Combination of SDS-PAGE and intact mass analysis for rapid determination of heterogeneities in monoclonal antibody therapeutics.

mAbs are currently mainstream in biopharmaceuticals, and their market has been growing due to their high target specificity. Characterization of heterogeneities in mAbs is performed to secure their quality and safety by physicochemical analyses. However, they require time-consuming task, which often strain the resources of drug development in pharmaceuticals. Rapid and direct method to determine the heterogeneities should be a powerful tool for pharmaceutical analysis. Considering the advantages of electrophoresis and MS, this study addresses the combination of SDS-PAGE and intact mass analysis, which provides direct, rapid, and orthogonal determination of heterogeneities in mAb therapeutics. mAb therapeutics that migrated in SDS-PAGE were recovered from gel by treatment with SDC-containing buffer. Usage of SDC-containing buffer as extraction solvent and ethanol-based staining solution enhanced the recovery of intact IgG from SDS-PAGE gels. Recovery of mAbs reached more than 86% with 0.2% SD. The heterogeneities, especially N-glycan variants in the recovered mAb therapeutics, were clearly determined by intact mass analysis. We believe that the study is important in pharmaceuticals‧ perspective since orthogonal combination of gel electrophoresis and intact mass analysis should be pivotal role for rapid and precise characterization of mAbs.

A validated simultaneous quantification method for vonoprazan (TAK-438F) and its 4 metabolites in human plasma by the liquid chromatography-tandem mass spectrometry.

Vonoprazan fumarate (TAK-438) is a potassium-competitive acid blocker which was approved in Japan for a treatment of acid-related diseases. In this study a simple and validated bioanalytical method, which can simultaneously determine vonoprazan (TAK-438F) and its four metabolites (M-I, M-II, M-III and M-IV-Sul) in human plasma, was developed. The method is based on protein precipitation and subsequent ultra-high performance liquid chromatography separation followed by tandem mass spectrometry detection. The mass spectrometric parameters for detection of TAK-438F, M-I, M-III and M-IV-Sul were modified from their optimum values in order to achieve a simultaneous quantification while retaining enough sensitivity and wide dynamic ranges for all the target analytes. The validity and robustness of the method was verified through a validation study as per the regulatory guidance on bioanalytical method validation. The calibration ranges are 0.1-100 ng/mL for TAK-438F and M-III, and 1-1000 ng/mL for M-I, M-II and M-IV-Sul using the 100 µL of human plasma. The total run time per sample is 5 min. The working solution for M-III was recommended to be prepared separately, especially for the long-term use, in order to avoid the instability of M-III in the mixed working solutions, which could cause the high consumption of reference standards. The established method was applied to clinical pharmacokinetic studies and concentrations of all the analytes in human plasma were successfully determined with high reproducibility ensured by incurred sample reanalysis, indicating the suitableness of the established method.

Studies on 1-alkylamine adduct formation in electrospray ionization mass spectrometry for quantitative analysis.

We investigated the formation of 1-alkylamine adduct ions using 1,2-, 1,3-, and 1,4-cyclohexanediol (CHD) as model compounds and the relationships of the peak intensity between the protonated molecules ([M+H]+), sodium adduct ions ([M+Na]+), and 1-alkylamine adduct ions ([M+A+H]+) of 16 model compounds using electrospray ionization mass spectrometry. When 1-octylamine was added to 1,2-, 1,3-, and 1,4-CHD solutions, the peak intensity of the 1-octylamine adduct ions ([M+Oct+H]+) was higher than those of [M+H]+ and [M+Na]+. The highest peak intensity of [M+Oct+H]+ was observed in 1,2-CHD, followed by 1,3-CHD and 1,4-CHD and this order was the same as that of [M+Na]+ for CHDs in the solution without 1-octylamine. These results suggest that the mechanism of formation of [M+Oct+H]+ is similar to that of [M+Na]+ and that adduct formation seems to occur between 1-octylamine and two oxygen atoms in CHD in a similar manner to [M+Na]+. Based on these results, 16 model compounds including CHDs were investigated with respect to the relationship between [M+Na]+ and [M+A+H]+. A positive correlation was observed between the peak intensities of [M+Na]+ and [M+A+H]+, supporting that the formation mechanism of [M+A+H]+ is potentially similar to the [M+Na]+ formation mechanism. These data indicate that a sensitivity enhanced quantitative analysis using [M+A+H]+ could be a feasible approach for compounds generating [M+Na]+.

Analytical method for determination of allylic isoprenols in rat tissues by liquid chromatography/tandem mass spectrometry following chemical derivatization with 3-nitrophtalic anhydride.

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method following chemical derivatization with 3-nitrophtalic anhydride was developed for the simultaneous determination of farnesol and geranylgeraniol in rat liver and testis. One analogue compound of the analytes, n-pentadecanol, was used as an internal standard (IS) for both analytes in this method. Rat tissues were disintegrated with 8% KOH ethanol solution, and then farnesol, geranylgeraniol and IS were extracted with a mixture of n-hexane-ethanol (98.5:1.5, v/v) in twice. Farnesol, geranylgeraniol and IS were then converted to 3-nitrophtalic derivatives of each analyte, and extracted with n-hexane. A turbo ion spray interface was used as the ionization source of LC-MS/MS and the analysis was performed in the multiple reaction monitoring (MRM) mode. The calibration curve at the spiked concentrations of 0.15-15 microg/g for both analytes showed good linearity. The method was precise; the relative standard deviations of the method for rat liver were not more than 13.4 and 5.4% for farnesol and geranylgeraniol, respectively, and those for rat testis were not more than 8.4 and 8.6% for farnesol and geranylgeraniol, respectively. The accuracies of the method for both rat liver and testis were good, with the deviations between the nominal concentration and calculated concentration of farnesol and geranylgeraniol typically being within 12.3 and 10.2%, respectively. This method provided reliable concentration levels for farnesol and geranylgeraniol in rat liver and testis.

TAK-652 inhibits CCR5-mediated human immunodeficiency virus type 1 infection in vitro and has favorable pharmacokinetics in humans.

The first small-molecule CCR5 antagonist, TAK-779, could not be developed as an anti-human immunodeficiency virus type (anti-HIV-1) agent because of its poor oral bioavailability. TAK-652 is an orally bioavailable TAK-779 derivative with potent anti-HIV-1 activity. TAK-652 inhibited the binding of RANTES (regulated on activation, normal T-cell expressed and secreted), macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta to CCR5-expressing cells at nanomolar concentrations. TAK-652 could also suppress the binding of monocyte chemotactic protein 1 (MCP-1) to CCR2b-expressing cells. However, its inhibitory effect on ligand binding to other chemokine receptors was limited. TAK-652 was active against CCR5-using (R5) HIV-1 but totally inactive against CXCR4-using (X4) HIV-1. The compound was active against R5 HIV-1 clinical isolates containing reverse transcriptase and protease inhibitor-resistant mutations, with a mean 50% effective concentration (EC50) and EC90 of 0.061 and 0.25 nM, respectively. In addition, recombinant R5 viruses carrying different subtype (A to G) envelope proteins were equally susceptible to TAK-652. A single oral administration of TAK-652 up to 100 mg was safe and well tolerated in humans. The compound displayed favorable pharmacokinetics, and its plasma concentration was 7.2 ng/ml (9.1 nM) even 24 h after the administration of 25 mg. Thus, TAK-652 is a promising candidate as a novel entry inhibitor of HIV-1.

Analytical method for ubiquinone-9 and ubiquinone-10 in rat tissues by liquid chromatography/turbo ion spray tandem mass spectrometry with 1-alkylamine as an additive to the mobile phase.

We investigated the application of 1-alkylamines, as additives to the mobile phase, to a quantification method for ubiquinone-9 (CoQ9) and ubiquinone-10 (CoQ10) in rat thigh muscle and heart using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the optimization of the analytical method, we found that 1-alkylamines mixed with CoQ9 and CoQ10 in the turbo ion sprayed solution formed the 1-alkylammonium adduct molecules of these compounds during the ionization process and that the intensity of the adduct ions was considerably higher than that of the protonated molecules ([M+H]+) of these compounds. Furthermore, we investigated a variety of 1-alkylamines in the mobile phase for LC-MS/MS analysis to select the most appropriate 1-alkylamine for higher sensitivities of CoQ9 and CoQ10. After these examinations, we found that methylamine was the most suitable additive for the mobile phase, allowing a 12.5-fold gain in signal intensity in the full ion mass spectrum compared with that without methylamine. The internal standard (IS) used was ubiquinone-11 (CoQ11) for each analyte. The analytes and IS were extracted with methanol from the tissue homogenates at neutral pH and were injected into an LC-MS/MS with a turbo ion spray interface. The calibration curves for CoQ9 (5-500 microg/g in thigh muscle and 50-10,000 microg/g in heart) and CoQ10 (1-500 microg/g in thigh muscle and 10-10,000 microg/g in heart) showed good linearity. The method was precise; the relative standard deviations of the method for rat thigh muscle were not more than 13.5 and 9.0% for CoQ9 and CoQ10, respectively, and those for rat heart were not more than 6.7 and 5.4% for CoQ9 and CoQ10, respectively. The accuracies of the method for both rat thigh muscle and heart were good, with the deviations between the nominal concentration and calculated concentration of CoQ9 and CoQ10 typically being within 12.3 and 4.3%, respectively. This method provided reliable concentration levels for CoQ9 and CoQ10 in rat thigh muscle and heart.

Synthesis of novel 4,1-benzoxazepine derivatives as squalene synthase inhibitors and their inhibition of cholesterol synthesis.

Modification of the carboxyl group at the 3-position and introduction of protective groups to the hydroxy group of the 4,1-benzoxazepine derivative 2 (metabolite of 1) were carried out, and the inhibitory activity for squalene synthase and cholesterol synthesis in the liver was investigated. Among these compounds, the glycine derivative 3a and beta-alanine derivative 3f exhibited the most potent inhibition of squalene synthase prepared from HepG2 cells (IC(50) = 15 nM). On the other hand, the piperidine-4-acetic acid derivative 4a, which was prepared by acetylation of 3j, was the most effective inhibitor of cholesterol synthesis in rat liver (ED(50) = 2.9 mg/kg, po). After oral administration, 4a was absorbed and rapidly hydrolyzed to deacylated 3j. Compound 3j was detected mainly in the liver, but the plasma level of 3j was found to be low. Compounds 3j and 4a were found to be competitive inhibitors with respect to farnesyl pyrophosphate. Further evaluation of 4a as a cholesterol-lowering and antiatherosclerotic agent is underway.

Highly sensitive liquid chromatography-tandem mass spectrometry method for quantification of a new bone anabolic agent, TAK-778, in human serum.

A liquid chromatography-tandem mass spectrometric (LC-MS-MS) method for the highly sensitive determination of a new bone-anabolic agent, TAK-778 in human serum was developed. The internal standard (I.S.) used was deuterated TAK-778. TAK-778 and I.S. were extracted from serum samples with diethyl ether at neutral pH. A turbo ion spray interface was used as the ion source of LC-MS-MS, and the analysis was performed in the selected reaction monitoring mode. The lower limit of quantification was 0.02 ng/ml when 0.4 ml of serum was used, and the standard curve was linear in the range of 0.02-10 ng/ml. The method was precise; the intra- and inter-day precision of the method was not more than 17.9%. The accuracy of the method was good with the deviations between added and calculated concentration of TAK-778 being typically within 9.0%.

Application of 1-alkylamines to a liquid chromatographic/turbo ionspray tandem mass spectrometric method for quantifying metabolites of a new bone anabolic agent, TAK-778, in human serum.

We investigated the application of alkylamines, as additives to the mobile phase, to a quantification method for the metabolites, M-III and M-IV, of TAK-778, which is a new bone anabolic agent, in human serum using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Prior to setting up the analytical method, we found that 1-alkylamines co-existing with M-III and M-IV in the turbo ionsprayed solution formed 1-alkylammonium adduct molecules of these metabolites during the ionization process, and the abundance of the adduct ions was considerably higher than that of protonated molecules ([M + H](+)s) of these metabolites. Based on these findings, we investigated a variety of 1-alkylamines and their spiked concentrations in the mobile phase for LC/MS/MS analysis to obtain higher sensitivities for the quantification of these metabolites. After these examinations, we found that 1-hexylamine at a final concentration of 0.05 mmol l(-1) was the most suitable additive for the mobile phase, and set the selected reaction monitoring (SRM) ions for the 1-hexylammonium adduct molecule and [M + H](+), allowing about a fivefold gain in the SRM chromatographic peak compared with that without 1-hexylamine. The adduct ion was considered to be formed by interaction between the amino group of 1-hexylamine and the phosphoryl group of M-III and M-IV. The internal standard (I.S.) used was deuterated M-III for each metabolite. The analytes and I.S. were extracted with diethyl ether from serum samples at neutral pH and injected into the LC/MS/MS system with a turbo ionspray interface. The limit of quantification for both analytes was 0.5 ng ml(-1) when 0.1 ml of serum was used, and the calibration curves were linear in the range 0.5-100 ng ml(-1). The method was precise; the intra- and inter-day precisions of the method were not more than 5.6%. The accuracy of the method was good, with deviations between added and calculated concentrations of M-III and M-IV being typically within 16.6%. This method provided reliable pharmacokinetic data for M-III and M-IV after the intramuscular administration of TAK-778 sustained-release formulation in humans.