Investigation into the genotoxic impurity, 1-methyl-4-nitrosopiperazine, in rifampicin.

This study assessed the presence of the genotoxic impurity 1-methyl-4-nitrosopiperazine (MNP) in 27 batches of rifampicin capsules obtained from 11 manufacturers in China. While they were below the temporary limit of 5 ppm set by the US Food and Drug Administration, the observed levels (0.33-2.36 ppm) exceeded the acceptable threshold of 0.16 ppm. Building upon preliminary findings and degradation experiments, we concluded that MNP is a by-product of the oxidative degradation of rifampicin or is introduced via oxidation or nitrosation during the synthesis process involving 1-methyl-4-aminopiperazine. The pathways of MNP formation were confirmed in this study. Furthermore, we observed that the addition of antioxidants, sealed storage, and selection of dominant crystal forms can aid in controlling MNP levels.

Research on storage stability differences between ceftriaxone sodium products.

The conditions and mechanisms leading to stability differences between ceftriaxone sodium products were examined to ensure drug quality and efficacy. We used a combination of powder X-ray diffraction and thermogravimetric analysis to examine the differences between preparations for injection from different pharmaceutical processes to elucidate the changed processes by exposing samples to different humidity and high-temperature conditions. Water loss or absorption due to varying environmental humidity levels did not adversely affect the crystal structure, but could lead to the reversible redistribution of hepta-hydrate in the unit cell of generic products, causing its stability change. The irreversible distribution of hydrate may occur when generic drugs stored at 25 °C, whereas the brand-name products remained stable at 40 °C. Therefore, generic ceftriaxone sodium and its powder preparations would be acceptable by better controlled sealing and storing under cool conditions during storage period to meet the efficacy and stability.

Proteomic analysis of Penicillin G acylases and resulting residues in semi-synthetic ß-lactam antibiotics using liquid chromatography - tandem mass spectrometry.

Penicillin G acylase (PGA), as a key enzyme, is increasingly used in the commercial production of semi-synthetic ß-lactam antibiotics (SSBAs). With the substitution of conventional chemical synthesis by emerging bioconversion processes, more and more PGAs fermented from different types of strains such as Escherichia coli (E. coli, ATCC 11105), Achromobacter sp. CCM 4824 and Providencia rettgeri (ATCC 31052) have been used in this kind of enzymatic processes. As an intermediate reaction catalyst, PGA protein and its presence in the final products may cause a potential risk of human allergic reaction and bring challenges for both quality and process controls. To achieve qualitative and quantitative analysis of PGAs and their residues in SSBAs, a tryptic digestion coupled with liquid chromatography - tandem mass spectrometry (LC-MS/MS) method was developed and proposed because of advantages like high selectivity and sensitivity. A suitable filter aided sample preparation (FASP) method was also used to remove matrix interference and to enrich the target PGA retained in the ultrafiltration membrane for an efficient enzymatic hydrolysis and subsequent accurate MS detection. Finally, twelve batches of PGAs from eight companies were identified and categorized into two types of strains (E. coli and Achromobacter sp. CCM 4824) using proteomic analysis. In total nine batches of five types of SSBAs (amoxicillin, cephalexin, cefprozil, cefdinir and cefaclor) from eight manufacturers were selected for investigation. Trace levels of PGA residual proteins ranging from 0.01 to 0.44 ppm were detected in six batches of different SSBAs which were far lower than the safety limit of 35 ppm reported by DSM, a manufacturer with expertise in the production of SSBAs by enzymatic processes. The developed FASP with LC-MS/MS method is superior to traditional protein assays in terms of selectivity, sensitivity and accuracy. Moreover, it could provide in-depth analysis of amino acid sequences and signature peptides contributing to assignment of the strain sources of PGAs. This method could become a promising and powerful tool to monitor enzymatic process robustness and reliability of this kind of SSBAs manufacturing.

Bridging the structure gap between pellets in artificial dissolution media and in gastro-intestinal tract in rats.

Changes in structure of oral solid dosage forms (OSDF) elementally determine the drug release and its therapeutic effects. In this research, synchrotron radiation X-ray micro-computed tomography was utilized to visualize the 3D structure of enteric coated pellets recovered from the gastrointestinal tract of rats. The structures of pellets in solid state and in vitro compendium media were measured. Pellets in vivo underwent morphological and structural changes which differed significantly from those in vitro compendium media. Thus, optimizations of the dissolution media were performed to mimic the appropriate in vivo conditions by introducing pepsin and glass microspheres in media. The sphericity, pellet volume, pore volume and porosity of the in vivo esomeprazole magnesium pellets in stomach for 2 h were recorded 0.47, 1.55 × 108 µm3, 0.44 × 108 µm3 and 27.6%, respectively. After adding pepsin and glass microspheres, the above parameters in vitro reached to 0.44, 1.64 × 108 µm3, 0.38 × 108 µm3 and 23.0%, respectively. Omeprazole magnesium pellets behaved similarly. The structural features of pellets between in vitro media and in vivo condition were bridged successfully in terms of 3D structures to ensure better design, characterization and quality control of advanced OSDF.

Applications of Physiologically Based Biopharmaceutics Modeling (PBBM) to Support Drug Product Quality: A Workshop Summary Report.

This report summarizes the proceedings for Day 3 of the workshop titled "Current State and Future Expectations of Translational Modeling Strategies toSupportDrug Product Development, Manufacturing Changes and Controls". From a drug product quality perspective, patient-centric product development necessitates the development of clinically relevant drug product specifications (CRDPS). In this regard, Physiologically Based Biopharmaceutics modeling (PBBM) is a viable tool to establish links between in-vitro to in-vivo data, and support with establishing CRDPS. The theme of day 3 was practical applications of PBBM to support drug product quality. In this manuscript, case studies from US FDA, EMA and pharmaceutical industry on applications of PBBM in drug product quality are summarized which include 1) regulatory agency's perspectives on establishing the safe space and achieving study waivers, 2) model-informed risk assessment on the effects of acid reducing agents, bridging of dissolution methods, food effect, and formulation selection, and 3) understanding clinical formulation performance. Breakout session discussions focused on four topics - 1) terminologies related to physiologically based modeling in support of drug product quality, 2) regulatory harmonization on evidentiary standards, 3) CRDPS approaches and 4) bridging between biorelevant and quality control (QC) dissolution methods.

A Raman imaging-based technique to assess HPMC substituent contents and their effects on the drug release of commercial extended-release tablets.

In this study, we tried to assess the substitute contents of HPMC used in commercial extended-release tablets directly by an innovative Raman imaging-based analysis technique and find their effects on the in vitro performance of these pharmaceuticals. Twenty-seven batches of metformin hydrochloride extended-release tablets from various sources were collected in the Chinese mainland market. While Raman imaging was used to qualitatively analyze the composition of the tablets, the MeO and HPO contents of HPMC were quantitatively assessed by a newly proposed calculation method based on the Raman intensity of corresponding characteristic band. Additionally, the dissolution test was performed to evaluate the relationship between HPMC substitution pattern and in vitro behavior. In sum, our findings indicate that the drug release rate can be downregulated by increasing the MeO content of HPMC, while the high HPO content would largely eliminate the variation of drug release profiles among batches.

Monitoring binding affinity between drug and α1-acid glycoprotein in real time by Venturi easy ambient sonic-spray ionization mass spectrometry.

A new approach for monitoring the binding affinity between drugs and alpha 1-acid glycoprotein in real time was developed based on a combination of drug-protein reaction followed by Venturi easy ambient sonic-spray ionization mass spectrometry determination of the free drug concentrations. A known basic drug, propranolol was used to validate the new built method. Binding constant values calculated by venturi easy ambient sonic-spray ionization mass spectrometry was in good accordance with a traditional ultrafiltration combined with high performance liquid chromatography method. Then six types of basic drugs were used as the samples to conduct the real time analysis. Upon injection of alpha 1-acid glycoprotein to the drug mixture, the ion chromatograms were extracted to show the changes in the free drug concentrations in real time. By observing the drop-out of six types of drugs during the whole binding reaction, the binding affinities of different drugs were distinguished. A volume shift validating experiment and an injection delay correcting experiment were also performed to eliminate extraneous factors and verify the reliability of our experiment. Therefore, the features of Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS) and the experimental results indicate that our technique is likely to become a powerful tool for monitoring drug-AGP binding affinity in real time.

The rational design of a highly sensitive and selective fluorogenic probe for detecting nitric oxide.

A rationally designed small-molecule fluorogenic probe for nitric oxide (NO) detection based on a new switching mechanism has been developed. Attaching a NO-responsive dihydropyridine pendant group to a fluorophore led to a probe that displays a very high sensitivity to NO concentrations down to the low nM range and a very high specificity to NO while being insensitive to other oxidative oxygen/nitrogen species that often interfere with the sensing of NO.

High throughput screening of high-affinity ligands for proteins with anion-binding sites using desorption electrospray ionization (DESI) mass spectrometry.

A high throughput screening system involving a linear ion trap (LTQ) analyzer, a house-made platform and a desorption electrospray ionization (DESI) source was established to screen ligands with a high affinity for proteins with anion-binding sites. The complexes were analyzed after incubation, ultrafiltration, washing, and displacement. A new anionic region inhibited dissociation (ARID) mechanism that was suitable for a protein with anion-binding site was proposed. We utilized the differences in detectable dissociation of protein-ligand complexes, combined with displacement experiments, to distinguish free ligands displaced from anion-binding sites from liberated ligands dissociated from nonspecific interactions. The method was validated by α1-acid glycoprotein (AGP) and (R), (S)-amlodipine. Site-specific enantioselectivity shown in our experiments was consistent with earlier studies. Obtaining all of the qualitative information of 15*3 samples in 2.3 min indicates that the analysis process is no longer the time-limiting step in the initial stage of drug discovery. Quantitative information verified that our method was at least a semiquantitative method.