Engineering batch and pulse refolding with transition of aggregation kinetics: An investigation using green fluorescent protein (GFP)

Pan, Siqi; Odabas, Nora; Sissolak, Bernhard; Imendörffer, Moritz; Zelger, Monika; Jungbauer, Alois; Hahn, Rainer

doi:10.1016/j.ces.2015.03.054

Cited by 9 publications

(1 citation statement)

References 37 publications

(73 reference statements)

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“…The reaction of the solubilized protein to the folding intermediates is considered to happen immediately, simplifying the model to the off-pathway model with the two reaction rates k r and k a (Hevehan and de Bernardez Clark 1997 ). The reaction of correct folding follows a first-order reaction while the aggregation follows a reaction with an order of two or higher (Dong et al 2004 ; Hevehan and de Bernardez Clark 1997 ; Kiefhaber et al 1991 ; Zettlmeissl et al 1979 ), or first order for low protein concentrations (Pan et al 2015 ). In a simple dilution by batch mode refolding process, Kiefhaber et al described the concentration of the solubilized and native protein with a second-order aggregation reaction as follows (Kiefhaber et al 1991 ), …”

Section: Models Of Refolding Processesmentioning

confidence: 99%

Advances in monitoring and control of refolding kinetics combining PAT and modeling

Pauk

Palanisamy

Kager

et al. 2021

Appl Microbiol Biotechnol

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Overexpression of recombinant proteins in Escherichia coli results in misfolded and non-active protein aggregates in the cytoplasm, so-called inclusion bodies (IB). In recent years, a change in the mindset regarding IBs could be observed: IBs are no longer considered an unwanted waste product, but a valid alternative to produce a product with high yield, purity, and stability in short process times. However, solubilization of IBs and subsequent refolding is necessary to obtain a correctly folded and active product. This protein refolding process is a crucial downstream unit operation—commonly done as a dilution in batch or fed-batch mode. Drawbacks of the state-of-the-art include the following: the large volume of buffers and capacities of refolding tanks, issues with uniform mixing, challenging analytics at low protein concentrations, reaction kinetics in non-usable aggregates, and generally low re-folding yields. There is no generic platform procedure available and a lack of robust control strategies. The introduction of Quality by Design (QbD) is the method-of-choice to provide a controlled and reproducible refolding environment. However, reliable online monitoring techniques to describe the refolding kinetics in real-time are scarce. In our view, only monitoring and control of re-folding kinetics can ensure a productive, scalable, and versatile platform technology for re-folding processes. For this review, we screened the current literature for a combination of online process analytical technology (PAT) and modeling techniques to ensure a controlled refolding process. Based on our research, we propose an integrated approach based on the idea that all aspects that cannot be monitored directly are estimated via digital twins and used in real-time for process control. Key points • Monitoring and a thorough understanding of refolding kinetics are essential for model-based control of refolding processes. • The introduction of Quality by Design combining Process Analytical Technology and modeling ensures a robust platform for inclusion body refolding.

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Section: Models Of Refolding Processesmentioning

confidence: 99%

Advances in monitoring and control of refolding kinetics combining PAT and modeling

Pauk

Palanisamy

Kager

et al. 2021

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

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Solubilization and Refolding of Inclusion Body Proteins

Singhvi

Panda

2022

Methods in Molecular Biology

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Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence

Igwe,

Müller,

Gisperg

et al. 2024

Anal Bioanal Chem

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Inclusion bodies (IBs) are protein aggregates formed as a result of overexpression of recombinant protein in E. coli. The formation of IBs is a valuable strategy of recombinant protein production despite the need for additional processing steps, i.e., isolation, solubilization and refolding. Industrial process development of protein refolding is a labor-intensive task based largely on empirical approaches rather than knowledge-driven strategies. A prerequisite for knowledge-driven process development is a reliable monitoring strategy. This work explores the potential of intrinsic tryptophan and tyrosine fluorescence for real-time and in situ monitoring of protein refolding. In contrast to commonly established process analytical technology (PAT), this technique showed high sensitivity with reproducible measurements for protein concentrations down to 0.01 g L$$^{-1}$$ - 1 . The change of protein conformation during refolding is reflected as a shift in the position of the maxima of the tryptophan and tyrosine fluorescence spectra as well as change in the signal intensity. The shift in the peak position, expressed as average emission wavelength of a spectrum, was correlated to the amount of folding intermediates whereas the intensity integral correlates to the extent of aggregation. These correlations were implemented as an observation function into a mechanistic model. The versatility and transferability of the technique were demonstrated on the refolding of three different proteins with varying structural complexity. The technique was also successfully applied to detect the effect of additives and process mode on the refolding process efficiency. Thus, the methodology presented poses a generic and reliable PAT tool enabling real-time process monitoring of protein refolding. Graphical abstract Real-time intrinsic fluorescence monitoring in protein refolding

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Engineering batch and pulse refolding with transition of aggregation kinetics: An investigation using green fluorescent protein (GFP)

Cited by 9 publications

References 37 publications

Advances in monitoring and control of refolding kinetics combining PAT and modeling

Advances in monitoring and control of refolding kinetics combining PAT and modeling

Solubilization and Refolding of Inclusion Body Proteins

Online monitoring of protein refolding in inclusion body processing using intrinsic fluorescence

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