Pharmeuropa bio & scientific notes最新文献

A generic approach for the analysis of counterions of pharmaceutical reference substances, which are established by the laboratory department of the European Pharmacopoeia (Ph. Eur.), was developed. A mixed-mode chromatography method using charged aerosol detection (CAD) published by Zhang et al. separating 25 commonly used pharmaceutical counterions was selected for this purpose. The method was validated in terms of specificity, repeatability, limits of quantification (LOQs), linearity and range according to ICH guideline Q2(R1) and the Technical Guide for the Elaboration of Monographs of the Ph. Eur. Moreover, the applicability of the method for the purpose of counterion identification and quantification in drug substances as well as for the control of inorganic ions as impurities was demonstrated using selected examples of Ph. Eur. reference standards and other samples of substances for pharmaceutical use (e.g. cloxacillin sodium, somatostatin). It was shown that for identification purposes of the parent substance as well as organic ions the chromatographic system can easily be coupled to a mass selective detector without any modification.

Following the heparin adulteration crisis, the European Pharmacopoeia (Ph. Eur.) Group of Experts on Biologicals (Group 6) considered a revision of the general chapter 2.7.5. Assay of heparin with regard to the assay of the anticoagulant activity of heparin in order to replace the clotting method with more specific chromogenic methods for anti-IIa and anti-Xa activities. An international collaborative study was carried out in 3 phases under the aegis of the Biological Standardisation Programme (BSP) of the Council of Europe and the European Commission in order to recalibrate Heparin sodium Biological Reference Preparation (BRP) batch 3 for these new assays. Phase 1 confirmed the feasibility of the project, but also indicated that the composition of the buffers affects the assay results, thereby highlighting the importance of using common assay procedures. Phase 2 consisted of a collaborative study involving 15 laboratories to calibrate the anti-IIa and anti-Xa activities of Heparin sodium BRP batch 3. The collaborative study confirmed that Heparin sodium BRP batch 3 is suitable for use as a reference preparation in the proposed chromogenic assays for unfractionated heparin. It also showed that the currently defined acceptance limits (90 % to 111 %) can be maintained in the revised Ph. Eur. texts. Phase 3 of the study collected data on the impact of the new unitage on the release of products marketed in Europe. The data from 5 manufacturers, who each reported results from both the clotting and chromogenic assays for a total of 23 batches, indicated that the replacement of the pharmacopoeial method is unlikely to cause batch release issues. Based on the results of this study, the Ph. Eur. Commission assigned Heparin sodium BRP batch 3 with a potency of 1000 IU/vial for both anti-IIa and anti-Xa activities in the chromogenic assays.

Determination of the molecular size distribution of vaccine products by high performance size exclusion chromatography coupled to refractive index detection is important during the manufacturing process. Partial elution of high molecular weight compounds in the void volume of the chromatographic column is responsible for variation in the results obtained with a reference method using a TSK G5000PWXL chromatographic column. GlaxoSmithKline Vaccines has developed an alternative method relying on the selection of a different chromatographic column with a wider separation range and the generation of a dextran calibration curve to determine the optimal molecular weight cut-off values for all tested products. Validation of this method was performed according to The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). The new method detected product degradation with the same sensitivity as that observed for the reference method. All validation parameters were within the pre-specified range. Precision (relative standard deviation (RSD) of mean values) was < 5 per cent (intra-assay) and < 10 per cent (inter-assay). Sample recovery was > 70 per cent for all polysaccharide conjugates and for the Haemophilus influenzae type B final container vaccine. All results obtained for robustness met the acceptance criteria defined in the validation protocol (≤ 2 times (RSD) or ≤ 2 per cent difference between the modified and the reference parameter value if RSD = 0 per cent). The new method was shown to be a suitable quality control method for the release and stability follow-up of polysaccharide-containing vaccines. The new method gave comparable results to the reference method, but with less intra- and inter-assay variability.

An international collaborative study was carried out for the establishment of replacement batches for the European Pharmacopoeia (Ph. Eur.) Somatropin Chemical Reference Substance (CRS) batch 2. The study was organised within the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the European Commission. Seventeen laboratories from Europe, North America, South America and Australia took part in the collaborative study. The study aimed at calibrating the somatropin content of 2 candidate preparations and demonstrating their suitability to serve as a reference substance in the tests for identification, for related proteins, for dimers and related substances of higher molecular mass (HMM), for charged variants distribution and for the assay of somatropin, as prescribed by the current Ph. Eur. monographs 0950 Somatropin bulk solution, 0951 Somatropin and 0952 Somatropin for injection. Based on the results summarised herein the Ph. Eur. Commission adopted in January 2012 candidate preparation b (cCRS-b, Sample D) as somatropin CRS batch 3 with an assigned content of 3.86 mg of somatropin monomer per vial, and candidate preparation a (cCRS-a, Sample C) as somatropin CRS batch 4 with an assigned content of 2.59 mg of somatropin monomer per vial.

This contribution provides an overview on the current legal requirements regarding limits for bromide and presents data on the actual bromide burden of commonly used herbal drugs. Evaluation of an extensive data base shows that results exceeding the limit of 50 mg/kg are found in specific plants which take up bromide to a high extent from the environment. Thus, positive findings of bromide in herbal drugs do not necessarily serve as a proof for methyl bromide treatment. Taking into account the ADI recommended by EMA and WHO, there are no toxicological concerns with regard to the intake of herbal teas, extracts or comminuted herbal drugs at therapeutic doses. Furthermore, the use of methyl bromide and other fumigants must be documented within the batch documentation. If stated in the batch documentation that no fumigation was carried out, it is not necessary to perform the test on bromide. In cases of a particular suspect and if toxicological concerns exist, additional testing can be performed in accordance with the limits set by Regulation (EC) No. 396/2005. For the above reasons, information obtained by performing the test on bromide is not significant for the assessment of quality. Therefore, it seems no longer necessary to maintain bromide in Ph. Eur. general chapter 2.8.13. Pesticide residues and it is recommended to delete it from Table 2.8.13.-1.

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).