Evaluation of candidate International Standards for meningococcal capsular polysaccharide groups W and Y.

Two candidate International Standards for meningococcal capsular group W and Y (MenW and MenY, respectively) polysaccharides were assessed for their suitability as quantitative standards in various physicochemical assays. The study was designed to evaluate the intended purpose of these standards, namely, to standardize the quantification of the respective polysaccharide content in meningococcal polysaccharide and conjugate vaccines and their intermediate components. Twelve laboratories from eleven different countries participated in the collaborative study of candidate preparations for International Standards for MenW and MenY polysaccharide (coded 16/152 and 16/206, respectively). Unitage was assigned using the Resorcinol assay. Our proposals, on the basis of data from the Resorcinol assay were: 1) candidate standard for MenW polysaccharide (16/152) to be assigned a content of 1.015 ± 0.071 mg MenW polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.13, corresponding to a 95 % level of confidence) and 2) candidate standard for MenY polysaccharide (16/206) be assigned a content of 0.958 ± 0.076 mg MenY polysaccharide per ampoule (expanded uncertainty with coverage factor k = 2.26, corresponding to a 95 % level of confidence). The amount of polysaccharide per ampoule remained consistent under all stability conditions over a 36-month period.

Development of a stable chemically cross-linked erythropoietin dimer for use in the quality control of erythropoietin therapeutic products.

Erythropoietin (EPO) is a glycoprotein hormone which promotes red cell replenishment and is also a global biotherapeutic medicine widely used to treat anaemia resulting, for example, from chemotherapy. Requirements of the European Pharmacopoeia stipulate that the level of dimer must be quantified in clinical EPO products (with a limit of 2%). Quantification is hampered by the lack of reference preparations containing stable measurable levels of EPO dimer, but the reproducible generation of a stable dimerised EPO preparation is challenging. We describe here the development of a lyophilised, chemically cross-linked EPO preparation, which has good stability and may be used for calibration and system suitability assurance for the size exclusion chromatographic separation of EPO preparations. Graphical abstract.

Investigating Alternative Container Formats for Lyophilization of Biological Materials Using Diphtheria Antitoxin Monoclonal Antibody as a Model Molecule.

When preparing biological reference materials, the stability of the lyophilized product is critical for long-term storage, particularly in order to meet WHO International Standards, which are not assigned expiry dates but are expected to be in use for several decades. Glass ampoules are typically used by the National Institute for Biological Standards and Control (NIBSC) for the lyophilization of biological materials. More recently, a clear need has arisen for the filling of smaller volumes, for which ampoules may not be optimal. We investigated the use of plastic microtubes as an alternative container for small volume fills. In this study, a recombinant diphtheria antitoxin monoclonal antibody (DATMAB) was used as a model molecule to investigate the suitability of plastic microtubes for filling small volumes. The stability and quality of the dried material was assessed after an accelerated degradation study using a toxin neutralization test and size exclusion HPLC. While microtubes have shown some promise in the past for use in the lyophilization of some biological materials, issues with stability may arise when more labile materials are freeze-dried. We demonstrate here that the microtube format is unsuitable for ensuring the stability of this monoclonal antibody.

Use of dynamic mechanical analysis (DMA) to determine critical transition temperatures in frozen biomaterials intended for lyophilization.

Dynamic mechanical analysis is widely used to determine glass transitions in solid state materials. However, here we demonstrate the application of DMA for the determination of glass transitions (Tg') in the frozen liquid state by means of a steel sample pocket. The use of the pocket allows frozen material to be analysed and glass transition events demonstrated. In addition, it allows weak glass transitions to be detected clearly in some complex formulations where they can be obscured by eutectic and other strong thermal events when other methods such as DSC or DTA are used. Classical excipients (trehalose, lactose, dextran) were analysed and shown to give reproducible Tg' values, though with values slightly higher than those obtained by DSC. Finally, several complex real biological materials, typical of those encountered when freeze drying biological and biopharmaceutical materials, were analysed and the potential value of DMA demonstrated to determine the relevant glass transition temperatures for use in cryobiology and freeze drying.

Collaborative study for the calibration of a replacement international standard for diphtheria toxoid adsorbed.

We present the results of a collaborative study for the characterization of a preparation of diphtheria toxoid adsorbed, and its calibration in terms of the 3rd International Standard (IS) for Diphtheria Toxoid Adsorbed. Calibration was performed using established World Health Organization (WHO) and European Pharmacopoeia (Ph. Eur.) protection models. Two candidate toxoid preparations were included in the study, one of which was adopted as a replacement Ph. Eur. Biological Reference Preparation (BRP, batch 4) in February 2009. The second candidate preparation was found to have a unitage of 213 IU/ampoule based on the calibration by in vivo bioassay in 19 laboratories in 16 countries, and was established as the 4th IS for Diphtheria Toxoid Adsorbed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2009. The study also assessed performance of the replacement standard in mouse and guinea pig serological assays which are used as alternative procedures for diphtheria potency testing. Participants tested both candidate preparations and potency was expressed in relative terms only. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays and that the Vero cell assay may be suitable for calibration of future replacement standards.

Vacuum-Induced Surface Freezing for the Freeze-Drying of the Human Growth Hormone: How Does Nucleation Control Affect Protein Stability?

In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum-induced surface freezing technique has been compared to conventional freezing, both with and without an annealing step. Size exclusion chromatography and cell-based potency assays have been used to characterize the formation of soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that controlled nucleation has a positive effect on both cycle performance and product homogeneity because of the formation of bigger ice crystals, and characterized by a narrower dimensional distribution. From the point of view of hGH stability, we observed that vacuum-induced surface freezing is not detrimental to the biological activity of the protein, or aggregate formation. In addition, the effect of 2 different formulations, including trehalose or cellobiose, on protein preservation was also considered for this study.

Freeze-drying cycle optimization for the rapid preservation of protein-loaded liposomal formulations.

Technology such as the use of microfluidics to generate liposomes has been well researched, yet the stabilisation of liposomal formulations is a major challenge to their greater implementation. To the best of our knowledge, this is the first study investigating the use of 96 well plates to freeze-dry ovalbumin (OVA) loaded neutral (DMPC:Chol and DSPC:Chol), anionic (DSPC:Chol:PS) and cationic (DSPC:Chol:DOTAP) liposomes. Through the use of high throughput screening, a freeze drying cycle was optimised; ramp freezing from from 4 °C to -45 °C, followed by primary drying at -30 °C and secondary drying at 30 °C under a vacuum of 0.1 mBar. These parameters maintained liposome physicochemical properties, with the liposomes remaining below 100 nm and were homogenous (polydispersity index of less than 0.2 post rehydration). Minimal leakage of the OVA protein was observed, with almost 100% OVA remaining encapsulated post rehydration of the formulations. Here we have identified a simple method that allows for the rapid screening and freeze-drying of a range of liposomal formulations.

Impact of controlled vacuum induced surface freezing on the freeze drying of human plasma.

During the freezing step of a typical freeze drying process, the temperature at which nucleation is induced is generally stochastically distributed, resulting in undesired within-batch heterogeneity. Controlled nucleation techniques have been developed to address this problem; these make it possible to trigger the formation of ice crystals at the same time and temperature in all the batch. Here, the controlled nucleation technique known as vacuum induced surface freezing is compared to spontaneous freezing for the freeze drying of human plasma, a highly concentrated system commonly stored in a dried state. The potency of Factor VIII (FVIII), a sensitive, labile protein present in plasma, and the reconstitution time of the dried cakes are evaluated immediately after freeze drying, and after 1, 3, 6 or 9 months storage at different degradation temperatures. We show that the application of controlled nucleation significantly reduces the reconstitution time and in addition helps to improve FVIII stability.

Multiple constitutional aetiological factors in bone marrow failure syndrome (BMFS) patients from north India.

BACKGROUND AND OBJECTIVES: A large number of patients diagnosed with bone marrow failure syndromes (BMFS), comprising aplastic anaemia (AA) and myelodysplastic syndromes (MDS), remain aetiologically uncharacterized worldover, especially in resource constrained set up. We carried out this study to identify a few constitutional causes in BMFS patients attending a tertiary care hospital in north India. METHODS: Peripheral blood lymphocyte cultures were performed (with and without clastogens) in a cohort of 135 consecutive BMFS patients, in order to detect Fanconi anaemia (FA), Down's syndrome (+21), trisomy 8 (+8) and monosomy 7 (-7). RESULTS: Constitutional factors were detected in 17 (12.6%) patients. FA defect was observed in 24.07 percent (13/54), 16.66 percent (1/6) and 2.85 percent (1/35) paediatric aplastic anaemia, paediatric MDS and adult MDS patients respectively. Down's syndrome was detected in 5.00 percent (2/40) adult aplastic anaemia patients. None of the patients revealed trisomy 8 or monosomy 7. INTERPRETATION AND CONCLUSION: Presence of an underlying factor determines appropriate management, prognostication, family screening and genetic counselling of BMFS patients. Special tests required to confirm or exclude constitutional aetiological factors are not available to majority of the patients in our country. Diepoxybutane (DEB) test yielded better results than mitomycin C (MMC) test in our experience.

Differential scanning fluorimetry: rapid screening of formulations that promote the stability of reference preparations.

When formulating a biopharmaceutical protein, its stability in the liquid state is critical. In addition, when preparing biological reference materials the stability, both when lyophilised and after reconstitution, needs to be determined. In order to optimise the stability in aqueous conditions (as indicated by Tmelt or denaturation point) the impact of different excipient choices should be evaluated. Micro differential scanning calorimetry is a well established method for these applications but can be time consuming even when an autosampler is used. Differential scanning fluorimetry (DSF) is a novel technique which measures the fluorescence of a dye when bound to the hydrophobic regions of a denatured protein. We have investigated these techniques for their suitability using alpha-1-protease inhibitor (A1PI) as a model system and found similar trends in terms of the impact of different excipients by both methods. DSF is a promising method and has advantages in terms of speed and quantities of biological material required and can be performed using a PCR instrument.

The serum C Peptide levels among the offsprings of the people with type 2 diabetes.

BACKGROUND: Insulin Resistance (IR) is a condition in which the cells of the body become resistant to the effect of insulin , that is, the normal response to a given amount of insulin is reduced. As a result, levels of insulin are needed in order for insulin to produce its effect. The incidence of diabetes in the offsprings of diabetic couples was more than the incidence of diabetes in the offsprings, of whom only a single parent was diabetic. AIMS AND OBJECTIVES: This study was done to assess the prevalence of insulin resistance in the offsprings of diabetic patients. MATERIAL AND METHODS: The present, cross sectional study conducted in the Teerthanker Mahaveer Medical College and Research Centre, Moradabad, U.P., india. The offsprings of diabetic subjects attended the General Medicine OPD and the Diabetic Clinic and they were also admitted in the indoor wards of the Department of Medicine. The study material consisted of 53 (35 males and 18 females) live offsprings of diabetics from 28 families. RESULTS: The mean c-peptide level in the offsprings of biparental diabetics was significantly higher than that in the offsprings of monoparental diabetics (p<0.01) and in the offsprings of non-diabetics (p<0.01). The frequency of the high c-peptide level was 38.1% in the offsprings of biparental diabetics, it was 21.1% in the offsprings of monoparental diabetics and it was 7.7% in the offsprings of non-diabetics. CONCLUSION: This study revealed that during the young ages of the offsprings of biparental diabetics, insulin resistance was common and that insulin resistance was more common in the obese, female offsprings of biparental diabetics.