Analytical characterization of in vitro refolding in the quality by design paradigm: Refolding of recombinant human granulocyte colony stimulating factor

• Published in: Journal of pharmaceutical and biomedical analysis 2016-07, Vol.126, p.124-131
• Main Authors: Pathak, Mili, Dixit, Shruti, Muthukumar, S., Rathore, Anurag S.
• Format: Article
• Language: English
• Subjects: Analytical characterization; Escherichia coli - metabolism; Ganulocyte colony stimulating factor (G-CSF); Granulocyte Colony-Stimulating Factor - chemistry; Granulocyte Colony-Stimulating Factor - metabolism; Humans; Inclusion Bodies - metabolism; Monitoring; Process Analytical Technology (PAT); Protein Refolding; Quality by Design (QbD); Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Technology, Pharmaceutical - methods
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2016.05.001

This paper aims to provide an approach to generate a deeper understanding of refolding of a therapeutic protein and then to use it for its optimal production commercially. Recombinant human granulocyte colony stimulating factor has been chosen as the model protein. Seven orthogonal analytical tools have been used to elucidate the refolding process. By strategically using these tools we have been able to segregate protein refolding into a series of well-defined sequence of events, starting from the unfolded random coil and ending with the uniquely folded metastable state. The study also suggests the choice of tools that can be used to monitor each event. The in-depth understanding has then been used to design an optimal refolding process, with the quality of the resulting therapeutic verified by several analytical tools as well as the cell based bioassay. We believe that this paper successfully demonstrates an approach to generate deeper understanding of the protein refolding process and then successfully implement it towards achieving consistent product quality during commercial manufacturing. [Display omitted] •Protein refolding has been broken into a series of well-defined sequence of events, starting from the unfolded random coil and ending with the uniquely folded metastable state.•Recombinant human granulocyte colony stimulating factor has been chosen as the model protein.•In-depth understanding has been used to design an optimal refolding process. Protein based therapeutics dominate most pharmaceutical pipelines today. For a therapeutic product to be effective, it is important that it is in its native form as slight modifications have been known to result in significantly different performance in the clinic. When expressed in hosts such as Escherichia coli, formation of inactive insoluble aggregates of proteins popularly known as inclusion bodies occurs in most cases. This necessitates the need for in vitro refolding to generate the native (and active) form of the therapeutic protein. This paper aims to provide an approach to generate a deeper understanding of refolding of a therapeutic protein and then to use it for its optimal production commercially. Recombinant human granulocyte colony stimulating factor has been chosen as the model protein. Seven orthogonal analytical tools have been used to elucidate the refolding process. By strategically using these tools protein refolding has been segregated into a series of well-defined sequence of events,