A European survey on donor deferral for allergy: Rationale and initial results of a survey in 35 countries.

Allergy accounts to near 0.5% of all reported transfusion adverse events. The responsibility of blood components themselves and - therefore - of blood donors is still questioned. The European Community undertook a large international survey to address the consistency and homogeneity of medical selection of blood donors with regard to the risk of allergy, and especially of transferring allergy to recipients. This short report presents the salient points of the survey, stressing that there is inconsistency in addressing the allergy question within countries or systems, with paths of improvement.

Collaborative study for the establishment of the WHO 3(rd) International Standard for Endotoxin, the Ph. Eur. endotoxin biological reference preparation batch 5 and the USP Reference Standard for Endotoxin Lot H0K354.

An international collaborative study was organised jointly by the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC), the United States Pharmacopeia (USP) and the European Directorate for the Quality of Medicines & HealthCare (EDQM/Council of Europe) for the establishment of harmonised replacement endotoxin standards for these 3 organisations. Thirty-five laboratories worldwide, including Official Medicines Control Laboratories (OMCLs) and manufacturers enrolled in the study. Three candidate preparations (10/178, 10/190 and 10/196) were produced with the same material and same formulation as the current reference standards with the objective of generating a new (3(rd)) International Standard (IS) with the same potency (10 000 IU/vial) as the current (2(nd)) IS, as well as new European Pharmacopoeia (Ph. Eur.). and USP standards. The suitability of the candidate preparations to act as the reference standard in assays for endotoxin performed according to compendial methods was evaluated. Their potency was calibrated against the WHO 2(nd) IS for Endotoxin (94/580). Gelation and photometric methods produced similar results for each of the candidate preparations. The overall potency estimates for the 3 batches were comparable. Given the intrinsic assay precision, the observed differences between the batches may be considered unimportant for the intended use of these materials. Overall, these results were in line with those generated for the establishment of the current preparations of reference standards. Accelerated degradation testing of vials stored at elevated temperatures supported the long-term stability of the 3 candidate preparations. It was agreed between the 3 organisations that batch 10/178 be shared between WHO and EDQM and that batches 10/190 and 10/196 be allocated to USP, with a common assigned value of 10 000 IU/vial. This value maintains the continuity of the global harmonisation of reference materials and unitage for the testing of endotoxins in parenteral pharmaceutical products. Based on the results of the collaborative study, batch 10/178 was established by the European Pharmacopoeia Commission as the Ph. Eur. Endotoxin Biological Reference Preparation (BRP) batch 5. The same batch was also established by the Expert Committee on Biological Standardisation (ECBS) of WHO as the WHO 3(rd) IS for Endotoxin. Batch 10/190 was adopted as the USP Endotoxin Reference Standard, lot H0K354 and vials from this same batch (10/190) will serve as the United States Food and Drug Administration (USFDA) Endotoxin Standard, EC-7.

HIV-1 core proteins expressed from recombinant vaccinia viruses.

The sequences encoding the core proteins p55, p25, and p18 of the human immunodeficiency virus (HIV-1) have been inserted into the vaccinia virus genome. Infection of cultured cells with the live recombinant viruses led to the expression of proteins that were recognized by sera from HIV-seropositive individuals. Immunization of mice with the recombinant virus expressing the HIV p25 protein and the p55 precursor yielded high levels of antibodies directed against the corresponding HIV antigens. The data obtained are discussed in terms of the possible use of these live recombinant viruses in the development of a strategy toward an AIDS vaccine.

Capillary electrophoresis for the control of impurities of rDNA somatropin.

In the European Pharmacopoeia, the monographs on somatropin, somatropin bulk solution and somatropin for injection prescribe a number of tests including "related substances", "dimer and related substances of higher molecular weight" and "isoform distribution" which are intended to control the levels of impurities in the substance. The aim of this study was to verify the robustness of a new method by capillary electrophoresis and to compare its performance with that of the existing test for "isoform distribution" by isoelectric focusing. It was demonstrated that the capillary electrophoresis method was superior to the method of isoelectric focusing. The interest of the CZE method consists in the resolution of related impurities that might be process specific and/or generated by the expression system.

Collaborative study for the recalibration of Ph. Eur. Heparin sodium BRP batch 3 for chromogenic potency assays.

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.

Collaborative study for the establishment of the 3rd international standard for neomycin.

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).

International collaborative study for the calibration of the Ph. Eur. somatropin chemical reference substance batches 3 and 4 (BSP108).

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.

Collaborative study for the establishment of the 3rd international standard for dihydrostreptomycin.

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.

Collaborative study for the establishment of the second international standard for vancomycin.

An international collaborative study has been organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish the World Health Organization (WHO) 2nd International Standard (IS) for Vancomycin. Twelve laboratories from 10 different countries participated. The potency of the candidate material, a freeze-dried preparation, was estimated by microbiological assays with sensitive micro-organisms. As the stocks of the 1st IS for Vancomycin had been completely depleted, in order to ensure continuity between consecutive batches of the WHO IS, the European Pharmacopoeia Chemical Reference Standard (CRS) for Vancomycin batch 2 was used as a standard. It had been calibrated in a large international collaborative study against the WHO 1st IS for Vancomycin. Based on the results of the study, the 2nd IS for Vancomycin was adopted at the meeting of the WHO Expert Committee on Biological Standardisation (ECBS) in 2010 with an assigned antimicrobiological activity of 109,700 IU per vial. The 2nd IS for Vancomycin is available from the EDQM.

Collaborative study for the establishment of replacement batches of heparin low- molecular-mass for assay biological reference preparations.

A collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM) in the context of the Biological Standardisation Programme (BSP), under the aegis of the Council of Europe and the European Commission, to establish replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay European Pharmacopoeia Biological Reference Preparation (BRP). The replacement batches of BRP are intended to be used in the assays for anti-Xa and anti-IIa activities, as described in the European Pharmacopoeia (Ph. Eur.) monograph Heparins, low-molecular-mass (0828). Three freeze-dried candidate batches were calibrated against the current International Standard (IS) for Heparin, lowmolecular- weight (2nd IS, 01/608). For the purpose of the continuity check between subsequent BRP batches, the current Heparin low-molecular-mass for assay BRP (batch 5) was also included in the test panel. Thirteen official medicines control and manufacturers laboratories from European and non-European countries contributed data. A central statistical analysis of the datasets was performed at the EDQM. On the basis of the results, the 3 candidate materials were assigned a potency of 104 IU/vial for the anti-Xa activity and 31 IU/vial for the anti-IIa activity. Taken into account the preliminary stability data and the results of this collaborative study, the 3 batches of candidate BRP were adopted in June 2010 by the Commission of the Ph. Eur. as Heparin low-molecular-mass for assay BRP batches 6, 7 and 8.

Collaborative study for the establishment of the second international standard for gramicidin.

An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish the WHO second International Standard (IS) for gramicidin as the stocks of the 1st IS, established in 1964, were close to depletion. The candidate material did not show any sign of potency loss when kept at elevated temperatures of + 4 °C, + 20 °C, + 37 °C and + 45 °C for 3 months. Six laboratories from 5 countries as well as the EDQM laboratory participated in the collaborative study. Potencies of the candidate material were estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 1st IS for gramicidin was used as standard. Based on the results of the study, the 2nd IS for gramicidin was adopted at the meeting of the WHO Expert Committee on Biological Standardization (ECBS) in 2008 with an assigned potency of 1070 International Units per mg (IU/mg). The 2nd IS for gramicidin is available from the EDQM.

Collaborative study for the establishment of the third international standard for nystatin.

Due to the depletion in stocks of the World Health Organization (WHO) 2nd International Standard (IS) for nystatin, an international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish a replacement batch. Seventeen laboratories participated in the collaborative study, performing the microbiological diffusion assay to estimate the potency of the candidate 3rd International Standard for nystatin. The 2nd International Standard for nystatin was used as a standard to ensure the continuity of the unitage. Follow-up accelerated degradation studies demonstrated that the IS is stable when at the customary storage temperature of - 20 °C. The 3rd IS for nystatin was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in 2006 with an assigned potency of 5710 International Units per mg (IU/mg). The 3rd IS for nystatin is available from the EDQM.

Collaborative study for the establishment of the second international standard for amphotericin B.

An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish the WHO second International Standard (IS) for amphotericin B as the stocks of the 1st IS, established in 1963, were close to depletion. The candidate material did not show any sign of potency loss when kept at elevated temperatures of + 4 degrees C, + 20 degrees C, + 37 degrees C and + 45 degrees C for 3 months. Ten laboratories from 8 countries participated in the collaborative study. Potencies of the candidate material were estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 1st IS for amphotericin B was used as the standard. Based on the results of the study, the 2nd IS for amphotericin B was adopted at the meeting of the WHO Expert Committee on Biological Standardisation (ECBS) in 2007 with an assigned potency of 944 International Units per mg (IU/mg). The 2nd IS for Amphotericin B is available from the EDQM.

Establishment of a biological reference preparation for hepatitis A vaccine (inactivated, non-adsorbed).

A reference standard calibrated in International Units (IU) is needed for the in vitro potency assay of hepatitis A vaccines prepared by formalin-inactivation of purified hepatitis A virus grown in cell cultures. Thus, a project was launched by the European Directorate for the Quality of Medicines & HealthCare (EDQM) to establish one or more non-adsorbed inactivated hepatitis A vaccine reference preparation(s) as working standard(s), calibrated against the 1st International Standard (IS), for the in vitro potency assay (ELISA) of all vaccines present on the European market. Four non-adsorbed liquid preparations of formalin-inactivated hepatitis A antigen with a known antigen content were obtained from 3 manufacturers as candidate Biological Reference Preparations (BRPs). Thirteen laboratories participated in the collaborative study. They were asked to use an in vitro ELISA method adapted from a commercially available kit for the detection of antibodies to hepatitis A virus. In-house validated assays were to be run in parallel, where available. Some participants also included commercially available hepatitis A vaccines in the assays, after appropriate desorption. During the collaborative study, several participants using the standard method were faced with problems with some of the most recent lots of the test kits. Due to these problems, the standard method did not perform satisfactorily and a high number of assays were invalid, whereas the in-house methods appeared to perform better. Despite this, the overall mean results of the valid assays using both methods were in agreement. Nonetheless, it was decided to base the assignment of the potency values on the in-house methods only. The results showed that all candidate BRPs were suitable for the intended purpose. However, based on availability of the material and on the results of end-product testing, 2 candidate reference preparations, Samples C and D, were selected. Both were from the same batch but filled on different days; no statistically significant difference in potency was observed. They were thus combined in 1 single batch. The candidate preparation (Sample C/D) was adopted at the June 2009 session of the European Pharmacopoeia (Ph. Eur.) Commission as the Ph. Eur. BRP batch 1 for hepatitis A vaccine (inactivated, non-adsorbed), with an assigned potency of 12 IU/ml for in vitro antigen content assays. Accelerated degradation studies have been initiated. The preliminary data show that the BRP is stable at the recommended storage temperature (< -50 degrees C). The BRP will be monitored at regular intervals throughout its lifetime.

A role for branchpoints in splicing in vivo.

The nucleotides immediately surrounding intron/exon junctions of genes transcribed by RNA polymerase B can be derived from 'consensus' sequences for donor and acceptor splice sites by only a few base changes. Studies in vivo have underlined the importance of these junction nucleotides for splicing. In higher eukaryotes, no evidence has been found for specific internal intron sequences involved in splicing. However, the recent discovery that, in vitro, introns are excised in a lariat form where the 5' end of the intron is joined via a 2'-5'-phosphodiester linkage to an A residue (branchpoint acceptor) close to the 3' end of the intron, suggests that internal intron sequences may nonetheless be important for splicing. Indeed, in yeast nuclear genes, the internal sequence 5'-TACTAAC-3' (or close homologue) is essential for splicing in vivo. A proposed consensus sequence for branchpoints in mammalian introns is 5'-CT(A/G)A(C/T)-3'. This sequence resembles the essential yeast internal sequence. Are branchpoints involved in the splicing of introns of higher eukaryotes in vivo? We show here that a branchpoint sequence from a human globin gene (5'-CTGACTCTCTCTG-3') greatly enhances the efficiency of splicing of a 'synthetic' intron in HeLa cells. A mutated branchpoint sequence, 5'-CTCCTCTCTCTG-3', in which the branchpoint acceptor nucleotide A has been deleted and the neighbouring purine G mutated to a C, does not exhibit this enhancing capability. We conclude that branchpoints have an important function in the splicing process in vivo.

Differential expression of preproenkephalin and transin mRNAs following oncogenic transformation: evidence for two classes of oncogene induced genes.

FR3T3 rat embryo fibroblast cells express preproenkephalin mRNA after transformation by polyoma virus middle T or Ha-ras oncogenes. This effect was not seen in another rat embryo fibroblast cell line (Rat-1) or in FR3T3 cells transformed by Rous sarcoma virus, bovine papilloma virus type I or SV40. The elevation in preproenkephalin mRNA levels is thus cell specific and oncogene specific. These results contrast with those obtained for transin mRNA, which was observed in both Rat-1 and FR3T3 cells transformed by a number of different oncogenes. The expression of transin RNA correlated with the expression of the transformed phenotype. We suggest that genes induced by oncogenes in a given cell will fall into two classes: those linked to the expression of the transformed phenotype (expression induced by all oncogenes conferring this phenotype) and those induced as a consequence of activation of a specific cellular signaling system (expression induced by a subset of oncogenes linked to the signaling system in question). This system may prove useful in distinguishing oncogene-induced events that are related to cellular transformation from those that are secondary to eliciting the transformed phenotype.

International collaborative study to evaluate methods for quantification of anti-D in immunoglobulin preparations.

BACKGROUND AND OBJECTIVES: The disadvantages of autoanalyser methodology for anti-D potency estimation have prompted the search for an alternative reference method. The aim of this study was to carry out a direct comparison of autoanalyser methodology, competitive enzyme-linked immunoassay (EIA) and flow cytometry. MATERIALS AND METHODS: The anti-D potencies of nine immunoglobulin preparations were estimated against the World Health Organization (WHO) International Reference Preparation for anti-D immunoglobulin, using the three different assay methods described above, in an international collaborative study involving 18 laboratories. RESULTS: No significant differences in potency estimates for five of nine samples were identified using the three different methods. For six of nine samples the mean potency estimates obtained using competitive EIA lay between those of the autoanalyser and flow cytometry. Flow cytometry gave the lowest estimates for seven of nine samples. The range of intralaboratory variability, expressed as percentage geometric coefficients of variation (% gcv) and excluding extreme values, was as follows: autoanalyser (eight laboratories), 0.4-17.0; competitive EIA (12 laboratories), 2.6-34.8; and flow cytometry (eight laboratories), 2.9-30.0. The range of interlaboratory variability (expressed as % gcv) was: autoanalyser (eight laboratories, eight samples), 10.8-17.6; competitive EIA (12 laboratories, nine samples), 10.3-17.3; flow cytometry (nine laboratories, eight samples), 6.2-16.1. CONCLUSIONS: The study did not show clear superiority of one method over the others. Both competitive EIA and flow cytometry are acceptable alternatives to autoanalyser methodology for polyclonal anti-D potency estimation. For monoclonal anti-D, further work is necessary to determine the most appropriate method for potency testing.

Epidermal growth factor or serum stimulation of rat fibroblasts induces an elevation in mRNA levels for lactate dehydrogenase and other glycolytic enzymes.

We have isolated cloned cDNAs corresponding to five mRNAs whose level is increased following stimulation of quiescent rat fibroblasts by either epidermal growth factor or serum. Partial sequencing followed by a computer search of data banks has shown that the cloned cDNAs correspond to mRNAs encoding proteins with extensive homology to lactate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, enolase, triose phosphate isomerase, and actin. The complete nucleotide sequence of a rat fibroblast lactate dehydrogenase is presented.

Recombinant technology as an alternative to chemical peptide synthesis: expression and characterization of HIV-1 Rev recombinant peptides.

Recombinant DNA technology has been widely used for the production of proteins in recent years. In this paper, we describe the expression and the purification of two specific peptides corresponding to parts of the human immunodeficiency virus Rev protein. The strategy of this method relies on the chemical synthesis of a pair of two complementary oligodeoxynucleotides corresponding to the coding region of the peptide of interest and the subsequent cloning into a prokaryotic expression vector. Transformation of Escherichia coli with these synthetic gene constructs yielded high production levels of recombinant protein in the bacteria. The recombinant protein was composed of two moieties, one corresponding to an "affinity handle" and the second corresponding to the peptide. Chemical cleavage of the fused protein followed by a combination of affinity chromatography and rp-HPLC led to rapid and convenient peptide purification. Peptide fused to the affinity handle as well as cleaved peptide were fully characterized by N-terminal microsequencing and mass spectrometry. The data presented demonstrate that although the major recombinant products had the expected amino acid composition, we detected unexpected processing such as alternative cleavage within the signal peptide, modified cysteines, and deamidations. These results emphasize the importance of the complete characterization of recombinant products by efficient analytical tools such as N-terminal microsequencing and mass spectrometry.

Evidence that HIV-1 Rev directly promotes the nuclear export of unspliced RNA.

The Rev trans-activator of human immunodeficiency virus type 1 (HIV-1) is a protein that regulates the simultaneous appearance in the cytoplasm of both spliced and unspliced forms of viral mRNAs from the same viral transcripts by way of recognition of a target sequence termed the Rev-responsive element (RRE). Whether Rev acts directly on RNA export or by inhibition of splicing, or both, is still a matter of debate. We have addressed this issue in Xenopus laevis oocytes by microinjecting RNA molecules containing RRE along with purified recombinant Rev protein into the oocyte nuclei. Adenovirus pre-mRNA containing an RRE in the intron was spliced equally well in the absence and presence of Rev protein. Only in the presence of Rev was non-spliced pre-mRNA exported from the nucleus; more surprisingly, the excised intron lariat (containing RRE) was also exported. Furthermore, an RRE-containing mRNA molecule that lacked intron sequences was also efficiently exported from the nucleus in a Rev-dependent manner. Therefore our results demonstrate that Rev can act directly at the level of nuclear export, independent of any inhibitory effect that it may exert on the splicing of pre-mRNA. Finally, our finding that the Rev mutant M10, shown previously to be inactive in human lymphoid cells, was also unable to export RRE-containing RNA molecules from oocyte nuclei suggests that one or more cellular factors, evolutionarily conserved between humans and Xenopus, interact with Rev in both cell systems to promote nuclear RNA export.