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Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R(2) > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%-8.0% and 0.34%-0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Drug-protein conjugates have been widely used for the cell-specific targeting of drugs to cells that can bind and internalize the proteinaceous carrier. For renal drug targeting, lysozyme (LZM) can be used as an effective carrier that accumulates in proximal tubular cells. We used capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF-MS) for the characterization of different drug-LZM conjugates. A recently developed prototype porous tip sprayer was employed for sheathless electrospray ionization (ESI) CE-MS interfacing. In order to prevent adsorption of LZM conjugates to the capillary wall, a positively charged polyethylenimine capillary coating was used in combination with a low-pH background electrolyte. Drug-LZM products had been prepared by first coupling BOC-l-methionine hydroxysuccinimide ester (BOCmet) to lysine residues of LZM followed by conjugation with the kinase inhibitors LY364947, erlotinib, or Y27632 via a platinum(II)-based linker. CE-TOF-MS of each preparation showed narrow symmetrical peaks for the various reaction products demonstrating that drug-LZM conjugates remained stable during the CE analysis and subsequent ESI. Components observed in the drug-LZM products were assigned based on their relative migration times and on molecular mass as obtained by TOF-MS. The TOF-MS data obtained for the individual components revealed that the preparations contained LZM carrying one or two drug molecules, next to unmodified and BOCmet-modified LZM. Based on relative peak areas (assuming an equimolar response for each component) a quantitative conjugate profile could be derived for every preparation leading to drug loading values of 0.4-0.6 mol drug per mole protein., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this study, a CE-MS method using a monolithic sol-gel concentrator for in-line solid-phase extraction (SPE) is evaluated for the analysis of methionine enkephalin in biological samples. Operational SPE parameters such as sample pH, loading volume, elution volume and composition have been studied. After optimization of the in-line preconcentration methodology, a 40-fold preconcentration was demonstrated for a methionine enkephalin test solution using a loading volume of 3200 nL. The method was linear in the range from 62.5 to 1000 ng/mL (R(2)>0.99). R.S.D. values for migration times and peak areas were 1.2% and 8.4%, respectively. Finally, the analysis of cerebrospinal fluid samples spiked with methionine enkephalin and deproteinized with perchloric acid (1:1, v/v) showed a detection limit (S/N=3) of approximately 1 ng/mL (ca. 5 nM). The recoveries of methionine enkephalin for three concentration levels (100, 10 and 1 ng/mL) were in the range of 74-91%, demonstrating the promising potential of the methodology for the analysis of biological samples.

In this study the possibility to deliver the acid-sensitive enzyme alkaline phosphatase (AP) from calf intestine (CIAP) to the intestinal system by oral administration was investigated. Tablets were prepared and in vitro evaluated. Final proof of concept studies were performed in rats. This acid labile enzyme is potentially useful in the treatment of sepsis, a serious condition during which endotoxins can migrate into the blood stream. The CIAP was freeze-dried with inulin and subsequently compacted into round biconvex tablets with a diameter of 4mm and a weight of 25-30 mg per tablet. The tablets were coated with an enteric coating in order to ensure their survival in the stomach. In vitro evaluation of tablets containing alkaline phosphatase from bovine intestine (BIAP) was the first step in the development. It was found that tablets without enteric coating dissolved rapidly in 0.10 M HCl with total loss of enzymatic activity of the alkaline phosphatase. Tablets that were coated were stable for at least 2 h in 0.10 M HCl, but dissolved rapidly when the pH was increased to 6.8. Furthermore, it was shown that the enzymatic activity of the released BIAP was fully preserved. The in vivo test clearly showed that the oral administration of enteric coated tablets resulted in the release of enzymatically active CIAP in the intestinal lumen of rats. The location of the enhanced enzymatic activity of AP in the intestines varied with the time that had passed between the administration of the tablets and the sacrificing of the rats. Also, the level of enzymatic activity increased with an increasing number of tablets that were administered.

Alkaline phosphatase (AP) is a potential therapeutic agent in the treatment of sepsis. In this paper the potential of capillary zone electrophoresis (CZE) for the monitoring of the degradation of placental alkaline phosphatase (PLAP) was investigated. To induce degradation PLAP samples were exposed to high temperatures, low and high pH and freeze-drying. The samples were then analyzed by CZE and enzymatic activity assay. Upon exposure to temperatures above 65 degrees C, PLAP lost its activity exponentially over time, while CZE revealed both a linear decrease of the area of the main peak and a rise of degradation products. At acidic pH the enzyme appeared to lose its activity. CZE revealed a decrease of the area of the main peak, but no degradation products could be detected. At pH 12 the enzymatic activity and the area of the main peak both decreased linearly over time and, in addition, formation of degradation products could be detected by CZE. Activity and CZE profile of PLAP remained unchanged upon freeze-drying in the presence of inulin. Prolonged storage of freeze-dried samples at room temperature caused a slight decrease of enzymatic activity, while the potential formation of oligomers was revealed by CZE analysis. The examples in this study show that, in combination with activity assays, CZE can provide useful complementary information, especially on the status of the protein and the presence of degradation products.

The aim of this study was to investigate the formulation of sugar glass stabilised alkaline phosphatase from bovine intestine (BIAP) into tablets. Two major subjects of tablet formulation were investigated. First, the compaction behaviour of the inulin sugar glass was investigated. Secondly, the effect of the compaction process on the physical stability of sugar glass stabilised BIAP was investigated, comparing inulin and trehalose glass. The tabletting properties of freeze-dried inulin without BIAP were studied first. Freeze-dried inulin conditioned at either 20 degrees C/0% relative humidity (RH) or 20 degrees C/45% RH was compacted at various pressures. As expected, the yield pressure of the material conditioned at 0% RH was higher (68 MPa) than after conditioning at 45% RH (39 MPa). Tablets made of the material stored at 0% RH showed severe capping tendency, especially at high compaction pressures. In contrast, material conditioned at 45% RH gave tablets without any capping tendency and a friability of less than 1%. Sugar glasses of BIAP and either inulin or trehalose were prepared by freeze-drying (BIAP/sugar 1/19 (w/w)). The material was subsequently compacted. Tablets and powders were stored at 60 degrees C/0% RH. The activity of the incorporated BIAP was measured at various time intervals. It was found that inulin was by far superior to trehalose as stabiliser of BIAP in tablets. The poor stabilising capacities of trehalose after compaction are explained by crystallisation of trehalose induced by the compaction process and moisture in the material. The results clearly show that inulin is an excellent stabiliser for BIAP. The tabletting properties are adequate, showing sufficient tablet strengths and low friability. Furthermore, the good (physical) stability of inulin glass with respect to exposure to high relative humidities makes it practical to work with.

The use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the rapid determination of apolipoprotein E (apoE) genotypes was studied. High resolution and sensitive detection of the concerned DNA restriction fragments was achieved using CE buffers with hydroxypropylmethylcellulose (HPMC) as sieving polymer and ethidium bromide (EB) as fluorescent intercalating agent. In order to achieve adequate resolutions in short analysis times, parameters such as concentration of HPMC and EB, separation voltage, and length and coating of the capillary were evaluated. Using a separation buffer with 0.8% (w/w) HPMC and 7 microM EB, characteristic DNA-fragment profiles could be obtained for all common apoE genotypes at an overall rate of ten samples per hour. The method allows direct injection of untreated PCR samples and the use of standard fused-silica capillaries which are effectively coated following a short, one-step rinse procedure. With a simple computerized algorithm based on migration-time ratios for pattern assignment, highly reliable apoE genotyping was achieved. Overall, in terms of speed, ease of use and objectivity the presented method provides a significant improvement over previously reported CE-based procedures for apoE genotyping., (Copyright 2002 Elsevier Science BV.)

Capillary electrokinetic separation techniques offer high efficiency and peak capacity, and can be very useful for the analysis of samples containing a large variety of (unknown) compounds. Such samples are frequently met in impurity profiling of drugs (detection of potential impurities in a pharmaceutical substance or product) and in general sample profiling (determination of differences or similarities between samples). In this paper, the potential, merits, and limitations of electrokinetic separation techniques for profiling purposes are evaluated using examples from literature. A distinction is made between impurity profiling, forensic profiling and profiling of natural products, and the application of capillary zone electrophoresis, micellar electrokinetic chromatography, and capillary electrochromatography in these fields is discussed. Attention is devoted to important aspects such as selectivity, resolution enhancement, applicability, detection, and compound confirmation and quantification. The specific properties of the various electrokinetic techniques are discussed and compared with more conventional techniques as liquid chromatography.

A general micellar electrokinetic chromatographic (MEKC) strategy for the impurity profiling of drugs was developed involving a sodium dodecyl sulfate (SDS) and a cetyltrimethylammonium bromide (CTAB) MEKC system. With this combination, in principle, each sample component passes the detector in at least one of the two MEKC systems provided that separation buffers of the same pH are used in both systems. In order to select the proper MEKC systems, the electroosmotic flow (EOF) and micelle migration time (t(mc)) were determined for separation buffers of several pH values, containing various amounts of surfactant and organic modifier. The selectivity of the MEKC systems was studied using a mixture of compounds with a wide range of physico-chemical properties. The final selection of two adequate MEKC systems for this approach was based on the requirements that the t(mc) (i.e., analysis time) of both systems was below 20 min and that the t(mc)/t(eof) ratio was above 3 or 2 for the SDS and CTAB system, respectively. Furthermore, the systems should provide high efficiency, exhibit differences in selectivity and use moderate concentrations of modifier and surfactant, so that, if needed, further optimization is possible. The selected MEKC systems contained 60 mM SDS or 10 mM CTAB, respectively, in a phosphate buffer (pH 7.5) with 10% acetonitrile. Some test compounds with extreme mobilities were used to demonstrate the suitability of the MEKC approach to detect each component of a sample. The potential of the proposed MEKC combination for impurity profiling was demonstrated by the analysis of fluvoxamine with several impurities at the 0.1% level.

Over the past years the coupling of liquid chromatography (LC) and Fourier-transform infrared spectrometry (FT-IR) has been pursued primarily to achieve specific detection and/or identification of sample constituents. Two approaches can be discerned in the combination of LC and FT-IR. The first and simpler approach is to use a flow cell through which the effluent from the LC column is passed while the IR spectra are continuously recorded. The second approach involves elimination of the LC solvent prior to IR detection using an interface which evaporates the eluent and deposits the analytes onto a substrate. This paper provides a general overview of flow-cell based IR detection and briefly discusses early solvent-elimination interfaces for LC-FT-IR. A more comprehensive description is given of interface systems which use spraying to induce rapid eluent evaporation, and which basically represent the state-of-the-art in LC-FT-IR. Finally, the interface systems suitable for reversed-phase LC are summarized and the perspectives of LC-FT-IR are discussed. The overview indicates that flow-cell LC-FT-IR has rather poor detection limits but can be useful for the specific and quantitative detection of major constituents of mixtures. Solvent-elimination techniques, on the other hand, provide much better sensitivity and enhanced spectral quality which is essential when unambiguous identification of low-level constituents is required.

The potential of micellar electrokinetic chromatography (MEKC) for the profiling of cocaine samples is described. An MEKC system containing sodium dodecyl sulfate (SDS) and methanol was optimized using a test mixture of cocaine, its common impurities (benzoylecgonine, norcocaine, tropacocaine, and trans-cinnamoylcocaine), and several degradation products. The effect of pH, percentage modifier, and concentration surfactant on the separation has been investigated. The optimal separation buffer for cocaine samples consisted of 75 mM SDS, 17.5% methanol, and 25 mM borate (pH 8.3) and was well suited to separate components of diverse polarity in one run. Various cocaine seizures have been analyzed with the MEKC system and their signatures were compared. The electrokinetic chromatograms obtained were characteristic, and differences and similarities among the samples could easily be observed. Several impurities were identified in the samples by means of migration times and comparison of recorded and library UV spectra. The composition of the samples was determined semiquantitatively using relative corrected peak areas.