomarkers in biological matrices.

LC-MS/MS bioanalysis of plasma 1, 14-tetradecanedioic acid and 1, 16-hexadecanedioic acid as candidate biomarkers for organic anion-transporting polypeptide mediated drug-drug interactions.

AIM: A robust LC-MS/MS assay was developed to quantify endogenous 1, 14-tetradecanedioic acid (TDA) and 1, 16-hexadecanedioic acid (HDA) in human plasma as potential biomarkers for evaluating drug-drug interactions mediated by the hepatic drug transporters, organic anion-transporting polypeptides. RESULTS: This assay was validated using fit-for-purpose approach over standard curve range of 2.5-1000 nM for TDA and HDA using analyte-free charcoal-stripped human plasma as the surrogate matrix. Chromatographic separation condition was successfully optimized to separate TDA from an interference peak while maintaining both analytes in neutral forms to minimize carryover issue. CONCLUSION: The described assay is currently applied to a clinical study for evaluating TDA/HDA as potential substitute biomarkers for drug-drug interaction studies.

UHPLC-MS/MS bioanalysis of human plasma coproporphyrins as potential biomarkers for organic anion-transporting polypeptide-mediated drug interactions.

AIM: Coproporphyrins (CP-I and CP-III) have been identified as possible biomarkers to predict human hepatic organic anion-transporting polypeptides-mediated-drug-interactions for a new drug entering clinical development. RESULTS: The method is applicable to quantify plasma CP-I and CP-III within 0.078-15.0 nM. The results identify and address a number of challenges encountered with porphyrin assays such as photodegradation and interferences. To overcome interferences from ubiquitous porphyrins, a surrogate matrix was used to prepare calibration standards. Quality controls were prepared in plasma and surrogate matrix to ensure parallelism between surrogate matrix and plasma. CONCLUSION: A robust UHPLC-MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug-drug interaction study.

Surface plasmon resonance as a tool for ligand-binding assay reagent characterization in bioanalysis of biotherapeutics.

Ligand-binding assay (LBA) performance depends on quality reagents. Strategic reagent screening and characterization is critical to LBA development, optimization and validation. Application of advanced technologies expedites the reagent screening and assay development process. By evaluating surface plasmon resonance technology that offers high-throughput kinetic information, this article aims to provide perspectives on applying the surface plasmon resonance technology to strategic LBA critical reagent screening and characterization supported by a number of case studies from multiple biotherapeutic programs.

Increased Sensitivity of HIV-1 p24 ELISA Using a Photochemical Signal Amplification System.

BACKGROUND: In this study we describe a photochemical signal amplification method (PSAM) for increasing of the sensitivity of enzyme-linked immunosorbent assay (ELISA) for determination of HIV-1 p24 antigen. The photochemical signal amplification method is based on an autocatalytic photochemical reaction of a horseradish peroxidase (HRP) substrate, orthophenylenediamine (OPD). METHODS: To compare the performance of PSAM-boosted ELISA with a conventional colorimetric ELISA for determination of HIV-1 p24 antigen we employed a PerkinElmer HIV-1 p24 ELISA kit, using conventional ELISA alongside ELISA + PSAM. RESULTS: In the present study, we show that PSAM technology allows one to increase the analytical sensitivity and dynamic range of a commercial HIV-1 p24 ELISA kit, with and without immune-complex disruption, by a factor of approximately 40-fold. CONCLUSIONS: ELISA + PSAM is compatible with commercially available microtiter plate readers, requires only an inexpensive illumination device, and the PSAM amplification step takes no longer than 15 min. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods. This method can be used for both commercially available and in-house ELISA tests, and has the advantage of being considerably simpler and less costly than alternative signal amplification methods.

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

Activation of neuronal defense mechanisms in response to pathogenic factors triggering induction of amyloidosis in Alzheimer's disease.

We present a new model for etiology of Alzheimer's disease (AD) which postulates early involvement of specialized neuroprotective mechanisms in the pathology of AD. These neuroprotective mechanisms work in concert to regulate metabolic homeostasis in healthy neuronal cells, but contribute to the distinctive cytopathic phenotype of neuronal degeneration in AD. According to this model, two molecular/genetic hallmarks of AD, amyloid-ß (Aß) deposition and tau hyperphosphorylation, are associated with neuronal mechanisms for dissipating thermal energy associated with high levels of protein synthesis in highly temperature-sensitive neuronal cells. Development of effective methods of AD treatment will require a better understanding of how this neuronal defense system is activated in response to cytopathological triggers in sporadic AD. The cause and effect link between synthesis and processing of amyloid-ß protein precursor (AßPP) and the AD terminal phenotype of neurofibrillary tangles and neuron loss involve the formation of Aß peptides that accumulate as oligomers, cannot be controlled by neurons, and are toxic to the surrounding neuronal membranes. We analyze experimental and clinical studies that have investigated the correlation between phosphorylation of some transport proteins and increased synthesis of proteins in neurons. We also review the evidence related to the possibility that protein hyperphosphorylation may be a byproduct of energetic imbalances in AD cells associated with high levels of protein synthesis, and that activation of defense systems, through which energy-rich molecules are eliminated from the site of protein synthesis and are sequestered to the peripheral neuronal areas, may bring about some of the distinctive morphological features of AD.

FAT10 modifies p53 and upregulates its transcriptional activity.

FAT10, also known as diubiquitin, has been implicated in the regulation of diverse cellular processes, including mitosis, immune response, and apoptosis. We seek to identify FAT10-targeted proteins, an essential step in elucidating the physiological function of FAT10. To this end, human FAT10 or its non-conjugatable derivative, FAT10ΔGG, was overexpressed in HEK293 cells. We observed a number of high molecular weight FAT10 conjugates in cells expressing wild-type FAT10, but not in FAT10ΔGG. The FAT10 conjugates are inducible by TNF-α and accumulated significantly when cells were treated with proteasome inhibitor, MG132. Among them, tumor suppressor p53 was found to be FATylated. The p53 transcriptional activity was found to be substantially enhanced in FAT10-overexpressing cells. In addition, overexpressing FAT10 in HEK293 cells also reduced the population of p53 which cross reacted with monoclonal anti-p53 antibody, PAB240, known to recognize only the transcriptionally inactive p53. FAT10 in the nucleus was found co-localized with p53 and altered its subcellular compartmentalization. Furthermore, overexpressing FAT10 led to a reduction in the size of promyelocytic leukemia nuclear bodies (PML-NBs) and altered their distribution in the nucleus. Based on these observations, a potential mechanism which correlates FATylation of p53 to its translocation and transcriptional activation is discussed.

Protein scission by metal ion-ascorbate system.

About 14 proteins were tested for specific oxidative scission catalyzed by metal ions in the presence of ascorbate and oxidizing agents (O(2) or hydrogen peroxide). Only four of them were degraded by Fe(3+)/Fe(2+)- ascorbate, twelve - by Cu(2+)/Cu(+)-ascorbate and two proteins (alpha- and beta-caseins) were degraded by Pd(2+) ions. The rate and the intensity of degradation are very different for various proteins. For the most of tested proteins only a small fraction of molecules was degraded. None of them was degraded completely. Two possible reasons of protein stability against oxidative degradation may be proposed as follows: either there is no metal binding site in a protein molecule, or metal binding ligands of protein undergo a rapid oxidative modification and the metal ion is released from the binding site. Human growth hormone was cut specifically at two sites by Cu(2+)/Cu(+)-ascorbate system. At least one of amino acid residues of this protein was modified by formation of reactive carbonyl.

Expression of SUMO-2/3 induced senescence through p53- and pRB-mediated pathways.

Three highly homologous small ubiquitin-related modifier (SUMO) proteins have been identified in mammals. Modifications of proteins by SUMO-1 have been shown to regulate transcription, nucleocytoplasmic transport, protein stability, and protein-protein interactions. Relative to SUMO-1, little is known about the functions of SUMO-2 or SUMO-3 (referred to as SUMO-2/3). Here, stable cell lines overexpressing processed forms of SUMO-2/3 (SUMO-2/3GG) as well as their non-conjugatable derivatives, SUMO-2/3DeltaGG, were established. Cells overexpressing SUMO-2/3GG showed a premature senescence phenotype as revealed by cellular morphology and senescence-associated galactosidase activity. The senescence pathway protein p21 was up-regulated in cells overexpressing SUMO-2/3GG. In contrast, cells overexpressing non-conjugatable forms of SUMO-2/3DeltaGG showed neither an apparent senescent phenotype nor elevated p21. Both p53 and pRB were found to be modified by SUMO-2/3. Site-directed mutagenesis studies showed that Lys-386 of p53, the SUMO-1 modification site, is also the modification site for SUMO-2/3. In addition, H2O2 treatment of untransfected cells caused an increase in p53 sumoylation by SUMO-2/3, whereas that by SUMO-1 remained unchanged. Moreover, knocking down tumor suppressor proteins p53 or pRB using small interfering RNA significantly alleviated the premature senescence phenotypes in SUMO-2/3GG overexpressing cells. Together, our results reveal that p53 and pRB can be sumoylated by SUMO-2/3 in vivo, and such modification of p53 and pRB may play roles in premature senescence and stress response.

A general approach for investigating enzymatic pathways and substrates for ubiquitin-like modifiers.

Ubiquitin-like modifiers (UBLs) contain ubiquitin homology domains and can covalently modify target proteins in a manner similar to ubiquitylation. In this study, we revealed a general proteomic approach to elucidate the enzymatic pathways and identify target proteins for three UBLs: SUMO-2, SUMO-3, and NEDD8. Expression plasmids containing the cDNAs of Myc/6xHis doubly-tagged processed or non-conjugatable forms of these UBLs were constructed. The constructed vectors were then used to transfect HEK 293 Tet-On cells, and stable cell lines expressing these UBLs and their mutants were established. The epitope-tagged proteins were purified by immunoprecipitation under native conditions or by affinity chromatography on nickel resin under denaturing conditions. Purified proteins were analyzed using liquid chromatography coupled with mass spectrometry (LC-MS/MS). Most of the E1-like activating enzymes, E2-like conjugating enzymes and the majorities of the known target as well as some previously unreported proteins for SUMO-2, SUMO-3, and NEDD8 pathways were identified.