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.

Identification of kynurenine and quinolinic acid as promising serum biomarkers for drug-induced interstitial lung diseases.

BACKGROUND: Drug-induced interstitial lung disease (DILD) is a lung injury caused by various types of drugs and is a serious problem in both clinical practice and drug development. Clinical management of the condition would be improved if there were DILD-specific biomarkers available; this study aimed to meet that need. METHODS: Biomarker candidates were identified by non-targeted metabolomics focusing on hydrophilic molecules, and further validated by targeted approaches using the serum of acute DILD patients, DILD recovery patients, DILD-tolerant patients, patients with other related lung diseases, and healthy controls. RESULTS: Serum levels of kynurenine and quinolinic acid (and kynurenine/tryptophan ratio) were elevated significantly and specifically in acute DILD patients. The diagnostic potentials of these biomarkers were superior to those of conventional lung injury biomarkers, Krebs von den Lungen-6 and surfactant protein-D, in discriminating between acute DILD patients and patients with other lung diseases, including idiopathic interstitial pneumonia and lung diseases associated with connective tissue diseases. In addition to identifying and evaluating the biomarkers, our data showed that kynurenine/tryptophan ratios (an indicator of kynurenine pathway activation) were positively correlated with serum C-reactive protein concentrations in patients with DILD, suggesting the potential association between the generation of these biomarkers and inflammation. Our in vitro experiments demonstrated that macrophage differentiation and inflammatory stimulations typified by interferon gamma could activate the kynurenine pathway, resulting in enhanced kynurenine levels in the extracellular space in macrophage-like cell lines or lung endothelial cells. Extracellular quinolinic acid levels were elevated only in macrophage-like cells but not endothelial cells owing to the lower expression levels of metabolic enzymes converting kynurenine to quinolinic acid. These findings provide clues about the molecular mechanisms behind their specific elevation in the serum of acute DILD patients. CONCLUSIONS: The serum concentrations of kynurenine and quinolinic acid as well as kynurenine/tryptophan ratios are promising and specific biomarkers for detecting and monitoring DILD and its recovery, which could facilitate accurate decisions for appropriate clinical management of patients with DILD.

Identification of the Stapled α-Helical Peptide ATSP-7041 as a Substrate and Strong Inhibitor of OATP1B1 In Vitro.

ATSP-7041, a stapled α-helical peptide that inhibits murine double minute-2 (MDM2) and MDMX activities, is a promising modality targeting protein-protein interactions. As peptides of molecular weights over 1000 Da are not usually evaluated, data on the drug-drug interaction (DDI) potential of stapled α-helical peptides remain scarce. Here, we evaluate the interaction of ATSP-7041 with hepatic cytochrome P450s (CYPs; CYP1A2, CYP2C9, CYP2C19, CYP3A4, and CYP2D6) and transporters (organic anion transporting polypeptides (OATPs; OATP1B1 and OATP1B3), P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP)). ATSP-7041 demonstrated negligible metabolism in human liver S9 fraction and a limited inhibition of CYP activities in yeast microsomes or S9 fractions. On the contrary, a substantial uptake by OATPs in HEK 293 cells, a strong inhibition of OATP activities in the cells, and an inhibition of P-gp and BCRP activities in reversed membrane vesicles were observed for ATSP-7041. A recent report describes that ALRN-6924, an ATSP-7041 analog, inhibited OATP activities in vivo; therefore, we focused on the interaction between ATSP-7041 and OATP1B1 to demonstrate that ATSP-7041, as a higher molecular weight stapled peptide, is a substrate and strong inhibitor of OATP1B1 activity. Our findings demonstrated the possibility of transporter-mediated DDI potential by high molecular weight stapled peptides and the necessity of their evaluation for drug development.

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.

Characterization of serotonin as a candidate biomarker of severity and prognosis of COVID-19 using LC/MS analysis.

The coronavirus disease 2019 (COVID-19) pandemic has been associated with high mortality worldwide. Owing to its complicated pathophysiology, diagnostic and prognostic biomarkers for effective patient management remain scarce. We analyzed kynurenine, tryptophan, and serotonin levels in the serum of patients with COVID-19 via liquid chromatography/mass spectrometry analysis. Serum serotonin levels were decreased in patients with more severe COVID-19, along with increased kynurenine and decreased tryptophan concentrations. Patients with moderate disease who subsequently worsened showed significantly lower serotonin concentrations compared with those who did not experience severe disease. Serum serotonin levels may represent a valuable biomarker for COVID-19 severity and prognosis.

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.

Measurement of sialic acid content is insufficient to assess bioactivity of recombinant human erythropoietin.

Assessment of biological potency and its comparison with clinical effects are important in the quality control of therapeutic glycoproteins. Animal models are usually used for evaluating bioactivity of these compounds. However, alternative methods are required to simplify the bioassay and avoid ethical issues associated with animal studies. Negatively charged sialic acid residues are known to be critical for in vivo bioactivity of recombinant human erythropoietin (rhEPO). In this study, we used capillary zone electrophoresis, a charge-based separation method, to estimate the sialic acid content for predicting in vivo bioactivity of rhEPO. In vivo bioactivities of rhEPO subfractions were measured and compared with sialylation levels. The results obtained indicated that in vivo bioactivity of rhEPO is not simply correlated with the sialylation level, which suggests that it is difficult to predict biological potency from the sialic acid content alone. N-Glycan moieties as well as sialic acid residues may have a significant impact on in vivo bioactivity of rhEPO.

Production of novel anti-recombinant human erythropoietin monoclonal antibodies and development of a sensitive enzyme-linked immunosorbent assay for detection of bioactive human erythropoietin.

Erythropoietin (EPO) is a growth factor, regulating the proliferation and differentiation of erythroid progenitor cells. In this study, we generated five monoclonal antibodies (mAbs) that reacted specifically with recombinant human EPO (rhEPO). Epitope exclusion and other experiments showed that the mAbs obtained were divided into two groups, differing in recognition sites for rhEPO: group 1 mAbs recognize the N-terminal region of rhEPO, whereas group 2 mAbs seem to recognize a conformation-dependent epitope. Although most of the previously reported anti-EPO antibodies that recognized the N-terminal region of EPO lacked the EPO-neutralizing activity, the group 1 mAbs obtained here had the rhEPO-neutralizing activity. Therefore, the group 1 mAbs may be useful for future study on structure-function relationship of EPO. One of the group 2 mAbs, 5D11A, showed the highest affinity for rhEPO with K(D) value 0.52 nM and had the highest rhEPO-neutralizing activity. Using this mAb, we developed a reproducible and sensitive enzyme-linked immunosorbent assay for the quantification of bioactive rhEPO.

Capillary electrophoresis with laser-induced fluorescence detection for detailed studies on N-linked oligosaccharide profile of therapeutic recombinant monoclonal antibodies.

Total N-linked oligosaccharide profiling method for recombinant monoclonal antibody (rmAb) using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and an approach for detailed structural analysis of N-linked oligosaccharide were developed. A CE-LIF method using 2-aminobenzoic acid (2-AA) as a fluorogenic reagent allowed sensitive detection of several minor peaks besides typical asialo-biantennary complex type oligosaccharides in the analysis of N-linked oligosaccharide from a commercial rmAb pharmaceutical, rituximab. These minor peaks were successfully assigned as sialo-biantennary complex type and high-mannose type oligosaccharides by comparison with the migration times of 2-AA derivatized oligosaccharides which were separately fractionated and determined by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In development of biopharmaceuticals, it is important to evaluate these minor oligosaccharides, because some of these minor glycans are likely to influence immunogenicity and clearance rate in vivo. The repetitive analysis using CE-LIF showed excellent precision in relative corrected peak areas. These results demonstrate that the present CE-LIF method is applicable for both structural characterization and quantitative profiling of N-linked oligosaccharides derived from rmAb pharmaceuticals. The present method will be a powerful tool for rapid, quantitative and exhaustive evaluation of N-linked oligosaccharides in various stages of rmAb pharmaceutical development such as clone selection, bioprocess control, and routine lot release testing to ensure product efficacy and consistency.

Methanol-induced tertiary and secondary structure changes of granulocyte-colony stimulating factor.

We have studied methanol-induced conformational changes in rmethuG-CSF at pH 2.5 by means of circular dichroism (CD), fluorescence and infrared (IR) spectroscopy, and 8-anilino-1-naphthalene sulfonic acid (ANS) binding. Methanol has little effect on the secondary and tertiary structures of rmethuG-CSF when its concentration is in the range of 0 to 20% (v/v). At 30% (v/v) methanol, rmethuG-CSF has ANS binding ability. In the methanol concentration range of 30 to 70% (v/v) the amount of alpha-helix decreases a little, and the tertiary structure decreases significantly. At methanol concentrations above 70% (v/v), a transition to a more helical state occurs, while there is little change in the tertiary structure, and no ANS binding ability. Thermal denaturation studies involving CD have demonstrated that as the methanol concentration increases the melting temperature and the cooperativity of transition decrease, and the transition covers a much wider range of temperature. It seems that the decreased cooperativity means an increase in the concentration of partially folded intermediate states during the unfolding of rmethuG-CSF.

Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method.

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

Effects of ionic strength on the thermal unfolding process of granulocyte-colony stimulating factor.

This paper reports the effect of ionic strength on the process of thermal unfolding of recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) at acid pH. We previously reported that the protein aggregates were formed at the highest temperature at pD 2.1 in the pD range of 5.5-2.1 and that the aggregation proceeded a little at pD 2.1 because of the strong repulsive interaction between the unordered structures that play the role of a precursor for the aggregation. In the present study temperature-dependent IR spectra and far-UV CD spectra were measured for rmethuG-CSF in aqueous solutions containing various concentrations of NaCl at acid pH. Second derivative and curve-fitting analysis were performed to examine the obtained IR spectra. The results revealed that the structure of rmethuG-CSF becomes less stable with increasing ionic strength at all pDs investigated (pD 2.1, 2.5, and 4.0). We have also demonstrated that, at pD 2.1, the temperature at which the protein aggregation starts becomes lower and that the amount of the aggregates becomes larger with the addition of NaCl. This is probably because the addition of NaCl masks the repulsive electrostatic interaction between the unordered structures.

An infrared spectroscopy study of acid stability and thermal unfolding process of granulocyte-colony stimulating factor.

Temperature-dependent (25-80 degrees C) infrared (IR) spectra were obtained for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over the pD range of 5.5-2.1 to investigate its thermal stability at various pDs. Second derivative, Fourier self-deconvolution, and curve-fitting analyses were performed to analyze the obtained spectra. These spectral analyses demonstrated that in the thermal unfolding process the alpha-helix structure of rmethuG-CSF partially changes to an unordered structure and then the unordered structure forms aggregates. The temperature-dependent IR spectra revealed that the structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 5.5-2.1. It has been suggested that the unordered structure formed before the marked structural change in the whole molecule is a perturbed form of the native structure of rmethuG-CSF and plays a role as a precursor for the aggregation. This alteration to the perturbed form is likely to be the first secondary structure change that occurs along the aggregation pathway. Of particular note is that the stability at pD 2.1 is slightly lower than that at pD 2.5, but that aggregates are formed at higher temperature at pD 2.1 than at pD 2.5, probably because the repulsive interaction between the unordered structure is stronger at pD 2.1.

Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: a method which can recover free oligosaccharides after analysis.

There are a large number of labeling methods for asparagine-type oligosaccharides with fluorogenic and chromophoric reagents. We have to choose the most appropriate labeling method based on the purposes such as mass spectrometry, high-performance liquid chromatography and capillary electrophoresis. Asparagine-type glycans are released from core proteins as N-glycosylamine at the initial step of the releasing reaction when glycoamidase F is employed as the enzyme. The N-glycosylamine-type oligosaccharides thus released by the enzyme are subjected to hydrolysis or mutarotation to form free-form oligosaccharides. In the detailed studies on the enzyme reaction, we found a condition in which the released N-glycosylamine-type oligosaccharides were exclusively present at least during the course of enzyme reaction, and developed a method for in situ derivatization of the glycosylamine-type oligosaccharides with 9-fluorenylmethyl chloroformate (Fmoc-Cl). The Fmoc labeled sialo- and asialo- (or high-mannose and hybrid) oligosaccharides were successfully analyzed on an amine-bonded polymer column and amide-silica column, respectively. The present method showed approximately 5 times higher sensitivities than that using 2-aminobenzoic acid (2-AA). The separation profile was similar to that observed using 2-AA method as examined by the analyses of carbohydrate chains derived from several glycoproteins including complex-type, high-mannose type and hybrid type of N-linked oligosaccharides. The labeled oligosaccharides were stable at least for several months when stored at -20 degrees C. Furthermore, it should be emphasized that the Fmoc-derivatized oligosaccharides could be easily recovered as free reducing oligosaccharides simply by incubation with morpholine in dimethylformamide solution. We obtained a pure triantennary oligosaccharide with 3 sialic acid residues as a free reducing form from fetuin in good yield after isolation of the corresponding Fmoc oligosaccharide followed by removing reaction of the Fmoc group. The proposed method will be useful for preparation of free oligosaccharides as standard samples at pmol-nmol scale from commercially available glycoproteins.

Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis.

Carbohydrate chains in glycoprotein pharmaceuticals have important roles for the expression of their biological activities. Therefore, development of an assessment method for the carbohydrate chains is an important parameter for quality control of glycoprotein pharmaceuticals such as newly developed therapeutic antibodies. In this report, we applied capillary electrophoresis with laser-induced fluorescence detection to the analysis of carbohydrate chains after releasing with glycoamidase followed by derivatization with 3-aminobenzoic acid. We found that four major oligosaccharides present in antibody pharmaceuticals were successfully separated with good resolution. The present method showed good precision in both migration times and relative peak areas, and gave comparable accuracy with that using a derivatization method with 8-aminopyrene-1,3,6-trisulfonate.