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In this commentary, we will provide a high-level introduction into LC-MS product characterization methodologies deployed throughout biopharmaceutical development. The ICH guidelines for early and late phase filings is broad so that it is applicable to diverse biotherapeutic products in the clinic and industry pipelines. This commentary is meant to address areas of protein primary sequence confirmation and sequence variant analysis where ambiguity exists in industry on the specific scope of work that is needed to fulfill the general guidance that is given in sections Q5b and Q6b. This commentary highlights the discussion and outcomes of two recent workshops centering on the application of LC-MS to primary structure confirmation and sequence variant analysis (SVA) that were held at the 2018 and 2019 CASSS Practical Applications of Mass Spectrometry in the Biotechnology Industry Symposia in San Francisco, CA and Chicago, IL, respectively. Recommendations from the conferences fall into two distinct but related areas; 1) consolidation of opinions amongst industry stakeholders on the specific definitions of peptide mapping and peptide sequencing for primary structure confirmation and the technologies used for both, as they relate to regulatory expectations and submissions and 2) development of fit-for-purpose strategy to define appropriate assay controls in SVA experiments., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Measurement of free thiols in antibody therapeutics is important for product development and assessment of critical quality attributes. Earlier studies demonstrated fast separation of free thiol variants of IgG1 using reversed-phase high performance liquid chromatography (RP-HPLC) with diphenyl resin. Here, we report using N-tert-butylmaleimide (NtBM) alkylation followed by RP-HPLC and online mass spectrometry for rapid total and domain-specific free thiol characterization of IgG1. By increasing hydrophobicity, NtBM alkylation improves separation of free thiol variants from disulfide-linked main peak species. The unique mass shift by NtBM alkylation offers unambiguous characterization of free thiol variants by online mass spectrometry. Variant peaks separated by RP-HPLC were antibody molecules containing two NtBM-alkylated cysteines, corresponding to IgG1 containing two free thiols before alkylation. Further characterization of the collected fractions of variants by peptide mapping revealed that each variant contained unpaired cysteines located in specific IgG1 domains (CH1, CH3, VH and VL domains). Total molecular-level and domain-specific free thiol content measured by this method correlate well with orthogonal differential alkylation peptide mapping analysis, which measures free thiol level at individual cysteine residues. This method provides high throughput quantitation of total and domain-specific free thiol content in IgG1 molecules, facilitating rapid, comprehensive product and manufacturing process characterization., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Matrix metalloproteinase-9 (MMP-9) plays an important role in the remodeling of the extracellular matrix in atherosclerosis plaques. Autophagy protects macrophages against the processes of vascular disease. Our research explores how autophagy plays roles in macrophages to secret MMP-9., Methods and Results: In response to increased doses of oxLDL or CQ we monitored the autophagic flux. Our results revealed that oxLDL was dynamically associated with autophagy and 100 μg/ml oxLDL blocked autophagic flux in THP-1 cells. Moreover p62/SQSTM1 knocking down and CQ respectively inhibited and increased MMP-9 transcriptional expression. These effects were mediated by inhibition of NF-κB., Conclusion: Abundant oxLDL blocked autophagic flux resulting in the aggregation of p62/SQSTM1. Then p62/SQSTM1 was involved in gene expression of MMP-9 via NF-κB-dependent signaling, and thus featuring novel plaque vulnerability properties of the atherosclerotic plaque. Understanding the mechanism that selectively modulates p62/SQSTM1 will provide a novel strategy for anti-atherogenesis., (Copyright © 2016. Published by Elsevier Inc.)

Diacylglycerol lipase α is the key enzyme in the formation of the most prevalent endocannabinoid, 2-arachidonoylglycerol in the brain. In this study we identified the catalytic triad of diacylglycerol lipase α, consisting of serine 472, aspartate 524 and histidine 650. A truncated version of diacylglycerol lipase α, spanning residues 1-687 retains complete catalytic activity suggesting that the C-terminal domain is not required for catalysis. We also report the discovery and the characterization of fluorogenic and chromogenic substrates for diacylglycerol lipase α. Assays performed with these substrates demonstrate equipotent inhibition of diacylglycerol lipase α by tetrahydrolipastatin and RHC-20867 as compared to reactions performed with the native diacylglycerol substrate. Thus, confirming the utility of assays using these substrates for identification and kinetic characterization of inhibitors from pharmaceutical collections., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Weekly PM2.5 samples were simultaneously collected at a semi-residential (Tsinghua University) and a downtown (Chegongzhuang) site in Beijing from August 2001 through September 2002. The ambient mass concentration and chemical composition of PM2.5 were determined. Analyses including elemental composition, water-soluble ions, and organic and elemental carbon were performed. The annual average concentrations of PM2.5 were 96.5 microg m(-3) and 106.9 microg m(-3) at CGZ and HU site, respectively. More than 80% of the PM2.5 mass concentrations were explained by carbonaceous species, secondary particles, crustal matters and trace elements at the two sites. Carbonaceous species were the most abundant components, constituting about 45% and 48% of the total PM2.5 mass concentrations at CGZ and THU site, respectively. SO4(2-), NO3- and NH4+ were three major ions, accounting for 37%, 23% and 20%, respectively, of the total mass of inorganic water-soluble ions.

The primary purpose of the present study was to compare the cardioprotective effects of the extract from radix stephaniae tetrandrae (RST) and its individual compounds, tetrandrine (Tet) and fanchinoline (Fan). Secondly, we also compared the cardiac effects of the individual compounds and the RST extract with those of verapamil, a classical Ca2+ channel blocker. The Langendorff isolated perfused rat heart preparation was used. Regional ischaemia and reperfusion was employed to induce myocardial infarct and arrhythmia. Infarct, arrhythmia, heart rate and coronary artery flow were determined in hearts treated with vehicle, RST extract, Tet, Fan, or verapamil. It was found that RST extract, of which only 9% was Tet, and Tet alone produced equally potent ameliorating effects on arrhythmia and infarct induced by ischaemia and reperfusion without further inhibiting ischaemia-reduced heart rate and coronary artery flow. Fan had no effects on arrhythmia and infarct induced by ischaemia and reperfusion; but it induced S-T segment elevation and further reduced heart rate and coronary artery flow during ischaemia. Verapamil also ameliorated the effects of ischaemia and reperfusion on arrhythmia and infarct. It should be noted that 1 microM verapamil, that produced comparable effects on infarct and arrhythmia to the RST extract and Tet, further inhibited heart rate during ischaemia. The results indicate that the RST extract produces equally potent cardioprotective and anti-arrhythmic effects as Tet alone. Both RST extract and Tet may be better choices for the treatment of arrhythmia and infarct induced by myocardial ischaemia and reperfusion than the classical Ca2+ channel blocker, verapamil as they do not further reduce heart rate during ischaemia.

The present study was designed to compare the cardiac actions of the extract and individual components, tetrandrine (Tet) and fangchinoline (Fan), of Radix stephaniae tetrandrae (RST). We measured the electrically induced [Ca2+]i transient in single rat ventricular myocytes and protein release following perfusion with a Ca2+ free solution (the Ca2+ paradox) from the isolated perfused rat heart, both of which are known to relate to Ca2+ influx. We found that Tet inhibited both electrically induced [Ca2+]i transient and protein release during the Ca2+ paradox, while Fan had no significant effects. The RST extract containing 9% Tet and 6% Fan by weight also affected the [Ca2+]i transient, and was only slightly, though significantly, less effective/potent than Tet alone. On the other hand, RST extract had a significantly greater inhibitory effect on protein release during the Ca2+ paradox than Tet alone. The observations suggest that the RST extract, which contains a mixture of components, may have more potent effects in the heart than its main active component.