Quantitative microcapillary electrophoresis immunoassay (mCE IA) for end-to-end analysis of pertactin within in-process samples and Quadracel® vaccine.

Protein concentration is an important attribute in the production of subunit or component-based vaccine antigens. Rigorous monitoring of protein concentration is required to identify potential areas for yield improvement. The current GMP method for quantitation is the plate-based ELISA which requires numerous hands-on steps and has low sensitivity in comparison to new microfluidic systems. To address this issue, a sensitive automated microCapillary Electrophoresis ImmunoAssay (mCE IA) method was developed to accurately separate and quantitate pertactin (PRN), an important antigen of the modern acellular Pertussis (aP) vaccine. PRN is reported to be a low-yielding antigen; thus, it is critical to observe its concentration throughout its manufacturing process. First, a primary antibody for PRN was identified to establish suitable immunoprobing conditions for detection of PRN over a wide linear dynamic range that spans 3 orders of magnitude. Next, the pre-adsorbed PRN Drug Substance (DS) was used as a reference standard to quantitate PRN samples against a calibration curve with adequate accuracy and precision. Four representative samples including three in-process steps and final adjuvanted drug product: Quadracel®, were examined to demonstrate the capability of mCE IA to quantitate PRN with high sensitivity and specificity. The matrices of the selected samples contain additional components (e.g. other proteins, growth factors, cell culture media, residual ammonium sulfate, and aluminum adjuvant) often making the quantitation of PRN challenging. The specificity and method linearity were demonstrated by spiking pre-adsorbed PRN DS into the four representative samples. In addition, it was shown that reportable concentrations of PRN for nine downstream process steps as analyzed by our method is comparable to concentrations obtained with ELISA. Most importantly, this study demonstrated that our method's quantitative accuracy is independent of matrix components, as each sample undergoes extensive dilution. This allows for seamless end-to-end analysis of PRN from fermenter harvest, through to complex downstream process samples to adjuvanted drug products. Finally, for the first time the developed and qualified mCE IA method was shown to quantify PRN throughout the entire manufacturing process to provide rapid feedback for process optimizations allowing for accurate yield and step-loss calculations.

High-precision quantitation of a tuberculosis vaccine antigen with capillary-gel electrophoresis using an injection standard.

Analysis of proteinogenic vaccine antigens in a quality control environment requires an accurate, precise, and reliable method for protein separation and quantitation. While having multiple advantages over the classical SDS-PAGE, capillary gel electrophoresis (CGE) has not yet become a standard tool in vaccine antigen analysis. Here we report on development of a CGE-based method for quantitative analysis of a tuberculosis vaccine fusion antigen protein, H4, currently in clinical trials. We demonstrate that our method can monitor antigen purity and relative quantity with greater precision and accuracy versus SDS-PAGE. In addition, due to use of direct light-absorbance detection, the CGE method is suitable for absolute quantitation, an application for which SDS-PAGE is limited due to the need for staining and limited dynamic range of detection. To further improve the performance of our quantitation method, we introduced Bovine Serum Albumin (BSA) as an injection standard to correct for signal variance associated with the injected sample volume. We found that, for our specific application, BSA was more appropriate as an injection standard versus one provided in a commercial kit, in terms of precision and accuracy for quantitation of H4. In addition to providing better method performance versus SDS-PAGE, CGE is also faster and less resource-intensive. We conclude that CGE should be considered as a replacement for traditional SDS-PAGE methods for vaccine antigen quantitation in a quality-control environment.

Electrochemical investigations of Tau protein phosphorylations and interactions with Pin1.

Phosphorylation of Tau by the protein kinase GSK-3ß was monitored by electrochemical impedance spectroscopy of immobilized Tau on gold surfaces. As a result of Tau phosphorylation, the film resistance decreases significantly due to conformational changes and reorganization of the immobilized phosphorylated Tau (pTau) protein, which in turn enables the interactions of pTau with the peptidyl-prolyl cis/trans isomerase, Pin1. Interactions are specific to phospho-Ser (pSer) and phospho-Thr (pThr) residues of pTau. Impedance changes occurred as a function of pTau-Pin1 interactions and are related to the amount of Pin1 bound, which resulted in an increase of the charge-transfer resistance, R(CT). Our results clearly indicate that the isomerase Pin1 interacts favorably with pSer/pThr-Pro residues in Tau, but does not bind non-phosphorylated Tau or phospho-Tyr residues in Tau films. Our study demonstrates the utility of electrochemical impedance studies to probe protein modifications and biomolecular interactions.

Hierarchical organization of ferrocene-peptides.

Hierarchical self-assembly of disubstituted ferrocene (Fc)-peptide conjugates that possess Gly-Val-Phe and Gly-Val-Phe-Phe peptide substituents leads to the formation of nano- and micro-sized assemblies. Hydrogen-bonding and hydrophobic interactions provide directionality to the assembly patterns. The self-assembling behavior of these compounds was studied in solution by using (1)H NMR and circular dichroism (CD) spectroscopies. In the solid state, attenuated total reflectance (ATR) FTIR spectroscopy, single-crystal X-ray diffraction (XRD), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM) methods were used. Spontaneous self-assembly of Fc-peptides through intra- and intermolecular hydrogen-bonding interactions induces supramolecular assemblies, which further associate and give rise to fibers, large fibrous crystals, and twisted ropes. In the case of Fc[CO-Gly-Val-Phe-OMe](2) (1), molecules initially interact to form pleated sheets that undergo association into long fibers that form bundles and rectangular crystalline cuboids. Molecular offsets and defects, such as screw dislocations and solvent effects that occur during crystal growth, induce the formation of helical arrangements, ultimately leading to large twisted ropes. By contrast, the Fc-tetrapeptide conjugate Fc[CO-Gly-Val-Phe-Phe-OMe](2) (2) forms a network of nanofibers at the supramolecular level, presumably due to the additional hydrogen-bonding and hydrophobic interactions that stem from the additional Phe residues.

Electrochemical investigations into Tau protein phosphorylations.

Hyperphosphorylation of Tau, a protein that stabilizes microtubules, leads to the breakdown of the microtubular structure and ultimately to the formation of neurofibrillar tangles within neurons. Here, we report monitoring of Tau phosphorylations electrochemically, using Tau protein films chemically linked to gold surfaces and 5'-γ-ferrocenyl (Fc) adenosine triphosphate (Fc-ATP) as a co-substrate. Fc-phosphorylation reactions of Tau are explored using the three protein kinases, glycogen synthase kinase (GSK-3ß), sarcoma (Src)-related kinase, and protein kinase A (PKA), which catalyze Fc-phosphorylation of different residues and regions within Tau. The kinetic parameters of the biochemical process (K(M) and V(max)) were determined.

Study of amyloid ß-peptide (Aß12-28-Cys) interactions with Congo red and ß-sheet breaker peptides using electrochemical impedance spectroscopy.

A surface-based approach is presented to study the interactions of Aß12-28-Cys assembled on gold surfaces with Congo red (CR) and a ß-sheet breaker (BSB) peptide. The various aspects of the peptide film have been examined using different electrochemical and surface analytical techniques. Cyclic voltammetry and electrochemical impedance spectroscopy (EIS) results using redox probes [Fe(CN)(6)](3-/4-) show that Aß12-28-Cys on gold forms a stable and reproducible blocking film. EIS analysis shows that CR and BSB have different effects on the electrochemical properties of Aß12-28-Cys films, presumably due to changes in the interactions between the film and CR and BSB. EIS results indicate that in the case of CR film resistance decreases significantly presumably due to better penetration of the solution-based redox probe into the film, whereas in the case of BSB, the film resistance increases. We interpret this difference to BSB being able to interact with the Aß12-28-Cys on the surface and presumably forming a film that presents a higher resistance for electron transfer from the redox probe to the solution.

Electrochemical "signal-on" reporter for amyloid aggregates.

The synthesis and characterization of four new Ferrocene (Fc) bioconjugates, bearing a podant (Lys)-Leu-Val-Phe-Phe motif, namely the hydrophobic sequence of amyloid-ß-peptides (Aß), is reported. The Fc-peptide conjugates are characterized by a reversible redox activity and the ability to undergo hydrophobic and hydrogen bonding interactions. Biomolecular interactions between Fc-bioconjugates with Aß(12-28) fragments were studied by circular dichroism (CD), transmission electron microscopy (TEM), and electrochemistry. All four Fc-peptides interacted favourable with Aß(12-28) and prevented fibril formation, the extent of which depended on the length of the peptide and the nature of the C-terminal group. The aggregates obtained for the Aß(12-28)/Fc-peptide mixtures range from short fibrils to spherical aggregates. We demonstrated that in solution the peptide sequence and peptide charge affect the biomolecular interactions. Fc-peptide interactions with immobilized Aß(12-28)-Cys films on Au surfaces were detected by measuring the current response of the Fc redox process. The formal redox potential, E(0), at ~440 (10) mV and i(pc)/i(pa) at 0.9 were observed characteristic for the monosubstituted Fc-derivative undergoing a one-electron redox process. On the surface, methyl ester-protected Fc-peptides (1 and 3) interacted only weakly with Aß(12-28)-Cys films, giving rise to minimal redox activity. In contrast, charged Fc-peptides (2 and 4) gave a significant electrochemical readout following the interaction with Aß(12-28)-Cys films. Interestingly, the Fc-peptide charge dictates the surface-assisted interactions, while hydrophobic and ionic effects contribute to the overall solution behaviour of the Fc-bioconjugates with Aß(12-28).

Synthesis, experimental and theoretical characterization of tetra dentate N,N'-dipyridoxyl (1,3-propylenediamine) salen ligand and its Co(III) complex.

The new tetra dentate dianionic H2PS (N,N'-dipyridoxyl (1,3-propylenediamine)) Schiff-base ligand and its octahedral Co(III) salen complex [Co(PS)(H2O)(CH3OH)]+CH3COO(-) were synthesized, where coordinating atoms of H2PS (N,N,O(-),O(-)) occupied equatorial positions with H2O and CH3OH as axial ligands. The nature of the H2PS and its complex were determined by elemental and spectrochemical (IR, UV-vis, 1H NMR and Mass) analysis. Also, the fully optimized geometries and vibrational frequencies of them together with the 1H NMR chemical shifts of H2PS have been calculated using density functional theory (B3LYP) method. Obtained structural parameters are in good agreement with the experimental data reported for similar compounds. The calculated and experimental results confirmed the suggested structures for the ligand and complex.