Naylor D.J.,Invion Ltd. |
Hunt B.,Hospira Adelaide Pty Ltd. |
Guidolin A.,Hospira Adelaide Pty Ltd. |
Hey A.W.,Hospira Adelaide Pty Ltd. |
And 12 more authors.
Process Biochemistry | Year: 2015
Human chaperonin 10 (Cpn10) and chaperonin 60 (Cpn60) fulfil an essential role in mitochondrial protein folding. Cpn10 is located in the mitochondrial matrix; however, it has also been detected in extra-mitochondrial compartments where it has demonstrated anti-inflammatory and immunoregulatory activity, suggesting a potential therapeutic value for the treatment of inflammatory and autoimmune disorders that corresponds with its recently proposed role as a resolution-associated molecular patterns (RAMPs) molecule. Ala-Cpn10, a recombinant, minimally modified Cpn10 synthesised in Escherichia coli, is formulated for use in clinical trials and pre-clinical studies with intravenous or subcutaneous administration. Herein, we report the development of a bioprocess for the production of ∼115 g of recombinant human Ala-Cpn10 from a 100 L E. coli fermentation with >99% purity (SDS-PAGE), <0.6 EU mg-1 endotoxin, <18 pg mg-1 DNA and 3.2% molecular variants. This bioprocess was achieved through a careful optimisation of the gene construct, the promoter system and the fermentation process. Recombinant Ala-Cpn10 produced in this process is active as a molecular chaperone indicating correct tertiary and quaternary structure and the stability profile indicates no significant changes with storage as a liquid for at least 3 years at 2-8 °C. The results of validated characterisation assays demonstrate that purified Ala-Cpn10 produced using the optimised process reported here is suitable for its intended purpose as an investigational drug product, and this material is currently being tested in a phase II study for efficacy, safety and impact on biomarkers in subjects with mildly active Systemic Lupus Erythematosus (SLE) under US IND 116156. © 2015 Elsevier Ltd.