title: Integrated continuous processing of proteins expressed as inclusion bodies: GCSF as a case study.
abstract: Affordability of biopharmaceuticals continues to be a challenge, particularly in developing economies. This has fuelled advancements in manufacturing that can offer higher productivity and better economics without sacrificing product quality in the form of an integrated continuous manufacturing platform. While platform processes for monoclonal antibodies have existed for more than a decade, development of an integrated continuous manufacturing process for bacterial proteins has received relatively scant attention. In this study, we propose an end-to-end integrated continuous downstream process (from inclusion bodies to unformulated drug substance) for a therapeutic protein expressed in Escherichia coli as inclusion body. The final process consisted of a continuous refolding in a coiled flow inverter reactor directly coupled to a three-column periodic counter-current chromatography for capture of the product followed by a three-column con-current chromatography for polishing. The continuous bioprocessing train was run uninterrupted for 26 h to demonstrate its capability and the resulting output was analyzed for the various critical quality attributes, namely product purity (>99%), high molecular weight impurities (<0.5%), host cell proteins (<100 ppm), and host cell DNA (<10 ppb). All attributes were found to be consistent over the period of operation. The developed assembly offers smaller facility footprint, higher productivity, fewer hold steps, and significantly higher equipment and resin utilization. The complexities of process integration in the context of continuous processing have been highlighted. We hope that the study presented here will promote development of highly efficient, universal, end-to-end, fully continuous platforms for manufacturing of biotherapeutics. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:998-1009, 2017.
authors.affiliation: Dept. of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Kateja
authors.affiliation: Dept. of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India.
authors.forename: Harshit
authors.initials: H
authors.lastname: Agarwal
authors.affiliation: Dept. of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India.
authors.forename: Vishwanath
authors.initials: V
authors.lastname: Hebbi
authors.affiliation: Dept. of Chemical Engineering, Indian Institute of Technology Delhi, New Delhi, India.
authors.forename: Anurag S
authors.initials: AS
authors.lastname: Rathore
mesh_terms: D019149:Bioreactors; D003377:Countercurrent Distribution; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D058849:Protein Refolding; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D016188:Receptors, Granulocyte Colony-Stimulating Factor
keywords: coiled flow inverter; continuous processing; periodic counter-current chromatography; refolding