Capillary zone electrophoresis tandem mass spectrometry detects low concentration host cell impurities in monoclonal antibodies

We have evaluated CZE‐ESI‐MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5‐point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE‐MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ～70‐min separation window (～90‐min total analysis duration) and ～300‐peak capacity. We also analyzed the sample using ultra‐performance LC‐MS/MS. CZE‐MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low‐level spiked proteins. Both methods detected all proteins spiked at ～100 ppm level with respect to the antibody.     

Accelerated tryptic digestion for the analysis of biopharmaceutical monoclonal antibodies in plasma by liquid chromatography with tandem mass spectrometric detection

Accelerated tryptic digestion of a therapeutic protein including microwave irradiation and thermal transfer by convection at 60 °C and 37 °C was investigated. An analytical setup was devised to follow the protein digestion rate using 1D gel electrophoresis and liquid chromatography coupled a triple quadrupole linear ion trap mass spectrometer. The formation kinetic of its tryptic peptides was monitored in the selected monitoring mode (LC-SRM/MS). Different digestion end points (e.g. 2, 5, 10, 15, 30 and 60 min) as well as an overnight digestion were tested using a therapeutic human monoclonal antibody (mAb) with the goal of its LC-SRM/MS quantification in human plasma. The peptides from the human mAb were generated at different rates and were classified into three categories: (1) the fast forming peptides, (2) the slow forming peptides and (3) the peptides degrading over time. For many monitored peptides, a heating temperature of 37 °C with a 750 rpm mixing applied for at least 30 min provided equivalent results to microwave-assisted digestion and generally allowed the achievement of an equivalent peptide concentration as an overnight digestion carried out at 37 °C. The disappearance of the protein of the heavy and light chains can be monitored by 1D gel electrophoresis but was found not to be representative of the final tryptic peptide concentrations. For quantitative purposes a stable isotope labeled version (¹³C₄, ¹⁵N₁) of the therapeutic protein was used. The labeled protein as internal standard was found to be very efficient to compensate for incomplete digestion or losses during sample preparation.     

Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry

Four methods were compared for analysis of host‐cell protein (HCP) impurities in a recombinant mAb. First, CZE‐MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 μg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE‐MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE‐ESI‐MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.     

On‐line solid‐phase extraction for liquid chromatography–mass spectrometry analysis of pesticides

Public concern about pesticides in food and water has increased dramatically in the last two decades. In order to guarantee consumers’ health and safety, analytical methods that could provide fast and reliable answers without compromising accuracy and precision are required. Sample treatment is probably the most tedious and time‐consuming step in many analytical procedures and, despite the significant advances in chromatographic separations and mass spectrometry techniques, sample treatment is still one of the most important parts of the analytical process for achieving good analytical results. Therefore, over the last years, considerable efforts have been made to simplify the stage and to develop fast, accurate, and robust methods that allow the determination of a wide range of pesticides without compromising the integrity of the extraction process. This review article intends to give a short overview of recently developed on‐line solid‐phase extraction, preconcentration, and clean‐up procedures for the determination of pesticides in complex matrices by liquid chromatography–mass spectrometry techniques.     

Antibody capture by mixed-mode chromatography: a comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins

We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of these mixed mode resins. Optimal conditions for antibody purification and host cell proteins (HCPs) elimination were determined and then directly up-scaled to laboratory columns. Then we used mass spectrometry to identify the major HCPs potentially coeluted with the antibody. Differences between the four resins in terms of amount, complexity and identity of the HCPs present in the elution fractions were investigated.     

Analysis of proteoglycans derived sulphated disaccharides by liquid chromatography/mass spectrometry

A method has been developed for the identification and quantitative determination of sulphated disaccharides derived from chondroitin sulphate (CS) and dermatan sulphate (DS) chains attached to proteoglycans (PGs). After digestion with Chondroitinase ABC, the pool of disaccharides can be directly separated by liquid chromatography on a porous graphitized carbon (PGC) column and identified by on-line electrospray mass spectrometry under negative ionization conditions. The relative intensities of the fragment ions obtained by MS/MS allow to distinguish the sulphate position. Calibration with standard disaccharides allows the quantification of the different isomers. The method showed good repeatability in terms of relative standard deviation (RSD < 2%) and linearity between 0.5 and 50 ng (total injected amount) for both 4- and 6-sulphated disaccharides. The limit of detection achieved in full scan mode was 0.1 ng. The methodology was applied to different types of biological samples obtained from patients suffering from chronic lung inflammation such as: lung tissue, bronchoalveolar lavage fluid (BALF), induced sputum and urine.     

Characterization of mixtures of biocidal oligoguanidines by capillary electrophoresis and high-performance liquid chromatography coupled to mass spectrometry

The polycondensation of guanidine hydrochloride and 2,2′-(ethylenedioxy)bis(ethylamine) leads to various types of oligomeric guanidines exhibiting a broad spectrum of biocidal activities. In the present work a reversed-phase high-performance liquid chromatographic method with a gradient consisting of aqueous 0.1% trifluoroacetic acid and acetonitrile as mobile phase has been developed to separate these oligoguanidines according to type and chain length. The combination with electrospray mass spectrometry allowed the identification of the various compounds. By this technique, some structures already suggested in the literature could be confirmed, and several additional oligoguanidines not yet reported could be identified. As a complementary technique, capillary zone electrophoresis was investigated. Best results were obtained with carrier electrolytes consisting of phosphoric acid in water/acetonitrile mixtures. Although the number of peaks that could be separated by the electrophoretic method was considerably lower than in case of the chromatographic method, capillary electrophoresis in combination with UV detection at 195 nm may still be a fast method suitable for quantitation of some of the major compounds and for monitoring the reaction rate during the polycondensation reaction.     

Complementary middle‐down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis – mass spectrometry

High‐resolution capillary zone electrophoresis – mass spectrometry (CZE‐MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle‐down and intact CZE‐MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post‐translational modifications (PTMs) and glycosylation structures. Middle‐down and intact CZE separations were performed in an acidified methanol‐water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle‐down analysis of the IdeS‐digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X‐deamidated, 1X‐deamidated, and non‐deamidated forms at baseline resolution. In the course of the middle‐down CZE‐MS analysis, separation of glycoforms of the F_C/2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE‐MS². Incorporation of TCEP‐HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE‐MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X‐glycosylated, 1X‐glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE‐MS represents a complementary approach to the more conventional liquid‐chromatography – mass spectrometry‐based approaches.     

Ultra-high capacity liquid chromatography chip/quadrupole time-of-flight mass spectrometry for pharmaceutical analysis

Nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) has attracted increasing interest in virtue of high sensitivity, low sample consumption, and minimal matrix effect. In this work a HPLC-Chip/quadrupole time-of-flight (Q-TOF) MS device with a new ultra-high capacity small molecule chip (UHC-Chip) which features a 500 nL enrichment column and a 150 mm × 75 μm analytical column, was evaluated with a drug mixture covering a wide range of polarities. Excellent chromatographic precision with 0.1-0.5% RSD for retention time and 1.7-9.0% RSD for peak area, low limit of detection, good chip-to-chip reproducibility and linearity were obtained by using this UHC-Chip. Compared with the standard HPLC-Chip with 40 nL trapping column, the UHC-Chip showed higher enrichment capability and hence gave a higher response in signal detection. Additionally, 4-30 times increase in sensitivity was obtained compared with conventional LC/MS, which indicated that UHC-Chip/MS was a valuable tool for the quantitative analysis of low level impurities and degradation products in pharmaceuticals. Moreover, satisfactory results obtained from trace drug analysis of serum samples further proved its practicality and potential for use in drug testing and development.     

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10–15 min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 μg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5 μg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation.     

Direct coupling of size exclusion chromatography and mass spectrometry for the characterization of complex monoclonal antibody products

The present study describes the possibilities offered by an innovative bioinert size exclusion chromatography column for size variant characterization of complex monoclonal antibody products. This size exclusion chromatography column includes a novel column hardware surface. The column was prepared from metallic hardware components that were treated to have prototype hydrophilically modified hybrid organic–inorganic silica surfaces called hybrid surface technology. This provides a significant reduction in nondesired hydrophobic and electrostatic interactions that can occur between column and analyte when performing size exclusion chromatography analysis with volatile mobile phase. Compared to a reference stainless-steel column packed with the same batch of packing material, peak tailing, band broadening, and above all recovery of high molecular weight species were distinctly improved for all types of monoclonal antibody products. Based on our observations, we found that 50 mM ammonium acetate in water was a suitable mobile phase offering good compromise in terms of liquid chromatography performance and mass spectrometry sensitivity. In addition, method repeatability (intra- and interday relative standard deviations) on elution times and high molecular weight species peak areas were found to be excellent. By using this innovative size exclusion chromatography material, the low and high molecular weight species contained in various stressed and nonstressed monoclonal antibody products were successfully characterized with mass spectrometry detection.     

Combining microwave-assisted extraction and liquid chromatography–ion-trap mass spectrometry for the analysis of hexabromocyclododecane diastereoisomers in marine sediments

An efficient microwave-assisted extraction (MAE) procedure coupled with high performance liquid chromatography–electrospray-ion-trap mass spectrometry (HPLC–ESI-ITMS) has been evaluated to determine hexabromocyclododecane diastereoisomers (α-, β- and γ-HBCD) in marine sediments. The composition of the LC mobile phase (consisting of water, methanol and acetonitrile) and the parameters of electrospray ionization (ESI) were evaluated to obtain chromatographic baseline separation and high sensitivity for the detection of these diastereoisomers. The effects of various operating parameters on the quantitative extraction of the HBCDs through MAE were systematically investigated. The three diastereoisomers were then quantitated by HPLC–ITMS employing ESI operated in the negative ionization mode. The HBCDs were extracted from the sediments through MAE using 40 mL of acetone/n-hexane (1/3, v/v) at 90 °C for 12 min. The limits of quantitation (LOQ) ranged from 25 to 40 pg/g (dry weight) in 5 g of the sediment samples. The recoveries of the HBCDs in spiked sediment samples ranged from 68 to 91% (relative standard derivation: 2–11%). The extraction efficiency of the MAE technique was also compared with Soxhlet extraction and pressurized liquid extraction.     

Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics

Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential ¹²C-/¹³C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N-Methyl-D-aspartic and tyrosine were identified. The changes of these biomarkers were likely due to the disruption of the endocrine system of silkworm by DDT. This work illustrates that the method of CIL LC-MS is useful to generate quantitative submetabolome profiles from a small volume of silkworm hemolymph with much higher coverage than conventional LC-MS methods, thereby facilitating the discovery of potential metabolite biomarkers related to EDC or other chemical exposure.     

Methodological aspects of an off‐line combination of preparative isotachophoresis and high‐performance liquid chromatography with mass spectrometry in the analysis of biological matrices

This work reports on some methodological aspects of an off‐line combination of preparative ITP and HPLC with mass spectrometric detection (pITP‐HPLC‐MS) and its potential applications to the analysis of high molecular mass compounds present in complex biological matrices from the analytical chemistry perspective. Lysozyme served as the model analyte and human saliva as the complex biological matrix in this study. A mixture of five low‐molecular mass compounds was found and successfully used in the pITP experiments as discrete spacers to isolate the analyte from the interferents present in the complex biological matrix and to minimize their disturbance effect on the final MS analysis. The experiments at the pITP stage were performed in the cationic mode. On‐column conductivity detectors were used for the detection of ITP zones. Lysozyme was found in the human saliva samples using just deconvolution of the MS data after background correction. The MS data obtained from HPLC‐MS analysis of pITP fractions exhibited the great analytical potential of the combination of pITP‐HPLC‐MS resulting from the ITP clean‐up effect as well as the ITP preconcentration of the analyte present at low concentration levels in complex biological matrices.     

Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins

The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC–MS) with the use of isotope analog standards.     

Construction of on-line supercritical fluid extraction with reverse phase liquid chromatography–tandem mass spectrometry for the determination of capsaicin

A column-switching system, composed of supercritical fluid extraction (SFE) and reverse phase liquid chromatography/mass spectrometry (RPLC/MS) was constructed for on-line extraction and reverse-phase separation of capsaicinoids in capsicum fruits.     

Imatinib (Gleevec, Glivec) tumour tissue analysis by measurement of sediment and by liquid chromatography tandem mass spectrometry

The analysis of the signal transduction inhibitor imatinib in patient tumour tissue using LC and MS/MS is described. The anticancer agent is eluted over RP-C18 within 2 mm together with its internal standard STI571-d8. Calibration curves were prepared in red blood cells (RBC). For quantitative isolation of the RBC, measurement of sediment was applied. There were no indications of signal suppression by substances originating in the biological matrix. The limit of determination in tumour tissue was in the range of those recorded for RBC and plasma. The assay is selective and sensitive, with its robustness favouring the experimental application in clinical oncology and its routine use in animal experiments. The LOD was 4.5 ng per gram in tumour tissue.     

Investigation of the biotransformation pathway of verapamil using electrochemistry/liquid chromatography/mass spectrometry - a comparative study with liver cell microsomes

The biotransformation pathway of verapamil, a widely prescribed calcium channel blocker, was investigated by electrochemistry (EC) coupled online to liquid chromatography (LC) and electrospray mass spectrometry (ESI-MS). Mimicry of the oxidative phase I metabolism was achieved in a simple amperometric thin-layer cell equipped with a boron-doped diamond (BDD) working electrode. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass data and additional MS/MS experiments. We were able to demonstrate that all of the most important metabolic products of the calcium antagonist including norverapamil (formed by N-demethylation) can easily be simulated using this purely instrumental technique. Furthermore, newly reported metabolic reaction products like carbinolamines or imine methides become accessible. The results obtained by EC were compared with conventional in vitro studies by conducting incubations with rat as well as human liver microsomes (RLMs, HLMs). Both methods showed good agreement with the data from EC/LC/MS. Thus, it can be noted that EC is very well-suited for the simulation of the oxidative metabolism of verapamil. In summary, this study confirms that EC/LC/MS can be a powerful tool in drug discovery and development when applied complementary to established in vitro or in vivo approaches.     

Single solid phase extraction method for the simultaneous analysis of polar and non-polar pesticides in urine samples by gas chromatography and ultra high pressure liquid chromatography coupled to tandem mass spectrometry

A new multiresidue method has been developed and validated for the simultaneous extraction of more than two hundred pesticides, including non-polar and polar pesticides (carbamates, organochlorine, organophosphorous, pyrethroids, herbicides and insecticides) in urine at trace levels by gas and ultra high pressure liquid chromatography coupled to ion trap and triple quadrupole mass spectrometry, respectively (GC-IT-MS/MS, UHPLC-QqQ-MS/MS). Non-polar and polar pesticides were simultaneously extracted from urine samples by a simple and fast solid phase extraction (SPE) procedure using C₁₈ cartridges as sorbent, and dichloromethane as elution solvent. Recovery was in the range of 60-120%. Precision values expressed as relative standard deviation (RSD) were lower than 25%. Identification and confirmation of the compounds were performed by the use of retention time windows, comparison of spectra (GC-amenable compounds) or the estimation of the ion ratio (LC-amenable compounds). For GC-amenable pesticides, limits of detection (LODs) ranged from 0.001 to 0.436 μg L⁻¹ and limits of quantification (LOQs) from 0.003 to 1.452 μg L⁻¹. For LC-amenable pesticides, LODs ranged from 0.003 to 1.048 μg L⁻¹ and LOQs ranged from 0.011 to 3.494 μg L⁻¹. Finally, the optimized method was applied to the analysis of fourteen real samples of infants from agricultural population. Some pesticides such as methoxyfenozide, tebufenozide, piperonyl butoxide and propoxur were found at concentrations ranged from 1.61 to 24.4 μg L⁻¹, whereas methiocarb sulfoxide was detected at trace levels in two samples.     

Multiresidue analysis of beta-agonists in bovine and porcine urine,feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry

The use of β-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of β-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 β-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCα and CCβ values of less than 0.2 and less than 0.5 μg/l respectively, for all β-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCβ values of less than 10.0 and less than 5.0 μg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC™) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of β-agonists.