Systematically amino acid analysis: Advancements in extinction coefficient determination for therapeutic proteins.

Biologicals developers often face challenges in accurately determining the extinction coefficient (EC) measurement. We have successfully improved the precision and robustness of the widely recognized amino acid analysis method for EC determination, through a stepwise optimization process. Extensive analyses based on 114 observations, covering eight proteins over three years were performed, with a maximum relative standard deviation of 1.5% among multiple analysts, and a maximum deviation of 2.8% from the theoretical EC across the eight given proteins examined.

Chitosan oligosaccharide attenuates acute kidney injury and renal interstitial fibrosis induced by ischemia-reperfusion.

Acute kidney injury (AKI) and renal interstitial fibrosis are global clinical syndromes associated with high morbidity and mortality. Renal ischemia-reperfusion (I/R) injury, which commonly occurs during surgery, is one of the major causes of AKI. Nevertheless, an efficient therapeutic approach for AKI and the development of renal interstitial fibrosis is still lacking due to its elusive pathogenetic mechanism. Here, we showed that chitosan oligosaccharide (COS), a natural oligomer polysaccharide degraded from chitosan, significantly attenuates I/R-induced AKI and maintains glomerular filtration function by inhibiting oxidative stress, mitochondrial damage, and excessive endoplasmic reticulum stress both in vitro and in vivo. In addition, long-term administration of COS can also attenuate the proliferation of myofibroblasts, mitigate extra cellular matrix deposition, and thus inhibit the transition of AKI to chronic kidney disease through participating in metabolic and redox biological processes. Our findings provide novel insights into the protective role of COS against acute kidney injury.

In-Depth Characterization of mAb Charge Variants by On-Line Multidimensional Liquid Chromatography-Mass Spectrometry.

In-depth characterization of charge heterogeneity is a pivotal step desired in the therapeutics antibody development. To this end, a novel on-line multidimensional liquid chromatography-mass spectrometry (MDLC-MS) method for charge variant characterization was developed to dig out potential risks on safety and efficacy. This method implemented 96-well plate fractionation and on-column preconcentration by multi-injection, thereby facilitating detection of charged species at low abundance. Eleven charge variants of mAb-A were preliminarily characterized by 2DLC(CEX × RP-C4)-MS. TRVHS and RVHS signal peptide variants of mAb-A were found in basic peaks of the CEX profile. The results supported process development in a timely manner, and the signal peptide-containing variants with potential immunogenicity were successfully removed by an optimized purification process. The retained seven charge variants of mAb-A were further characterized by 4DLC(CEX × RP-C4 × Trypsin×RP-C18)-MS. Post-translational modifications including deamidation, cyclization of N-terminal glutamine, C-terminal lysine truncation as well as proline amidation, and methionine oxidation were identified, and their potential risks were evaluated. Biological activity of the seven charge variants was evaluated by 2DLC (CEX × FcγRIIIa). Increased FcγRIIIa receptor binding affinity was observed in the acidic variants. The MDLC-MS detection can be completed in 72 h with 1.25 mg of mAb, demonstrating to be sample-economic, time-effective, and labor-saving. It provided a powerful and timely tool for charge variant characterization and met the aggressive timeline desired for antibody development.

Demonstrating analytical similarity of a biosimilar HLX04 to bevacizumab with a panel of state-of-the-art methods and tiering of quality attributes.

Therapeutical monoclonal antibodies are structurally and functionally complex, whereas the innovator's manufacturing processes are proprietary. With respect to the similarity assessment, a proposed biosimilar product needs to demonstrate a side-by-side comparison between the reference product (RP) and candidate product in terms of physicochemical properties and biological activities, as well as nonclinical and clinical outcomes. Here, a comprehensive analytical similarity assessment was performed for in-depth comparison of HLX04, China-sourced Avastin® (CN-Avastin®), and Europe-sourced Avastin® (EU-Avastin®) following a tier-based quality attribute (QA) evaluation. A series of orthogonal and state-of-the-art analytical techniques were developed for the assessment. Ten lots of HLX04 were compared with 29 lots bevacizumab RP. Referred to the characterization results, HLX04 is highly similar to the RPs with respect to physicochemical properties and biological functions. In addition, HLX04 was found with similar stability and degradation behaviors upon multiple stressed conditions to bevacizumab. Minor differences were observed in glycosylation, aggregates, FcγRIIIa(F), and FcγRIIIa(V) binding activities; nevertheless, they were evaluated and demonstrated not to impact clinical outcomes. According to the reported clinical results, the totality of evidence, including the pharmacokinetic, efficacy, safety, and immunogenicity, further shows that HLX04 is similar to CN-/EU-Avastin®.

Simultaneous Analysis of Fatty Alcohols, Fatty Aldehydes, and Sterols in Thyroid Tissues by Electrospray Ionization-Ion Mobility-Mass Spectrometry Based on Charge Derivatization.

In this work, we developed a rapid and high-sensitivity method for simultaneous analyses of fatty alcohols, fatty aldehydes, and sterols by combining the optimized derivatization reaction with electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS). Pyridine and thionyl chloride were used as derivatization reagents as they were easily removed after the derivatization reaction and could generate permanently charged tags on different functional groups including hydroxyls and aldehydes. Through this one-step derivatization reaction, the sensitivity of detection for fatty alcohols, fatty aldehydes, and sterols was significantly increased. Moreover, the introduction of ion mobility spectrometry (IMS), offering an additional resolution power, ensured more sensitive and accurate detection of derivative products without increasing analytical time. Being connected with high-performance liquid chromatography, more than 15 kinds of compounds were analyzed within 4 min. Relative quantification using peak intensity ratios between d0-/d5-labeled ions were subsequently applied for analyzing these 15 kinds of compounds in human thyroid carcinoma and para-carcinoma tissues. The results showed significant differences in content of some analytes between these two kinds of tissues (p < 0.05). The correlations between most of the analytes in thyroid carcinoma tissues are better than the correlations in para-carcinoma tissues.

Celastrol mitigates high glucose-induced inflammation and apoptosis in rat H9c2 cardiomyocytes via miR-345-5p/growth arrest-specific 6.

BACKGROUND: Celastrol (Cel) has been corroborated as an anti-inflammatory and anti-apoptotic agent in multiple cell damage models. However, the protective effect of Cel in high glucose (HG)-induced cardiomyocyte injury is still unclear. The present study aimed to determine whether Cel can mitigate HG-stimulated cardiomyocyte injury via regulating the miR-345-5p/growth arrest-specific 6 (Gas6) signaling pathway. METHODS: Cardiomyocytes were exposed to normal glucose (NG; 5 mmol/l) or HG (30 mmol/l) and then administered with Cel. Cell counting kit-8 and flow cytometry assays were used to detect cell proliferative activity and apoptosis. mRNA and protein expression were analyzed using a quantitative reverse transcriptase-polymerase chain reaction and western blotting, respectively. A bioinformatics algorithm and a luciferase reporter gene assay were used to determine whether Gas6 is a direct target of miR-345-5p. RESULTS: The present study confirmed the inhibitory effects of Cel in HG-induced inflammation in cardiomyocytes. Moreover, Cel exhibited the ability to antagonize HG-induced cardiomyocyte apoptosis and suppress the elevated Bax/Bcl-2 ratio elicited by HG stimulation. Intriguingly, Cel treatment revoked the HG-triggered repression of Gas6 protein expression, and Gas6 loss-of-function accelerated HG-induced cardiomyocyte apoptosis. HG-triggered up-regulation of miR-345-5p expression was depressed following Cel treatment. Importantly, we validated that Gas6 is a direct target of miR-345-5p. Transfection with miR-345-5p inhibitors restrained HG-induced release of pro-inflammatory cytokines and cell apoptosis. CONCLUSIONS: The findings of the present study demonstrate that Cel administration antagonized HG-induced cardiomyocyte apoptosis and inflammation through up-regulating Gas6 expression by restraining miR-345-5p.

Charged tag founded in N-(1-chloroalkyl)pyridinium quaternization for quantification of fatty aldehydes.

N-(1-chloroalkyl)pyridinium quaternization was developed for the derivatization of fatty aldehydes. Differing from common pre-charged reagents, non-charged pyridine and thionyl chloride were designed to add permanently charged tag on aldehydes. Pyridine was far less competitive than charged derivatives in ionization. Thionyl chloride in excess was quenched by deionized water, converting into less residual sulfur dioxide bubbles. Thus solutions could be tested directly by mass spectrometry without further post-treatments. Pyridine-d5 labeled fatty aldehydes were prepared as internal standards. Mixed derivatives were then analyzed by high performance liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analytical parameters including reaction yield, stability, precision, linearity, and detection limits (LODs < 0.3 pg mL(-1)) were carefully validated. This method facilitated the analysis low content (ng mL(-1)) levels of free aliphatic aldehydes (C6C18) in human thyroid carcinoma and para-carcinoma tissue with a simple pretreatment procedure. Content of long chain nonvolatile aldehydes (C10C18) remarkably increased in thyroid carcinoma tissues (p < 0.05).

Glutamatergic and central cholinergic dysfunction in the CA1, CA2 and CA3 fields on spatial learning and memory in chronic cerebral ischemia-Induced vascular dementia of rats.

Chronic cerebral ischemia (CCI) is associated with cognitive decline in aging, vascular dementia and Alzheimer's disease. Substantial evidence has shown that chronic cerebral ischemia may cause cognitive impairment, but the underlying neurobiological mechanism is poorly understood so far. In the present study, we used a rat model of chronic cerebral ischemia by permanent bilateral common carotid artery occlusion (BCCAO) to investigate the alterations of glutamatergic and central cholinergic dysfunction, and their causal relationship with the cognitive deficits induced by chronic cerebral ischemia. We found that BCCAO rats exhibited spatial learning and memory impairments dysfunction 3 month after BCCAO. Meanwhile, vGluT levels as well as glutamatergic and central cholinergic positive neurons in the hippocampus CA1-3 field significantly decreased. The protection of glutamergic and cholinergic neurons or regulating glutamate and central cholinergic levels in hippocampal subregion may have beneficial effects on cognitive impairments associated with the possible mechanism in CCI-induced vascular dementia.

The effect of Scutellaria baicalensis stem-leaf flavonoids on spatial learning and memory in chronic cerebral ischemia-induced vascular dementia of rats.

Flavonoids have been shown to improve cognitive function and delay the dementia progression. However, the underlying mechanisms remain elusive. In the present study, we examined the effect of Scutellaria baicalensis stem-leaf total flavonoids (SSTFs) extracted from S. baicalensis Georgi on spatial learning and memory in a vascular dementia (VaD) rat model and explored its molecular mechanisms. The VaD rats were developed by permanent bilateral occlusion of the common carotid artery. Seven days after recovery, the VaD rats were treated with either 50 or 100 mg/kg of SSTF for 60 days. The spatial learning and memory was evaluated in the Morris water maze (MWM) test. The tau hyperphosphorylation and the levels of the related protein kinases or phosphatases were examined by western blot analysis. In VaD rats, SSTF treatment at 100 mg/kg significantly reduced the escape latency in training trial in MWM test. In the probe trial, SSTF treatment increased the searching time and travel distance in the target quadrant. SSTF treatment inhibited the tau phosphorylation in both cortex and hippocampus in VaD rats. Meanwhile, SSTF reduced the activity of glycogen synthase kinase 3ß and cyclin-dependent kinase 5 in VaD rats. In contrast, SSTF treatment increased the level of the protein phosphatase 2A subunit B in VaD rats. SSTF treatment significantly improved the spatial cognition in VaD rats. Our results suggest that SSTF may alleviate tau-hyperphosphorylation-induced neurotoxicity through coordinating the activity of kinases and phosphatase after a stroke. SSTF may be developed into promising novel therapeutics for VaD.

Dialectal and gender differences in nasalance for a Mandarin population.

The purpose of this study was to determine whether there are dialectal and gender related differences in nasalance of main Mandarin vowels and three sentences in 400 Chinese normal adults. The mean nasalance score difference for dialect and gender was significant (p < .001) in all speech materials. For different dialects, the average nasalance scores show that Chongqing > Beijing > Shanghai > Guangzhou for the nasal sentence, oro-nasal sentence, /a/, /i/ and /u/. In addition, the average nasalance scores of females were higher than those of males for all speech materials in all dialects. The clinical significance of this study can be helpful in making nasalance clinical decisions for Chinese people with cleft palate, hearing disorders and dysarthria with resonance disorders. It also shows the theoretical and socio-cultural features for linguists considering dialects and gender.

Improving detection sensitivity of amino acids in thyroid tissues by using phthalic acid as a mobile phase additive in hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry.

In this work, 0.08 mmol L(-1) of phthalic acid was introduced as a mobile phase additive to quantify free amino acids (AAs) by hydrophilic interaction liquid chromatography (HILIC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The addition of phthalic acid significantly increased the signal intensity of protonated AA ions, resulting from the decrease of the relative abundance of AA sodium adducts. Meanwhile, the chromatographic peak shapes of AAs were optimized. As a consequence, there was a noticeable increase in the sensitivity of detection for AAs. The limits of detection (LOD) and quantification (LOQ) of the AAs ranged from 0.0500 to 20.0 ng mL(-1) and from 0.100 to 50.0 ng mL(-1), respectively, which were 4-50 times lower compared to the values measured without the addition of phthalic acid. The enhanced detection and separation of AAs were obtained by merely adding phthalic acid to the mobile phase without changing other conditions. Eventually, this simple method was validated and successfully applied to the analysis of twenty-four kinds of free AAs in human thyroid carcinoma and para-carcinoma tissues, demonstrating a significant increase of most AAs in thyroid carcinoma tissues (p<0.05).

N-alkylpyridinium quaternization combined with liquid chromatography-electrospray ionization-tandem mass spectrometry: A highly sensitive method to quantify fatty alcohols in thyroid tissues.

A highly sensitive method was developed for the identification and quantification of fatty alcohols in biological tissues. In the presence of pyridine-d0 and triflic anhydride (Tf2O), fatty alcohols were converted into permanently charged N-alkylpyridinium ions. Stable isotope-labeled derivatives were generated by pyridine-d5 and added as internal standard (IS). The mixture was analyzed by liquid chromatography coupled to positive electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This method was optimized and validated in terms of reaction time, derivatization efficiency, stability, desalting, and ion suppression effect. Besides, fatty alcohols exhibited good linear relationship (r(2)>0.993) over the concentration range of 10 ngmL(-1)-1 µgmL(-1). The limits of detection (LODs) were lowered from previously reported 0.1 ngmL(-1) to 0.25 pgmL(-1). Precision (RSD%<15.6%), accuracy (93.0-107.2%), matrix effect, and recovery (in thyroid tissues) were validated as well. Finally, this method was applied for the analysis of ten even carbon-numbered fatty alcohols (C8-C24) in human thyroid carcinoma and para-carcinoma tissues, revealing a significant decrease of fatty alcohols (free and esterified) in thyroid carcinoma tissues (p<0.05).

Application of isotope-based carboxy group derivatization in LC-MS/MS analysis of tissue free-fatty acids for thyroid carcinoma.

Free fatty acids (FFAs) analysis of human tissue is challenging due to the low abundance of tissue lipids and interference from the complex matrix. Based on our previously reported on 2,4-dimethoxy-6-piperazin-1-yl pyrimidine (DMPP) derivatization toward carboxy group, we have further applied DMPP derivatization method in the assay of tissue FFAs in patients with thyroid carcinoma after method optimization including sample pretreatment, and derivatization condition. d6-DMPP labeled mixture of FFAs standard was used as the internal standard (IS). The d0-DMPP labeled samples (spiked with IS) were subsequently used for lipids profiling by selective reaction monitoring (SRM) detection. The DMPP derivatization combined with SRM not only allowed trace analysis of FFAs in complex matrix due to the extremely high sensitivity, but also minimized the errors in quantitation because of the very similar property between d0-DMPP derivatized samples and d6-DMPP derivatized IS. Eighteen kinds of FFAs were detected successfully with the increased level in carcinoma tissue compared with the normal tissue, which may be the results of abnormality in the metabolism of fatty acid synthases and lipids. Application of the DMPP based FFAs derivatization is expected to provide a promising tool for the investigation of lipids in vivo, especially for the research of lipid metabolism.

Copper-catalyzed di- and trifluoromethylation of α,ß-unsaturated carboxylic acids: a protocol for vinylic fluoroalkylations.

Dual action: the Lewis acid CuF(2) ⋅2 H(2)O efficiently catalyzes the reaction between electrophilic fluoroalkylating agents and α,ß-unsaturated carboxylic acids by dually activating both reactants, thus affording di- and trifluoromethyl alkenes in high yields with excellent E/Z selectivity.