Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry

High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats.

In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market.     

Cobalt determination in serum and urine by electrothermal atomic absorption spectrometry

Cobalt determinations in biological fluids are of great interest in biological or toxicological research programs. Cobalturia is often chosen as an indicator for a biological monitoring program in occupational exposure to cobalt dusts. The method described here derives from the IUPAC reference method for nickel determination. It enables cobaltemia and cobalturia to be measured in small samples (1 mL). The mean usual values for cobalt in biological fluids are very low (2.7 nmol L⁻¹ for serum and 6.7 nmol L⁻¹ for urine), and therefore, thus require an analytical procedure with preconcentration and extraction. The sample is mineralized by wet acid digestion. After digestion, inorganic cobalt is extracted in form of ammonium pyrrolidine dithiocarbamate complex into isobutyl methyl ketone and measured in the organic layer by electrothermal atomic absorption spectrometry. The analytical parameters are described in detail. The extraction output is about 99%. The detection limits are 1.93 and 1.89 nmol L⁻¹ for serum and urine, respectively. Sensitivity (expressed as the concentration that gives a 0.044 absorbance) is 3.4 nmol L⁻¹ for serum and 3.3 nmol L⁻¹ for urine. Within-run precision ranged between 3.9 and 2.5% (coefficients of variation) for serum and 4.2 and 1.1% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. Between-run precision ranged between 4.3 and 3.3% (coefficients of variation) for serum and 4.2 and 2.3% for urine, at 87 and 136 nmol L⁻¹ levels, respectively. At very low concentration, 5.7 nmol L⁻¹ for serum and 2.5 nmol L⁻¹ for urine, the between-run precision is, respectively, 19.5 and 28%. Linearity is effective between 0 and 272 nmol L⁻¹. Interferences and matrix effects are negligible for urine, serum, or plasma samples without hemoglobin. The method is easily applicable for routine determinations.     

Determination of trace elements in porcine brain by inductively coupled plasma-mass spectrometry,electrothermal atomic absorption spectrometry,and instrumental neutron activation analysis

Methods have been developed for the analyses of trace metals in various areas of porcine brains, (temporal, parietal, frontal cortex, both right and left hemispheres). Determinations were carried out using inductively coupled plasma-mass spectrometry (ICP-MS) and electrothermal atomic absorption spectrometry (ETAAS). The elements investigated were Li, Mn, Cu, Zn, Cd, Hg, and Pb by ICP-MS and Cu, Cd, and Mn by ETAAS. For determination by ICP-MS, a method of standard additions calibration coupled with internal standards was used, and for ETAAS, standard additions calibrations were prepared. The accuracy of all methods was determined using NIST and IAEA certified reference material. A small number of pig brains were analyzed by instrumental neutron activation analysis for Cr, Co, Cs, Fe, Rb, Se, Sc, Sb, and Zn using the comparator method of analysis. Four separate NIST standard reference materials have been used to examine the validity of the comparator method.     

Determination of chromium in cerebrospinal fluid using electrothermal atomisation atomic absorption spectrometry

A rapid method to determine the chromium content of cerebrospinal fluid (CSF) samples using electrothermal atomization atomic absorption spectrometry (ETA-AAS) with deuterium-arc background correction is described. The chromium concentration in CSF was evaluated by the standard addition method. Sample dilution (1 + 1) with 0.25% (m/v) Triton X-100 and 4.5% (v/v) HNO₃ gave the best combination of sensitivity, reproducibility, and low blank reading compared with dilution using other solvents. Within-batch reproducibility was 3.2% for 20 CSF samples, between-batch reproducibility was 4.7%. CSF samples from 43 healthy volunteers collected in a manner designed to avoid contamination yielded chromium concentrations of 14.6 ± 6.3 ng mL⁻¹.     

Metallothionein separation and analysis by reversed phase high performance liquid chromatography coupled with graphite furnace atomic absorption spectrometry

Abstract

The feasibility of using reversed-phase high performance liquid chromatography (RP-HPLC) for the separation of metallothioneins (MTs) and subsequent determination of cadmium in MTs by graphite furnace atomic absorption spectrometry (GFAAS) in rabbit kidney and liver has been studied. RP-HPLC was used to isolate, characterise and quantitate liver and kidney MT isoforms. The MTs were eluted from a radially compressed C18 column with a neutral sodium phosphate buffer and detected by UV absorbance at 254 nm. Rabbit liver MTs was found to be comprised of seven distinct isoforms with five of which were found to be subspecies of the MT-I isoform. Rabbit kidney MTs exhibited only two predominant isoforms. A standard calibration curve was constructed using purified rabbit kidney MT-I and MT-II which demonstrated excellent linear correlation between peak height and the quantity of MT injected into the column. Recovery of MT from RP-HPLC was found to exceed 90%. Kidney and liver tissues from rabbit by feeding low levels of cadmium in diets was assayed using the RP-HPLC analysis of cytosol samples. Feeding stable cadmium in the diet resulted in the deposition of MT in the kidney rather than in the liver. The cadmium content in MT isoforms was determined by GFAAS. Less than 10% of the total cadmium in kidney was associated with MTs.     

Separation and quantitation of metallothioneins by high-performance liquid chromatography coupled with atomic absorption spectrophotometry

A rapid, reproducible, and sensitive high-performance liquid chromatography (HPLC) method for the determination of the concentrations of metallothionein-I (MT-I) and metallothionein-II (MT-II) in rat liver has been developed. Metallothioneins (MTs) were separated and quantitated by anion-exchange high-performance liquid chromatography coupled with atomic absorption spectrophotometry (AAS). Purified rat liver MT-I and MT-II, used as standards for developing the method, were easily resolved, eluting at 7.5 and 10.4 min, respectively. To establish standard curves, protein concentrations of solutions of the purified MTs were determined by the Kjeldahl method for the determination of nitrogen, after which the standards were saturated with Cd (final concentration of 50 ppm Cd). Rat liver cytosols obtained from untreated and Cd- or Zn-treated rats were prepared for HPLC-AAS analysis by saturation with Cd (50 ppm Cd) followed by heat denaturation (placing in a boiling water bath for 1 min). Based on the method of standard additions, recovery of MTs exceeded 95% and repeated injection of a sample yielded a coefficient of variance of approximately 2%. A detection limit of 5 micrograms MT/g liver was established for the method. Only MT-II was detected in untreated rats, whereas following exposure to Cd or Zn, both forms of MTs were detected. Concentrations of total MTs in liver of untreated and Cd- or Zn-treated rats were also determined by the Cd/hemoglobin radioassay (which fails to distinguish MT-I from MT-II) and indicated that results obtained with the HPLC-AAS method compared favorably to the Cd/hemoglobin radioassay. Thus, the HPLC-AAS method for quantitating MT-I and MT-II offers the advantage of determining the concentrations of both proteins in tissues and should be useful for studying the regulation of MT-I and MT-II.     

Distribution of elements binding to molecules with different molecular weights in aqueous extract of Antarctic krill by size-exclusion chromatography coupled with inductively coupled plasma mass spectrometry

The distribution of silver, arsenic, cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, selenium and zinc binding to species with different molecular weight in aqueous extract of krill was studied by on-line size-exclusion chromatography (SEC)/inductively coupled plasma mass spectrometry (ICP-MS). The extract was fractionated in three fractions with different molecular weight (MW) ranges (>20,000 relative molecular mass (rel. mol. mass), 2000-20,000 rel. mol. mass and <2000 rel. mol. mass), which were further analyzed by SEC with columns having different optimum fractionation ranges in order to obtain more detailed information about the MW distribution of the elements. Various distribution profiles for the target elements among different MW ranges were observed. The results obtained indicated that manganese, zinc, silver, cadmium and lead species were mostly distributed in the higher MW range (>20,000 rel. mol. mass). In the case of chromium, iron, cobalt, arsenic and selenium, most of them bind to species with lower MW (<2000 rel. mol. mass). Only copper and nickel species was predominantly present in middle MW range (2000-20,000 rel. mol. mass). Further speciation of arsenic compounds in the small MW fraction was carried out with anion exchange chromatography (AEC) coupled with ICP-MS. The results showed that the dominant arsenic species in this fraction is As(III) (63% of extractable arsenic), while As(V) (13%) and two unknown arsenic species (19% and 5%, respectively) are present in lower amounts.     

Physicochemical screening for chemical stabilizer of erythropoietin to prevent its aggregation

Recombinant protein aggregation is a problematic issue and can provoke immunological response. The aim of this study was to analyze the stability of erythropoietin (EPO), as a therapeutic protein expressed in mammalian cells, in the presence of different chemicals and find a specific stabilizer for EPO. The effects of several chemicals, including mannitol, betaine, trehalose, taurine, linoleic acid, beta-cyclodextrin, copper sulfate, spermidine, maltose, maltodextrin, sucrose, dextran, beta-alanine, myo-inositol, and cysteine, on protein stabilization through the thermally induced aggregation of EPO were monitored. Based on the results of turbidity assay for thermal aggregation, three different patterns were observed for protein stability of active pharmaceutical ingredient of EPO, namely, accelerated, dose-dependent, and inhibitory behaviors for aggregate formation due to treatment with spermidine, mannitol, and betaine, respectively. According to circular dichroism outcomes, EPO treatment with betaine and spermidine resulted in different helical contents of the secondary structure. Dynamic light scattering experiments indicated that treating EPO with betaine resulted in less protein aggregation due to freeze and thaw stresses. Betaine was able to stabilize EPO and inhibit its aggregation, as opposed to spermidine that induced protein aggregation.     

Determination of cadmium in blood, plasma, and urine by electrothermal atomic absorption spectrophotometry after isolation by anion-exchange chromatography

The determination of cadmium in whole blood, urine, or plasma by atomic absorption using electrothermal atomization is described. In preparation for atomic absorption analysis, cadmium was concentrated on an anion-exchange column, significantly lowering the limit of detection and allowing for the first time the accurate and precise determination of plasma cadmium concentrations in persons/animals with low-level cadmium exposures. Recovery of 109Cd from spiked whole blood, plasma, and urine into supernatants of nitric acid-deproteinated samples averaged 99, 100, and 95%, respectively. Anion-exchange isolation of the anionic chlorocadmium complex removed 99.8% of the major elements associated with a deproteinated whole blood sample. The recovery of 109Cd from the anion-exchange column was 92.2 +/- 0.9% (mean +/- SE, N = 35). The separation of cadmium from constituents in blood, urine, or plasma in this manner allowed comparison of unknown samples to aqueous standards with a defined acid matrix using commercially available acids. The mean intra-assay coefficient of variation (CV) was 12 +/- 3% (mean +/- SE, N = 6) for blood, plasma, and urine samples having cadmium concentrations of 0.1-0.8 microgram/liter. The interassay CV was 13% (N = 7) for a blood sample containing 0.6 microgram Cd/liter. The recovery of known amounts of cadmium added to blood, plasma, and urine in the range of 0.2 to 5.0 micrograms Cd/liter was 97 +/- 6% (mean +/- SE, N = 4).     

High-performance affinity chromatography: a powerful tool for studying serum protein binding

High-performance affinity chromatography (HPAC) is a method in which a biologically-related ligand is used as a stationary phase in an HPLC system. This approach is a powerful means for selectively isolating or quantitating agents in complex samples, but it can also be employed to study the interactions of biological systems. In recent years there have been numerous reports in which HPAC has been used to examine the interactions of drugs, hormones and other substances with serum proteins. This review discusses how HPAC has been used in such work. Particular attention is given to the techniques of zonal elution and frontal analysis. Various applications are provided for these techniques, along with a list of factors that need to be considered in their optimization and use. New approaches based on band-broadening studies and rapid immunoextraction are also discussed.     

Iron in granulocytes of nephrotic patients determined by Zeeman electrothermal atomic absorption spectrophotometry

One of the major threats to patients undergoing maintenance hemodialysis is an increased susceptibility to infections caused by microorganisms, among whichYersinia orListeria are frequently recovered. In patients receiving hemodialysis, iron overload owing to multiple transfusions plays an important role in the mechanisms underlying the susceptibility to bacterial infections, partially mediated by impaired neutrophil function. In order to assess the true role of iron at the cellular level, an AAS method was developed to measure the iron content of granulocytes. Iron levels in the granulocytes were determined in an apparently healthy population and in a population of iron-overloaded renal hemodialysis patients. Granulocytes were isolated by a method modified from Böyum. The analyses were performed using pyrocoated graphite tubes, and in one of the char steps, oxygen was used to facilitate ashing. The mean iron level found in the granulocytes from apparently normal persons was 4.07 fg/cell (n=17) with a CV of 44%; the mean value for the dialysis patient group was 4.59 fg/cell (n=8) with a CV of 37%. There was no significant difference between the two groups,p=0.70.     

Measurement of endogenous lithium levels in serum and urine by electrothermal atomic absorption spectrometry: a method with potential clinical applications

A highly sensitive flameless atomic absorption method has been adapted for the determination of endogenous trace lithium levels in serum and urine. With ammonium nitrate as the only matrix modifier, serum levels of Li as low as 0.03 mumol/liter are measured accurately and there is no requirement for standard additions. The need for background correction during analysis was clearly established, and tungsten and Zeeman-effect background corrections were compared. The tungsten correction offered superior sensitivity and linearity of standards. Recoveries in urine and serum average 94.8 +/- 7.7 and 95.3 +/- 6.1% (+/- SD), respectively. The endogenous serum Li levels were 0.16 +/- 0.08 mumol/liter for normal subjects dwelling in the Denver metropolitan area. The mean 24-h excretion rate was 5.24 +/- 1.4 mumol/day. The mean fractional excretion of endogenous Li (clearance Li/clearance creatinine) was 23.2 +/- 3.0%, a value similar to values published for exogenously administered Li and measured by conventional methods.     

Selenium measurement in human plasma with Zeeman effect electrothermal atomic absorption spectrometry: sample stability and calibration method

The dual aim of the present study is the investigation of the stability of plasma samples for selenium determination with time and temperature and the assessment of the calibration method. A comparative study is performed, using two calibration methods: standard addition to each sample and matrix matched curve. Our findings show that, in general, significant differences in the selenium content are observed when comparing the results obtained with these two calibration methods. Plasma samples stored at -20 degrees C are stable relative to the selenium content for a period of at least one year.     

Determination of zinc in tissues of normal and dystrophic mice using electrothermal atomic absorption spectrometry and slurry sampling

An electrothermal atomic absorption spectrometric procedure for zinc determination in animal tissues is first optimized and then used to measure the metal content of different tissues from normal and dystrophic mice. The procedure involves minimal manipulation of the sample to overcome the severe contamination problems normally associated with the measurement of low zinc levels. The samples (recommended amount 10 mg) are slurried in 2 ml of a 10-mM tetramethylammonium hydroxide solution containing 0.1 g/100 ml silicone antifoam. After mild heating at 60 degrees C for 10 min and homogenization for 1 min, the suspensions are submitted to a 10-fold dilution and then injected into the electrothermal atomizer. Calibration is carried out using aqueous standard solutions of zinc prepared in the same suspension media. The reliability of the whole procedure is verified using three certified reference materials.     

Survey of albumin purification methods for the analysis of albumin-organic toxicant adducts by liquid chromatography-electrospray ionization-tandem mass spectrometry

HSA has been shown to react with many organic toxicants to form adducts that are useful biomarkers for exposure. Albumin isolation is an important first step for the analysis of these protein-toxicant adducts. We tested several approaches to isolate albumin from serum treated with an electrophilic organic toxicant known to form adducts with albumin, i.e., sulfur mustard agent (HD) (2,2'-dichloroethyl sulfide), in order to evaluate these techniques as purification methods. To select the most efficient isolation strategy, methods were evaluated using gel electrophoresis, total protein quantitation, and peptide-adduct identification by MS. Results suggest that the albumin-rich fractions obtained can be used to identify exposure by quantitating the albumin adducts to electrophilic organic toxicants such as HD. The HiTrap Blue HP albumin isolation system appears to display the most promising results for purifying albumin to detect HD-adducts, exhibiting high purification efficiency, satisfactory albumin recovery, promising specificity, and a higher loading capacity for serum.     

A study of the distribution of aluminium in human placental tissues based on alkaline solubilization with determination by electrothermal atomic absorption spectrometry

Aluminium (Al) is a nonessential element known to induce neurotoxic effects, such as dialysis dementia, in patients on hemodialysis, with compromised kidney function. The role of Al in the progression of some neurodegenerative diseases, such as Alzheimer's disease (AD), is controversial, and remains unclear. The effects of Al on other vulnerable populations, such as fetuses and infants, have been infrequently studied. In the present study, Al has been measured in human placenta samples, comprising ～160 each of placenta bodies, placenta membranes, and umbilical cords, using electrothermal atomic absorption spectrometry (ETAAS) after atmospheric pressure digestion with tetramethylammonium hydroxide (TMAH) and ethylenediaminetetraacidic acid (EDTA). The sensitivity, or characteristic mass (m(0)), for Al at the 309.3-nm line was found to be 30 ± 4 pg. The instrumental detection limit (IDL) (3s) for Al in solution was calculated as 0.72 μg L(-1) while the method detection limit (MDL) (3s) was 0.25 μg g(-1). Accuracy was assessed through analysis of quality control (QC) materials, including certified reference materials (CRMs), in-house reference materials (RMs), and spike recovery experiments, of varying matrices. Placental tissue analyses revealed geometric mean concentrations of approximately 0.5 μg g(-1) Al in placenta bodies (n = 165) and membranes (n = 155), while Al concentrations in umbilical cords (n = 154) were about 0.3 μg g(-1). Al was detected in 95% of placenta bodies, and 81% of placenta membranes, but only in 46% of umbilical cords.     

Detection of new urinary exemestane metabolites by gas chromatography coupled to mass spectrometry

Exemestane is an aromatase enzyme complex inhibitor. Its metabolism in humans is not fully described and there is only one known metabolite: 17β-hydroxyexemestane. In this work, excretion studies were performed with four volunteers aiming at the detection of new exemestane metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) after enzymatic hydrolysis and liquid-liquid extraction. Urine samples collected from four volunteers were analyzed separately. The targets of the study were mainly the 6-exomethylene oxidized metabolites. Two unreported metabolites were identified in both free and glucuconjugated urine fractions from all four volunteers, both of them were the result of the 6-exomethylene moiety oxidation: 6ξ-hydroxy-6ξ-hydroxymethylandrosta-1,4-diene-3,17-dione (metabolite 1) and 6ξ-hydroxyandrosta-1,4-diene-3,17-dione (metabolite 2). Furthermore, only in glucoconjugated fractions from all volunteers, one metabolite arising from the A-ring reduction was identified as well, 3ξ-hydroxy-5ξ-androst-1-ene-6-methylene-17-one (metabolite 3). The molecular formulae of all these metabolites were ascertained by the determination of exact masses using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS). Moreover, all metabolites were confirmed using an alternative derivatization with methoxyamine and MSTFA/TMS-imidazole.     

Methods to measure the intracellular concentration of unlabeled compounds within cultured cells using liquid chromatography/tandem mass spectrometry

A high-throughput and sensitive liquid chromatography/tandem mass spectrometry assay was established to detect total unlabeled hepatitis C virus inhibitor concentrations in replicon cells. The intracellular concentrations determined by this assay correlated well with concentrations obtained using radiolabeled compound. Some compounds accumulated inside the cells, with concentrations up to 300-fold higher than the input concentration. Confocal microscopic evaluation of two fluorescent-tagged inhibitors confirmed high accumulation inside the cells, sequestered inside vesicles within the cytoplasm. Incubation of cells with compound at 4 °C revealed that nonspecific binding to the outside of the cell membrane and to the cell culture plate occurred for some compounds. Therefore, the total concentration of compound extracted at 37 °C was reduced by the amount that was nonspecifically bound at 4 °C to yield the amount of compound inside the cells. A modification of the protocol was used for compounds with low intracellular concentrations in which cells were harvested with trypsin-EDTA prior to extraction. This eliminated the nonspecific binding to the cell culture plate and decreased the overall background of the assay. This assay was used to understand differences in cellular potency between compounds and the effects of serum proteins on the metabolic stability of compounds during incubation with cells.     

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.     

Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry

With the growing interest for peptides and proteins in different kinds of fields, e.g. pharmacy, clinical diagnostics or food industry, the quantification of these compounds is becoming more and more important. Quantitative analysis of these analytes in biological matrices, however, remains a challenging task, due to the complexity of both the matrix and the analytical characteristics of these large bio-molecules. Liquid chromatography coupled to (tandem) mass spectrometry (LC-MS or LC-MS/MS) is the preferred analytical technique for peptide analysis as it allows very selective and sensitive measurements. This article summarizes the numerous published LC-MS applications for the quantification of peptides in biological matrices and discusses all different issues herewith concerned. This includes chromatographic aspects as the selection and effects of mobile and stationary phase, flow rate and temperature, as well as mass spectrometric characteristics such as ionization and detection modes, collision-induced dissociation of peptides and factors influencing the mass spectrometric response. For both techniques the main properties of all described methods have been listed, creating a comprehensive overview with the peptide analytes divided into different classes. Likewise, all other issues concerned with quantitative bioanalysis have been evaluated in detail, including extensive consideration of several different applied sample pre-treatment techniques and reflection of subjects as the choice for an internal standard and assay validation. Furthermore, several issues which are of particular interest for the quantitative bioanalysis of peptide compounds like peptide adsorption and degradation have been regarded.