The effects of manufacture processes on post-translational modifications of bioactive proteins in pertussis vaccine.

Because the increasing morbidity of pertussis in all age groups worldwide, the quality of pertussis vaccines has aroused a common concern. To improve the quality of pertussis vaccine in research and production, the effects of manufacture processes on post-translational modifications (PTMs) of bioactive proteins in pertussis vaccine were investigated by a liquid chromatography quadruple - time of flight mass spectrometer (LC-Q-TOF) method in this study. The main bioactive proteins in pertussis vaccine studied include pertussis toxin (PT), pertactin (PRN) and filamentous hemagglutinin (FHA). The main manufacture processes focused are fermentation techniques, purification techniques and storage conditions. The results show that FHA and PRN are rather stable against PTM as only deamidation (Asn) was detected, which is believed to be due to their larger sizes of the bioactive proteins. For PT, however, all the manufacture processes studied have shown significant effects on types and sites of PTMs. Modifications of oxidation and demethylation (Met) occurred in the PT proteins produced by B. pertussis strain Tohama and stored in suspension in saline solution. However, they were not observed in the PT samples produced from stain CS and stored in powders. Carbamylation (Arg) on multiple sites (in S3, S4 and S5) was observed in the PT produced from 5th generation strain CS of B. pertussis. The high abundance ratio of carbamylation modification was potentially a negative effect on the detoxification of PT, since unmodified Lys was the active site for detoxification. The results obtained in this study provide information for making protection strategies against PTMs in pertussis vaccine in manufacture and storage.

Quantitative determination of bioactive proteins in diphtheria tetanus acellular pertussis (DTaP) vaccine by liquid chromatography tandem mass spectrometry.

A liquid chromatography tandem mass spectrometry method (LC-MS/MS) was developed to determine simultaneously the bioactive proteins including pertussis toxin (PT) subunits, filamentous hemagglutinin (FHA), pertactin (PRN) and fimbriae (FIM) in diphtheria, tetanus and acellular pertussis combined vaccine (DTaP). The trypsin digestion conditions were investigated in detail using PT reference to achieve satisfactory results in detection of the peptides on LC-MS/MS with a Bio-C18 column. The performance of the described method was evaluated using reference proteins and the results showed a wide linear range (0.15-24 ng µL-1), a high sensitivity (0.038 ng. µL-1 for FHA) and a good precision (RSD of peak area <3.3%). This novel LC-MS/MS method was applied to determine PT subunits, FHA, PRN and FIM in DTaP vaccines, a total of ten batches, obtained from five manufacturers. The results revealed clearly that batch-to-batch consistency of the DTaP vaccines in terms of the protein amounts was stable, while those from manufacturers were varied significantly. On the other hand, the amount of bioactive proteins in component DTaP vaccines was generally higher than those in co-purified DTaP vaccines. The described LC-MS/MS method was compared with Chinese Pharmacopeia method (Lowry method) and it was found that FHA and PRN amounts measured by the two methods were in good agreement. The LC-MS/MS method could provide the amounts of PT subunits. However, the Lowry method could not differentiate the subunits. The LC-MS/MS method was not only more selective and sensitive, but it can be used to determine simultaneously different bioactive proteins in complex matrix-formulated vaccines. The method was extended successfully in other purposes, such as the effect of detoxification on bioactive proteins and characterization of PT references from four organizations worldwide.

Overexpression of HexCer and LacCer containing 2-hydroxylated fatty acids in cholangiocarcinoma and the association of the increase of LacCer (d18:1-h23:0) with shorter survival of the patients.

Alteration of glycosphingolipid (GSL) synthesis is observed in many types of cancer. In this study, we have analyzed the expression of sphingolipids and GSLs in cholangiocarcinoma (CCA) tissues and adjacent normal liver tissues. Neutral lipids were extracted from tissue samples using mild-alkaline treatment method followed by TLC and LC-MS analysis. The expression of ceramides, hexosylceramides (HexCer), and lactosylceramides (LacCer) was altered in CCA tissues, 61.1% (11/18) of them showing an increase whereas 38.9% (7/18) showing a decrease, compared with the adjacent normal tissue. Cers and GSLs containing 2-hydroxylated fatty acids except one LacCer molecular species were overexpressed in CCA tissues, and the increase of LacCer (d18:1-h23:0) was correlated with shorter survival of CCA patients, suggesting the involvement of GSL synthesis and fatty acid hydroxylation in progression of CCA. Taken together, we have demonstrated in this study the increase of GSL synthesis and fatty hydroxylation in CCA, which probably be used as a target for CCA treatment.

A novel two-dimensional liquid chromatography - Mass spectrometry method for direct drug impurity identification from HPLC eluent containing ion-pairing reagent in mobile phases.

In this study, a novel two dimensional liquid chromatography - mass spectrometry (2D-LC-MS) method with use of a weak anion exchange column between the 1st DLC RP column and the 2nd DLC RP column (RP1-WAX-RP2) was developed and applied to identify drug impurities from MS incompatible mobile phases containing sodium 1-octanesulfonate and non-volatile buffer. The 1st DLC conditions follow exactly the original standard HPLC method recorded in Chinese Pharmacopeia (ChP), European Pharmacopeia (EP) or US Pharmacopeia (USP). An impurity fraction was collected with a built-in sample loop (100 µL) and transferred to the WAX column where 1-octanesulfonate and phosphate were trapped and removed. While, the impurity and other cations were eluted to the 2nd D column (RP2) for separation and identification by connected IT-TOF MS. Methods were programmed and applied to identify impurities in two generic drugs, sulpiride (hydrophilic drug with logP 0.57) and dobutamine (hydrophobic drug with logP 3.6). The results indicate that the methods based on RP1-WAX-RP2 column configuration offer a feasible solution for direct impurity identification in generic drug product or API without needs of off-line desalting from the MS incompatible mobile phases containing ion-pairing reagent and non-volatile buffer.

A fast and robust hydrophilic interaction liquid chromatography tandem mass spectrometry method for determining methylpentose, hexose, hexosamine and hexonic acid in pneumococcal polysaccharide vaccine hydrolysates.

The conventional UV/Vis spectroscopy methods recommended by the European Pharmacopoeia (EP) for determining hexosamine, hexonic acid and methylpentose in pneumococcal polysaccharide vaccine (PPSV) hydrolates are time-consuming due to derivatization process (typically, an analysis cycle is more than 4 h) and improvements of selectivity and precision of the methods are in demand. In this study, a new approach based on hydrophilic interaction liquid chromatography and triple quadrupole mass spectrometry (HILIC-MS/MS) was optimized to overcome the drawbacks of the EP methods for simultaneous determination of methylpentose, hexose, hexosamine and hexonic acid in PPSV hydrolysates. The chromatographic, MS and sample hydrolysis conditions were systematically investigated. A zwitterionic column, Click Cys, using a gradient elution with a mobile phase of 10 mM ammonium formate (pH 4.3) in acetonitrile from 72% to 21% in 6 min was applied for separating the targets, which exhibited low column bleeding, easy equilibration and long-term stability. The HILIC-MS/MS method showed a high sensitivity (LOD = 0.98 µg L-1 for hexonic acid), a good repeatability (RSD of peak area less than 1.669%), accuracy (92.9%-104.2%), recovery (97.6%-99.3%) and a wide linear range. The RSD of retention time obtained from more than 3000 injections in three months was less than 1.64%. The new method was compared with the EP method for determining hexosamine in 23 serotypes of PPSV hydrolysates. The results indicated that the new HILIC-MS/MS method was highly selective, accurate, stable and extremely fast due to without need of derivatization, as compared to the conventional EP methods.

Immunochromatographic determination of bacopaside I in biological samples.

We describe a novel immunochromatographic method for qualitative and quantitative analyses of bacopaside I, a bioactive constituent in Bacopa monnieri (L.) Wettst in biological samples. The assay was performed on polyethersulfone membrane using a polyclonal antibody raised against bacopaside I. The finalised method could quantitatively determine bacopaside I in the range of 31.3-1000.0ng and the detection and quantification limits were 1.0 and 31.3ng, respectively. The percentage recoveries of bacopaside I in blood and urine were nearly 100% indicating the accuracy of the extraction. The method was then applied for the determination of this compound in rat serum, urine and feces after an oral dose of 15mg/kg body weight. About 4% of the ingested dose of bacopaside I was detected in rat feces but none was detected in serum and urine which accorded with results from liquid chromatography tandem mass spectrometry. The accuracy, selectivity, sensitivity of the method are appropriate for in vivo pharmacokinetic studies.

Rapid identification of gallotannins from Chinese galls by matrix-assisted laser desorption/ionization time-of-flight quadrupole ion trap mass spectrometry.

Chinese gall, a conventional traditional Chinese medicine, contains high levels of gallotannins. A rapid method for direct analysis of the gallotannins without using any troublesome sample pretreatments was developed using matrix-assisted laser desorption/ionization time-of-flight quadrupole ion trap mass spectrometry (MALDI-QIT-TOF MS) to successfully identify the gallotannin components in the crude extract of Chinese galls within several minutes. The high quality of the MS and MS(2) spectra acquired clearly showed that hydrolysable tannins in Chinese galls were identified as a series of the gallotannins with degrees of polymerization (DP) of 4-11 galloyl units. The MS(2) data indicated that the identified gallotannins with DP of 4-7 galloyl units had clear fragmentation with loss of 1-5 galloyl units which were further deprived of 1-3 water moieties. This technique may be used for rapid evaluation and screening of hydrolysable tannins in medicinal plants.

Phenolic antioxidants (hydrolyzable tannins, flavonols, and anthocyanins) identified by LC-ESI-MS and MALDI-QIT-TOF MS from Rosa chinensis flowers.

Rosa chinensis (Yuejihua) is a well-known ornamental plant, and its flowers are commonly used in traditional Chinese medicine. Methanolic crude extracts of dried R. chinensis flowers were used for simultaneous determination of phenolic constituents by liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS). A total of 36 known and unknown phenolics were identified as hydrolyzable tannins, flavonols, and anthocyanins, mainly including gallotannins (mono-, di-, or trigalloylglucopyranosides), ellagitannins, quercetin, quercetin/kaempferol mono- and diglycosides, and cyanidin/pelargonidin diglycosides. MALDI-QIT-TOF MS was applied not only to verify most phenolics isolated and identified by LC-MS but also to tentatively identify two ellagitannins (rugosins B and C) not isolated and unidentified by LC-MS. This study is the first to demonstrate the rapid and successful use of MALDI-QIT-TOF MS and LC-MS to directly and simultaneously identify phenolics in the crude extracts of R. chinensis flowers without any purification. The antioxidant activity of the crude extracts from R. chinensis flowers was also measured with three assay methods. The results showed that the phenolic antioxidants from R. chinensis flowers exhibited very strong radical scavenging effect and antioxidant power. High levels of flavonols and hydrolyzable tannins might be important bioactive principles in the dried R. chinensis flowers.