Oversulphated Chondroitin Sulphate (OSCS) and Dermatan Sulphate (DS) in unfractionated heparins can be identified by nuclear magnetic resonance spectrometry (NMR). The limit of detection (LoD) of OSCS is 0.1% relative to the heparin content. This LoD is obtained at a signal-to-noise ratio (S/N) of 2000:1 of the heparin methyl signal. Quantification is best obtained by comparing peak heights of the OSCS and heparin methyl signals. Reproducibility of less than 10% relative standard deviation (RSD) has been obtained. The accuracy of quantification was good.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 1, the World Health Organisation (WHO) 3rd International Standard, Human (IS, 96/854) and the FDA Standard for human blood coagulation Factor IX concentrate have been available since 1996, following their establishment by a common collaborative study. Due to dwindling stocks of all three standards, a new WHO-EDQM-FDA tri-partite collaborative study was launched to establish replacement batches. Thirty laboratories from fourteen countries took part in the collaborative study to assign potency values to candidate preparations. Three candidates, one of recombinant and two of human plasma-derived origins, were assayed against the 3rd IS for Blood Coagulation Factor IX, Concentrate, Human (96/854). The 3rd IS for Blood Coagulation Factors II, VII, IX and X, Plasma, Human (99/826) was also included to evaluate the relationship between the factor IX plasma and concentrate unitage. Thirty-two sets of clotting assay results and two sets of chromogenic assay data were analysed. There was a significant difference in potency estimates by these two methods for the recombinant candidate (sample B) and the plasma IS (sample P). Similar potency values were obtained for the plasma derived products (monoclonal antibody- and chromatography-purified factor IX, samples C and D) by clotting and chromogenic assays. For the clotting assays, intra-laboratory variability (GCV) was found to range from 0.5 - 21.7%, with the GCV for the majority of laboratories being less than 10%. Good inter-laboratory agreement, with the majority of the GCV being less than 10% (GCV range = 4.7 - 10.6 %) was also obtained. The mean potency values estimated by the clotting assay using plasma as pre-diluent (as directed by the Ph. Eur. general chapter method) did not differ from values obtained using buffer. Taking into account the preliminary stability data, the intra- and inter-laboratory variability, and the differences between the clotting and chromogenic assay results, sample C (07/182) was established as the Human coagulation factor IX concentrate BRP batch 2, with a potency value of 7.9 IU/ampoule assigned with clotting assay results. As an outcome of this tri-partite collaborative study, the same sample C (07/182) has also been adopted as the 4th International Standard for Blood Coagulation Factor IX, Concentrate, Human by the Expert Committee on Biological Standardisation (ECBS) of the World Health Organisation (WHO), and as the replacement batch for the reference standard for Human coagulation factor IX concentrate by the FDA.

The European Pharmacopoeia (Ph. Eur.) and the World Health Organization (WHO) require the performance of extensive quality control testing including a potency test before a vaccine batch is released for human use. Whole cell pertussis (wP) vaccine potency is assessed by a mouse protection test (MPT) based on the Kendrick test. This test compares the vaccine dose necessary to protect 50% of mice against the effect of a lethal intracerebral dose of Bordetella pertussis and the dose of a suitable reference vaccine needed to give the same protection level. Due to the large variability in the results of this test and the severe distress which is inflicted on the many animals involved, its replacement by an alternative method is highly desirable. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM) of the Council of Europe, in collaboration with the WHO and the In-vitro toxicology Unit/European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission (EC) Joint Research Centre-Institute for Health and Consumer Protection (JRC-IHCP), wP vaccine specialists from all over the world were invited to present an overview of candidate alternatives at a symposium organised in Geneva (Switzerland) in March 2005. Although no alternative method was found suitable for immediate implementation of batch potency control, the Pertussis Serological Potency Test (PSPT), initially developed in mice and recently transferred to guinea pigs (gps), was identified as a model of interest. Using the PSPT in gps to test several components of combined vaccines such as Diphtheria-Tetanus-wP vaccines in the same animal series would allow further implementation of the European 3Rs policy to batch potency control, by additional method refinement and reduction of animal use. The present study evaluated 2 features of the serological response to wP vaccination: 1) the overall antibody response as measured by a "whole cell" ELISA (PSPT-wC-ELISA) which uses the B. pertussis 18323 challenge strain prescribed for the MPT to coat the assay plates and 2) the functional neutralising antibodies to pertussis toxin (PT, one of the main virulence factors of B. pertussis), as measured by the Chinese Hamster Ovary (CHO) cell assay. The results showed that 1) the gp model can be used for wP vaccine potency testing; 2) despite good repeatability and precision, the CHO cell assay did not generate results comparable to the MPT. Moreover, the CHO cell assay showed significant differences in the ability of wP vaccines to induce neutralising anti-PT antibodies, which did not correlate to the overall antibody response evaluated by PSPT-wC-ELISA; 3) comparable potencies were obtained in the MPT and the PSPT-wC-ELISA. This study, supported by the previous ones correlating the PSPT-wC-ELISA in mice with the MPT, confirms that PSPT-wC-ELISA in gps is a promising approach for batch release potency testing of wP vaccines for which consistency in production has already been demonstrated by the MPT. However, a large scale validation study is required prior to the adoption of PSPT-wC-ELISA as a compendial reference method for wP vaccines batch release control.

A collaborative study was run by the European Directorate for the Quality of Medicines and HealthCare (EDQM) under the aegis of the Biological Standardisation Programme (BSP) to establish replacement batches of the current Prekallikrein activator in albumin Biological Reference Preparation (BRP) batch 1, the stocks of which were dwindling. Candidate BRP replacement batch 2 and batch 3 were assayed against the 2nd World Health Organization International Standard for Prekallikrein activator, human (2nd IS) and the Prekallikrein activator in albumin BRP batch 1. The candidate batches were manufactured from the same starting material as the current Biological Reference Preparation and the 2nd IS. They consisted of a 20 % solution of albumin lyophilised under the same conditions as the Prekallikrein activator in albumin BRP batch 1. Sixteen laboratories participated in the collaborative study and were requested to assay the candidates by their routine method, complying with the European Pharmacopoeia (Ph. Eur.) general method 2.6.15 for the determination of prekallikrein activator content. A central statistical analysis was performed at the EDQM using in-house calculations of prekallikrein activator contents provided by the participating laboratories. On the basis of the results of this study, which confirmed the assigned potency of 29 IU/vial of Prekallikrein activator in albumin BRP batch 1, the 2 candidate materials were assigned a potency of 30 IU/vial. The 2 candidates were adopted by the Ph. Eur. Commission in March 2008 as Ph. Eur. Prekallikrein activator in albumin Biological Reference Preparation batch 2 and batch 3.

The European Pharmacopoeia (Ph. Eur.) monograph on Erythropoietin concentrated solution (1316) specifies that identification and assay are performed using pharmacopoeial methods requiring the use of a reference preparation. To replace the current erythropoietin Biological Reference Preparation (BRP) of Ph. Eur., in 2006, the European Directorate for the Quality of Medicines undertook a collaborative study designed to establish a replacement batch. In order to guarantee continuity, the formulation of the candidate batch was similar to that of previous batches (1 and 2). The methods chosen to qualify the new standard were those included in the current monograph. The study was defined to allow calibration of the candidate by in vivo bioassay in terms of the current World Health Organization (WHO) International Standard (IS) and to assign a unitage. The suitability of the candidate preparation to serve as a reference standard for the other pharmacopoeial analytical procedures was also investigated. The collaborative study involved 16 laboratories from Europe, Australia, Canada, China, Japan, South Korea and the United States of America. Participants carried out biological and physicochemical assays on the candidate erythropoietin BRP batch 3 (cBRP3), using batch 2 (BRP2) and where necessary the 2nd World Health Organization International Standard (WHO 2nd IS) for recombinant erythropoietin as the reference standards. It was demonstrated that the replacement batch is appropriate for use as erythropoietin BRP in the context of the control of erythropoietin concentrated solutions according to the Ph. Eur. monograph (1316). However as regards the potency of BRP2 and cBRP3 in the mouse bioassay unexpected observations were made. Direct calibration of BRP2 against the WHO 2nd IS yielded, in all laboratories, results that were systematically higher than the potency of 32,500 IU/vial assigned by direct calibration against the WHO 2nd IS in the former study. It was therefore recommended to assign the potency of cBRP3 against BRP2, using the average of all results that were not considered as outlying obtained in the collaborative study, in order to guarantee continuity of unitage between the successive BRP batches. The outcome of the study enabled the Ph. Eur. Commission to establish the proposed standard as 'erythropoeitin BRP batch 3' (BRP3). BRP3 was established in June 2007 for use as a reference preparation for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 35,280 IU/vial, the identification by capillary zone electrophoresis, by polyacrylamide gel electrophoresis, immunoblotting and peptide mapping and as a reference for checking the system suitability of size-exclusion chromatographic procedures used in the test for dimers and related substances of higher molecular mass in the Ph. Eur. monograph (1316).

An international collaborative study involving fourteen laboratories has taken place, organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) with National Institute for Biological Standards & Control (NIBSC) (in its capacity as a World Health Organisation (WHO) Laboratory for Biological Standardisation) to provide supporting data for the establishment of replacement batches of Heparin Low-Molecular-Mass (LMM) for Calibration Chemical Reference Substance (CRS), and of the International Reference Reagent (IRR) Low Molecular Weight Heparin for Molecular Weight Calibration. A batch of low-molecular-mass heparin was donated to the organisers and candidate preparations of freeze-dried heparin were produced at NIBSC and EDQM. The establishment study was organised in two phases: a prequalification (phase 1, performed in 3 laboratories in 2005) followed by an international collaborative study (phase 2). In phase 2, started in March 2006, molecular mass parameters were determined for seven different LMM heparin samples using the current CRS batch and two batches of candidate replacement material with a defined number average relative molecular mass (Mn) of 3,700, determined in phase 1. The values calculated using the candidates as standard were systematically different from values calculated using the current batch with its assigned number-average molecular mass (Mna) of 3,700. Using raw data supplied by participants, molecular mass parameters were recalculated using the candidates as standard with values for Mna of 3,800 and 3,900. Values for these parameters agreed more closely with those calculated using the current batch supporting the fact that the candidates, though similar to batch 1 in view of the production processes used, differ slightly in terms of molecular mass distribution. Therefore establishment of the candidates was recommended with an assigned Mna value of 3,800 that is both consistent with phase 1 results and guarantees continuity with the current CRS batch. In phase 2, participants also determined molecular weight parameters for the seven different LMM heparin samples using both the 1st IRR (90/686) and its Broad Standard Table and the candidate World Health Organization (WHO) 2nd International Standard (05/112) (2nd IS) using a Broad Standard Table established in phase 1. Mean molecular weights calculated using 2nd IS were slightly higher than with 1st IRR, and participants in the study indicated that this systematic difference precluded establishment of 2nd IS with the table supplied. A replacement Broad Standard Table has been devised on the basis of the central recalculations of raw data supplied by participants; this table gives improved agreement between values derived using the 1st IRR and the candidate 2nd IS. On the basis of this study a recommendation was made for the establishment of 2nd IS and its proposed Broad Standard Table as a replacement for the 1st International Reference Reagent Low Molecular Weight Heparin for Molecular Weight Calibration. Unlike the 1st IRR however, the candidate material 2nd IS is not suitable for use with the method of Nielsen. The candidate materials were established as heparin low-molecular-mass for calibration batches 2 and 3 by the Ph. Eur. Commission in March 2007 and as 2nd IS low-molecular-weight heparin for molecular weight calibration (05/112) by the Expert Committee on Biological Standardization in November 2007.

An international collaborative study was organised to replace the current European Pharmacopoeia biological reference preparation for heparin sodium. The project was organised by the European Directorate for the Quality of Medicines & HealthCare in the frame of its Biological Standardisation Programme. A suitable candidate batch representative of the quality of heparin products currently marketed in Europe was donated to the EDQM and included in a collaborative study involving 19 laboratories from 10 European countries, the Americas, Australia and the Council of Europe. Laboratories were requested to perform their routine assays following the prescriptions of the Ph. Eur. for the assay and the identification of unfractionated heparin and for the assay of protamine. The results made it possible to demonstrate that the candidate batch was suitable for its intended use and it was therefore established by the European Pharmacopoeia Commission as the Ph. Eur. heparin sodium BRP batch 3 in June 2007.

The biological nature of vaccines imposes a permanent risk for contamination with extraneous agents. Therefore, testing of vaccines for freedom from extraneous agents is essential in the manufacturing process and quality control. Relevant methods for testing for extraneous agents of avian viral vaccines are specified in the monographs of the European Pharmacopoeia (Ph. Eur.). Currently, most of these methods involve the use of embryonated eggs or chickens. Polymerase chain reaction (PCR) is a widely used and suitable tool for the amplification and detection of extraneous nucleic acids. Different PCR assays have been developed for the application in routine testing of veterinary vaccines. However, before introduction of new methods in monographs of the Ph. Eur., they must undergo validation. Here we report about a pre-validation study performed in Official Medicines Control Laboratories (OMCLs). Diluted samples of avian infectious laryngotracheitis, avian infectious bronchitis and avian infectious bursal disease viruses have been analysed using standardised procedures and reagents. The study demonstrated that PCR methods can be transferred to other laboratories. The results also show that further work is warranted for full validation of the method.

In 2004, the Office International des Epizooties (OIE) Expert Surveillance Panel on equine influenza recommended that the American lineage component (H3N8) of equine influenza vaccines (A/eq/Newmarket/1/93-like) be updated to an A/eq/South Africa/4/03-like virus. As a consequence the common European Pharmacopoeia (Ph. Eur.) - OIE reference for equine influenza subtype 2 American-like antiserum had to be complemented by an antiserum raised in horses against an A/eq/South Africa/4/03 strain. An international collaborative study run by the European Directorate for the Quality of Medicines (EDQM) in the frame of its Biological Standardisation Programme (BSP) under the aegis of the Ph. Eur. and the OIE was organised. The study was aimed at evaluating a candidate reference horse anti-serum using the single radial haemolysis (SRH) and haemagglutination inhibition (HI) tests. The standard was to be established for use in immunogenicity and batch potency assay of equine influenza vaccines as a Ph. Eur. BRP and for use in clinical diagnostic tests as an OIE-approved International Standard. The evaluation performed in the collaborative study enabled the suitability of the candidate to be demonstrated and an SRH value to be assigned. The candidate was adopted as a BRP by the Ph. Eur. Commission and approved by the OIE Biological Standards Commission as an International Standard Serum in June and September 2006, respectively.

The European Directorate for the Quality of Medicines (EDQM) supplies Chemical Reference Substances (CRS) for Interferon (IFN) alfa-2a (CRS I0320300) and for IFN alfa-2b (CRS I0320301) for specified physicochemical tests. However, no information is provided as to their biological activity. In contrast, the World Health Organization (WHO) provides the 2nd International Standards (IS) for IFN alfa-2a (code 95/650) and for IFN alfa-2b (code 95/566), with activity defined in International Units (IU) for calibration of biological activity of preparations of IFN. We have compared the EDQM CRSs with the WHO ISs in two bioassay systems, one measuring the anti-proliferative activity in the Daudi cell line and the other measuring a reporter gene activation in an A549 cell line. In each of these assay systems, the CRSs gave dose - response relations, which were similar to those for the WHO ISs. Estimates of relative activity for each CRS, in terms of the respective IS, showed specific biological activity for the CRSs of the same order as the nominal specific activity for the ISs. However, the estimates of relative activity were not consistent between the two assays systems, emphasizing the need for calibration within each system, if the CRS were to be used as a working standard for bioassays. For structure-activity studies, both physicochemical and biological activity characterisation are required for the same biopharmaceutical preparation. CRS I0320300 and CRS I0320301 may prove useful as working standards for some bioassay systems.

The study is a contribution to the EDQM's efforts to meet some of the expectations of the 3 Rs: Replacement, Reduction and Refinement of animal assays as proposed by Russell and Burch in 1959 and adopted by the European Union in 1986, and specifically to validate alternative assays to replace, for batch-release purposes, the European Pharmacopoeia (Ph. Eur.) in vivo direct challenge procedures for the potency determination of diphtheria toxoid vaccines. The study results may be used in support of the replacement of the multi-dilution direct challenge procedures in different animal models by a single dilution serology test, where appropriate, and to use sera from the same animals for potency testing of several components in combined vaccines. With regard to the latter, the present study explores the possibility of testing both diphtheria and tetanus toxoid potencies using serum from the same animals.

European Pharmacopoeia (Ph. Eur.) general chapter 2.6.7. Mycoplasma requires for the culture test reference strains of mycoplasma field isolates with fewer than 15 passages for validation and run control and in the test for inhibitory substances. Low passage field isolates of 5 mycoplasma strains (Mycoplasma hyorhinis, Mycoplasma synoviae, Mycoplasma fermentans, Mycoplasma orale and Acholeplasma laidlawii) have been prepared for this purpose and a small scale collaborative study involving European laboratories was carried out to confirm the suitability of the material for the intended purpose. Strains were prepared as 1 ml samples in frozen format and are stored below -60 degrees C. Each laboratory determined a titre for the material on their in-house media. A secondary part of the study also compared the growth of prediluted samples on the different culture media. Results of the study confirm that the material is suitable for use as a biological reference preparation (BRP) and an estimated titre has been provided for each strain based on the results of the study. It was noted that differences in the culture media used in the different laboratories did not have a detrimental effect on titre estimation. The estimated titre is intended as a guide for users to validate the use of the reference material in house. The candidate BRPs were adopted by the European Pharmacopoeia Commission on June 28, 2006 and are available for use from EDQM. A revision to chapter 2.6.7, including reference to the use of nucleic acid amplification techniques (NAT) was also adopted in June 2006 and will appear in the European Pharmacopoeia version 5.8 in January 2007 and come into force the 1st of July 2007. While it was not part of the study a number of participants also performed in-house NAT assays on the study material. Preliminary findings from these studies are presented.

An international collaborative study was organised to establish a European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) and United States (US) Food and Drug Administration (FDA) reference preparation for the test for anti-D (anti-Rho) antibodies in human normal immunoglobulin for intravenous administration (IGIV). A candidate positive control (IGIV+anti-D) and negative control IGIV were compared to corresponding World Health Organization (WHO) International Reference Reagents using a direct haemagglutination reference method. Sixteen (16) laboratories participated in the collaborative study. Further to completion of the study, the materials assayed in the study were granted the status of Ph. Eur. and US FDA reference preparations for controlling the levels of anti-D in IGIV.

A study was carried out by the European Directorate for the Quality of Medicines (EDQM) as part of the joint Biological Standardisation Programme of the Council of Europe and the European Commission with the aim to establish replacement batches of the European Pharmacopoeia (Ph. Eur.) human immunoglobulin Biological Reference Preparation (BRP) batch 2. Twenty-eight laboratories participated in this study. The suitability of the candidate reference preparations to serve as working references in the tests for distribution of the molecular size, anticomplementary activity and Fc function, in accordance with the specifications of the Ph. Eur. monographs Human normal immunoglobulin for intravenous administration (0918), Human normal immunoglobulin (0338) and Anti-T lymphocyte immunoglobulin for human use, animal (1928) was demonstrated. The candidates were therefore established as human immunoglobulin BRP batch 3 and Human immunoglobulin (molecular size) BRP batch 1. The prescribed use of the latter BRP is limited to the test for distribution of molecular size.

A project was run for the establishment of replacement batches of the European Pharmacopoeia (Ph. Eur.) Somatropin Chemical Reference Substance (CRS) batch 1. Twenty two laboratories from 16 countries took part in a collaborative study aimed at demonstrating the suitability of the candidate reference preparations to serve as working references in the tests for identification by peptide mapping and capillary electrophoresis (CE); related proteins, dimers and related substances of higher molecular mass; charged variants distribution; and/or for the assay of somatropin, as performed in accordance with the specifications of the current Ph. Eur. monographs 0950 Somatropin bulk solution, 0951 Somatropin and 0952 Somatropin for injection. Further to the completion of the study the Ph. Eur. Commission adopted one candidate in March 2006 as somatropin CRS batch 2 (with an assigned content of 1.69 mg somatropin monomer per vial) and the second one in June 2006 as somatropin/desamidosomatropin resolution mixture CRS batch 1 (prescribed use of the latter standard is restricted to the test for related proteins).