Protein identification by syringe pump-driven reversed-phase LC-MS/MS

In typical mass spectrometry-based protein identification using peptide fragmentation fingerprinting, front-end separation plays a critical role in successful peptide sequencing. This separation step demands a great deal of time and usually is the rate-limiting step for the whole process. Here we provide an alternative separation method, based on a simple nanoflow delivery system, that is able to shorten the separation time considerably. This system consists of a 25-mul syringe connected to a manually packed reversed-phase mini-capillary column that can be directly coupled to an electrospray ionization tandem mass spectrometer. A syringe pump is then used to deliver the peptide mixtures at a nanoscale flow rate. We examined the efficiency and efficacy of this method by analyzing the tryptic peptides of bovine serum albumin and of 10 Escherichia coli proteins separated by two-dimensional gel electrophoresis (2DE). The results showed that identification of each protein could be achieved successfully within 25 min by using the disposable mini-capillary column. Moreover, all 2DE-separated E. coli proteins were identified at high confidence levels. Together, our data suggest that this method is a suitable option for mass spectrometry-based protein identification.     

Ambient mass spectrometry employing a DART ion source for metabolomic fingerprinting/profiling: a powerful tool for beer origin recognition

A metabolomic fingerprinting/profiling generated by ambient mass spectrometry (MS) employing a direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight mass spectrometry (TOFMS) was employed as a tool for beer origin recognition. In a first step, the DART–TOFMS instrumental conditions were optimized to obtain the broadest possible representation of ionizable compounds occurring in beer samples (direct measurement, no sample preparation). In the next step, metabolomic profiles (mass spectra) of a large set of different beer brands (Trappist and non-Trappist specialty beers) were acquired. In the final phase, the experimental data were analyzed using partial least squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and artificial neural networks with multilayer perceptrons (ANN-MLP) with the aim of distinguishing (i) the beers labeled as Rochefort 8; (ii) a group consisting of Rochefort 6, 8, 10 beers; and (iii) Trappist beers. The best prediction ability was obtained for the model that distinguished the group of Rochefort 6, 8, 10 beers from the rest of beers. In this case, all chemometric tools employed provided ≥95% correct classification. The current study showed that DART–TOFMS metabolomic fingerprinting/profiling is a powerful analytical strategy enabling quality monitoring/authenticity assessment to be conducted in real time.     

Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry

Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel.     

Bioorganic Research of Galactites tomentosa Moench. Honey Extracts: Enantiomeric Purity of Chiral Marker 3‐Phenyllactic Acid

Thistle (Galactites tomentosa Moench.) honey organic extracts were obtained by headspace solid‐phase microextraction (HS‐SPME) and ultrasonic solvent extraction (USE) and analyzed by gas chromatography (GC‐FID and GC‐MS) for the first time. Most abundant headspace compounds were terpenes, particularly linalool derivatives (hotrienol was predominant with a range of 38.6–57.5%). 3‐Phenyllactic acid dominated in the solvent extracts (77.4–86.4%) followed by minor percentages of other shikimate pathway derivatives. After determination of an adequate enantioseparation protocol on Chirallica PST‐4 column, the honey solvent extracts were analyzed by high‐performance liquid chromatography (HPLC). The chiral analysis revealed high enantiomeric excess (>95%) of (–)‐3‐phenyllactic acid in all samples. Therefore, previous findings of chemical markers of thistle honey were extended, providing new potential for advanced chemical fingerprinting (optical pure chemical marker). Chirality 26:405–410, 2014. © 2014 Wiley Periodicals, Inc.     

Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification

An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold.     

On-line extraction using an alkyl-diol silica precolumn for racemic citalopram and its metabolites in plasma: Results compared with solid-phase extraction methodology

Sample preparation is usually the most critical and time consuming step when using HPLC for drug analysis in biological matrixes. Sample extracts have to be clean considering both chromatographic interferences and column maintenance. To meet some of these criteria a fully automated on-line extraction (OLE) analysis method was developed for the antidepressant drug citalopram and its two demethylated metabolites, using an RP-C4-ADS extraction column. A comparison between the new OLE method and an off-line solid-phase extraction method showed that the two methodologies were equal in analytical precision but that the OLE method was faster and therefore superior in sample capacity per day.     

Structural identification of the metabolites for strictosamide in rats bile by an ion trap-TOF mass spectrometer and mass defect filter technique

We report herein, a facile metabolite identification workflow on the antimicrobial strictosamide, which is derived from accurate mass measurement by a hybrid ion trap-TOF mass spectrometer. In step 1, the parent drug and metabolites in rat bile were separated on an HPLC column followed by ion trap-TOF mass spectrometer analysis after a single oral dose of 50mg/kg strictosamide. In step 2, mass defect filter technique, which enables high-resolution mass spectrometers to be utilized for detecting drug metabolites based on well-defined mass defect ranges, was used to find metabolites in the mass spectrum. In step 3, the differences of accurate masses and their mass fragmentation pattern among the parent drug and metabolites used to assign structures for the metabolites successfully. As a result, five metabolites of strictosamide were found in rat bile, and all the metabolites were reported for the first time.     

Antibody purification from CHO cell supernatant using new multimodal membranes

This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG₁ following a size‐exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two‐step MMM chromatography process achieved high selectivity for capturing hIgG₁ from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was >98% and HCP levels were <20 ppm for both purification methods. In addition, hIgG₁ could be eluted from the MMM chromatography column at neutral pH, which is important for limiting the formation of aggregates; although slow elution dilutes the product. Overall, this paper shows that MMMs are highly effective for capture step purification of proteins and should be considered when Protein A cannot be used, e.g., for pH sensitive mAbs or proteins lacking an Fc binding domain. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:658–665, 2017     

Direct coupling of expanded bed adsorption with a downstream purification step

In the course of developing a cost-effective, scaleable process for the purification of a recombinant protein from Chinese hamster ovary (CHO) suspension cell culture, we investigated direct capture of this molecule using expanded bed adsorption (EBA). EBA combines clarification, purification, and concentration of the product into a single step. The unclarified bioreactor material was directly applied to a STREAMLINE 25 column containing an affinity STREAMLINE adsorbent. This work focused on simplifying the EBA operations and minimizing the overall processing time by running the EBA column unidirectionally, eluting in the expanded bed mode, and coupling the EBA column directly with ion exchange or hydrophobic interaction chromatography. Unidirectional EBA was clearly a simpler unit operation and did not require the use of specialized equipment. The increase in the elution pool volume was insignificant, especially when the EBA column was eluted directly onto the downstream column. Scale-down was simple and could be automated. Coupling of unidirectional EBA with a downstream purification step reduced processing time, equipment requirements and cost.     

Large scale demonstration of a process analytical technology application in bioprocessing: Use of on‐line high performance liquid chromatography for making real time pooling decisions for process chromatography

Process Analytical Technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community because of the potential for continuous real time quality assurance resulting in improved operational control and compliance. In previous publications, we have demonstrated feasibility of applications involving use of high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) for real‐time pooling of process chromatography column. In this article we follow a similar approach to perform lab studies and create a model for a chromatography step of a different modality (hydrophobic interaction chromatography). It is seen that the predictions of the model compare well to actual experimental data, demonstrating the usefulness of the approach across the different modes of chromatography. Also, use of online HPLC when the step is scaled up to pilot scale (a 2294 fold scale‐up from a 3.4 mL column in the lab to a 7.8 L column in the pilot plant) and eventually to manufacturing scale (a 45930 fold scale‐up from a 3.4 mL column in the lab to a 158 L column in the manufacturing plant) is examined. Overall, the results confirm that for the application under consideration, online‐HPLC offers a feasible approach for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes. The observations demonstrate that the proposed analytical scheme allows us to meet two of the key goals that have been outlined for PAT, i.e., “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions”. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Use of large particles support for fast analysis of methadone and its primary metabolite in human plasma by liquid chromatography-mass spectrometry

A bioanalytical method was developed for the quantitation of methadone (MTD) and its primary metabolite, (EDDP) in plasma. The extraction step was performed within a capillary column packed with large particles (35x0.3 mm I.D.; d(p) 30 micrometer) at high flow-rate conditions (450 microliter/min). The separation was performed on a microbore analytical column (55x2 mm I.D.; d(p) 3 micrometer) coupled to a mass spectrometer (MS). This procedure was based on a column-switching unit. Analytes of interest were retained on the precolumn by hydrophobic interactions and backflushed from the precolumn to the analytical column. The detection was carried out with a MS single quadrupole equipped with an electrospray interface. The total analysis time was 6 min. The limits of quantification were evaluated at 10 and 25 ng/ml for MTD and EDDP, respectively. At this level, good accuracies were obtained for both analytes with repeatability values less than 18%.     

Development of a micro-turbulent flow chromatography focus mode method for drug quantitation in discovery bioanalysis

An online turbulent flow chromatography method coupled to tandem mass spectrometry (TFC-MS/MS) has been developed within our bioanalytical group, suited to the analysis of mid to late stage discovery compounds. A dual column configuration utilising isocratic focusing of the analyte upon the analytical column maintained an excellent peak shape for a large proportion of compounds encountered and enabled consistent quantitation to sub-nanogram concentrations (<15 pg on column). Furthermore, the low sample injection volume coupled with rapid column washing using basic and acidic mobile phases, has proved advantageous in removing sample carryover and also the overall exposure to biological material; favourable for good system robustness. All the data discussed were generated with a method cycle time of 5 min providing accurate quantitation (acceptance criteria based upon FDA method validation guidelines) with multiple analytes and biological matrices.     

Reactors for continuous primary beer fermentation using immobilised yeast

A continuous multistage column bioreactor with fluidised beds and continuous gas-lift bioreactor system with immobilised yeast Saccharomyces cerevisiae was developed for the first step of wort fermentation. The system of gas-lift reactor with yeast entrapped in calcium pectate beads was stable for 5 weeks by the optimal residence time of 12.75h and produced beer with a composition and flavour profile similar to that of beer produced by batch fermentation. Concentration of diacetyl was less than 0.1mg/l.     

Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture

High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25–42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 × 10⁶ cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption ≥96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2775, 2019.     

新疆雪莲的高效液相指纹图谱及液-质联用分析

建立新疆雪莲药材的高效液相指纹图谱。以Luna C18为分析柱,用乙腈-0.1%醋酸梯度洗脱,获得分离度较好的新疆雪莲药材HPLC分析条件。通过10批样品的分析和对照,标示出12个共有峰,相似度分析大于0.92。最后通过HPLC-UV-MS/MS联用分析鉴定出其中7个化学成分。该方法可为新疆雪莲药材指纹图谱和质量分析方法的建立提供参考。     

Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.     

Caprylic acid‐induced impurity precipitation from protein A capture column elution pool to enable a two‐chromatography‐step process for monoclonal antibody purification

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA‐induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high‐molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host–cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15–25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5–1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA‐based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1515–1525, 2015     

Identification of calnexin as a binding protein for Amadori-modified glycated albumin

Albumin modified by Amadori glucose adducts (glycated albumin) selectively binds to glomerular mesangial cells and triggers signal transduction processes that modulate cellular function. To identify glycated albumin binding proteins, we applied membrane extracts prepared from murine mesangial cells to a column of lysine-Sepharose followed by application to an affinity column of fructosyllysine-Sepharose. This procedure yielded an approximately 90 kDa polypeptide that immunoreacted with Amadori-modified but not carbohydrate-free albumin. MALDI mass fingerprinting matched 9 out of 25 peptides with calnexin, and amino acid analysis showed homology with this transmembrane calcium-binding protein of the calreticulin family. These results indicate that one of the mesangial cell receptors for glycated albumin is a calnexin-like protein.     

Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and other cationic antimicrobial peptides from complex culture medium

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.