02682nas a2200205   4500008004100000022001400041245013800055210006900193260000900262300001400271490000600285520194300291100002002234700001602254700001702270700002202287700002102309700002002330856012602350       2024                        eng d  a2470-134300aMechanistic modeling of size exclusion chromatography-assisted in vitro refolding of the recombinant biosimilar teriparatide (PTH-34)0 aMechanistic modeling of size exclusion chromatographyassisted in  cJAN   a3204-32160 v93 a<p>
	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-degrees 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.</p>
1 aUghade, Santosh1 aRana, Sunil1 aNadeem, Mohd1 aKumthekar, Rupali1 aMahajani, Sanjay1 aBhambure, Rahul  uhttp://library.ncl.res.in/content/mechanistic-modeling-size-exclusion-chromatography-assisted-vitro-refolding-recombinant