Continuous protein refolding and purification by two-stage periodic counter-current chromatography

Rajendran, Vivek; Ponnusamy, Ananthi; Pushpavanam, S.; Jayaraman, Guhan

doi:10.1016/j.chroma.2023.463938

Cited by 2 publications

(2 citation statements)

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“…On-column protein refolding can be achieved using ion exchange chromatography, size exclusion chromatography, and metal affinity chromatography. , Chromatography-assisted protein refolding is advantageous over dilution-based refolding, as it can separate contaminants, quickly remove denaturants, and refold the recombinant proteins at higher concentrations. Many reports have shown that size exclusion chromatography can be extensively used as a refolding tool for various model proteins expressed in the form of inclusion bodies (IBs) . It has been hypothesized that using size exclusion chromatography for protein folding reduces aggregate formation by gradually removing denaturants and separating folding intermediates due to different diffusion properties while passing through the column .…”

Section: Introductionmentioning

confidence: 99%

“…Many reports have shown that size exclusion chromatography can be extensively used as a refolding tool for various model proteins expressed in the form of inclusion bodies (IBs). 25 It has been hypothesized that using size exclusion chromatography for protein folding reduces aggregate formation by gradually removing denaturants and separating folding intermediates due to different diffusion properties while passing through the column. 22 Boris et al showed in their report that hen egg white lysozyme was refolded using size exclusion chromatography; when the initial load concentration was low, the aggregation reaction for lysozyme was reduced; however, with a higher concentration load, an elevated aggregation reaction was observed.…”

Section: Introductionmentioning

confidence: 99%

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Mechanistic Modeling of Size Exclusion Chromatography-Assisted In Vitro Refolding of the Recombinant Biosimilar Teriparatide (PTH-34)

Ughade,

Rana,

Nadeem

et al. 2024

ACS Omega

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In vitro protein refolding is one of the critical unit operations in manufacturing recombinant peptides expressed using Escherichia coli as host cells. This study is focused on designing size exclusion chromatography-assisted in vitro refolding process for biosimilar recombinant parathyroid hormone. Inclusion bodies (IBs) of recombinant parathyroid hormone were solubilized at higher pH, and in vitro refolding was performed using size exclusion chromatography. In the first part of the investigation, DoE-based empirical optimization was performed to achieve a higher refolding yield for a biosimilar recombinant parathyroid hormone. The effect of solubilized inclusion body (IB) feed volume, concentration of IBs, and residence time on in vitro refolding of recombinant teriparatide was studied using the Box−Behnken design. Size exclusion chromatography (SEC)-assisted in vitro refolding was performed at 8 °C at pH 10.5 by using 20 mM Tris buffer. The maximum refolding yield of 98.12% was achieved at feed volume (12.5% of CV) and 20 mg/mL inclusion body (IB) concentration with a residence time of 50 min and a purity of 66.1% based on densitometric analysis using SDS-PAGE. In the latter part of the investigation, the general rate mechanistic model framework for size exclusion chromatography was developed and validated with the experimental results. The developed model helped in the accurate prediction of the elution volumes and product yield. The developed model also helps to predict the elution performance of a scalable column a priori. Post in vitro refolding, the formation of the native peptide structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structures. The developed hybrid process development approach is a valuable tool toachieve high-yield, scalable refolding conditions for recombinant proteins without disulfide bonds.

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