Impurities In Preparations Research Articles

Mutagenicity assessment of two potential impurities in preparations of 5-amino-2,4,6 triiodoisophthalic acid, a key intermediate in the synthesis of the iodinated contrast agent iopamidol

5-Aminoisophthalic acid and 5-nitroisophthalic acid (5-NIPA) are potential impurities in preparations of 5-amino-2,4,6-triiodoisophthalic acid, which is a key intermediate in the synthesis of the iodinated contrast agent iopamidol. We have studied their mutagenicity in silico (quantitative structure-activity relationships, QSAR) and by the bacterial reverse mutation assay (Ames test). First, the compounds were screened with the tools Derek Nexus™ and Leadscope®. Both compounds were flagged as potentially mutagenic (class 3 under ICH M7). However, contrary to the in silico prediction, neither chemical was mutagenic in the Ames test (plate incorporation method) with or without S9 metabolic activation.

A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands.

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

Evaluation of synthetic formaldehyde and methanol assimilation pathways in Yarrowia lipolytica

BackgroundCrude glycerol coming from biodiesel production is an attractive carbon source for biological production of chemicals. The major impurity in preparations of crude glycerol is methanol, which is toxic for most microbes. Development of microbes, which would not only tolerate the methanol, but also use it as co-substrate, would increase the feasibility of bioprocesses using crude glycerol as substrate.ResultsTo prevent methanol conversion to CO2 via formaldehyde and formate, the formaldehyde dehydrogenase (FLD) gene was identified in and deleted from Yarrowia lipolytica. The deletion strain was able to convert methanol to formaldehyde without expression of heterologous methanol dehydrogenases. Further, it was shown that expression of heterologous formaldehyde assimilating enzymes could complement the deletion of FLD. The expression of either 3-hexulose-6-phosphate synthase (HPS) enzyme of ribulose monosphosphate pathway or dihydroxyacetone synthase (DHAS) enzyme of xylulose monosphosphate pathway restored the formaldehyde tolerance of the formaldehyde sensitive Δfld1 strain.ConclusionsIn silico, the expression of heterologous formaldehyde assimilation pathways enable Y. lipolytica to use methanol as substrate for growth and metabolite production. In vivo, methanol was shown to be converted to formaldehyde and the enzymes of formaldehyde assimilation were actively expressed in this yeast. However, further development is required to enable Y. lipolytica to efficiently use methanol as co-substrate with glycerol.

Use of capillary electrophoresis for the determination of impurities in preparations of fluorine-18 labelled PET radiopharmaceuticals

Fluorine-18 labelled radiotracers are the most common diagnostic agents employed in positron emission tomography (PET). Despite well-established quality control (QC) procedures, introduction of new synthetic methods demands continuous development of analytical methodology. Here we propose capillary electrophoresis (CE) as a simple, fast and cost-efficient analytical method, allowing for the evaluation of potentially toxic impurities, including transition metals, in the formulated preparations. Method developed was applied in routine QC procedures for several clinically relevant radiotracers.

Patch testing of patients allergic to Disperse Blue 106 and Disperse Blue 124 with thin‐layer chromatograms and purified dyes

Former investigations have demonstrated that patch test preparations containing Disperse Blue (DB) 106 and DB 124 are impure. To investigate the significance of impurities in preparations of disperse dyes with regard to contact allergy. 21 patients allergic to DB 106 and/or DB 124 were tested with dilution series of commercial and purified DB 106 and DB 124. All patients were additionally tested with thin-layer chromatography (TLC) strips made from the commercial preparations containing DB 106 and 18 of them also with TLC strips containing DB 124. 13 of 21 and 12/18 patients tested positively to the strips of DB 106 and DB 124, respectively. Among these patients, 4/13 and 5/12, respectively, did not react to the main spot but reacted to other spots. Dilution series of purified DB 106 and DB 124 were positive in 10 patients each, while 16 and 15 patients, respectively, tested positively to the dilution series of the corresponding commercial disperse dye. Approximately 25% of the patients, diagnosed as contact allergic to DB 106 and DB 124, only reacted to impurities in the patch test preparations. This has implications for diagnosis and prevention.

A PCR-based semiquantitative assay of DNA impurities in recombinant protein preparations

A semiquantitative assay of DNA impurities in preparations of human recombinant insulin is described. The assay is based on the detection of a fragment of the ampicillin-resistant gene within the producer strain DNA by PCR. The analysis of PCR products of the studied preparations and PCR products containing known amounts of E. coli total DNA enabled a quantitative determination of the producer strain DNA content in the preparations under study. The sensitivity of the method is 7 pg of E. coli DNA per 10 microg of human recombinant insulin. The high sensitivity of the method allows us to recommend it for the quantitative determination of DNA content in recombinant preparations that do not inhibit PCR.

An Anionic Impurity in Preparations of Cytochrome c Interferes with Assays of Cationic Catalysts of the Dismutation of the Superoxide Anion Radical

An Anionic Impurity in Preparations of Cytochrome c Interferes with Assays of Cationic Catalysts of the Dismutation of the Superoxide Anion Radical

An anionic impurity in preparations of cytochrome e interferes with assays of cationic catalysts of the dismutation of the superoxide anion radical

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Limited enzymatic digestion for the determination of the quantities of minor diastereomeric impurities in preparations of RMP-7, a peptide containing a reduced peptide bond

RMP-7 is a bradykinin analogue containing all “ l” amino acids and a reduced dipeptide bond between amino acids eight and nine. This reduced dipeptide bond [4-Me-Tyr-Ψ(CH 2NH)-Arg] is created under synthetic conditions which could result in inversions of the chiral centers of either 4-Me-Tyr or Arg. Stereoisomers of RMP-7 would be expected to have altered biological specificity. Current chromatographic methods are not sufficiently sensitive to distinguish the anticipated stereoisomeric variants of the intact molecule. Therefore we have devised an analytical method based on limited enzymatic digestion of the compound followed by reversed-phase HPLC analysis of the peptide fragments. Using this method we have been able to carry out precise and reliable quantitative analysis of the stereoisomeric content of different batches of peptide prepared for biological testing.

Impurities in Preparations of Acetabularia Chloroplasts

IN a recent description of photosynthesis by preparations of Acetabularia mediterranea chloroplasts in an artificial leaf1, it was suggested that the stability and impermeability of these chloroplasts are due to qualities of the chloroplast membrane. Recent work in this laboratory (F. Winkenbach and R. G. S. Bidwell, in preparation for publication) suggests that these chloroplasts, together with the small quantities of cytoplasm previously noted2,3, are enveloped in the form of droplets by a membrane-like structure. It is therefore possible that the stability and impermeability of Acetabularia chloroplasts are due to the presence of this membrane. Varying the proportion of contamination does not appreciably alter the distribution of 14C among products of photosynthesis, so that it seems unlikely that the cytoplasmic contamination is directly involved in the observed photosynthetic metabolism.

Beta hemolysin: a persistent impurity in preparations of staphylococcal nuclease and enterotoxin.

Purified staphylococcal nuclease and enterotoxin B from several sources contained beta hemolysin whose physicochemical resemblances to the other two proteins make its elimination difficult.

Beta Hemolysin: a Persistent Impurity in Preparations of Staphylococcal Nuclease and Enterotoxin

Purified staphylococcal nuclease and enterotoxin B from several sources contained beta hemolysin whose physicochemical resemblances to the other two proteins make its elimination difficult.

A polarographic method for the detection of protein impurities in preparations of tobacco mosaic virus

A polarographic method for the detection of protein impurities in preparations of tobacco mosaic virus