Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis.

The space of advanced therapeutic modalities is currently evolving in rapid pace necessitating continuous improvement of analytical quality control methods. In order to evaluate the identity of nucleic acid species in gene therapy products, we propose a capillary electrophoresis-based gel free hybridization assay in which fluorescently labeled peptide nucleic acids (PNAs) are applied as affinity probes. PNAs are engineered organic polymers that share the base pairing properties with DNA and RNA but have an uncharged peptide backbone. In the present study, we conduct various proof-of-concept studies to identify the potential of PNA probes for advanced analytical characterization of novel therapeutic modalities like oligonucleotides, plasmids, mRNA, and DNA released by recombinant adeno-associated virus. For single-stranded nucleic acids up to 1000 nucleotides, the method is an excellent choice that proved to be highly specific by detecting DNA traces in complex samples, while having a limit of quantification in the picomolar range when multiple probes are used. For double-stranded samples, only fragments that are similar in size to the probe could be quantified. This limitation can be circumvented when target DNA is digested and multiple probes are used opening an alternative to quantitative PCR.

Stronger together: Analytical techniques for recombinant adeno associated virus.

With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC). Here, we evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two modes of capillary electrophoresis (CE) for their ability to analyze the DNA encapsidated by rAAVs. While TEM and AUC are well-established methods for rAAV, capillary gel electrophoresis (CGE) has been just recently proposed for viral genome sizing. The data presented reflect that samples are very complex, with various DNA species incorporated in the virus, including small fragments as well as DNA that is larger than the targeted transgene. CGE provides a good insight in the filling of rAAVs, but the workflow is tedious and the method is not applicable for the determination of DNA titer, since a procedure for the absolute quantification (e.g., calibration) is not yet established. For estimating the genome titer, we propose a simplified capillary zone electrophoresis approach with minimal sample preparation and short separation times (<5 min/run). Our data show the benefits of using the four techniques combined, since each of them alone is prone to delivering ambiguous results. For this reason, a clear view of the rAAV interior can only be provided by using several analytical methods simultaneously.

Methionine oxidation of proteins analyzed by affinity capillary electrophoresis in presence of silver(I) and gold(III) ions.

Oxidative damage of biopharmaceuticals during manufacturing and storage is a key concern throughout pharmaceutical development. However, few simple and robust analytical methods are available for the determination of oxidation sites. Here, the potential of affinity capillary electrophoresis (ACE) in the separation of proteins with oxidized methionine (Met) residues is shown. Silver(I) and gold(I) ions have the attribute to selectively form complexes with thioethers over sulfoxides. The addition of these ions to the BGE leads to a selective complexation of Met residues and, thus, to a change of charge allowing separation of species according to the different oxidation states of Met. The mechanisms of these interactions are discussed and binding constants for peptides containing Met with silver(I) are calculated. Additionally, the proposed method can be used as an indicator of oxidative stress in large proteins. The presented technique is easily accessible, economical, and has rapid analysis times, adding new approaches to the analytical toolbox of Met sulfoxide detection.

Capillary gel electrophoresis of therapeutic oligonucleotides--analysis of single- and double-stranded forms.

Recently, several therapeutic double-stranded (ds) oligonucleotides (ODNs) are in pharmaceutical development. During quality control, these therapeutic molecules have to be characterized with respect to their identity, their content and their impurity profile. It follows that the ds molecule as well as its process- and product-related impurities have to be quantified. The single strands are considered as process as well as product-related impurities in the ds drug substance. Applying well known, conventional, single-base resolution CE-CGE systems developed for the quality control of single-stranded antisense ODNs in the early 1990s, it turned out that the ds ODNs under investigation are migrating in broad, splitted peaks between the peaks reaction zones are observed. It follows that the quantification of the single strands in the drug substance as well as quantification of other product-related impurities, e.g. n-1; n-2 (loss of one and two bases (n), respectively) etc., are not possible without adaptation of the test system. The paper shows how the test system was adjusted in order to determine single-stranded strands as well as ds strands next to each other quantitatively in the ds drug substance under investigation.

Quantification of single-stranded nucleic acid and oligonucleotide interactions with metal ions by affinity capillary electrophoresis--Part II.

The interactions between tetranucleotides or heptanucleotides and inorganic cations have been measured by affinity CZE. The variation in migration behavior with increasing concentrations of divalent cations (Ca(2+), Mg(2+) and Ni(2+)) in the running buffer was investigated and quantified by the calculation of binding constants for mononuclear and multinuclear interactions. In addition to these fundamental studies of binding equilibria, the effect of sequence and the position of the guanine transition metal-binding site in the oligonucleotide have been investigated.

Pharmaceutical applications of isoelectric focusing on microchip with imaged UV detection.

For the first time, the application of a commercial Shimadzu microchip electrophoresis system MCE-2010 equipped with an imaging UV detector for isoelectric focusing (IEF) of therapeutic proteins is reported. By proper adjustment of the pH gradient, samples with pI values ranging from 2.85 to 10.3 can be focused to the imaged part of the separation channel. Three therapeutic proteins (hirudin, erythropoietin, and bevacizumab) have been successfully focused on the microchip, and the results have been compared to conventional capillary IEF in terms of peak profile, pI values, and reproducibility.

Peak splitting in the CE separation of enantiomers caused by organic solvents in the sample.

Two robust chiral standard separation systems were developed for the analysis of the chiral purity of chemically different model compounds applied in homogeneous asymmetric hydrogenation catalysis. Sulfated CDs were used as chiral selectors as they allow the analysis of neutral, acidic as well as basic compounds in the same electrophoretic system. Poorly water-soluble amines were dissolved in different organic solvent/buffer mixtures. Reproducibly, depending on the amount of organic solvent in the sample solution, peak splitting occurred and/or more peaks than expected were observed, implying impure model compounds. The dependence of the "chiral purity" on experimental parameters, e.g., kind and amount of sample solvent, length of injection plug, inner surface modification of the capillary, kind of sulfated CD, hydrophobicity, and basicity of the analytes, etc. was investigated. It is gathered that different equilibrium constants of the strong binding basic analytes and highly sulfated CD complex in the organic phase of the injection plug and the aqueous electrolyte phase are resulting in two different mobility zones for each enantiomer. It follows that each enantiomer is showing two peaks instead of one. Experimental strategies are shown to avoid these peak splitting/artificial impurity effects and obtain the "real" chiral purity picture of the samples.

Electrophoretic affinity measurements on microchip. Determination of binding affinities between diketopiperazine receptors and peptide ligands.

ACE on a microchip (MC-ACE) is introduced as a fast and reliable method to determine binding affinities. It is based on monitoring the change in the ionic mobility of a receptor upon binding to a ligand, or vice versa. The method is complementary to other standard methods for binding affinity determinations, like isothermal titration calorimetry (ITC), NMR, UV-Vis spectroscopy, etc. and allows for affinity studies of weak to strong binding interactions. The method is attractive since it principally allows for the analysis of the binding affinity of multiple receptors to a given ligand and requires comparatively small quantities of the binding partners (particularly in comparison to affinity measurements on capillary). We demonstrate the applicability of MC-ACE for the determination of the binding affinities between acid-rich diketopiperazine receptors and basic tripeptides in aqueous solution.

Determination of cationic neurotransmitters and metabolites in brain homogenates by microchip electrophoresis and carbon nanotube-modified amperometry.

An electrophoretic method for simultaneous determination of catecholamines and their O-methoxylated metabolites on the microchip as well as in the capillary is presented. A complex separation system employing sodium dodecyl sulfate (SDS) micelles, dendrimers forming a second pseudostationary phase and borate complexation is needed for the satisfactory separation of the selected compounds on the short migration length. A carbon nanotube-modified working electrode has been applied for the sensitive amperometric detection with submicromolar detection limits. The applicability of this new method for the analytics of real samples is demonstrated by analysis of mouse brain homogenate on the microchip and human urine by capillary electrophoresis.

Quantification of single-stranded nucleic acid and oligonucleotide interactions with metal ions by affinity capillary electrophoresis: part I.

The interactions between oligonucleotides and inorganic cations have been measured by capillary zone electrophoresis. With increasing concentrations of divalent cations (Ca(2+), Mg(2+), Mn(2+) and Ni(2+)) in the running buffer, the migration behavior was evaluated by calculation of the binding constants. Besides these fundamental studies of binding equilibria, different buffer components, tris(hydroxymethyl)aminomethane and 3-(N-morpholino)propanesulfonic acid, have been investigated and their effects on metal ion binding quantified.

Enzymatic sensitivity enhancement of biogenic monoamines on a chip.

Detection of biogenic monoamines in nanomolar concentrations is of great importance for probing the brain chemistry and for their analytics in biological fluids. The sensitivity enhancement of amperometric detection of neurotransmitters (NTs) and their metabolites after their electrophoretic separation on a microchip is presented and is based on coupled enzymatic reactions. The current response of the analyte is amplified by cyclic oxidation on a gold electrode mediated by reduced nicotinamide dinucleotide coenzyme and glucose oxidase enzyme present in the electrophoresis buffer. Using this approach, detection limits of about 10 nM for NTs and their metabolites can be reached.

Modified Hadamard transform microchip electrophoresis.

Sensitivity is a crucial point in the development applications for medicine or environmental samples in which the analytes are present in the nanomolar range. Besides further technical development of detection systems, the multiplex sample injection technique can be applied for enhancing the signal-to-noise ratio. Hadamard transform is easily applied to microchip electrophoresis due to the fact that sample injection is generally achieved through cross, double-tee, or tee injector structures. This paper reports the first demonstration of a modified Hadamard transform electrophoresis on a microchip by using an amperometric detector. Contrary to the previous Hadamard applications, the resolution (number of points per unit of time) of electropherograms obtained is independent of the number of injections.

Affinity capillary electrophoresis on microchips.

The present study shows that the application of the method of affinity capillary electrophoresis (ACE) to investigate interactions between ligands and their substrates can be realized on microchips. With ACE it is possible to characterize non-covalent molecular interactions (complexation and partition equilibria). Binding constants (K(B)) provide a measured value of the affinity of a ligand molecule to a substrate, which is basic information for the understanding of hormones, drugs and their targets, e.g. receptors in the human body. A microchip electrophoresis instrument equipped with a UV-detector and a home-built chip-station with electrochemical detection were used. ACE could be achieved with model solutions of neurotransmitters using sulfated beta-cyclodextrin (sCD) as substrate in a background buffer. This paper describes the advantages of microchip-ACE (MC-ACE) to traditional affinity capillary electrophoresis on a capillary. The results show that MC-ACE has great potential as a tool for fast scanning of interactions and to calculate binding constants of ligands with their substrates.

Enzyme-catalyzed amperometric oxidation of neurotransmitters in chip-capillary electrophoresis.

The determination of biogenic monoamines by enzyme-catalyzed oxidation after electrophoretical separation on a microfluidic chip decreases their detection limits significantly. An amperometric system with a chemically amplified response for neurotransmitters and their metabolites is presented. The principle is the rapid cyclic oxidation of the analyte on the amperometric detector in the presence of the redoxactive enzyme glucose oxidase in the capillary electrophoresis buffer. With this approach, detection limits in the range of 10(-7)-10(-8) M could be reached. Because of the good linearity between the current response and the concentration of catecholamines and their metabolites at concentrations up to 300 microM, this method is attractive for the analytical detection at low concentration levels such as in biological fluids.

Chiral on-chip separations of neurotransmitters.

The fast on-chip determination of a range of compounds of relevance in the study and treatment of neurological disorders is demonstrated. These include dopamine and its metabolites methoxytyramine, homovanillic acid, noradrenaline, adrenaline, normetanephrine, and metanephrine, its artificial precursors DOPA and tyrosine; and the related compounds DOPS and CDOPA. Two runs are needed for the determination of these compounds. The enantiomers of adrenaline, noradrenaline, and dopamine can be separated in a buffer containing the novel combination of a cyclodextrin and a dendrimer. The isomers of homovanillic acid, DOPA, CDOPA, methoxytyramine, metanephrine, and normetanephrine, which were found to interact more weakly with cyclodextrins, could be separated in approximately 3 min with a buffer containing a cyclodextrin and a crown ether. To our knowledge, this is the first report of a fast chiral separation of such a complex mixture on an electrophoresis chip. Detection was carried out amperometrically; derivatization of the analytes is not necessary.