Advances and applications of capillary electromigration methods in the analysis of therapeutic and diagnostic recombinant proteins - A Review.

This review provides a comprehensive overview of methodological advances and applications of CE in the analysis and characterization of recombinant therapeutic and diagnostic proteins over the past two decades. The first part of the review discusses various aspects of biotechnological protein production and the related effects on the final product. This covers upstream processes, e.g., selection and transfection of host cells, up-scaling of cell cultures and cultivation conditions, as well as downstream processing and a discussion of future trends in biotechnological manufacturing. This part is essential for relating biotechnological production to analytical challenges and requirements in order to provide a holistic insight. In this context, the influence of manufacturing steps on the quality of the final drug substance/product is discussed in terms of related post-translational modifications of the target molecule with a major focus on glycosylation pattern and conformational effects. Particular attention is given to host cell specific and non-human modifications affecting the efficacy and safety of recombinant products. Endowed with this propaedeutic knowledge, the major part of the review discusses the manifold contributions of different CE techniques to the development and optimization of the manufacturing process, to the evaluation and characterization of the final drug product and their role in quality control. Different CE techniques, such as CZE, capillary gel electrophoresis (CGE), (imaged) capillary isoelectric focusing ((i)CIEF), µChipCE, CE-Western blot, affinity CE (ACE), and CE-MS are discussed including a brief introduction in the respective separation and hyphenation principle as well as their applications in the analysis of different recombinant biologics together with recent strategies. The addressed analyte portfolio comprises a vast variety of recombinant proteins with molecular masses from 4.1 kDa up to 20.3 MDa (for recombinant virus-like particles), and a pI range from 2.0 to 11.2. Antibodies are not explicitly covered in the survey. The review is complemented by compiling validation aspects and proposed suitability tests in order to assure the feasibility of methods to industrial and pharmaceutical needs.

Bottom-Up Analysis of Proteins by Peptide Mass Fingerprinting with tCITP-CZE-ESI-TOF MS After Tryptic Digest.

The identification of proteins in samples of moderate to complex composition is primarily done by bottom-up approaches. Therefore, proteins are enzymatically digested, mostly by trypsin, and the resulting peptides are then separated prior to their transfer to a mass spectrometer. The following protocol portrays a bottom-up method, which was optimized for the application of CZE-ESI-TOF MS. Protein denaturation is achieved by addition of 2,2,2-trifluoroethanol (TFE) and heat treatment. Afterwards, disulfide bonds are reduced with tris-(2-carboxyethyl)phosphine (TCEP) and subsequently alkylated with iodoacetamide (IAA). The tryptic digest is performed in an ammonium bicarbonate buffer at pH 8.0. The digested protein sample is then concentrated in-capillary by transient capillary isotachophoresis (tCITP) with subsequent CZE separation of tryptic peptides in an acidic background electrolyte. Hyphenation to a time-of-flight (TOF) mass spectrometer is carried out by a triple-tube coaxial sheath flow interface, which uses electrospray ionization (ESI). Peptide identification is done by peptide mass fingerprinting (PMF). The protocol is outlined exemplarily for a model protein, i.e., bovine ß-lactoglobulin A.

Intact and middle-down CIEF of commercial therapeutic monoclonal antibody products under non-denaturing conditions.

A two-step CIEF with chemical mobilization was developed for charge profiling of the therapeutic mAb rituximab under non-denaturing separation conditions. CIEF of the intact mAb was combined with a middle-down approach analyzing Fc/2 and F(ab´)2 fragments after digest with a commercial cysteine protease (IdeS). CIEF methods were optimized separately for the intact mAb and its fragments due to their divergent pIs. Best resolution was achieved by combining Pharmalyte (PL) 8-10.5 with PL 3-10 for variants of intact rituximab and of F(ab´)2 fragments, respectively, whereas PL 6.7-7.7 in combination with PL 3-10 was used for Fc/2 variants. Charge heterogeneity in Fc/2 dominates over F(ab´)2 . In addition, a copy product of rituximab, and adalimumab were analyzed. Both mAbs contain additional alkaline C-terminal lysine variants as confirmed by digest with carboxypeptidase B. The optimized CIEF methods for intact mAb and Fc/2 were tested for their potential as platform approaches for these mAbs. The CIEF method for Fc/2 was slightly adapted in this process. The pI values for major intact mAb variants were determined by adjacent pI markers resulting in 9.29 (rituximab) and 8.42 (adalimumab). In total, seven to eight charge variants could be distinguished for intact adalimumab and rituximab, respectively.

Boiling down the cysteine-stabilized LTP fold - loss of structural and immunological integrity of allergenic Art v 3 and Pru p 3 as a consequence of irreversible lanthionine formation.

BACKGROUND: Non-specific lipid transfer proteins (LTPs) are important allergens in fruits, pollen, vegetables, nuts and latex. Due to their compact structure, LTPs are highly resistant to heat treatment. Here, Art v 3 from mugwort pollen and Pru p 3 from peach were used as model allergens to in-depth investigate structural and immunological properties upon thermal treatment at different buffer conditions. METHODS: Recombinant Art v 3 and Pru p 3 were purified from E. coli and incubated at 95 °C up to 120 min using sodium phosphate buffer pH 3.4 or 7.3. Physicochemical properties of allergens were analyzed in circular dichroism spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, size exclusion chromatography, and mass spectrometry. The crystal structure of Art v 3.0201 was determined to 1.9 Šresolution. IgG and IgE binding was investigated in ELISA using murine and LTP allergic patients' sera. RESULTS: Highly pure and homogenous recombinant allergens were obtained from bacterial production. The crystal structure of Art v 3.0201 revealed an antiparallel four helix bundle with a C-terminal extension mediating an asymmetric, transient dimer interface and differently sized cavities. Both allergens showed high thermal stability at acidic conditions. In contrast, extensive heat treatment in neutral buffer induced irreversible structural changes due to lanthionine-based cysteine rearrangement. This fostered loss of the typical α-helical structure, increased molecular size and abrogation of IgG and IgE binding epitopes. Pru p 3 lost its structural integrity at shorter heat stress duration than Art v 3, which did however only partially affect the molecule's IgE binding epitopes. CONCLUSION: During thermal treatment, susceptibility to structural changes of the LTP-fold is highly dependent on the surrounding environment but also on intrinsic features of individual LTPs. This is a crucial fact to consider when processing LTP-containing food or food products as this will directly influence their allergenic potential.

Monitoring of Deamidation and Lanthionine Formation in Recombinant Mugwort Allergen by Capillary Zone Electrophoresis (CZE)-UV and Transient Capillary Isotachophoresis-CZE-Electrospray Ionization-TOF-MS.

The response to thermal stress is an important parameter relevant for characterizing the biological activity and long-term stability of recombinant proteins, which may show irreversible, pH dependent structural changes under these conditions. We selected the recombinant pollen allergen of mugwort ( Artemisia vulgaris) rArt v 3.0201 as a relevant model to study structural changes due to thermal and pH stress by means of capillary zone electrophoresis (CZE)-UV and capillary zone electrophoresis (CZE)-electrospray ionization (ESI)-TOF-MS. Therefore, this recombinant protein was exposed to 95 °C under acidic (pH 3.4) and slightly alkaline (pH 7.3) conditions for up to 120 min. CZE-UV data showed a continuous degradation of the allergen accompanied by the gradual formation of several reaction products. Characterization of novel allergen variants occurring at longer migration times was done via CZE-ESI-TOF-MS using in-capillary transient capillary isotachophoresis (tCITP) preconcentration to facilitate the identification of minor variants. MS data revealed various modifications of rArt v 3.0201 in response to heating. Variants with deamidations and sulfur-related modifications including both yield and loss of sulfur were identified at increased migration times. Desulfurization produced allergen variants with up to four lanthionines that replaced initial disulfide bonds. In addition, mass spectra revealed shifts in the charge state distribution which indicate concomitant conformational alterations. Moreover, several low-abundant oxidized variants were identified. With extended thermal stress, the portfolio of variants increased and progressively shifted toward rArt v 3.0201 with high lanthionine content. The kinetics of conversion and the complexity of variant composition were pH dependent and increased under alkaline conditions.

Analytical Cascades of Enzymes for Sensitive Detection of Structural Variations in Protein Samples.

Protein function critically depends on structure. However, current analytical tools to monitor consistent higher-order structure with high sensitivity, as for instance required in the development of biopharmaceuticals, are limited. To complement existing assays, we present the analytical cascade of enzymes (ACE), a method based on enzymatic modifications of target proteins, which serve to exponentially amplify structural differences between them. The method enables conformational and chemical fingerprinting of closely related proteins, allowing for the sensitive detection of heterogeneities in protein preparations with high precision. Using this method, we detect protein variants differing in conformation only, as well as structural changes induced by diverse covalent modifications. Additionally, we employ this method to identify the nature of structural variants. Moreover, the ACE method should help to address the limited reproducibility in fundamental research, which partly relates to sample heterogeneities.

Top-down and bottom-up characterization of nitrated birch pollen allergen Bet v 1a with CZE hyphenated to an Orbitrap mass spectrometer.

Tyrosine (Tyr) residues of the major pollen allergen of birch Betula verrucosa, Bet v 1a, were nitrated by peroxynitrite. This modification enhances the allergenicity. Modified tyrosines were identified by analyzing intact allergen variants in combination with top-down and bottom-up approaches. Therefore, a laboratory-built sheath-liquid assisted ESI interface was applied for hyphenation of CE to an Orbitrap mass spectrometer to localize individual nitration sites. The major focus was on identification of primary nitration sites. The top-down approach unambiguously identified Tyr 5 as the most prominent modification site. Fragments from the allergen core and the C-terminal part carried up to three potential nitration sites, respectively. Thus, a bottom-up approach with tryptic digest was used as a complementary strategy which allowed for the unambiguous localization of nitration sites within the respective peptides. Nitration propensity for individual Tyr residues was addressed by comparison of MS signals of nitrated peptides relative to all cognates of homolog primary sequence. Combined data identified surface exposed Tyr 5 and Tyr 66 as major nitration sites followed by less accessible Tyr 158 whereas Tyr 81, 83 and 150 possess a lower nitration tendency and are apparently modified in variants with higher nitration levels.

Effects of ß-Carotene and Its Cleavage Products in Primary Pneumocyte Type II Cells.

ß-Carotene has been shown to increase the risk of developing lung cancer in smokers and asbestos workers in two large scale trails, the Beta-Carotene and Retinol Efficacy Trial (CARET) and the Alpha-Tocopherol Beta-carotene Cancer Prevention Trial (ATBC). Based on this observation, it was proposed that genotoxic oxidative breakdown products may cause this effect. In support of this assumption, increased levels of sister chromatid exchanges, micronuclei, and chromosomal aberrations were found in primary hepatocyte cultures treated with a mixture of cleavage products (CPs) and the major product apo-8'carotenal. However, because these findings cannot directly be transferred to the lung due to the exceptional biotransformation capacity of the liver, potential genotoxic and cytotoxic effects of ß-carotene under oxidative stress and its CPs were investigated in primary pneumocyte type II cells. The results indicate that increased concentrations of ß-carotene in the presence of the redox cycling quinone dimethoxynaphthoquinone (DMNQ) exhibit a cytotoxic potential, as evidenced by an increase of apoptotic cells and loss of cell density at concentrations > 10 µM. On the other hand, the analysis of micronucleated cells gave no clear picture due to the cytotoxicity related reduction of mitotic cells. Last, although CPs induced significant levels of DNA strand breaks even at concentrations ≥ 1 µM and 5 µM, respectively, ß-carotene in the presence of DMNQ did not cause DNA damage. Instead, ß-carotene appeared to act as an antioxidant. These findings are in contrast with what was demonstrated for primary hepatocytes and may reflect different sensitivities to and different metabolism of ß-carotene in the two cell types.

Advanced portrayal of SMIL coating by allying CZE performance with in-capillary topographic and charge-related surface characterization.

A successive multiple ionic polymer layer (SMIL) coating composed of four layers improved the capillary electrophoretic separation of a recombinant major birch pollen allergen and closely related variants when poly(acrylamide-co-2-acrylamido-2-methyl-1-propansulfonate) (55% PAMAMPS) replaced dextran sulfate as terminal SMIL layer. 55% PAMAMPS decelerated the electroosmotic flow (EOF) due to its lower charge density. Atomic force microscopy (AFM) was used to investigate SMIL properties directly on the inner capillary surface and to relate them to EOF measurements and results of associated CZE separations of a mixture of model proteins and peptides that were performed in the same capillary. For the first time, AFM-based biosensing topography and recognition imaging mode (TREC) under liquid conditions was applied for a sequential characterization of the inner surface of a SMIL coated capillary after selected treatments including pristine SMIL, SMIL after contact with the model mixture, after alkaline rinsing, and the replenishment of the terminal polyelectrolyte layer. A cantilever with tip-tethered avidin was used to determine the charge homogeneity of the SMIL surface in the TREC mode. SMIL coated rectangular capillaries with 100 µm internal diameter assured accessibility of the inner surface for this cantilever type. Observed changes in CZE performance and EOF mobility during capillary treatment were also reflected by alterations in surface roughness and charge distribution of the SMIL coating. A renewal of the terminal SMIL layer restored the original surface properties of SMIL and the separation performance. The alliance of the novel TREC approach and CZE results allows for an improved understanding and a comprehensive insight in effects occurring on capillary coatings.

Mapping molecular adhesion sites inside SMIL coated capillaries using atomic force microscopy recognition imaging.

Capillary zone electrophoresis (CZE) is a powerful analytical technique for fast and efficient separation of different analytes ranging from small inorganic ions to large proteins. However electrophoretic resolution significantly depends on the coating of the inner capillary surface. High technical efforts like Successive Multiple Ionic Polymer Layer (SMIL) generation have been taken to develop stable coatings with switchable surface charges fulfilling the requirements needed for optimal separation. Although the performance can be easily proven in normalized test runs, characterization of the coating itself remains challenging. Atomic force microscopy (AFM) allows for topographical investigation of biological and analytical relevant surfaces with nanometer resolution and yields information about the surface roughness and homogeneity. Upgrading the scanning tip to a molecular biosensor by adhesive molecules (like partly inverted charged molecules) allows for performing topography and recognition imaging (TREC). As a result, simultaneously acquired sample topography and adhesion maps can be recorded. We optimized this technique for electrophoresis capillaries and investigated the charge distribution of differently composed and treated SMIL coatings. By using the positively charged protein avidin as a single molecule sensor, we compared these SMIL coatings with respect to negative charges, resulting in adhesion maps with nanometer resolution. The capability of TREC as a functional investigation technique at the nanoscale was successfully demonstrated.

Nitration of ß-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice.

BACKGROUND: We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins. OBJECTIVE: The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy. DESIGN: BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen ß-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood. RESULTS: A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation. CONCLUSION: Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.

Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants.

One pressure and three chemical mobilization strategies have been optimized and tested for two-step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1-10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3-10 with Pharmalyte 5-6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5-6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. l-Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. l-Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed.

Reversible biofunctionalization of surfaces with a switchable mutant of avidin.

Label-free biosensors detect binding of prey molecules (″analytes″) to immobile bait molecules on the sensing surface. Numerous methods are available for immobilization of bait molecules. A convenient option is binding of biotinylated bait molecules to streptavidin-functionalized surfaces, or to biotinylated surfaces via biotin-avidin-biotin bridges. The goal of this study was to find a rapid method for reversible immobilization of biotinylated bait molecules on biotinylated sensor chips. The task was to establish a biotin-avidin-biotin bridge which was easily cleaved when desired, yet perfectly stable under a wide range of measurement conditions. The problem was solved with the avidin mutant M96H which contains extra histidine residues at the subunit-subunit interfaces. This mutant was bound to a mixed self-assembled monolayer (SAM) containing biotin residues on 20% of the oligo(ethylene glycol)-terminated SAM components. Various biotinylated bait molecules were bound on top of the immobilized avidin mutant. The biotin-avidin-biotin bridge was stable at pH ≥3, and it was insensitive to sodium dodecyl sulfate (SDS) at neutral pH. Only the combination of citric acid (2.5%, pH 2) and SDS (0.25%) caused instantaneous cleavage of the biotin-avidin-biotin bridge. As a consequence, the biotinylated bait molecules could be immobilized and removed as often as desired, the only limit being the time span for reproducible chip function when kept in buffer (2-3 weeks at 25 °C). As expected, the high isolectric pH (pI) of the avidin mutant caused nonspecific adsorption of proteins. This problem was solved by acetylation of avidin (to pI < 5), or by optimization of SAM formation and passivation with biotin-BSA and BSA.

Separation and characterization of nitrated variants of the major birch pollen allergen by CZE-ESI-µTOF MS.

A CZE-ESI-TOF MS method has been optimized for the separation and identification of nitrated variants of the major birch pollen allergen from Betula verrucosa, isoform 1a (Bet v 1a). In-house nitration of recombinant Bet v 1a was done by peroxynitrite. As a BGE, 10 mmol/L ammonium bicarbonate with pH 7.50 provided best resolution. Nebulizer gas pressure and sheath liquid flow rate of 0.4 bar and 6 µL/min, respectively, maintained CZE selectivity and constituted stable electrospray conditions. A sheath liquid composition of 75% v/v methanol with 0.1% v/v formic acid in ultrapure water resulted in highest signal intensities. Alternatively, methanol could be replaced by 50% v/v isopropanol. Two modified allergen products derived from reaction mixtures that contained different amounts of the nitration reagent were compared by the elaborated CZE-ESI-TOF MS method. Up to twelve different Bet v 1a variants with one- to sixfold nitration could be distinguished. Several allergen fractions of equivalent nitration grade were resolved. Their different migration times indicate site-specific nitration with concomitant differences in pI and maybe also in hydrodynamic radius. The method allows for a characterization of in-house nitrated allergen samples that are intended for testing the postulated enhanced allergenicity of nitrated Bet v 1a variants.

Beta-adrenergic stimulation suppresses phagocytosis via Epac activation in murine microglial cells.

Endogenous noradrenaline presumably prohibits neuroinflammation by stimulation of ß-adrenergic receptor-dependent suppression of the production of inflammatory mediators. Using the microglial cell line, BV-2, as well as primary murine microglial cells, we show here that the ß-adrenergic agonist, isoproterenol, suppresses uptake of hydrophobic polystyrene microspheres. The number of cells showing a specific number of engulfed microspheres followed a Poisson distribution. Isoproterenol decreased the number of engulfed particles per cell and the number of cells showing at least one incorporated particle. Elevation of intracellular cAMP by activation of adenylyl cyclase activity with forskolin, suppression of phosphodiesterase activity with 3-isobutyl-1-methylxanthine (IBMX), or application of the membrane-permeable cAMP analog, 8-bromo-cAMP, suppressed particle uptake. The protein kinase A inhibitor, H-89, did not prevent isoproterenol-dependent suppression of particle engulfment. However, activation of exchange protein activated by cAMP (Epac), specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, with the Epac1-specific cAMP analog, 8-pCPT-2'-O-Me-cAMP, mimicked the suppressive effect of isoproterenol on particle uptake. Our results suggest that ß-adrenergic receptor stimulation suppresses particle uptake in microglia by cAMP-dependent activation of Epac.

Successive multiple ionic polymer layer coated capillaries in the separation of proteins - recombinant allergen variants as a case study.

A successive multiple ionic polymer layer (SMIL) coating consisting of two pairs of poly(diallyldimethylammonium chloride) and dextran sulfate (DS) layers was applied for the separation of recombinant products of the major birch pollen allergen Betula verrucosa (Bet v 1a). The combination with volatile ammonium bicarbonate buffer at pH 6.70 offers the possibility for future MS hyphenation. The negative net charge of allergens required DS as terminal SMIL layer. The EOF was accelerated from 3.17x10(-8) m(2) V(-1) s(-1) in uncoated to 4.52x10(-8) m(2) V(-1) s(-1) in SMIL capillaries. Fresh prepared SMIL capillaries showed slight EOF acceleration due to gradual re-organization of SMIL structure until stabilization was achieved. Dry storage of SMIL capillaries prevented fluctuations in EOF and migration times and improved coating durability. However, the gradual reconstitution of entangled SMIL layers affected efficiency, but was cured by a 10 mmol/L NaOH rinsing step. Durability of SMIL capillaries in MS-applicable dimension was confirmed for > 70 runs and in total 42 h of voltage application with average intra-day precision of 0.22 and 0.79% and inter-day-precision of 0.91 and 1.17% for migration times of EOF and Bet v 1a, respectively. Final SMIL coating allowed for the separation of Bet v 1a, a hypoallergenic isoform and carbamylated variants with 150,000-685,000 plates.

Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening.

BACKGROUND: The incidence of allergic diseases in developed countries has been increasing constantly in the last 2 decades. The only curative treatment available thus far is specific immunotherapy (SIT). The problematic side effects that can occur during SIT with allergen extracts led to the search for safer alternatives, such as recombinant hypoallergenic proteins with reduced allergenic potential and preserved T-cell immunogenicity. OBJECTIVE: We created hypoallergenic variants of the allergens Bet v 1a and Phl p 5b by using in silico mutation and screening and characterized the biochemical and immunologic properties of selected mutant proteins. METHODS: Knowledge-based potentials were used to estimate structural changes of the protein structures under sequence variation. IgE antibodies and their cross-linking capacity were determined by using a basophil release assay, binding of human birch pollen-specific IgE was investigated by means of ELISA, and ELISPOT assays were performed to examine the T-cell immunogenicity. RESULTS: Selected mutated proteins showed significantly reduced IgE-binding and cross-linking ability but retained their T cell-stimulating properties. Immunization with the hypoallergenic mutants induced blocking antibodies against murine and human IgE epitopes. CONCLUSION: In silico calculation and selection of mutations that render a protein hypoallergenic represents a novel and rapid tool to create candidate molecules for SIT with recombinant allergens.

Confirmation of immuno-reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity-CIEF.

Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample.

Protein attachment onto silica surfaces--a survey of molecular fundamentals, resulting effects and novel preventive strategies in CE.

This review addresses the fundamentals governing the adsorption of individual protein molecules onto the surface of fused-silica capillaries, the protein aggregation to adsorbate clusters and their final accretion to monolayers with subsequent stratification to protein multilayers. The attention in CE protein separation has primarily been focused on (i) tuning the BGE including the buffer type, ionic strength, pH and additives, (ii) tailored post-rinse procedures to detach adhered protein residues and (iii) the optimization of capillary wall shielding in order to reduce protein attachment. Improvements in protein separation as well as related adverse effects are mainly discussed on the basis of parameters known to become deteriorated in case of protein adhesion, e.g. repeatability of the EOF and of migration times, peak width, theoretical plate numbers, resolution and asymmetry factor. However, knowledge of the molecular principles controlling protein adsorption onto silica surfaces is indispensable for separation optimization. Furthermore, it facilitates troubleshooting and the interpretation of undesired concomitant phenomena. This review comprehensively discusses protein adsorption models derived from surface chemistry primarily in terms of their relevance for CE, clearly showing that the adsorption process in its complexity is only partially revealed by models, which address single or binary protein solutions. In a further section theoretical concepts and surface models are related to surface phenomena encountered in CE. The final part of the review surveys recent concepts for prevention of protein adhesion, thereby addressing capillary treatment, favorable buffer types, dynamic and adhesive semi-permanent coating strategies covering the literature from 2000-2008.

Validation of capillary zone electrophoresis and capillary isoelectric focusing separations optimized for the characterization of two recombinant products of the birch pollen allergen Bet v 1a.

CZE and CIEF separation systems, both developed previously for a quality control of two recombinant products of the major birch pollen allergen Bet v 1a of Betula verrucosa, were validated including aspects of the International Conference on Harmonization. One product contained carbamylated variants as impurities. Linearity of response was confirmed by Mandel's fitting test between 0.028 and 1.90 mg/mL for CZE and between 0.016 and 0.26 mg/mL for CIEF. Repeatability and intermediate precision were evaluated for the effective mobility (mu(eff)) in CZE, for relative mobilization time in CIEF and the peak area ratio of Bet v 1a. LOQ for Bet v 1a was between 10 and 23 microg/mL for both methods. Evaluation of robustness for CZE revealed susceptibility of micro(eff) of Bet v 1a to alterations in of buffer pH and separation temperature. Selectivity was impaired by an increase in temperature, pH, and buffer concentration. In addition, pH variations influenced the separation profile of impurities. For CIEF, the ratio of narrow pH range carrier ampholytes is the critical parameter to retain robustness. Results demonstrate the suitability of both separation systems to discriminate between nonmodified Bet v 1a and carbamylated variants in the selected recombinant allergen products.