Interlaboratory evaluation of LC-MS-based biomarker assays.

Validation of biomarker assays is crucial for effective drug development and clinical applications. Interlaboratory reproducibility is vital for reliable comparison and combination of data from different centers. This review summarizes interlaboratory studies of quantitative LC-MS-based biomarker assays using reference standards for calibration curves. The following points are discussed: trends in reports, reference and internal standards, evaluation of analytical validation parameters, study sample analysis and normalization of biomarker assay data. Full evaluation of these parameters in interlaboratory studies is limited, necessitating further research. Some reports suggest methods to address variations in biomarker assay data among laboratories, facilitating organized studies and data combination. Method validation across laboratories is crucial for reducing interlaboratory differences and reflecting target biomarker responses.

Multi-laboratory evaluation of immunoaffinity LC-MS-based glucagon-like peptide-1 assay.

Background: Although the fit-for-purpose approach has been proposed for biomarker assay validation, practical data should be compiled to facilitate the predetermination of acceptance criteria. Methods: Immunoaffinity LC-MS was used to analyze glucagon-like peptide-1 as a model biomarker in six laboratories. Calibration curve, carryover, parallelism, precision, relative accuracy and processed sample stability were evaluated, and their robustness among laboratories was assessed. The rat glucagon-like peptide-1 concentrations in four blinded samples were also compared. Results: The obtained results and determined concentrations in the blinded samples at all laboratories were similar, with a few exceptions, and robust, despite the difference in optimization techniques among laboratories. Conclusion: The results provide insights into the predefinition of the acceptance criteria of immunoaffinity LC-MS-based biomarker assays.

Development and multicenter validation of an LC-MS-based bioanalytical method for antisense therapeutics.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

A multilaboratory validation study of LC/MS biomarker assays for three lysophosphatidylcholines.

Aim: Although the fit-for-purpose approach has been proposed for validation procedures and acceptance criteria for biomarker assays, practical biomarker assays to facilitate clinical application and regulatory documents on biomarker assays remain limited. Materials & methods: We assigned six independent laboratories and selected three lysophosphatidylcholines (LPCs): LPC(16:0), LPC(18:0) and LPC(18:1) as model biomarkers. Using LC-MS, the following key validation parameters were evaluated: calibration curve, carryover, parallelism, precision and relative accuracy and these values were similar among all laboratories. Further, we determined LPC levels in six lots of rat plasma at unknown concentrations and compared them among the laboratories. Conclusion: Our multilaboratory validation and reproducibility data are useful for the development of future biomarker assay validation procedures, as well as regulatory documents.

Renadirsen, a Novel 2'OMeRNA/ENA® Chimera Antisense Oligonucleotide, Induces Robust Exon 45 Skipping for Dystrophin In Vivo.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients' DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2'-O,4'-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2'-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2'OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.

Bioanalysis of therapeutic monoclonal antibody by peptide adsorption-controlled LC-MS.

Aim: We aimed to develop an easy, low-cost and versatile mass spectrometric method for the bioanalysis of a therapeutic monoclonal antibody (mAb) in human serum that employs peptide adsorption-controlled (PAC)-LC/MS using selected reaction monitoring mode (LC-MS/MS-SRM). Materials & methods: Rituximab was used as a model mAb. To apply the method to human serum samples, a peptide of the complementarity-determining region was selected as the surrogate peptide. The usefulness of PAC-LC-MS/MS-SRM was evaluated by a collaborative study. Results: The calibration curve ranged from 0.5 (or 1.0) to 1000.0 µg/ml. The selectivity, linearity, accuracy and precision met the predefined acceptance criteria. Conclusion: Our method could be a useful bioanalytical method for the quantification of mAbs in clinical samples.

Development of a bioanalytical method for an antisense therapeutic using high-resolution mass spectrometry.

Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5-250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.

Generic MS-based method for the bioanalysis of therapeutic monoclonal antibodies in nonclinical studies.

Aim: A generic bioanalytical method was developed to quantify therapeutic IgG1 monoclonal antibodies (mAbs) in mouse sera by combining an easy sample preparation method with LC/MS using selected reaction monitoring. Materials & methods: Rituximab and trastuzumab were used as model mAbs. A synthetic stable isotope-labeled peptide or a stable isotope-labeled mAb was used as an internal standard. The method feasibility was evaluated by a collaborative study involving six laboratories. Results: The calibration curve ranged from 1.0 to 1000.0 µg/ml (correlation coefficient >0.99). The validation parameters including selectivity, linearity of calibration curve, accuracy and precision met the predefined acceptance criteria. Conclusion: Our method is a useful bioanalytical method for the quantification of therapeutic IgG mAbs in nonclinical animal studies.

ABCE1 Acts as a Positive Regulator of Exogenous RNA Decay.

The 2'-5'-oligoadenylate synthetase (OAS)/RNase L system protects hosts against pathogenic viruses through cleavage of the exogenous single-stranded RNA. In this system, an evolutionally conserved RNA quality control factor Dom34 (known as Pelota (Pelo) in higher eukaryotes) forms a surveillance complex with RNase L to recognize and eliminate the exogenous RNA in a manner dependent on translation. Here, we newly identified that ATP-binding cassette sub-family E member 1 (ABCE1), which is also known as RNase L inhibitor (RLI), is involved in the regulation of exogenous RNA decay. ABCE1 directly binds to form a complex with RNase L and accelerates RNase L dimer formation in the absence of 2'-5' oligoadenylates (2-5A). Depletion of ABCE1 represses 2-5A-induced RNase L activation and stabilizes exogenous RNA to a level comparable to that seen in RNase L depletion. The increased half-life of the RNA by the single depletion of either protein is not significantly affected by the double depletion of both proteins, suggesting that RNase L and ABCE1 act together to eliminate exogenous RNA. Our results indicate that ABCE1 functions as a positive regulator of exogenous RNA decay rather than an inhibitor of RNase L.

Observation of Clinically Relevant Drug Interaction in Chimeric Mice with Humanized Livers: The Case of Valproic Acid and Carbapenem Antibiotics.

BACKGROUND AND OBJECTIVE: Human in vitro and dog in vitro/in vivo researches indicate that the drug-drug interaction (DDI) of decreased plasma valproic acid (VPA) concentration by co-administration of carbapenem antibiotics is caused by inhibition of acylpeptide hydrolase (APEH)-mediated VPA acylglucuronide (VPA-G) hydrolysis by carbapenems. In this study, we investigated VPA disposition and APEH activities in TK-NOG chimeric mice, whose livers were highly replaced with human hepatocytes, to evaluate the utility of this animal model and the clinical relevance of the DDI mechanism. METHODS: VPA and VPA-G concentrations in plasma, urinary excretion of VPA-G and APEH activity in humanized livers were measured after co-administration of VPA with meropenem (MEPM) to chimeric mice. RESULTS: After co-administration with MEPM to the chimeric mice, plasma VPA concentration more rapidly decreased than without the co-administration. An increase in plasma AUC and urinary excretion of VPA-G was also observed. APEH activity in humanized livers was strongly inhibited even at 24 h after co-administration of MEPM to the chimeric mice. CONCLUSION: The DDI of VPA with carbapenems was successfully observed in chimeric mice with humanized livers. The DDI was caused by long-lasting inhibition of hepatic APEH-mediated VPA-G hydrolysis by carbapenems, which strongly supports the APEH-mediated mechanism of the clinical DDI. This is the first example showing the usefulness of chimeric mice with humanized livers for evaluation of a DDI via non-cytochrome P450 enzyme.

2'-O-Methyl RNA/Ethylene-Bridged Nucleic Acid Chimera Antisense Oligonucleotides to Induce Dystrophin Exon 45 Skipping.

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disease characterized by dystrophin deficiency from mutations in the dystrophin gene. Antisense oligonucleotide (AO)-mediated exon skipping targets restoration of the dystrophin reading frame to allow production of an internally deleted dystrophin protein with functional benefit for DMD patients who have out-of-frame deletions. After accelerated US approval of eteplirsen (Exondys 51), which targets dystrophin exon 51 for skipping, efforts are now focused on targeting other exons. For improved clinical benefits, this strategy requires more studies of the delivery method and modification of nucleic acids. We studied a nucleotide with a 2'-O,4'-C-ethylene-bridged nucleic acid (ENA), which shows high nuclease resistance and high affinity for complementary RNA strands. Here, we describe the process of developing a 2'-O-methyl RNA(2'-OMeRNA)/ENA chimera AO to induce dystrophin exon 45 skipping. One 18-mer 2'-OMeRNA/ENA chimera (AO85) had the most potent activity for inducing exon 45 skipping in cultured myotubes. AO85 was administered to mdx mice without significant side effects. AO85 transfection into cultured myotubes from 13 DMD patients induced exon 45 skipping in all samples at different levels and dystrophin expression in 11 patients. These results suggest the possible efficacy of AO-mediated exon skipping changes in individual patients and highlight the 2'-OMeRNA/ENA chimera AO as a potential fundamental treatment for DMD.

In vivo inhibition of acylpeptide hydrolase by carbapenem antibiotics causes the decrease of plasma concentration of valproic acid in dogs.

1. Our previous in vitro studies suggest that inhibition of the acylpeptide hydrolase (APEH) activity as valproic acid glucuronide (VPA-G) hydrolase by carbapenems in human liver cytosol is a key process for clinical drug-drug interaction (DDI) of valproic acid (VPA) with carbapenems. Here, we investigated whether in vivo DDI of VPA with meropenem (MEPM) was caused via inhibition of APEH in dogs. 2. More rapid decrease of plasma VPA levels and increased urinary excretion of VPA-G were observed after co-administration with MEPM compared with those after without co-administration, whereas the plasma level and bile excretion of VPA-G showed no change. 3. Dog VPA-G hydrolase activity, inhibited by carbapenems, was mainly located in cytosol from both the liver and kidney. APEH-immunodepleted cytosols lacked VPA-G hydrolase activity. Hepatic and renal APEH activity was negligible even at 24 h after dosing of MEPM to a dog. 4. In conclusion, DDI of VPA with carbapenems in dogs is caused by long-lasting inhibition of APEH-mediated VPA-G hydrolysis by carbapenems, which could explain the delayed recovery of plasma VPA levels to the therapeutic window even after discontinuation of carbapenems in humans.

Evaluation of peptide adsorption-controlled liquid chromatography-tandem mass spectrometric (PAC-LC-MS/MS) method for simple and simultaneous quantitation of amyloid ß 1-38, 1-40, 1-42 and 1-43 peptides in dog cerebrospinal fluid.

To evaluate the usefulness of the peptide adsorption-controlled liquid chromatography-tandem mass spectrometry (PAC-LC-MS/MS) for reproducible measurement of peptides in biological fluids, simultaneous quantitation of amyloid ß 1-38, 1-40, 1-42 and 1-43 peptides (Aß38, Aß40, Aß42 and Aß43) in dog cerebrospinal fluid (CSF) was tried. Each stable isotope labeled Aß was used as the internal standard to minimize the influence of CSF matrix on the reproducible Aß quantitation. To reduce a loss of Aß during the pretreatment procedures, the dog CSF diluted by water-acetic acid-methanol (2:6:1, v/v/v) was loaded on PAC-LC-MS/MS directly. Quantification of the Aß in the diluted dog CSF was carried out using multiple reaction monitoring (MRM) mode. The [M+5H(5+)] and b(5+) ion fragment of each peptide were chosen as the precursor and product ions for MRM transitions of each peptide. The calibration curves were drawn from Aß standard calibration solutions using PAC-LC-MS/MS. Analysis of dog CSF samples suggests that the basal concentration of Aß38, Aß40, Aß42 and Aß43 in dog CSF is approximately 300, 900, 200 and 30 pM, respectively. This is the first time Aß concentrations in dog CSF have been reported. Additionally, the evaluation of intra- and inter-day reproducibility of analysis of Aß standard solution, the freeze-thaw stability and the room temperature stability of Aß standard solution suggest that the PAC-LC-MS/MS method enables reproducible Aß quantitation.

Development of a pretreatment method for amyloid beta-protein analysis based on the effect of acetic acid on the dissolution of plasma polypeptides.

During the evaluation of a pretreatment method for the simultaneous quantification of four amyloid beta-protein fragments in transgenic mice plasma by a new gradient system, we have found that acetic acid has potency to completely dissolve plasma polypeptides in the presence of an organic solvent. Based on this observation, we designed a simple pretreatment method using an ultrafiltration membrane. An analysis of the filtrate obtained by this method suggests the possibility that acetic acid inhibits the interaction between amyloid beta-protein fragments and plasma polypeptides, which leads to a higher recovery of the amyloid beta-protein fragments from mouse plasma. In addition, higher dilution of mouse plasma using a dilution solution produced higher recovery as well. The highest recovery of amyloid beta-protein 1-38, 1-40, 1-42 and 1-43 fragments was 101.7, 94.9, 96.2 and 84.8%, respectively. Furthermore, calibration curves with the lower limit of quantification of 0.65 nM were successfully constructed with good accuracy using the developed method. Consequently, a pretreatment method using an ultrafiltration membrane is a powerful tool to determine the amyloid beta-protein fragments in transgenic mice plasma containing an abundance of plasma polypeptides such as albumin.

Superiority of a new gradient system utilizing a critical threshold for the adsorption capacity of polypeptides to column packing when compared with a standard gradient system for the simultaneous and quantitative analysis of polypeptides.

Compared with a standard gradient system, the new gradient system which we developed has a major advantage because it permits a wide range of acetonitrile content, e.g. more than the critical threshold, in the polypeptide solution and allows the quantitative analysis of the polypeptide with satisfactory analytical precision. Additionally, this new gradient system allows the enhancement of the sensitivity of the polypeptide analysis proportionate to the increased volume of solution loaded with the same levels of precision. In contrast, when using a standard gradient system it is difficult to analyze a polypeptide quantitatively with good precision due to either adsorption to various materials or to irregular change in the ratio between a retained and a passed peak of the polypeptide. Additionally, the appearance of a passed peak results in a loss in the sensitivity of the polypeptide analysis, although no adsorption of a polypeptide to various materials occurs in a solution with acetonitrile content more than the critical threshold. Consequently, the new gradient system is effective for the simultaneous and quantitative analysis of different polypeptides with good precision and without any loss of sensitivity due to either adsorption to various materials or the appearance of a passed peak.

Elution mechanism of polypeptides in reversed-phase liquid chromatography based on the critical threshold of organic solvent to induce abrupt change in adsorption capacity to the column packing.

Adsorption capacity of polypeptides to the column packing in a solution containing multiple organic solvents was found to be expressed by means of an fn value, which is the sum of the ratios of the content of each organic solvent in the solution to the critical content of each organic solvent to cause abrupt change in the adsorption capacity, and to change abruptly at the point where the fn value becomes 1. Additionally, our results indicate that each polypeptide is eluted by the eluent containing a specific organic solvent content regardless of gradient elution rate in reversed-phase liquid chromatography, and that total organic solvent content in the eluent containing polypeptides is equal to the critical content. Considering the power law relationship between the retention times and the gradient elution rates, our results suggest that the elution of each polypeptide in reversed-phase liquid chromatography is mainly controlled by abrupt change in the adsorption capacity induced by change in the organic solvent content of the eluent during a gradient elution process, and that the abrupt change repeats across the critical threshold while a polypeptide moves through the column, and as a result, each polypeptide is concentrated in the eluent with the critical threshold.

Highly sensitive and quantitative analysis of polyeptides using a new gradient system based on an abrupt change in adsorption of polypeptide to the reversed-phase column packing.

During a study of 100 microL aliquots of urocortin containing various acetonitrile contents, we hypothesized that a change in the acetonitrile content in the solution across a specific content of acetonitrile (critical threshold) causes an abrupt change in adsorption capacity to the column packing. Circular dichroism measurements suggest that the conformational change induced by acetonitrile in the solution causes the abrupt change in adsorption capacity, and this solvent-induced conformational change is reversible across the critical threshold. This hypothesis can apply to various polypeptides with molecular weights range from 1007 to 6789 and to other organic solvents. A new gradient system utilizing the instant recovery of the adsorption capacity across the critical threshold was designed, and applied to the analysis of a 100 microL aliquot of various polypeptide solutions. The results suggest that use of a solution containing organic solvents more than the critical threshold allows successful dilution of polypeptides up to picomolar concentration range without any loss due to its adsorption during handling procedures and loading onto the LC system, and that a new gradient system enables quantitative analysis of polypeptides at picomolar concentrations in such solutions.