Rahul Bhambure

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.

Background: L‐Asparaginase is an essential enzyme for the food and biopharmaceutical industry. The stability, however, of L‐asparaginase is widely known to be an issue. Commercial manufacturing of any biopharmaceutical involves hold‐ups during processing, and can result in product loss if stability is an issue, as is the case with L‐asparaginase. This interplay of product intermediate stability and process design is the focus of this investigation.Methods and Results: In this study, we propose a strategy to simultaneously increase the refolding yield and stability of refolded L‐asparaginase so as to improve overall process yield. Using one variable at a time (OVAT) experiments, urea (6 M), solubilized inclusion bodies (15 mg/ml), refolding method (step dilution), and pH (8.6) were identified as significant process parameters. A design of experiment (DOE)‐based optimization was then performed for the refolding step. The net outcome was more than a three‐fold increase in enzyme recovery (i.e., 4.90 IU/ml) compared to unoptimized conditions (i.e., 1.26 IU/ml). Further, the L‐asparaginase process intermediate was found to be stable for more than a week at room temperature and 2–8°C, while the unoptimized sample was stable at 2–8°C but did not show any activity at room temperature after 72 h.Conclusions: The current study elucidates how process intermediate stability needs to be given due consideration during process optimization, particularly for products such as L‐asparaginase which are labile.

Post-translational modifications remarkably regulate proteins’ biological function. Small molecules such as reactive thiols, metabolites, and drugs may covalently modify the proteins and cause structural changes. This study reports the covalent modification and noncovalent interaction of insulin and captopril, an FDA-approved antihypertensive drug, through mass spectrometric and computation-based approaches. Mass spectrometric analysis shows that captopril modifies intact insulin, reduces it into its “A” and “B” chains, and covalently modifies them by forming adducts. Since captopril has a reactive thiol group, it might reduce the insulin dimer or modify it by reacting with cysteine residues. This was proven with dithiothreitol treatment, which reduced the abundance of captopril adducts of insulin A and B chains and intact Insulin. Liquid chromatography tandem mass spectrometric analysis identified the modification of a total of four cysteine residues, two in each of the A and B chains of insulin. These modifications were identified to be Cys6 and Cys7 of the A chain and Cys7 and Cys19 of the B chain. Mass spectrometric analysis indicated that captopril may simultaneously modify the cysteine residues of intact insulin or its subunits A and B chains. Biophysical studies involving light scattering and thioflavin T assay suggested that the binding of captopril to the protein leads to the formation of aggregates. Docking and molecular dynamics studies provided insights into the noncovalent interactions and associated structural changes in insulin. This work is a maiden attempt to understand the detailed molecular interactions between captopril and insulin. These findings suggest that further investigations are required to understand the long-term effect of drugs like captopril.

BACKGROUND: Continuous multicolumn Protein A chromatography offers various advantages for capture stage purification of monoclonal antibody therapeutics, like higher productivity and resin capacity utilization, lower buffer consumption, small footprint, etc. Due to the complexity of the continuous process, experimental optimization is time‐consuming and cost‐intensive. This investigation proposes a hybrid process development approach integrating experimental and mechanistic modeling for time‐ and cost‐effective development and optimization of continuous Protein A affinity chromatography.RESULTS: Productivity and capacity utilization of the continuous CaptureSMB process under varying operating conditions were predicted using the Chromatography Analysis and Design Toolkit (CADET) framework and validated with experimental results. Effects of critical process parameters like feed concentration (c₀), loading breakthrough (s) and residence time (RT) on productivity and capacity utilization were evaluated. Model predictions were validated using the experimental results proving the reliability and feasibility of the modeling approach. At 15.00 ± 0.20 mg mL^–1 feed model mAb concentration, the model‐based approach predicted the best performance giving 27.56 g L^–1 h^–1 productivity (RT = 2 min, s = 80%) using MabSelect™ PrismA resin. This productivity prediction was validated by performing the continuous CaptureSMB experiment at the same operating conditions and was found to be 25.59 g L^–1 h^–1 with a root mean square error of 1.96.CONCLUSIONS: The hybrid process development and optimization approach for CaptureSMB purification of biosimilar mAb purification is demonstrated and validated using the experimental and model‐based framework. The observed results showcase the effectiveness of the proposed hybrid process development approach, enabling a better understanding of the chromatography process and parameters affecting the CaptureSMB process. © 2021 Society of Chemical Industry (SCI).