Pharmeuropa bio & scientific notes最新文献

The goal of the project was to standardise a new in vitro method in replacement of the existing standard method for the determination of hepatitis A virus antigen content in hepatitis A vaccines (HAV) marketed in Europe. This became necessary due to issues with the method used previously, requiring the use of commercial test kits. The selected candidate method, not based on commercial kits, had already been used for many years by an Official Medicines Control Laboratory (OMCL) for routine testing and batch release of HAV. After a pre-qualification phase (Phase 1) that showed the suitability of the commercially available critical ELISA reagents for the determination of antigen content in marketed HAV present on the European market, an international collaborative study (Phase 2) was carried out in order to fully validate the method. Eleven laboratories took part in the collaborative study. They performed assays with the candidate standard method and, in parallel, for comparison purposes, with their own in-house validated methods where these were available. The study demonstrated that the new assay provides a more reliable and reproducible method when compared to the existing standard method. A good correlation of the candidate standard method with the in vivo immunogenicity assay in mice was shown previously for both potent and sub-potent (stressed) vaccines. Thus, the new standard method validated during the collaborative study may be implemented readily by manufacturers and OMCLs for routine batch release but also for in-process control or consistency testing. The new method was approved in October 2012 by Group of Experts 15 of the European Pharmacopoeia (Ph. Eur.) as the standard method for in vitro potency testing of HAV. The relevant texts will be revised accordingly. Critical reagents such as coating reagent and detection antibodies have been adopted by the Ph. Eur. Commission and are available from the EDQM as Ph. Eur. Biological Reference Reagents (BRRs).

An international collaborative study was organised to establish the World Health Organization (WHO) 3(rd) International Standard (IS) for neomycin. Ten laboratories from different countries participated in the collaborative study. The potency of the candidate material, a freeze-dried preparation, was estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 2(nd) IS for neomycin was used as a standard. Based on the results of the study, the 3(rd) IS for neomycin was adopted at the meeting of the WHO Expert Committee on Biological Standardization (ECBS) in 2012 with an assigned potency of 19,050 IU per vial. The 3(rd) IS for neomycin is available from the European Directorate for the Quality of Medicines & HealthCare (EDQM).

Based on experimental results of aflatoxin analysis as well as information from literature, this contribution discusses the likelihood of aflatoxin contamination in fresh medicinal plants. As cultivation and collection of medicinal plants in accordance with Good Agricultural and Collection Practice (GACP) and the local climatic conditions minimise aflatoxin contamination and, as fresh raw material is normally processed immediately, aflatoxin contamination of fresh medicinal plants from Central European countries is extremely unlikely. As a result of the risk-based approach to aflatoxin testing, 3 options are proposed depending on the origin of the material and the plant parts used: no testing, skip lot testing or routine testing.

Higher order structure, including conformation, is considered a critical quality parameter of therapeutic proteins, and is mandatory information in development of first use and bio-similar therapeutic protein drugs, the assumption being that the biological activity of a protein is directly dependent on its adoption of a 'correct' conformation. Studies on the relationship between conformation and activity depend on the ability to induce conformational changes in proteins, and conventional approaches such as thermal or chemical denaturation are incompatible with bioactivity measurements. To explore the relationship between bio-activity and conformational studies, we have studied variants of the therapeutic protein filgrastim (rec met huGCSF) which have been mutated by the replacement of helical alanine residues with glycine, to destabilise the conformation of the molecule. In the GCSF A-G mutant series studied, single conformation-destabilising amino-acid substitutions significantly reduced the biological activity. These effects were not, however correlated with changes in secondary structure measurable by far-UV Circular Dichroism (CD) spectroscopy. Only the more extensively mutated double and triple substitutions showed measurable reductions in alpha-helical structure by CD. We conclude that in this system, GCSF does not readily adopt a reduced-activity altered conformational state which can be detected by low resolution techniques such as CD. In contrast, reductions in biological activity do reflect reductions in conformational stability, possibly caused by time-dependent degradation of the protein in the cell-proliferation bioassay. Although not a formal model of biosimilarity, we suggest that our results could inform the regulatory process in determining appropriate experimental approaches to meeting regulatory requirements for higher order structural analysis of therapeutic proteins.

The current European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation batch 3 (BRP3) for Human Immunoglobulin was established in 2005. Stocks of this BRP are dwindling and a replacement batch is needed to serve as working standard in the tests for distribution of molecular size by HPLC, anticomplementary activity (ACA) and Fc function, in accordance with the requirements of the Ph. Eur. monographs Human normal immunoglobulin (0338) and Human normal immunoglobulin for intravenous administration (0918). The European Directorate for the Quality of Medicines & HealthCare (EDQM) carried out a project (BSP099) to establish replacement batches for this BRP. The project was run in 2 phases, a prequalification phase (Phase 1) and an international collaborative study (Phase 2) involving 19 laboratories. Three batches of candidate materials of various sizes, Samples A, B and C, were procured from 2 different manufacturers on the European market. Based on the results of the study, Sample A was shown to be suitable as a reference standard for the ACA test and for molecular size determination by HPLC, whereas Samples B and C were demonstrated to be suitable for the Fc function test and for the molecular size determination by HPLC. All 3 BRPs are to be used in conjunction with the monographs Human normal immunoglobulin (0338) and Human normal immunoglobulin for intravenous administration (0918). The BRPs were adopted by the Ph. Eur. Commission at its 141st session in November 2011 as official Ph. Eur. Human Immunoglobulin BRPs for ACA and molecular size Batch 1 (Sample A) and Fc function and molecular size Batch 1 and Batch 2 (Samples B and C respectively).

To assess the risk of heavy metal contamination of homoeopathic mother tinctures, 9 plant species and 1 fungus used in the manufacture of homoeopathic medicaments were investigated. Mother tinctures were prepared according to the manufacturing procedures described in the European Pharmacopoeia. The original herbal drug and the material processed during production were analysed for their cadmium (Cd), lead (Pb) and mercury (Hg) content. The plant components were harvested from 2 sites contaminated by different heavy metal exposure pathways and compared with reference material from Deutsche Homöopathie-Union (DHU). It was confirmed that in all cases a significant depletion of all heavy metals studied occurred during manufacture of the mother tincture, regardless of the starting material used. In all cases, most of the heavy metal content was retained in the press cake; low levels only were detected in the mother tincture. None of the mother tinctures of plant origin exceeded the required limits, not even those of plant starting materials originating from highly contaminated sites. Substantial heavy metal concentrations could only be detected in the mother tincture of the fungus Amanita muscaria, calculated from the dry weight of the starting material. According to the results obtained, a risk-based approach to heavy metal assessment is suggested where permanent control is focused on the heavy metals accumulating in organisms such as fungi.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

An unknown bromhexine hydrochloride (BRH) degradation product in BRH oral solutions (finished products) was potentially related to the purity of this API. Several degradation experiments were conducted and its identity and formation were investigated using LC-DAD and LC-DAD-MS/MS. Using the LC method described in the Ph.Eur monograph BRH the degradation product was observed at RRTBRH 0.1 and the specified impurities A-D were ruled out as candidates. Impurity E was initially not considered as a candidate as EDQM reported an expected RRTBRH of 1.8. Still, the LC-DAD-MS/MS results were consistent with the M+ ion for impurity E and its expected fragment ions. Therefore, standard addition was carried out using the Ph. Eur. method which confirmed that the degradation product at RRT 0.1 was impurity E. Upon changing the column type to a column described in the knowledge database, impurity E eluted at an RRT of 1.5. Nevertheless, both columns met all of the criteria in the monograph. The formation of impurity E was even observed in BRH solutions without added reagents. As the conversion from BRH to impurity E requires a source of carbon, we suggest that one BRH molecule degrades through a radical mechanism to a reactive species which subsequently is quenched by another BRH molecule producing impurity E. We suggest the transparency list for BRH to be more explicit on the formation of impurity E, its RRT and the permissible LC columns.

An international collaborative study was organised to establish the World Health Organization (WHO) 3rd International Standard (IS) for dihydrostreptomycin. Eleven laboratories from different countries participated in the collaborative study. The potency of the candidate batch, a freeze-dried preparation, was estimated by microbiological assays with sensitive microorganisms. To ensure continuity between consecutive batches of the WHO IS, the 2nd IS for dihydrostreptomycin was used as standard. Based on the results of the study, the 3rd IS for dihydrostreptomycin was adopted at the meeting of the WHO Expert Committee on Biological Standardisation (ECBS) in 2011 with an assigned anti-microbiological activity of 19425 International Units (IU) per vial. The 3rd IS for dihydrostreptomycin is available from the EDQM.

NMR spectrometry has many analytical applications; for instance, the identification of known substances; the structure elucidation of unknown ones; the quantification of APIs, impurities, solvent and water; kinetic studies, stereochemistry determinations, and the analyses of complex mixtures as in metabonomics. NMR spectrometry has the potential to substitute or complement existing analyses that are performed on APIs. In this work, 4 different NMR analyses were done on 2 APIs: fluvastatin sodium and benzalkonium chloride with good results.