Ph Eur Research Articles

Controlling chloroform content to a safe level in a pharmaceutical oral solution of chloral hydrate

Abstract Chloral hydrate is a halogenated hydrocarbon with sedative and hypnotic properties and has been used for its medicinal properties for many years. Its solubility and permeability determine it as a Biopharmaceutical Classification System class 1 compound, and as such is ideal to be pharmaceutically presented as an aqueous oral liquid presentation. Its primary route of degradation is by hydrolysis to form formic acid and chloroform and hence licensed pharmaceutical presentations require validated methods to analyse these degradants. The content of the toxic chloroform degradation product was determined via a head-space gas chromatograph with a flame ionisation detector, and its content was controlled to a safe pharmaceutically acceptable level. This can be achieved by solution pH optimisation in the oral solution, incorporating a buffering system to control the rate of this degradation mechanism. Where other behavioural and pharmacological therapies have failed for the treatment of short-term severe insomnia, a chloral hydrate 500 mg/5 ml oral solution presentation with an optimised safety profile has been developed as a medication for prescription. Extensive analytical and formulation development work has arrived at a room temperature stable oral solution, with a multiyear expiry date. Key findings: The use of Chloral hydrate in medicinal practice is often associated with high levels of the carcinogen chloroform as a degradation product. The research described herein has shown that the rate of formation of chloroform can be reduced by controlling the impact that the pH has on the stability of the oral solution. The Ph Eur monograph for Chloral hydrate oral solution (unlicensed medicines) does not include a criterion for chloroform quantification, hence Specials manufacturers of this oral solution are potentially risking the safety of patients without appropriate control of chloroform. This research has used validated analytical techniques to support the detection of chloroform, and other possible impurities, thus ensuring a safe product can be provided to the patient.

Intraspecific Variability of Stinging Nettle (Urtica dioica L.).

The purpose of the work was to determine the intraspecific variability of the stinging nettle, in respect of the mass of leaves and their chemical composition, including the content of phenolic compounds and assimilative pigments. The objects of the study were 10 populations of nettle, originating from the eastern and southern part of Poland. The results obtained indicate a high level of variability between and within the populations investigated but not strictly related to their geographical locations. The mass of the leaves ranged from 0.19 to 0.28 kg dry weight (DW)/plant (Coefficient of variation (CV) = 16.33%). Using HPLC-DAD, four phenolic acids were detected, i.e., caffeoylmalic (570.97-1367.40 mg/100 g DW), chlorogenic (352.79-1070.83 mg/100 g DW), neochlorogenic (114.56-284.77 mg/100 g DW) and cichoric (58.31-189.52 mg/100 g DW) acids, with the last one differentiating populations to the highest degree (CV = 48.83%). All of the analyzed populations met the requirements of the European Pharmacopoeia (Ph Eur 10th) concerning the minimum content of caffeoylmalic and chlorogenic acids in nettle leaves (not less than 0.3%). Within the flavonoid fraction, two compounds were identified, namely rutoside (917.05-1937.43 mg/100 g DW, CV = 21.32%) and hyperoside (42.01-289.45 mg/100 g DW; CV = 55.26%). The level of chlorophyll a ranged from 3.82 to 4.49 mg/g DW, chlorophyll b from 1.59 to 2.19 mg/g DW, while the content of carotenoids varied from 2.34 to 2.60 mg/100 g DW. Given all the traits investigated, the level of a population's polymorphism (CV) was visibly higher within a population than between populations. Population no. 4 was distinguished by the highest mass of leaves, and the highest content of rutoside, while population no. 2 was distinguished by the highest content of hyperoside, caffeoylmalic and chlorogenic acid.

Aseptic process validation of [18F]Sodium Fluoride radiopharmaceutical in-house production

Sodium fluoride ([18F]NaF) is a PET radiopharmaceutical for vizualization of the skeletal system and microcalcification. In the originally designed in-house method, [18F]NaF is recovered in aqueous solution after cyclotron irradiation, sterilized by passage through a 0.22 µm sterile filter and dispensed under aseptic conditions. To ensure the microbiological safety of drugs produced under aseptic conditions, validation of aseptic procedures is always recommended. This is essential for radiopharmaceuticals because most of them are released for administration before any sterility test can be completed due to their radioactive nature. This study reports the validation of the aseptic process applied to the internal production of [18F]NaF carried out in two phases: testing the number of viable microorganisms in radiopharmaceutical product prior to sterilization and process simulation studies (media fill tests). We found that all samples were sterile and the endotoxin concentration was well below the maximum acceptable level reported in the Ph Eur. monograph on [18F]NaF. The results confirmed that the entire production process of [18F]NaF can be carried out under strictly aseptic conditions following the validated procedures preserving the sterility of the final product.

Aseptic process validation of [18F]Sodium Fluoride radiopharmaceutical in-house production

Sodium fluoride ([18F]NaF) is a PET radiopharmaceutical for vizualization of the skeletal system and microcalcification. In the originally designed in-house method, [18F]NaF is recovered in aqueous solution after cyclotron irradiation, sterilized by passage through a 0.22 µm sterile filter and dispensed under aseptic conditions. To ensure the microbiological safety of drugs produced under aseptic conditions, validation of aseptic procedures is always recommended. This is essential for radiopharmaceuticals because most of them are released for administration before any sterility test can be completed due to their radioactive nature. This study reports the validation of the aseptic process applied to the internal production of [18F]NaF carried out in two phases: testing the number of viable microorganisms in radiopharmaceutical product prior to sterilization and process simulation studies (media fill tests). We found that all samples were sterile and the endotoxin concentration was well below the maximum acceptable level reported in the Ph Eur. monograph on [18F]NaF. The results confirmed that the entire production process of [18F]NaF can be carried out under strictly aseptic conditions following the validated procedures preserving the sterility of the final product. Keywords: [18F]NaF, radiopharmaceutical, validation, aseptic process

The development and validation of a high performance liquid chromatography method to determine the radiochemical purity of [177Lu]Lu-HA-DOTA-TATE in pharmaceutical preparations

Lutetium-177 [177Lu] tetra-azacyclododecanetetra-acetic acid [DOTA]-(Tyr3)-octreotate [TATE] ([177Lu]Lu-DOTA-TATE) is a radiopeptide used for peptide receptor radionuclide therapy in patients with neuroendocrine tumours (NETs). This radiopeptide is made by labelling the ligand octreotate with Lutetium-177 using the linker DOTA. After labelling, and before clinical application quality control of the radiopeptide is needed and the radiochemical purity is assessed. Acceptance limits for radiochemical purity should be within 90–110% of the label claim for radiopharmaceuticals for diagnostic use and within 95–105% of the label claim for radiopharmaceuticals for therapeutic use. Moreover, the amount of unlabelled [177Lu]LuCl3 cannot exceed 2% of the radioactive dose. Since no monograph is available for [177Lu]Lu-DOTA-TATE in the European Pharmacopeia (Ph Eur), this article describes the development and validation of a high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection and radiodetection. A Waters Acquity Arc UHPLC system equipped with a Waters 2998 photodiode array (PDA) detector was used coupled to a Berthold Lb 514 Flowstar detector equipped with a BGO-X gamma measuring cell. A reversed phase Symmetry Shield C18 column (4.6 mm × 250 mm, 5 µm) was used for chromatographic separation. A flow of 1.5 mL/min was maintained during analysis, using 0.1% TFA in water as mobile phase A and 0.1% TFA in ACN as mobile phase B. The retention time was around 1.7 min and 13.5 min for [177Lu]LuCl3 and [177Lu]Lu-HA-DOTA-TATE, respectively. Stock solutions of [177Lu]LuCl3 were made by serial dilution and were injected to test for linearity, accuracy and precision, carry over and signal-to-noise ratio. A [177Lu]Lu-HA-DOTA-TATE sample was prepared and injected to determine the carry over. The results showed that the method is linear over a range of 0.300–130 MBq/mL, which covers the range for clinical samples, provided that the clinical sample is diluted ten times before analysis. The LLOQ can be measured accurately even after dilution, with a signal-to-noise ratio of at least 5. In short, the method is accurate, precise and sensitive and can be implemented as part of the quality control of [177Lu]Lu-HA-DOTA-TATE.

Development of an updated assay for prekallikrein activator in albumin and immunoglobulin therapeutics.

BackgroundPrekallikrein activator (PKA) is a contaminating enzyme found in therapeutic albumin and immunoglobulin products. The level is commonly measured using methods such as that defined by the European Pharmacopoeia (Ph Eur) with traceability to the WHO International Standard for PKA. This method generally works well, but problems are sometimes observed.Materials and methodsA simplified one‐step method has been developed to replace the existing Ph Eur two‐step method which consists of kallikrein generation followed by kallikrein measurement using a chromogenic substrate. Analysis of data from the one‐stage method is simplified by the use of a dedicated online app.ResultsThe one‐stage method was validated against the current Ph Eur method using batches of albumin and immunoglobulins. Problem batches of immunoglobulins were investigated using the one‐stage method. Improved methodology using true initial rate determinations and use of acid‐treated prekallikrein substrate (PKS) helped understand and reduce artefactual results.ConclusionsThe one‐stage method and associated app streamline real‐time determination of PKA and promote good principles of enzyme assays to limit substrate depletion, while also conserving expensive PKS. Blanking steps and reproducibility are simplified.

New sensitive method for HEPES quantification in 68Ga-radiopharmaceuticals

BackgroundThe introduction of a GMP-certified 68Ga-generator spurred the application of 68Ga-radiopharmaceuticals. Several radiosynthesis of 68Ga-radiopharmaceuticals are more efficient and robust when performed with 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) buffer, which is considered as an impurity in the quality control (QC) procedure. Thus, prior to clinical use, QC must be conducted to ensure that HEPES does not exceed the maximum dose of 200 μg/V Injected as described in European Pharmacopoeia (Ph Eur) for edotreotide. However, when applying the thin-layer chromatography (TLC) method described in the Ph Eur to quantify the HEPES amount present in the 68Ga-octreotide or in the remaining 68Ga-radiopharmaceuticals that were tested, no amount was detectable after 4 min of iodine incubation. Here we tested our modified TLC method and validate a new high-performance liquid chromatography (HPLC) method to quantify HEPES in 68Ga-radiopharmaceuticals and compare it to the TLC-method described in Ph Eur. In addition, samples collected from various institutes were tested to evaluate whether the synthesis of different 68Ga-radiopharmaceuticals or the use of different synthesis methods could affect the amounts of HEPES.ResultsHEPES could not be detected by the TLC method described in the Ph Eur within 4 min incubation in an iodine-saturated chamber. As for our modified TLC method, only after 2 h, spots were only visible > 1 mg/mL. The HPLC method had a limit-of-quantification (LOQ) of 3 μg/mL and a limit-of-detection (LOD) of 1 μg/mL. From the three 68Ga-radiopharmaceuticals tested, only in the [68Ga]Ga-NODAGA-Exendin samples exceeding amounts of HEPES were found and its concentration in the [68Ga]Ga-NODAGA-Exendin was significantly higher, when compared to [68Ga]Ga-DOTATOC and [68Ga]Ga-PSMA-11.ConclusionThe TLC method described in Ph Eur and our modified TLC method may not be sufficiently sensitive and thus unsuitable to use for QC release. The new HPLC method was sensitive, quantitative, reproducible and suitable for QC release. With this method, we were able to determine that some 68Ga-radiopharmaceuticals may exceed the HEPES limit of 200 μg/ V Injected. This new analytical system would allow correcting for the maximum injected dose in order not to exceed this amount.

Tablets of soy protein-alginate microparticles with Lactobacillus casei 01: Physicochemical and biopharmaceutical properties

The aim of the study was to develop direct-compress-tablets of microencapsulated probiotic Lactobacillus casei 01 in soy protein-alginate microparticles and excipients able to provide probiotic delivery near the colon. Considering their physicochemical properties, all series of tablets prepared met the requirements of the Ph Eur 9.0. The compaction of the probiotic loaded microparticles caused viability decrease up to 1.5 log cycles. The tablets containing Methocel K100M showed higher potential for preserving probiotic viability in simulated gastrointestinal fluids within 4 h and retarding its release in the intestinal stage, maintaining the required minimum of viable probiotic cells above 107 CFU per gram of tablet. In addition, acceptable storage stability (viability of probiotic above 106 CFU/g) at 25?C/60% relative humidity for 42 days was observed. In conclusion, novel tablet dosage forms of microencapsulated L. casei 01 were prepared with high potential for preserving probiotic viability in simulated gastrointestinal fluids and retarding its release in the lower intestine. Further research is needed to optimize the formulation and process parameters in order to obtain tablets with probiotic viability over long storage periods.

Formulation design and development of matrix diffusion controlled transdermal drug delivery of glimepiride.

BackgroundThe present work was conducted to prepare and evaluate transdermal patches with optimization of suitable polymeric blend of poly(meth) acrylates (Eudragit®) (Ammonio Methacrylate Copolymer Ph Eur) for sustained transdermal delivery of glimepiride.MethodPolymeric matrix transdermal films were prepared by using Ammonio Methacrylate Copolymer Ph Eur RL 100 and Ammonio Methacrylate Copolymer Ph Eur RS 100 as the film former, and dibutyl phthalate (30% w/w) as the plasticizer. Patches were characterized by physical appearance, thickness, weight variation, folding endurance, percentage erosion, swelling index, moisture content, and moisture uptake capacity. Fourier transform infrared spectroscopic studies and differential scanning calorimetry analysis of physical mixtures of contents were performed to identify any chemical and physical interaction between drug and excipients. Five different enhancers (isopropyl myristate [IPM], Span® 80, Tween® 20, eucalyptus oil, and limonene) were employed at three different concentrations of polymer (2%, 5%, and 10% w/w) in order to enhance permeation through rabbit skin. In vitro drug release studies were performed at pH 7.4, and scanning electron microscopy was conducted to elucidate surface morphology before and after the drug release. In vitro permeation studies through rabbit skin were performed on Franz diffusion cells and permeation kinetics followed the Higuchi model.ResultsResults of in vitro permeation studies revealed that these enhancers not only increased drug release but also augmented the skin permeation of glimepiride.ConclusionIPM was the most effective enhancer with the highest permeation flux of 51.763 μg/cm2/hr, and the enhancement effect of different enhancers on glimepiride permeation through rabbit skin was in the rank order of IPM > eucalyptus oil > Span® 80 > Tween® 20> limonene.

In-use microbiological assessment of caffeine citrate 10 mg/mL oral solution

ObjectivesThis study was conducted to investigate the microbial contamination of caffeine citrate 10 mg/mL oral solution (CCOS) during a simulated in-use test in a clinical environment.MethodsA real-time in-use simulation study...

European Pharmacopoeia: An Optimal Guarantee For The Quality Of Herbals

The European Pharmacopoeia (Ph Eur) lays down mandatory quality and standards for the production and control of medicines in Europe. The standards of the Ph Eur are elaborated, permanently updated and revised by the Group of Experts such as the Phytochemistry Groups 13A and 138 and the TCM Working Party.

Pharmacopoeia Monographs for Chinese Herbal Drugs for Quality Assurance

The consistent request of European patients for a safe access to Chinese Materia Medica (CMM) was the basis, almost 10 years ago, to accept and implement the respective quality monographs for the European Pharmacopoeia (Ph Eur). The safe use of TCM herbal drugs relies on the correct definition of the material in question and further a detailed pharmacognostic botanical and chemical identification. Good agricultural and collection practice (GACP) and all the necessary quality parameters based on Pharmacopoeia standards are an essential prerequisite. However, the existing quality standards of the Chinese Pharmacopoeia (Ch P), even in their English version, could not be directly adopted, due to significant differences between European- and the Chinese Pharmacopoeian legal requirements. Consequently, an experimental re-examination and modification, followed by corresponding validation procedures as well as in some cases, introduction of new analytical methods had to be applied to pre-existing Ch P TCM herbal drug monographs prior to an acceptance by the Ph Eur Commission.

The role of the European Pharmacopoeia (Ph Eur) in Quality Control of Traditional Chinese Herbal Medicine in European Member States

In order to assure the safety and efficacy of the Chinese Medicines in Europe, the quality of TCM herbals should be guaranteed so that they can be freely imported in the European Union and other Western European Countries which are signatories of the European Pharmacopoeia Convention. Consequently, new Ph Eur TCM herbal drug Monographs should be elaborated, based on preexisting Monographs in the Chinese Pharmacopoeia (ChP) 2010. Such a program has been inaugurated in 2005 by the Ph Eur Groups of Experts 13 A and B (Phytochemistry). Since then good progress has been made, elaborating of about one third of the originally proposed 100 TCM herbals being identified as important monographs for the European Market. Taking into account the many challenges still laying ahead, the establishment of a specialized Working Party (WP) on TCM with specialists and experts from many EU Member States has been decided by the Ph Eur Commission in 2008 which is highly active ever since in the examination and elaboration of new TCM herbal drug monographs, primarily to assure the safety of the European patient and further to provide quality parameters extremely important for all registration and licensing procedures of the respective National Authorities all over Europe. This paper is a survey of results and difficulties obtained so far which has been encountered meanwhile in the elaboration process by the Ph Eur TCM WP of these monographs and will discuss these in detail. Moreover the role of Ph Eur TCM monographs in the European community is addressed.

TCH-012 Differences in Purity Between Biosimilar Filgrastims and Copy Biological Filgrastims

Background Biosimilars are follow-on versions of peptide biological drugs, and differences in manufacturing and formulation can result in variations in physicochemical and clinical profiles. The European Medicines Agency (EMA) has...

TCH-018 Formulation of an Oral Solution Containing “Potion JouliÈ” Phosphorus to Counteract the Shortage of Phosphoneuros

Background In October 2011 a shortage of Phosphoneuros became apparent. This oral solution containing phosphorus is prescribed in the treatment of diseases where phosphorus intake is essential. None of the...

HPTLC detection: an appropriate preparation of spraying and dipping solutions

In thin-layer chromatography, for the purpose of post-chromatographic derivatization, the specific derivatization reagent solution can be transferred onto the plate by spraying or dipping. This TLC step of detection, or derivatization, was studied with a view to elaborating a standardized approach. The focus was on the solvent used to prepare the derivatization solutions, and also on the concentrations of these reagent solutions. For this purpose, qualitative results from both spraying and dipping experiments were compared. As herbal raw material, four flavonoid-containing drugs were used: ginkgo leaf, birch leaf, passion flower, and hawthorn leaf and flower. The specific derivatization reagent was natural product/macrogol 400; on the one hand dissolved in methanol, and on the other hand dissolved in ethyl acetate/dichlormethane [1]. The spraying as well as dipping procedures were carried out with appropriate automatic devices. Qualitative results showed that, in each case, derivatization reagents dissolved in ethyl acetate/dichlormethane showed concise zones, and weakly pronounced zones were also clearly visible. By contrast, dipping the plate into a derivatization solution containing methanol led partly to indistinct zones. Thus, post-chromatographic derivatization by dipping required an adaptation of the original spraying reagent composition, i.e. specific solvent and concentration, to obtain comparable results between spraying and dipping. Such standardization of TLC steps may be helpful in the goal of harmonizing Ph Eur monographs.

Spectrophotometric determination of acteoside in the herbal drugs Plantaginis lanceolatae folium and Plantaginis majoris folium

Plantaginis lanceolatae folium (Plantago lanceolata L., Plantaginaceae) and Plantaginis majoris folium (P. major L.) are well known herbal drugs that have been used for treating inflammation of the mouth and throat. Chemical investigations have detected various constituents. Acteoside as phenylethanoid is a major phenolic constituent and reportedly has antiphlogistic, antihepatotoxic, antioxidant, spasmolytic, antimetastatic and antiviral activity. According to the requirement of Ph Eur Plantaginis lanceolatae folium should contain not less than 1.5% of total ortho-dihydroxycinnamic acid derivatives expressed as acteoside calculated with reference to the dried drug.

IL CONTROLLO DI QUALITÀ NELL’IMPIEGO DELLA PCR APPLICATA ALLA DETERMINAZIONE QUALITATIVA DELL’HCV-RNA.

Detection of hepatitis C virus (HCV) RNA in samples of plasma/serum has become an essential part of the diagnosis and management of HCV-infected patients. Qualitative HCV-RNA tests are used to identify acute HCV infections as well as chronic HCV carriers.In recent years,a variety of commercial and non commercial test systems have been developed for this purpose. Each of these methods is calibrate with proprietary standards and exhibits its own sensitivity (detection limit) and specificity. Obviously, laboratories performing HCV-RNA test should report accurate and reliable results regardless of the type of assay used.Where commercial kit are used for part of or the complete analytical procedure, documented validation points already covered by the kit manufacturer can substitute for the validation by the user.Nevertheless, the performance of the kit with respect to its intended use has to be demonstrated by the user. One of the best ways to assess the performance of individual laboratories for validation of qualitative HCV-RNA test is determine: 1. Specificity. In order to validate the specificity of the analytical procedure, at least 100 HCV-RNA-negative plasma pools should be tested and shown to be non-reactive. 2. Positive cut-off point (detection limit/sensitivity).The positive cut-off point (as defined in the Ph Eur General Method 2. 6. 21) is the minimum number of the target sequences per volume sample which can be detected in 95% of test runs.A dilution series of a working reagent or reference material, which has been calibrated against the WHO HCV International Standard (96/790), should be tested on different days to examine variation between test runs.At least 3 independent dilution series should be tested with a sufficient number of replicates at each dilution to give a total number of 24 test results for each dilution to enable a statistical analysis of the results; 3. Robustness.To demonstrate robustness, at least 20 HCV-RNA negative plasma pools (selected at random) and spiked with HCV-RNA to a final concentration of 3 times the previously determined 95% cut-off value should be tested and found positive; 4. Cross-contamination error.Cross-contamination prevention should be demonstrated by the accurate detection of a panel of at least 20 samples consisting of alternate samples of negative plasma pools and negative plasma pools spiked with high concentrations of HCV (at least 102 x the 95% cut-off value or at least 104 IU/ml).

Il controllo di qualità nell’impiego della PCR applicata alla determinazione qualitativa dell’HCV-RNA

Detection of hepatitis C virus (HCV) RNA in samples of plasma/serum has become an essential part of the diagnosis and management of HCV-infected patients. Qualitative HCV-RNA tests are used to identify acute HCV infections as well as chronic HCV carriers.In recent years,a variety of commercial and non commercial test systems have been developed for this purpose. Each of these methods is calibrate with proprietary standards and exhibits its own sensitivity (detection limit) and specificity. Obviously, laboratories performing HCV-RNA test should report accurate and reliable results regardless of the type of assay used.Where commercial kit are used for part of or the complete analytical procedure, documented validation points already covered by the kit manufacturer can substitute for the validation by the user.Nevertheless, the performance of the kit with respect to its intended use has to be demonstrated by the user. One of the best ways to assess the performance of individual laboratories for validation of qualitative HCV-RNA test is determine: 1. Specificity. In order to validate the specificity of the analytical procedure, at least 100 HCV-RNA-negative plasma pools should be tested and shown to be non-reactive. 2. Positive cut-off point (detection limit/sensitivity).The positive cut-off point (as defined in the Ph Eur General Method 2. 6. 21) is the minimum number of the target sequences per volume sample which can be detected in 95% of test runs.A dilution series of a working reagent or reference material, which has been calibrated against the WHO HCV International Standard (96/790), should be tested on different days to examine variation between test runs.At least 3 independent dilution series should be tested with a sufficient number of replicates at each dilution to give a total number of 24 test results for each dilution to enable a statistical analysis of the results; 3. Robustness.To demonstrate robustness, at least 20 HCV-RNA negative plasma pools (selected at random) and spiked with HCV-RNA to a final concentration of 3 times the previously determined 95% cut-off value should be tested and found positive; 4. Cross-contamination error.Cross-contamination prevention should be demonstrated by the accurate detection of a panel of at least 20 samples consisting of alternate samples of negative plasma pools and negative plasma pools spiked with high concentrations of HCV (at least 102 x the 95% cut-off value or at least 104 IU/ml).

Collaborative study for the establishment of a European Phamacopoeia biological reference preparation for Bordetella pertussis mouse antiserum for serological potency testing of acellular pertussis vaccines

A collaborative study was organised by the European Directorate For the Quality of Medicines (EDQM) to assess the suitability of a candidate mouse antiserum as a European Pharmacopoeia Biological reference preparation (BRP) for acellular pertussis vaccine potency testing. The candidate antiserum was obtained by immunising mice with a five-component acellular pertussis vaccine: pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN) and Fimbrial 2/Fimbrial 3 (Fim 2&3). The study has been divided into two separate phases. Phase I was a pre-qualification study including three laboratories. This phase was aimed at pre-qualifying the candidate BRP (cBRP) and at documenting the impact of differences in the antibody detection methodology enzyme linked immunosorbent assay (ELISA) procedures on results of pertussis antisera calibration versus the currently used standard US standard pertussis antiserum (mouse) Lot 1 (SPAM-1) (United States Food and Drug Administration (USFDA) reference serum) and the cBRP. As no significant difference between the antibody titres determined by using the different ELISA methodologies was found, a large-scale study enrolling 13 laboratories (Phase II) was carried out, each participant performing its in-house methodology. Its aim was to calibrate the cBRP (in terms of the SPAM-1 reference) and to demonstrate its equivalence or superiority to internal references. The study showed that there was no difference in positive sera titres expressed relative to their corresponding internal reference (homologous situation) or the proposed standard (heterologous situation) reference. The cBRP can, therefore, reliably act as replacement for the in-house reference preparations. Further analysis of the outcome of this study enabled to assign to the cBRP a potency of 39, 138, 34 and 56 ELISA unit per millilitre, respectively, to its anti-PT, anti-FHA, anti-PRN and anti-Fim 2&3 antibody contents. The cBRP has been adopted by the European Pharmacopoeia Commission at its June 2000 session as Bordetella pertussis mouse anti-serum Ph Eur. BRP batch 1.