Inclusion Body Processing Research Articles

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

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\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^{-1}$$\\end{document}. 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 abstractReal-time intrinsic fluorescence monitoring in protein refolding

Inclusion Bodies in Ionic Liquids

The pivotal role of proteins in pharmaceuticals is challenged by stability issues, making the study of inclusion bodies—a source of insoluble protein aggregates—increasingly relevant. This review outlines the critical procedures in inclusion body processing, focusing on ’mild solubilization concepts’ and refolding methodologies. Attention is afforded to the emerging role of ionic liquids with unique and tunable physicochemical properties in optimizing protein unfolding and refolding processes. The review critically assesses the existing literature at the intersection of inclusion bodies and ionic liquids, identifying recent advancements, potential applications, and avenues for future research. This comprehensive analysis aims to elucidate the complexities in efficient protein processing from inclusion bodies.

Inclusion Bodies: Status Quo and Perspectives.

Multiple E. coli cultivations, producing recombinant proteins, lead to the formation of inclusion bodies (IBs). IBs historically were considered as nondesired by-products, due to their time- and cost-intensive purification. Nowadays, many obstacles in IB processing can be overcome. As a consequence, several industrial processes with E. coli favor IB formation over solubleproduction options due to the high space time yields obtained. Within this chapter, we discuss the state-of-the art biopharmaceutical IB process, review its challenges, highlight the recent developments and perspectives, and also propose alternative solutions, compared to the state-of-the art processing.

Intensification of Inclusion Body Processing via Temperature-Based Refolding.

Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been observed in protein expression systems such as E. coli, yeast, and higher eukaryotes. To recover soluble recombinant proteins in their native state, IB are commonly first solubilized with a high concentration of denaturant. This is followed by concurrent denaturant removal or reduction and a transition into a refolding-favorable chemical environment to facilitate the refolding of solubilized protein to its native state. Due to the high concentration of denaturant used, conventional refolding approaches can result in dilute products and are buffer inefficient. To circumvent the limitations of conventional refolding approaches, a temperature-based refolding approach which combines a low concentration of denaturant (0.5M guanidine hydrochloride, GdnHCl) with a high temperature (95°C) during solubilization was proposed. In this chapter, we describe a temperature-based refolding approach for the recovery of core streptavidin (cSAV) from IB. Through the temperature-based approach, intensification was achieved through the elimination of a concentration step which would be required by a dilution approach and through a reduction in buffer volumes required for dilution or denaturant removal. High-temperature treatment during solubilization may have also resulted in the denaturation and aggregation of undesired host-cell proteins, which could then be removed through a centrifugation step resulting in refolded cSAV of high purity without the need for column purification. Refolded cSAV was characterized by biotin-binding assay and SDS-PAGE, while purity was determined by RP-HPLC.

The impact of technical failures on recombinant production of soluble proteins in Escherichia coli: a case study on process and protein robustness

Technical failures lead to deviations in process parameters that can exceed studied process boundaries. The impact on cell and target protein is often unknown. However, investigations on common technical failures might yield interesting insights into process and protein robustness. Recently, we published a study on the impact of technical failures on an inclusion body process that showed high robustness due to the inherent stability of IBs. In this follow-up study, we investigated the influence of technical failures during production of two soluble, cytosolic proteins in E. coli BL21(DE3). Cell physiology, productivity and protein quality were analyzed, after technical failures in aeration, substrate supply, temperature and pH control had been triggered. In most cases, cell physiology and productivity recovered during a subsequent regeneration phase. However, our results highlight that some technical failures lead to persistent deviations and affect the quality of purified protein.

Scalable High-Performance Production of Recombinant Horseradish Peroxidase from E. coli Inclusion Bodies.

Horseradish peroxidase (HRP), an enzyme omnipresent in biotechnology, is still produced from hairy root cultures, although this procedure is time-consuming and only gives low yields. In addition, the plant-derived enzyme preparation consists of a variable mixture of isoenzymes with high batch-to-batch variation preventing its use in therapeutic applications. In this study, we present a novel and scalable recombinant HRP production process in Escherichia coli that yields a highly pure, active and homogeneous single isoenzyme. We successfully developed a multi-step inclusion body process giving a final yield of 960 mg active HRP/L culture medium with a purity of ≥99% determined by size-exclusion high-performance liquid chromatography (SEC-HPLC). The Reinheitszahl, as well as the activity with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 3,3′,5,5′-tetramethylbenzidine (TMB) as reducing substrates, are comparable to commercially available plant HRP. Thus, our preparation of recombinant, unglycosylated HRP from E. coli is a viable alternative to the enzyme from plant and highly interesting for therapeutic applications.

High pressure homogenization is a key unit operation in inclusion body processing

Recombinant protein production in E. coli often leads to the formation of inclusion bodies (IBs). Although downstream processing of IBs has the reputation of being a great hurdle, advantages of IBs can be substantial. Highly pure recombinant protein with the possibility of correctly folded structures and an easy separation from cell matter are decisive factors that make IB processes so interesting. Product yield, purity and biological activity of the refolded protein are the responses to evaluate an IB process. The objective of this case study was to develop a refolding process in an integrated manner. The effects of the unit operations 1) homogenization, 2) IB wash and 3) IB solubilisation as well as their interdependencies were analyzed. We revealed interesting factor interactions between homogenization and IB wash, as well as homogenization and solubilisation, which would be overlooked if the single unit operations were investigated individually. Furthermore, we found that homogenization was a key unit operation for IB processing. By changing the conditions during homogenization only, the product yield, purity and biological activity of the refolded product was affected 2-fold, 1.2-fold and 2.5-fold, respectively.

The impact of technical failures during cultivation of an inclusion body process

In biotechnological processes, technical failures in the upstream process often lead to batch loss. It is of great interest to investigate the empirical impact of technical failures to understand and mitigate their impact accurately and reduce economic damage. We investigated the impact in the upstream and downstream of a recombinant antibody fragment inclusion body production process chain to provide integrated empirical data and knowledge. First, we provided a reproducible process chain that yielded high inclusion body content, high specific product titer, and a refolding yield of 30%. The inclusion body downstream proved to be of high reproducibility. Through the intended introduction of technical failures, we were not only able to shed more light on the empirical responses in the upstream and downstream, but also on process-boosting parameters that would have been neglected. Herein, a short increase in temperature during the cultivation clearly increased the refolding yield.

GroEL/ES mediated the in vivo recovery of TRAIL inclusion bodies in Escherichia coli

Inclusion body (IB) formation generates substantial bio-waste in the pharmaceutical industry and remains a major challenge for heterologous protein expression. Although chaperones can be co-expressed to improve soluble protein yield, their contribution to IB processing in vivo has not been thoroughly studied. Here, a GroEL-GroES co-expressing strain and a deficient strain were constructed to study the in vivo recovery of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The interaction between GroEL/ES and TRAIL was simulated by molecular docking and identified by co-immunoprecipitation. The in vitro cytotoxicity of TRAIL IBs before and after in vivo recovery was subsequently determined by MTT assay. Additionally, IB structures were measured by Fourier transform infrared (FT-IR) spectroscopy and fluorescence spectroscopy. The results showed that after in vivo refolding, IBs retained lower levels of anti-tumor activity and fewer native-like β-sheet structures. Fewer recoverable polypeptides were trapped in IBs after GroEL/ES co-expression and refolding in vivo. Therefore, GroEL/ES mediated the in vivo recovery of TRAIL IBs in Escherichia coli. These results may identify potential uses for IBs and provide additional insight into the detailed mechanisms of in vivo protein recovery.

Teaching an old pET new tricks: tuning of inclusion body formation and properties by a mixed feed system in E. coli

Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.

Integrated process development—a robust, rapid method for inclusion body harvesting and processing at the microscale level

ABSTRACTEscherichia coli stores large amounts of highly pure product within inclusion bodies (IBs). To take advantage of this beneficial feature, after cell disintegration, the first step to optimal product recovery is efficient IB preparation. This step is also important in evaluating upstream optimization and process development, due to the potential impact of bioprocessing conditions on product quality and on the nanoscale properties of IBs. Proper IB preparation is often neglected, due to laboratory-scale methods requiring large amounts of materials and labor. Miniaturization and parallelization can accelerate analyses of individual processing steps and provide a deeper understanding of up- and downstream processing interdependencies. Consequently, reproducible, predictive microscale methods are in demand. In the present study, we complemented a recently established high-throughput cell disruption method with a microscale method for preparing purified IBs. This preparation provided results comparable to laboratory-scale IB processing, regarding impurity depletion, and product loss. Furthermore, with this method, we performed a “design of experiments” study to demonstrate the influence of fermentation conditions on the performance of subsequent downstream steps and product quality. We showed that this approach provided a 300-fold reduction in material consumption for each fermentation condition and a 24-fold reduction in processing time for 24 samples.

An Integrated Downstream Process Development Strategy along QbD Principles.

The development, optimization, and analysis of downstream processes are challenged by a high number of potentially critical process parameters that need to be investigated using lab-scale experiments. These process parameters are spread across multiple unit operations and potentially show interactions across unit operations. In this contribution, we present a novel strategy for bioprocess development that considers the risk of parameter interactions across unit operations for efficient experimental design. A novel risk assessment tool (interaction matrix) is introduced to the Quality by Design (QbD) workflow. Using this tool, the risk of interaction across unit operations is rated. Subsequently, a design of experiments (DoE) across unit operations is conducted that has the power to reveal multivariate interdependencies. The power of the presented strategy is demonstrated for protein isolation steps of an inclusion body process, focusing on the quality attribute inclusion body purity. The concentration of Triton X-100 in the course of inclusion body (IB) purification was shown to interact with the g-number of the subsequent centrifugation step. The presented strategy targets a holistic view on the process and allows handling of a high number of experimental parameters across unit operations using minimal experimental effort. It is generically applicable for process development along QbD principles.

High‐throughput system for determining dissolution kinetics of inclusion bodies

Efficient solubilization is a crucial step during inclusion body processing and dissolving conditions were usually empirically established. Here we describe a new methodology for rapid screening of solubilization conditions and evaluation of dissolution kinetics in microtiter plates. Increase of protein in solution over time was directly related to decrease of turbidity measured by absorbance at 600 nm. Dissolution kinetics of inclusion bodies were described by a first-order reaction kinetics, which was used for drug dissolution modeling. Reaction constants were in the range of 0.01-0.03 s(-1) for buffer conditions providing sufficient solubilization power. This method is not limited to the screening of optimal buffer conditions for solubilization and can be applied for studying other parameters involved in the solubility of IBs, such as pI of the protein, influence of fermentation conditions, influence of initial protein concentration, and more.

A crosslinked inclusion body process for sialic acid synthesis

The propensity of a recombinant protein produced in bacteria to aggregate has been assumed to be unpredictable, and inclusion bodies have been thought of as wasted cell material. However, a target protein can be purposely driven to inclusion bodies, which demonstrate full cell tolerable activity. Sialic acid aldolase, N-terminally fused with the cellulose-binding module of Clostridium cellulovorans, was almost quantitatively physiologically aggregated into active inclusion bodies. These inclusion bodies were entrapped in alginate beads and crosslinked by glutaraldehyde. The immobilized biocatalyst generated by this crosslinked inclusion bodies (CLIB) technology was used in a repetitive batch protocol for sialic acid production that was monitored on-line by flow calorimetry. The required substrate, N-acetyl- d-mannosamine, was obtained by partially improved alkaline epimerization.

Simplified inclusion body processing

Simplified inclusion body processing

A simplified bioprocess for human alpha‐fetoprotein production from inclusion bodies

A simple and effective Escherichia coli (E. coli) bioprocess is demonstrated for the preparation of recombinant human alpha-fetoprotein (rhAFP), a pharmaceutically promising protein that has important immunomodulatory functions. The new rhAFP process employs only unit operations that are easy to scale and validate, and reduces the complexity embedded in existing inclusion body processing methods. A key requirement in the establishment of this process was the attainment of high purity rhAFP prior to protein refolding because (i) rhAFP binds easily to hydrophobic contaminants once refolded, and (ii) rhAFP aggregates during renaturation, in a contaminant- dependent way. In this work, direct protein extraction from cell suspension was coupled with a DNA precipitation-centrifugation step prior to purification using two simple chromatographic steps. Refolding was conducted using a single-step, redox-optimized dilution refolding protocol, with refolding success determined by reversed phase HPLC analysis, ELISA, and circular dichroism spectroscopy. Quantitation of DNA and protein contaminant loads after each unit operation showed that contaminant levels were reduced to levels comparable to traditional flowsheets. Protein microchemical modification due to carbamylation in this urea-based process was identified and minimized, yielding a final refolded and purified product that was significantly purified from carbamylated variants. Importantly, this work conclusively demonstrates, for the first time, that a chemical extraction process can substitute the more complex traditional inclusion body processing flowsheet, without compromising product purity and yield. This highly intensified and simplified process is expected to be of general utility for the preparation of other therapeutic candidates expressed as inclusion bodies.

The economics of inclusion body processing

Many recombinant proteins are often over-expressed in host cells, such as Escherichia coli, and are found as insoluble and inactive protein aggregates known as inclusion bodies (IBs). Recently, a novel process for IB extraction and solubilisation, based on chemical extraction, has been reported. While this method has the potential to radically intensify traditional IB processing, the process economics of the new technique have yet to be reported. This study focuses on the evaluation of process economics for several IB processing schemes based on chemical extraction and/or traditional techniques. Simulations and economic analysis were conducted at various processing conditions using granulocyte macrophage-colony stimulating factor, expressed as IBs in E. coli, as a model protein. In most cases, IB processing schemes based on chemical extraction having a shorter downstream cascade demonstrated a competitive economic edge over the conventional route, validating the new process as an economically more viable alternative for IB processing.

Production of enzymatically active ketosteroid isomerase following insoluble expression inEscherichia coli

Enzymatically active Delta(5)-3-ketosteroid isomerase (KSI) protein with a C-terminus his(6)-tag was produced following insoluble expression using Escherichia coli. A simple, integrated process was used to extract and purify the target protein. Chemical extraction was shown to be as effective as homogenization at releasing the inclusion body proteins from the bacterial cells, with complete release taking less than 20 min. An expanded bed adsorption (EBA) column utilizing immobilized metal affinity chromatography (IMAC) was then used to purify the denatured KSI-(His(6)) protein directly from the chemical extract. This integrated process greatly simplifies the recovery and purification of inclusion body proteins by removing the need for mechanical cell disruption, repeated inclusion body centrifugation, and difficult clarification operations. The integrated chemical extraction and EBA process achieved a very high purity (99%) and recovery (89%) of the KSI-(His(6)), with efficient utilization of the adsorbent matrix (9.74 mg KSI-(His(6))/mL adsorbent). Following purification the protein was refolded by dilution to obtain the biologically active protein. Seventy-nine percent of the expressed KSI-(His(6)) protein was recovered as enzymatically active protein with the described extraction, purification, and refolding process. In addition to demonstrating the operation of this intensified inclusion body process, a plate-based concentration assay detecting KSI-(His(6)) is validated. The intensified process in this work requires minimal optimization for recovering novel his-tagged proteins, and further improves the economic advantage of E. coli as a host organism.

Coupling of chemical extraction and expanded-bed adsorption for simplified inclusion-body processing: optimization using surface plasmon resonance.

Integration of the chemical extraction of recombinant inclusion-body protein from Escherichia coli, and its recovery by metal-affinity expanded-bed adsorption (IMAC-EBA) under denaturing conditions, was investigated. The viral coat protein L1 with a hexa-histidine tag was expressed in Escherichia coli HMS174(DE3) as a model protein. Interference of released host DNA with adsorbent fluidization in the EBA step was solved by selective precipitation using spermine and low-speed centrifugation. However, the capacity and selectivity of the adsorbent for L1 remained lower than anticipated. The binding of L1 to immobilized Ni(2+) was therefore studied in detail using surface plasmon resonance (SPR). The Tris buffer and ethylene-diamine tetraacetic acid (EDTA) used in the extraction mixture were found to interfere significantly with the L1-Ni(2+) interaction. The SPR studies suggest that L1 binding could be improved by replacing the Tris buffer with HEPES and by adding CaCl(2) to inactivate the EDTA. The modified chemical extraction conditions resulted in effective L1 extraction from cytoplasmic inclusion bodies, at high cell density (OD(600 )= 80) and without the use of reducing agent, into a medium optimized for subsequent IMAC recovery. The modified buffer conditions resulted in an improved binding capacity and a good L1 purification factor (12.7) and recovery yield (71%). This work demonstrates that it is possible to reduce the complexity and hence the cost associated with traditional processes used to prepare purified denatured protein, ready for refolding, from cytoplasmic inclusion bodies.

Continuous processing of fusion protein expressed as an Escherichia coli inclusion body

In this study we develop the components of an integrated process for the continuous extraction and purification of a histidine-tagged fusion protein expressed as an inclusion body in Escherichia coli. Lac21 was selected as a model peptide and was expressed as a fusion to ketosteroid isomerase. A purification strategy was developed on a 1-ml batch column before successful scale-up and transfer to a continuous purification system, having a bed volume of 240 ml. Preliminary experiments proved cleavage of the fusion protein. The use of chemical extraction and continuous chromatography gives a flowsheet far superior to the traditional methods for inclusion body processing.