Demonstration of Equivalence of Generic Glatiramer Acetate and Copaxone®.

The objective of the current work was to demonstrate the equivalence of Mylan's glatiramer acetate (GA) to that of the reference product Copaxone® (COP) using the four criteria for active pharmaceutical ingredient sameness as established by the US Food and Drug Administration (FDA). The reaction scheme used to produce Mylan's glatiramer acetate (MGA) was compared with that of COP, determined from publicly available literature. Comparative analyses of MGA and COP were performed for physicochemical properties such as amino acid composition and molecular weight distributions. Spectroscopic fingerprints were obtained using circular dichroism spectroscopy. Structural signatures for polymerization and depolymerization including total diethylamine (DEA) content, relative proportions of DEA-adducted amino acids, and N-and C-terminal amino acid sequences were probed with an array of highly sensitive analytical methods. Biological activity of the products was assessed using validated murine Experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. MGA is produced using the same fundamental reaction scheme as COP and was shown to have equivalent physicochemical properties and composition. Analyses of multiple structural signatures demonstrated equivalence of MGA and COP with regard to polymerization, depolymerization, and propagational shift. Examination of the impact on prevention and treatment of EAE demonstrated equivalence of MGA and COP with respect to both activity and toxicity, and thereby provided confirmatory evidence of sameness. A rigorous, multi-pronged comparison of MGA and COP produced using an equivalent fundamental reaction scheme demonstrated equivalent physicochemical properties, structural signatures for polymerization and depolymerization, and biological activity as evidenced by comparable effects in EAE. These studies demonstrate the equivalence of MGA and COP, establishing active ingredient sameness by the US Food and Drug Administration (FDA) criteria for GA, and provide compelling evidence that the FDA-approved generic MGA can be substituted for COP for the treatment of patients with relapsing-remitting MS.

Development and validation of a cell based assay for the detection of neutralizing antibodies against recombinant insulins.

Recombinant biopharmaceuticals can induce generation of anti-drug antibodies, which could potentially neutralize therapeutic drug activity. In this report, we describe development and validation of a cell-based assay for detection of neutralizing antibodies (Nab) against insulin and insulin analogues. In order to achieve clinically meaningful sensitivity the method used an early signalling event, insulin induced insulin receptor phosphorylation as the endpoint. Percentage insulin receptor phosphorylation in cell lysates was measured using ECL based ELISA. Presence of neutralizing antibodies (Nab) in samples will inhibit insulin induced receptor phosphorylation and consequently lead to a reduction in the percentage of phosphorylated insulin receptor. Additionally, usage of human insulin receptor overexpressing recombinant CHO cell line further improved the assay sensitivity by reducing the fixed drug (EC50) concentration used for induction of receptor phosphorylation. To ensure adequate free drug tolerance a pre-treatment step was introduced, where serum samples underwent acid dissociation and charcoal extraction before drug incubation. In order to distinguish ADA positive samples containing true Nab from samples containing non-antibody phosphorylation inhibitory serum factors, a confirmatory tier was integrated based on immunodepletion using protein AGL mix. Assay parameters including determination of screening and confirmatory cut-points, intra and inter assay precision, selectivity, specificity and stability were assessed during validation in accordance with recent regulatory guidelines and white papers. The advantage of selecting insulin receptor phosphorylation as assay endpoint made the assay capable of detecting Nab against insulin and insulin analogues.

2009 White Paper on recent issues in regulated bioanalysis from the 3rd Calibration and Validation Group Workshop.

The 3rd Calibration and Validation Group Workshop on Recent Issues in Regulated Bioanalysis was organized by the Calibration and Validation Group as a 1.5-day full immersion workshop for contract research organizations, pharmaceutical companies and regulatory agencies to discuss several 'hot' topics concerning bioanalytical issues and regulatory challenges. A consensus was reached among panelists and attendees on many points regarding method validation of small molecules.

The 2nd Calibration and Validation Group workshop on recent issues in good laboratory practice bioanalysis.

This event was organized by the Calibration and Validation Group (a scientific nonprofit organization based in Toronto, Canada) as a 1.5-day workshop for contract research organizations and pharmaceutical companies involved in providing bioanalytical data for bioavailability, bioequivalence, pharmacokinetic and comparability studies.

Development and validation of a selective and sensitive bioanalytical procedure for the quantitative determination of gaboxadol in human plasma employing mixed mode solid phase extraction and hydrophilic interaction liquid chromatography with tandem mass spectroscopic detection.

A selective and sensitive hydrophilic interaction liquid chromatography tandem mass spectrometric bioanalytical method for the quantitative determination of gaboxadol in human heparinized plasma was developed and validated. Gaboxadol and the stable isotope labeled internal standard were extracted from plasma by mixed mode solid phase extraction and analyzed on an Asahipak NH2P HPLC column with a mobile phase composed of 70% acetonitrile and 30% ammonium acetate (20 mM, pH 4). The analytes were detected by a SCIEX API 4000 triple quadropole instrument using turbo electrospray ionization and multiple reaction monitoring negative mode. The method was validated over the concentration range of 0.5-100 ng/mL. The intra-day precision of the assay, as measured by the coefficient of variation (CV%), was within 4%. The intra-day assay accuracy was found to be within 2.2% of the nominal concentration for all the standards. The average recovery of gaboxadol was about 87% and the ion suppression was approximately 8%. To eliminate late eluters including the glucuronides, a "front cut" column switching procedure was added to the chromatographic system. The effectiveness of the column switching in eliminating the absolute matrix effect caused by late eluters was demonstrated by the low variation (CV<3.5%) in the peak areas of the internal standard during the assessment of the inter-day precision and accuracy and no significant relative matrix effect was observed as illustrated by the excellent intra-day precision (CV<1.5%) from the assessment of standard samples prepared in five different lots of control plasma. The described bioanalytical method has been successfully utilized for the analysis of gaboxadol in post-dose samples (>8000) from various clinical studies. Inter-day precision and accuracy were assessed from the daily mean (n=2) of QC values from 52 runs, i.e. more than 3000 samples. The inter-day precision of the assay, based on the coefficient of variation of QC, ranged from 2.1 to 5.1%. The inter-day assay accuracy was found to be within 4% of the nominal concentration for all QC samples.