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.

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.

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.

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.

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.

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.

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.