New Approach and Practical Modelling of Bead Milling Process for the Manufacturing of Nanocrystalline Suspensions.

Stirred media milling is the main technology for producing colloidal nanocrystalline suspensions. A number of studies have been reported on the effect of different operating parameters for lab, pilot, and industrial scales. However, typical milling tool box that can be used to support candidate from selection up to phase III clinical supplies can involve different mill configurations. This article describes a parametric study and milling kinetic modelling of the different mills. The impact of active pharmaceutical ingredient (API) type and process parameters on milling kinetics was determined. The milling kinetics were modeled using an empirical model which allows for predicting and simulation of milling kinetics of stirred annular and pin mills. The proposed model was found to accurately fit milling kinetics whatever the API considered, technology employed, and the process parameters used for milling. Moreover, the model was found to be able to ensure the process transfer from one mill to another.

Analytical and statistical comparability of generic enoxaparins from the US market with the originator product.

Low-molecular-weight heparins (LMWHs) are complex anticoagulant drugs, made from heparin porcine mucosa starting material. Enoxaparin sodium manufactured by Sanofi is one of the most widely prescribed LMWHs and has been used since 1993 in the USA. In 2010, US Food and Drug Administration approval for supplying generic enoxaparin was granted to Sandoz and subsequently to Amphastar. Little is known, however, of the differences in composition of these preparations. In this study, samples from several batches of generic enoxaparins were purchased on the US market and analyzed with state of the art methodologies, including disaccharide building blocks quantification, nuclear magnetic resonance (NMR), and a combination of orthogonal separation techniques. Direct high-performance liquid chromatography analysis of the different enoxaparin batches revealed distinct process fingerprints associated with each manufacturer. Disaccharide building block analysis showed differences in the degree of sulfation, the presence of glycoserine derivatives, as well as in proportions of disaccharides. Results were compared by statistical approaches using multivariate analysis with a partial least squares discriminant analysis methodology. The variations were statistically significant and allowed a clear distinction to be made between the enoxaparin batches according to their manufacturer. These results were further confirmed by orthogonal analytical techniques, including NMR, which revealed compositional differences of oligosaccharides both in low- and high-affinity antithrombin fractions of enoxaparin.

Reproducibility of the anti-Factor Xa and anti-Factor IIa assays applied to enoxaparin solution.

Enoxaparin is a widely used subcutaneously administered antithrombotic agent comprising a complex mixture of glycosaminoglycan chains. Owing to this complexity, its antithrombotic potency cannot be defined by physicochemical methods and is therefore evaluated using an enzymatic assay of anti-Xa and anti-IIa activity. Maintaining consistent anti-Xa activity in the final medicinal product allows physicians to ensure administration of the appropriate dosage to their patients. Bioassays are usually complex and display poorer reproducibility than physicochemical tests such as HPLC assays. Here, we describe the implementation of a common robotic platform and standard release potency testing procedures for enoxaparin sodium injection (Lovenox, Sanofi, Paris, France) products at seven quality control sites within Sanofi. Qualification and analytical procedures, as well as data handling, were optimized and harmonized to improve assay reproducibility. An inter-laboratory study was performed in routine-release conditions. The coefficients of variation for repeatability and reproducibility in assessments of anti-Xa activity were 1.0% and 1.2%, respectively. The tolerance interval in reproducibility precision conditions, expressed as percentage potency, was 96.8-103.2% of the drug product target of 10,000 IU/ml, comparing favorably with the United States of America Pharmacopeia specification (90-110%). The maximum difference between assays in two different laboratories is expected to be 4.1%. The reproducibility characteristics of anti-IIa activity assessments were found to be similar. These results demonstrate the effectiveness of the standardization process established and allow for further improvements to quality control in Lovenox manufacture. This process guarantees closeness between actual and target potencies, as exemplified by the results of release assays obtained during a three-year period.

Relationship between HPLC precision and number of significant figures when reporting impurities and when setting specifications.

The rounding of an analytical result is a process that should take into account the uncertainty of the result, which is in turn assessed during the validation exercise. Rounding rules are known in physical and analytical chemistry since a long time, but are often not used or misused in pharmaceutical analysis. The paper describes the theoretical background of the most common rules and their application to fix the rounding of results and specifications. The paper makes use of uncertainty values of impurity determination acquired during studies of reproducibility and intermediate precision with regards to 22 impurities of drug substances or drug products. As a general rule, authors propose the use of sound and well-established rounding rules to derive rounding from the results of the validation package.

A colorimetric 96-well microtiter plate assay for the determination of urate oxidase activity and its kinetic parameters.

Urate oxidase (E.C.1.7.3.3; uricase, urate oxygen oxidoreductase) is an enzyme of the purine breakdown pathway that catalyzes the oxidation of uric acid in the presence of oxygen to allantoin and hydrogen peroxide. A 96-well plate assay measurement of urate oxidase activity based on hydrogen peroxide quantitation was developed. The 96-well plate method included two steps: an incubation step for the urate oxidase reaction followed by a step in which the urate oxidase activity is stopped in the presence of 8-azaxanthine, a competitive inhibitor. Hydrogen peroxide is quantified during the second step by a horseradish peroxidase-dependent system. Under the defined conditions, uric acid, known as a radical scavenger, did not interfere with hydrogen peroxide quantification. The general advantages of such a colorimetric assay performed in microtiter plates, compared to other methods and in particular the classical UV method performed with cuvettes, are easy handling of large amounts of samples at the same time, the possibility of automation, and the need for less material. The method has been applied to the determination of the kinetic parameters of rasburicase, a recombinant therapeutic enzyme.