Direct quantitative detection of host cell residual DNA in recombinant Filgrastim by qPCR.

Host cell residual DNA is considered as an impurity in recombinant biopharmaceuticals. This study aimed to develop a direct qPCR method to quantify E. Coli residual DNA in recombinant Filgrastim. The specific primers were designed to amplify E. Coli's 16S-rDNA genomic region, which encodes the 16S-rRNA. The developed qPCR method showed that the designed primer has specifically amplified the target genome without any secondary reaction. The designed primer was also able to amplify the target gene as a representative of residual DNA in the drug matrix. Results show that the amount of residual DNA in Filgrastim is undetectable.

Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR.

PURPOSE: Filgrastim is the generic name for recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF). It is marketed under the brand name Neupogen® by Amgen. Since this product has lost patent protection, many biosimilar versions have been approved or are in the process of filing for market authorization throughout the world. Here we show that NMR spectroscopy can be used to assess the three-dimensional structure of the active ingredient in the formulated approved product Neupogen®. METHODS: Recombinant metHuG-CSF was prepared in E. coli and isotopically enriched with (13)C and (15) N isotopes. NMR spectroscopy was used to study the effects of excipients on the conformation. RESULTS: The effects of pH variation on the amide chemical shifts suggest the presence of cation-pi interactions between His-79 and Trp-118, and His-156-Trp-58-His-52 that stabilizes the conformation at low pH. This may be associated with a small local conformational change. The NMR data showed that polysorbate does not interact significantly with filgrastim thus allowing the collection of spectra in the presence of 20 times the formulation concentration in the sample. However, at higher detergent concentrations a reduction of signal intensity is observed. Conclusions The NMR fingerprint assay applied to filgrastim (Neupogen® and a CRS from the European Pharmacopeia (EP)) provided residue specific information of the structure of the drug substance. In addition to current methods, the ability to assess the conformation with a high degree of resolution can greatly facilitate comparability exercises.