Propensities of Fatty Acid-Modified ASOs: Self-Assembly vs Albumin Binding.

We conducted a biophysical study to investigate the self-assembling and albumin-binding propensities of a series of fatty acid-modified locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers specific to the MALAT1 gene. To this end, a series of biophysical techniques were applied using label-free ASOs that were covalently modified with saturated fatty acids (FAs) of varying length, branching, and 5'/3' attachment. Using analytical ultracentrifugation (AUC), we demonstrate that ASOs conjugated with fatty acids longer than C16 exhibit an increasing tendency to form self-assembled vesicular structures.

Liquid crystal phase formation and non-Newtonian behavior of oligonucleotide formulations.

Viscosity behavior of liquid oligonucleotide therapeutics and its dependence on formulation properties has been poorly studied to date. We observed a high increase in viscosity and solidification of therapeutic oligonucleotide formulations with increasing oligonucleotide concentration creating challenges during drug product manufacturing. In this study, we characterized the viscosity behavior of three different single strand DNA oligonucleotides based on oligonucleotide concentration and formulation composition.

Chemical, Analytical and Pharmacokinetic Characterisation of RO7304898, an API Consisting of Two Rapidly Interconverting Diastereoisomers.

Purpose: Exploration of the chemical, analytical and pharmacokinetic properties of the API, RO7304898, an allosteric EGFR inhibitor, intended to be developed as a mixture of two rapidly interconverting diastereoisomers with composition ratio of approximately 1:1.

Methods: Assessment of diastereoisomer stereochemistry, interconversion rates, binding to EGFR protein, metabolic stability and in vivo PK in Wistar-Han rats was conducted.

Results: The two diastereoisomers of the API undergo fast interconversion at physiologically relevant pH and direct EGFR binding studies revealed diastereoisomer B to be the active moiety.

The development and qualification of liquid adsorption chromatography for poloxamer 188 characterization.

Poloxamer 188 (P188) is formulated in proteinaceous therapeutics as an alternative surfactant to polysorbate because of its good chemical stability and surfactant properties, which enable interfacial protection, preventing visible and sub-visible particle formation. However, due to the nature of polymer heterogeneity and limited analytical approaches to resolve the superimposed components of P188, the impact of its quality variance on protein stability is still not well understood. In this study, we developed an analytical method to evaluate the components of P188 as a function of the length of polypropylene oxide (PPO), by maintaining polyethylene oxide (PEO) at the critical point of adsorption (CPA) to eliminate its chromatographic interference.

Protein NMR of biologicals: analytical support for development and marketed products.

Application of NMR spectroscopy to derive in-depth characterization of structure and dynamical properties of biomolecules is well established nowadays in many laboratories. Most of these methods rest on the availability of protein labeled with stable isotopes like C and N. In this report examples are presented on the application of NMR spectroscopic methods to characterize biopharmaceutical proteins in cases no isotope labeled material are available.

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs.

Unexpected Solubility Enhancement of Drug Bases in the Presence of a Dimethylaminoethyl Methacrylate Copolymer.

The methacrylate copolymer Eudragit EPO (EPO) has previously shown to greatly enhance solubilization of acidic drugs via ionic interactions and by multiple hydrophobic contacts with polymeric side chains. The latter type of interaction could also play a role for solubilization of other compounds than acids. The aim of this study was therefore to investigate the solubility of six poorly soluble bases in presence and absence of EPO by quantitative ultrapressure liquid chromatography with concomitant X-ray powder diffraction analysis of the solid state.

NMR and MS Methods for Metabolomics.

Metabolomics, also often referred as "metabolic profiling," is the systematic profiling of metabolites in biofluids or tissues of organisms and their temporal changes. In the last decade, metabolomics has become more and more popular in drug development, molecular medicine, and other biotechnology fields, since it profiles directly the phenotype and changes thereof in contrast to other "-omics" technologies. The increasing popularity of metabolomics has been possible only due to the enormous development in the technology and bioinformatics fields.

A Systematic Study of Molecular Interactions of Anionic Drugs with a Dimethylaminoethyl Methacrylate Copolymer Regarding Solubility Enhancement.

The methacrylate-copolymer Eudragit EPO (EPO) has raised interest in solubility enhancement of anionic drugs. Effects on aqueous drug solubility at rather low polymer concentrations are barely known despite their importance upon dissolution and dilution of oral dosage forms. We provide evidence for substantial enhancement (factor 4-230) of aqueous solubility of poorly water-soluble anionic drugs induced by low (0.

P and H NMR Studies of the Molecular Organization of Lipids in the Parallel Artificial Membrane Permeability Assay.

The parallel artificial membrane permeability assay (PAMPA) has emerged as a widely used primary in vitro screen for passive permeability of potential drug candidates. However, the molecular structure of the permeation barrier (consisting of a filter-supported dodecane-egg lecithin mixture) has never been characterized. Here, we investigated the long-range order of phospholipids in the PAMPA barrier by means of P static solid-state NMR.

Crystal Structures of the Human Doublecortin C- and N-terminal Domains in Complex with Specific Antibodies.

Doublecortin is a microtubule-associated protein produced during neurogenesis. The protein stabilizes microtubules and stimulates their polymerization, which allows migration of immature neurons to their designated location in the brain. Mutations in the gene that impair doublecortin function and cause severe brain formation disorders are located on a tandem repeat of two doublecortin domains.

NMR and MS methods for metabonomics.

Metabonomics, also often referred to as "metabolomics" or "metabolic profiling," is the systematic profiling of metabolites in bio-fluids or tissues of organisms and their temporal changes. In the last decade, metabonomics has become increasingly popular in drug development, molecular medicine, and other biotechnology fields, since it profiles directly the phenotype and changes thereof in contrast to other "-omics" technologies. The increasing popularity of metabonomics has been possible only due to the enormous development in the technology and bioinformatics fields.

Degradation of polysorbates 20 and 80: studies on thermal autoxidation and hydrolysis.

The purpose of this work was to study the mechanistic pathways of degradation of polysorbates (PS) 20 and PS80 in parenteral formulations. The fate of PS in typical protein formulations was monitored and analyzed by a variety of methods, including (1)H NMR, high-performance liquid chromatography/evaporative light scattering detection, and ultraviolet-visible spectroscopy. Oxidative degradation of PS in neat raw material was studied using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and headspace gas chromatography-mass spectrometry.

Automatic compatibility tests of HSQC NMR spectra with proposed structures of chemical compounds.

A recently introduced similarity measure is extended here for comparing two-dimensional spectra. Its applicability is demonstrated with heteronuclear single-quantum correlation (HSQC) NMR spectra. For testing the compatibility of a spectrum with the proposed chemical structure, first, the spectrum is predicted on the basis of that structure and then, the proposed comparison algorithm is applied.

Evaluation of the immuno-stimulatory potential of stopper extractables and leachables by using dendritic cells as readout.

Recombinant protein pharmaceuticals may bear some risks and undesirable side effects, such as the appearance of immunogenic reactions. The increased incidence of antibody-mediated pure red cell aplasia (PRCA) outside the United States after administration of a human serum albumin (HSA)-free EPREX (recombinant human erythropoietin alpha) formulation was explained with the generation of rubber stopper related leachables, possibly acting as immunogenic adjuvants. In our study, we have investigated the potential of extractable and leachable preparations of three different pharmaceutical relevant stoppers to generate a "danger signal" in a dendritic cell assay.

Application of metabonomics in a comparative profiling study reveals N-acetylfelinine excretion as a biomarker for inhibition of the farnesyl pathway by bisphosphonates.

In this work, the results of metabolic profiling of urine from a preclinical comparative profiling study with the two biphosphonates ibandronate and zoledronate are reported. Toxicological assessment showed very different effects for the two compounds. Ibandronate did not cause major signs of toxicity, whereas zoledronate elicited hepatotoxicity and nephrotoxicity.

Metabolic profiling technologies for biomarker discovery in biomedicine and drug development.

The state-of-the-art of nuclear magnetic resonance spectroscopy, mass spectrometry and statistical tools for the acquisition and evaluation of complex multidimensional spectroscopic data in metabolic profiling is reviewed in this article. The continuous evolution of the sensitivity, precision and throughput has made these technologies powerful and extremely robust tools for application in systems biology, pharmaceutical and diagnostics research. Particular emphasis is also given to the collection and storage of biological samples that are subjected to metabolite profiling.

Application of metabonomics in a compound ranking study in early drug development revealing drug-induced excretion of choline into urine.

Selecting drug candidates based on toxicity is an important step in early drug development. In this case study, it is shown how metabonomics is applied to a ranking study, in which drug candidates with equal pharmacological activities are selected based on least toxic side effects. The metabonomic analyses were carried out on an animal study that followed an established protocol for pilot toxicology/ranking studies in rats, however, not specifically modified for a metabonomic assessment.

Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabonomics.

For the analysis of the spectra of complex biofluids, preprocessing methods play a crucial role in rendering the subsequent data analyses more robust and accurate. Normalization is a preprocessing method, which accounts for different dilutions of samples by scaling the spectra to the same virtual overall concentration. In the field of 1H NMR metabonomics integral normalization, which scales spectra to the same total integral, is the de facto standard.

Metabolite projection analysis for fast identification of metabolites in metabonomics. Application in an amiodarone study.

The assignment of significantly changed NMR signals, which were identified with the help of multivariate models, to individual metabolites in biofluids is a manual and tedious task requiring knowledge in chemometrics and NMR spectroscopy. Metabolite projection analysis, introduced in this work, allows automatic linking of multivariate models with metabolites by skipping the level of manual NMR signal identification. The method depends on the projection of sets of metabolite NMR spectra from a database into PCA or PLS models of NMR spectra of biofluid samples.

Structural, kinetic, and thermodynamic analysis of the binding of the 40 kDa PEG-interferon-alpha2a and its individual positional isomers to the extracellular domain of the receptor IFNAR2.

Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS).

Structural and biophysical characterization of the 40 kDa PEG-interferon-alpha2a and its individual positional isomers.

The human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFNalpha(2a).

A high-throughput NMR-based ee-assay using chemical shift imaging.

The throughput of a previously described NMR-based -assay has been increased by a factor of at least 4 as a consequence of adapting the system to chemical shift imaging; by using a 19-capillary system the enantiomeric purity of 5600 samples can be measured per day.

Automation of biomolecular NMR screening.

For NMR based screening we review equipment needed for automated preparation of samples and acquisition of a large number of data sets. Hardware connecting lab-bench and NMR spectrometer is described. We focus on software used for automated calculation of the similarity between spectra - a prerequisite for the identification of test compounds interacting with a target-molecule.

High-resolution capillary tube NMR. A miniaturized 5-microL high-sensitivity TXI probe for mass-limited samples, off-line LC NMR, and HT NMR.

A new triple-resonance (TXI) (1H, 13C, 15N) high-resolution nuclear magnetic resonance (NMR) capillary probe with 2.5-microL NMR-active sample volume (V(obs)) was built and tested for applications with mass- and volume-limited samples and for coupling of microbore liquid chromatography to NMR. This is the first microliter probe with optimized coil geometry for use with individual capillary tubes with an outer diameter of 1 mm.