A Comparison of Two Manufacturing Processes for an Antithrombin Concentrate

Abstract number: P0695

Chenou C, Bihoreau N, Sauger A, Siret L, Just O, Chtourou S

Introduction:  The antithrombin manufacturing process has been modified by the implementation of two additional safety steps: depth filtration and 15 nm nanofiltration. Moreover, a new affinity chromatography gel has been implemented. To evaluate the impact of these modifications on product quality, pharmacokinetic and analytical comparability studies were performed.

Material and methods:  Three batches from current and modified processes that conformed to European Pharmacopoeia specifications were analysed. The following biochemical characterisations were performed: peptide mapping of the primary structure, Circular Dichroism (CD) of the secondary structure, isofocusing electrophoresis for microheterogeneity analyses, and capillary gel electrophoresis to determine protein glycosylation. Product purity was evaluated by SDS-PAGE and immunochemical assays for major co-purified proteins. The pharmacokinetic profiles for both products were determined after a single IV administration of 100 IU/kg in rabbits.

Results:  Qualitative analyses of heterogeneity demonstrated that the isoform profile was identical for both proteins. The three major isoforms had isoelectric points of 5.09, 5.05 and 4.91, and seven minor isoforms were identified. The glycosylation profiles generated by qualitative and quantitative analyses of carbohydrate residues were similar. The peptide maps generated for both products were superimposible. The CD spectra demonstrated the comparability of secondary structures. The combined sum for the co-purified proteins was 0.1% using the modified process and 0.8% for the former process and thus demonstrated the very high purity achieved. The pharmacokinetic profiles showed elimination kinetics comparable with the half life (t_1/2) in the order of 21.0 hours for both products.

Conclusion:  Together, all analytical and pharmacokinetic data generated during studies enabled the non-clinical comparability of the two products to be concluded.