Improved process analytical technology for protein a chromatography using predictive principal component analysis tools

Protein A chromatography is widely employed for the capture and purification of antibodies and Fc‐fusion proteins. Due to the high cost of protein A resins, there is a significant economic driving force for using these chromatographic materials for a large number of cycles. The maintenance of column performance over the resin lifetime is also a significant concern in large‐scale manufacturing. In this work, several statistical methods are employed to develop a novel principal component analysis (PCA)‐based tool for predicting protein A chromatographic column performance over time. A method is developed to carry out detection of column integrity failures before their occurrence without the need for a separate integrity test. In addition, analysis of various transitions in the chromatograms was also employed to develop PCA‐based models to predict both subtle and general trends in real‐time protein A column yield decay. The developed approach has significant potential for facilitating timely and improved decisions in large‐scale chromatographic operations in line with the process analytical technology (PAT) guidance from the Food and Drug Administration (FDA). Biotechnol. Bioeng. 2011; 108:59–68. © 2010 Wiley Periodicals, Inc.     

Online integrity monitoring in the Protein A step of mAb Production Processes—increasing reliability and process robustness

The purification of recombinant proteins and antibodies using large packed‐bed columns is a key component in most biotechnology purification processes. Because of its efficiency and established practice in the industry, column chromatography is a state of the art technology with a proven capability for removal of impurities, viral clearance, and process efficiency. In general, the validation and monitoring of chromatographic operations—especially of critical process parameters—is required to ensure robust product quality and compliance with health authority expectations. One key aspect of chromatography that needs to be monitored is the integrity of the packed bed, since this is often critical to achieving sufficient separation of protein species. Identification of potential column integrity issues before they occur is important for both product quality and economic efficiency. In this article, we examine how transition analysis techniques can be utilized to monitor column integrity. A case study on the application of this method during a large scale Protein A capture step in an antibody purification process shows how it can assist with improving process knowledge and increasing the efficiency of manufacturing operations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:383–390, 2014     

HPLC MS/MS method for quantification of meprobamate in human plasma: application to 24/7 clinical toxicology

We described the development and full validation of rapid and accurate liquid chromatography method, coupled with tandem mass spectrometry detection, for quantification of meprobamate in human plasma with [(13)C-(2)H(3)]-meprobamate as internal standard. Plasma pretreatment involved a one-step protein precipitation with acetonitrile. Separation was performed by reversed-phase chromatography on a Luna MercuryMS C18 (20 mm×4 mm×3 μm) column using a gradient elution mode. The mobile phase was a mix of distilled water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The selected reaction monitoring transitions, in electrospray positive ionization, used for quantification were 219.2→158.2 m/z and 223.1→161.1m/z for meprobamate and internal standard, respectively. Qualification transitions were 219.2→97.0 and 223.1→101.1 m/z for meprobamate and internal standard, respectively. The method was linear over the concentration range of 1-300 mg/L. The intra- and inter-day precision values were below 6.4% and accuracy was within 95.3% and 103.6% for all QC levels (5, 75 and 200 mg/L). The lower limit of quantification was 1 mg/L. Total analysis time was reduced to 6 min including sample preparation. The present method is successfully applied to 24/7 clinical toxicology and demonstrated its usefulness to detect meprobamate poisoning.     

Development of a liquid chromatography/negative-ion electrospray tandem mass spectrometry assay for the determination of cilnidipine in human plasma and its application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) with methyl-t-butyl ether (MTBE) followed by high-performance liquid chromatography (HPLC) with negative-ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of cilnidipine in human plasma using benidipine as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 491.1>121.8 for cilnidipine and m/z 504.2>122.1 for IS, respectively. Analytes were chromatographed on a CN column by isocratic elution using 10mM ammonium acetate buffer-methanol (30:70, v/v; adjusted with acetic acid to pH 5.0). Results were linear (r2=0.99998) over the studied range (0.1-20ng/ml) with a total LC-MS/MS analysis time per run of 3min. The developed method was validated and successfully applied to a cilnidipine bioequivalence study in 24 healthy male volunteers.     

Measuring the kinetics of membrane phase transitions

This article presents a brief review of literature on the physical chemistry of lipid phase transitions with emphasis on their kinetic properties. The theoretical foundations of perturbation techniques, and specifically the volume-perturbation technique are discussed in some detail. These are presented as a rationale for, and introduction to, a volume-perturbation kinetic calorimeter that we have constructed for measurement of the kinetics of lipid phase transitions. The instrument has been applied to study the gel-liquid crystalline phase transition in a variety of phospholipid bilayer systems. The design and implementation of the volume-perturbation calorimeter are presented along with a discussion of the techniques of data analysis. Finally, we present typical results obtained with this methodology for a multilamellar vesicle dispersion of dipalmitoylphosphatidylcholine.     

The analysis of thiamin and its derivatives in whole blood samples under high pH conditions of the mobile phase

In this study a protocol for the analysis of thiamin and thiamin coenzymes in whole blood was developed. Thiamin and its coenzymes are analyzed by reversed phase liquid chromatography (RPLC), precolumn derivatisation with alkaline potassium ferricyanide and fluorescence detection, all at pH 10. Under these relatively high pH conditions the detectability of the analytes and the robustness of the method were substantially improved. The use of a high pH resistant RPLC column was a crucial step in developing this analysis method. Reproducibility, linearity, recovery, detection limit and column robustness were investigated. The within-batch CV was <2.5%, the between-batch CV <4.5%. The method was linear far above the physiological relevant concentration level. Recovery was almost 100% on an average. The limit of quantification was 1 nmol/l. The robustness of the RPLC column proved to be very high. Up to 1500 injections hardly any substantial changes in retention times and efficiency were observed. In summary: Using a high pH resistant RPLC column resulted in a robust, sensitive and precise method for the analysis of total Vitamin B1 and especially of TDP.     

Analysis of sugars in traditional Chinese drugs

This review is presented of chromatography and electromigration methods currently in use to determine sugars in traditional Chinese drugs: gas chromatography (GC), high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel column chromatography (GCC), paper chromatography (PC) and thin layer chromatography (TLC), capillary electrophoresis (CE) and gel electrophoresis (GEP). The detection methods combined with above separation methods including ultra-violet, mass spectra, fluorescent light, refractive index (RI), electrochemical detection are also described. For the complicacy of structural analysis of polysaccharides in traditional Chinese drugs, the hyphenation procedures concerned with this analysis are introduced in this article too.     

Direct analysis of carbohydrates in animal plasma by ion chromatography coupled with mass spectrometry and pulsed amperometric detection for use as a non-invasive diagnostic tool

The present paper demonstrates that electrochemical detection (ECD) coupled to ion chromatography and electrospray ionization tandem mass spectrometry (IC-ECD-ESI/MS/MS) can be used to rapidly estimate some indications of the health status of organisms. The lactulose to mannitol ratio (L/M) is used as a non-invasive assay to investigate small intestinal absorption pathways and mucosal integrity. In the present study, an evaluation of the negative effects of nonsteroidal anti-inflammatory drug meloxicam perorally administrated to a group of dogs was carried out by determining the lactulose/mannitol index using the IC-ECD-ESI/MS/MS hyphenated technique. According to the results of the study, meloxicam altered gastrointestinal permeability. Coenzyme Q(10) (CoQ(10)) was tested to determine if it could prevent meloxicam induced gastrointestinal damage and it was found that CoQ(10) could be an effective preventive treatment. Furthermore, plasma glucose concentration level was determined to be an indirect indicator of the oxidative state in the blood. To find out the beneficial effects of a double antioxidant combination (α-lipoic acid (ALA) and CoQ(10)) on the total glucose level in chickens, ALA and CoQ(10) were provided as food additives in factory farm raised chicken. The results of the pilot study indicate that the glucose level in the plasma of chickens group fed with CoQ(10) and ALA was significantly decreased compared to the control group. Ion chromatography (IC) utilizing pulsed amperometric detection (PAD) was compared to ion chromatography coupled with tandem mass spectrometry (MS/MS) as an analytical tool for monitoring the carbohydrate level in biological fluids. In electrochemical detection, the newly developed two-pulse waveform successfully withstands matrix effects in biological samples. Continuous on-line desalting of the high salt concentrations used as the eluent for carbohydrate separation from the anion-exchange column allows coupling of IC and MS techniques. A make-up solution (0.5mM LiCl) was delivered prior to MS detection for efficient ionization of eluted carbohydrates. Method validation showed that both used techniques are practically comparable and some advantages of each are presented.     

High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3-methyl5-pyrazolone derivatives

We found that 1-phenyl-3-methyl-5-pyrazolone reacts with reducing carbohydrates almost quantitatively to yield 2:1 compounds having no stereoisomers, which strongly absorb the uv light at 245 nm and are easily oxidizable on a glassy carbon electrode. Reverse-phase partition chromatography on a column of Capcell Pak C18 with uv or electrochemical detection allowed rapid analysis of aldoses and N-acetylhexosamines with the detection limit of 1 pmol or 100 fmol, respectively. This method proved especially useful for analysis of component monosaccharides of glycorproteins. It was also shown to be valid for separation of reducing oligosaccharides; maltodextrins with a degree of polymerization up to 19 were similarly derivatized and separated on this stationary phase.     

Measurement of H2S in Crude Oil and Crude Oil Headspace Using Multidimensional Gas Chromatography,Deans Switching and Sulfur-selective Detection

A method for the analysis of dissolved hydrogen sulfide in crude oil samples is demonstrated using gas chromatography. In order to effectively eliminate interferences, a two dimensional column configuration is used, with a Deans switch employed to transfer hydrogen sulfide from the first to the second column (heart-cutting). Liquid crude samples are first separated on a dimethylpolysiloxane column, and light gases are heart-cut and further separated on a bonded porous layer open tubular (PLOT) column that is able to separate hydrogen sulfide from other light sulfur species. Hydrogen sulfide is then detected with a sulfur chemiluminescence detector, adding an additional layer of selectivity. Following separation and detection of hydrogen sulfide, the system is backflushed to remove the high-boiling hydrocarbons present in the crude samples and to preserve chromatographic integrity. Dissolved hydrogen sulfide has been quantified in liquid samples from 1.1 to 500 ppm, demonstrating wide applicability to a range of samples. The method has also been successfully applied for the analysis of gas samples from crude oil headspace and process gas bags, with measurement from 0.7 to 9,700 ppm hydrogen sulfide.     

New method for the chiral evaluation of mirtazapine in human plasma by liquid chromatography

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method was developed for the enantioselective analysis of the new antidepressant drug mirtazapine in human plasma. The procedure involved liquid-liquid extraction using toluene, followed by liquid chromatography coupled to UV detection at 292 nm. The chromatographic separation of the (+)-(S)- and (-)-(R)-enantiomers of mirtazapine was achieved on a Chiralpak AD column (250 mm x 4.6 mm, 10 microm particle size) protected with a CN guard column, using hexane-ethanol (98:2, v/v) plus 0.1% diethylamine as the isocratic mobile phase, at a flow rate of 1.2 ml/min. The total analysis time was less than 12 min per sample. The recoveries of (+)-(S)- and (-)-(R)-mirtazapine were in the 88-111% range with a linear response over the 6.25-625 ng/ml concentration range for both enantiomers. The quantification limit (LOQ) was 5 ng/ml. Within-day and between-day assay precision and accuracy were studied at three concentration levels (10, 50 and 250 ng/ml). For both mirtazapine enantiomers, the coefficients of variation (CV) and deviation from the theoretical value were lower than 15% at all concentration levels. The method proved to be suitable for pharmacokinetic studies.     

Pyrolysis Gas Chromatography of Pseudomonas and Acinetobacter Species

Low resolution pyrolysis gas chromatography was performed on five species of Pseudomonas and two species of Acinetobacter. Conventional species groups could be differentiated by canonical variates analysis, and visual examination suggested that subspeciation would have been possible with more data.
Carbowax 20M has been used as the stationary phase for the majority of published work on pyrolysis gas chromatography of micro-organisms. However, with this material baseline resolution was poor, analysis times were long and column deterioration was relatively rapid. Peak area reproducibility on a single column averaged 10%, but it proved impossible to achieve quantitatively similar pyrograms on a new column. These serious drawbacks of Carbowax 20M limit the usefulness of this stationary phase for pyrolysis gas chromatography of micro-organisms.     

An HPLC procedure for separating polyphosphoinositides on hydroxylapatite

We describe a method for separation of several phosphoinositides by high performance liquid chromatography (HPLC) for the purposes of identification, analysis, and possible purification of cell phospholipid extracts. The phosphoinositides were separated on an hydroxylapatite column using a solvent system consisting of tetrahydrofuran-ethanol-water with a gradient of triethylamine phosphate ranging from 1 to 100 mM. Increasing triethylamine phosphate concentrations over a series of isocratic steps resulted in the resolution of [3H]phosphoinositide standards (phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4,5-bisphosphate) and their lyso-derivatives. The eluted peaks were collected and quantitated using scintillation counting, and the samples of the eluates were subjected to further analysis by thin-layer chromatography to verify their integrity and identity.     

Differentiation between indoleacetic Acid and the citrus auxin by column chromatography

A gradient elution column chromatography technique and a step-wise technique succeeded in differentiating between IAA and the citrus auxin. IAA was eluted ahead of the citrus auxin in both systems. The highest Avena curvature ever obtained from the citrus auxin occurred after the auxin had passed through the 2 purification techniques and a paper chromatography step. This is probably due to the elimination of inhibitors. Fluorometric assay, Ehrlich's reaction, thin-layer chromatography, and biological assay were used for the detection of IAA or citrus auxin in the column eluates.     

Site-specific incorporation of the 1-hexanol-1,N6-etheno-2'-deoxyadenosine adduct into oligodeoxyribonucleotides

Modified oligonucleotides that contain the hydrophobic 1-hexanol-1,N(6)-etheno-2'-deoxyadenosine adduct have been synthesized using a mild solid phase phosphoramidite chemistry. The presence and the integrity of the modified nucleoside in the synthetic oligomers were confirmed by electrospray ionization and MALDI mass spectrometry measurements together with analysis of the complete enzymatic hydrolysate by high performance liquid chromatography coupled to UV and fluorescent detection techniques.     

Quantitative determination of atractylenolide III in rat plasma by liquid chromatography electrospray ionization mass spectrometry

Atractylenolide III is a major active component in Atractylodes macrocephala. This paper describes a simple, rapid, specific and sensitive method for the quantification of atractylenolide III in rat plasma using a liquid-liquid extraction procedure followed by liquid chromatography mass spectrometric (LC-MS) analysis. A Kromasil 3.5 microm C(18) column (150 mm x 2.00 mm) was used as the analytical column. Linear detection responses were obtained for atractylenolide III concentration ranging from 5 to 500 ng L(-1). The precision and accuracy data, based on intra-day and inter-day variations over 5 days were within 10.29%. The lower limit of quantitation for atractylenolide III was 5 ng mL(-1), using 0.1 mL plasma for extraction and its recoveries were greater than 85% at the low, medium and high concentrations. The method has been successfully applied to a pharmacokinetic study in rats after an oral administration of atractylenolide III with a dose of 20.0 mg kg(-1). With the lower limits of quantification at 5 ng mL(-1) for atractylenolide III, this method was proved to be sensitive enough for the pharmacokinetics study of atractylenolide III.     

An automated SPE/LC/MS/MS method for the analysis of cocaine and metabolites in whole blood

As laboratories are called upon to develop novel, fast, and sensitive methods, here we present a completely automated method for the analysis of cocaine and its metabolites (benzoylecgonine, ecgonine methyl ester, ecgonine and cocaethylene) from whole blood. This method utilizes an online solid-phase extraction (SPE) with high performance liquid chromatographic separation and tandem mass spectrometric detection. Pretreatment of samples involve only protein precipitation and ultracentrifugation. An efficient online solid-phase extraction (SPE) procedure was developed using Hysphere MM anion sorbent. A gradient chromatography method with a Gemini C6-Phenyl (50mmx3.00mm i.d., 5microm) column was used for the complete separation of all components. Analysis was by positive ion mode electrospray ionization tandem mass spectrometry, using multiple reaction monitoring (MRM) to enhance the selectivity and sensitivity of the method. For the analysis, two MRM transitions are monitored for each analyte and one transition is monitored for each internal standard. With a 30-microL sample injection, linearity was analyte dependent but generally fell between 8 and 500ng/mL. The limits of detection (LODs) for the method ranged from 3 to 16ng/mL and the limits of quantitation (LOQs) ranged from 8 to 47ng/mL. The bias and precision were determined using a simple analysis of variance (ANOVA: single factor). The results demonstrate bias as <7%, and %precision as <9% for all components at each QC level.     

Polyamines as cancer markers: applicable separation methods

Spermine, spermidine, putrescine and cadaverine are aliphatic amines widely spread in the human body. Their concentrations together with their acetyl conjugates increase significantly in the biological fluids and the affected tissues of cancer patients. Their concentrations decrease with the improvement in the patient’s condition on multiple therapy. Various chromatographic techniques are frequently used in monitoring concentrations of di- and polyamines in cancer. Among these techniques, thin-layer chromatography and liquid chromatography using pre- or postcolumn derivatization, separating on a reversed-phase or an ion-exchange column are the most commonly used. Besides, high-resolution capillary column gas chromatography (GC) is increasingly used over packed column GC, and in recent years, capillary zone electrophoresis has also gained some importance in polyamine determinations. The review examines the prospects and the limitations of polyamines as cancer markers using chromatographic and electrophoretic techniques.     

Determination of sumatriptan succinate in human plasma by high-performance liquid chromatography with coulometric detection and utilization of solid-phase extraction

Sumatriptan succinate (the analyte) and naloxone (the internal standard) were extracted from plasma with a solid-phase extraction technique. Chromatography and detection were performed by isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The standard curve was linear over the range 0–100 ng/ml of sumatriptan succinate in plasma. The reproducibility (as defined by the coefficient of variation, C.V.) over the range of the standard curve was 4.9–7.3%. The recovery averaged 83%. The sensitivity was 0.25 ng of sumatriptan on column (allowing a concentration of 0.5 ng/ml to be determined from a 1-ml plasma sample volume). Plasma profiles of the analyte following subcutaneous (s.c.) administration in eight normal male volunteers, are presented.