Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling

doi:10.1016/j.chroma.2013.10.039

Journal of Chromatography A

Volume 1319, 6 December 2013, Pages 107-117

https://doi.org/10.1016/j.chroma.2013.10.039 Get rights and content

Highlights

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A chromatographic refolding method which saves up to 99% refolding buffer.
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The methods shifts refolding equilibrium.
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The method can be connected to a purification step.

Abstract

An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from N^pro fusion pep6His and N^pro fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes – one using urea for IB dissolution, the other one using NaOH for IB dissolution – and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes.

Introduction

Protein expression as inclusion bodies provides high yield [1] and purity as high as 90% [2] and this outweighs the disadvantage of the time consuming refolding process. Further improvements may arise from process integration of inclusion body solubilization and refolding on a matrix. A continuous process is the basis for the reduction of footprint, a further increase of productivity, and the possibility of buffer recycling. In protein refolding, the process of correct protein refolding usually is in competition with protein aggregation, where aggregation typically follows a higher order of reaction, at least an order of two or higher [2], [3], [4]. Thus, refolding is commonly performed at concentrations below 0.5 g/L. Proteins in inclusion bodies can be dissolved to extremely high concentrations by chaotropic agents and then diluted into refolding buffer for refolding [5]. Refolding of proteins at dilute concentration is typically performed as a batch operation. Low protein concentrations during refolding are required to achieve reasonable yields, which depend on the folding kinetics and the thermodynamic folding equilibrium and the yields may range from as low as 10% up to 90%, depending on the individual protein [6]. Common to all refolding processes based on batch dilution are high buffer consumption and large reactor volumes, and consequently, low productivities. An alternative to batch dilution refolding is an on-column process also termed matrix-assisted refolding (MAR) where the protein is refolded in the presence of a stationary phase which influences the refolding reaction. Different chromatographic methods have been used for MAR including ion exchange [7], [8], [9], hydrophobic interaction [10], [11], [12], affinity [13], [14], [15], and size exclusion chromatography [2], [16], [17]. Size exclusion chromatography (SEC) is a simple technique for matrix-assisted refolding of proteins. The protein is loaded at high concentrations on the column and eluted using a decreasing chaotrope gradient [16], [18], [19], [20], [21] or refolding buffer. It has been hypothesized that aggregate formation is reduced by the spatial separation within the pores of the SEC beads of aggregated, partly folded, and folded proteins due to the differences in size and the effective diffusivity of unfolded proteins, folded proteins, and aggregates [18]. Hence, the refolding equilibrium is shifted toward the refolded proteins and higher refolding yields are obtained.

The combination of matrix-assisted refolding with continuous processing might increase refolding yield and will intensify the process toward higher productivity and throughput, and lower buffer consumption.

Several continuous refolding processes using SEC have been described. Pressurized continuous annular chromatography (P-CAC) was used for the continuous refolding of α-lactalbumin and lysozyme [22], [23] and simulated moving bed (SMB) for the continuous refolding of lysozyme [24], [25]. Unfolded proteins were continuously separated from chaotropic compounds and transferred to refolding buffer during migration. Continuous refolding with SMB using chaotropes for IB dissolution results in a zone with high chaotrope concentration which affords continuous unfolding of the proteins.

Herein, we present a new concept for a matrix-assisted refolding SMB size exclusion chromatography (MSS) process where the IBs are dissolved with NaOH instead of chaotropes and the folding intermediates and folded proteins are continuously separated from aggregated proteins. This new process has the advantages that the SMB system can be operated isocratically which enables a closed-loop configuration with refolding buffer used as the desorbent buffer and the use of tangential flow filtration (TFF) for recycling of the refolding buffer of the raffinate.

For process evaluation, we used two N^pro fusion peptides, EDDIE-pep6His and EDDIE-MCP-1. N^pro autoprotease fusion proteins and their mutations are self-cleaving fusion proteins which are used for the production of recombinant peptides and proteins in Escherichia coli [2], [26], [27], [28] and are expressed in large amounts in inclusion bodies. During refolding, the autoprotease N^pro becomes active and cleaves itself off from the target protein or peptide leaving it with an authentic N-terminus [29], [30].

IB dissolution with NaOH had to be performed continuously to prevent protein degradation. Therefore, we designed a continuous dissolution reactor with subsequent filtration that involved a tubular reactor design.

Based on the actual refolding data for the two model proteins, we compared the throughput, productivity, and buffer consumption of our MSS process to two batch dilution refolding processes, one using urea for IB dissolution, the other using NaOH for IB dissolution.

Section snippets

Equipment and chemicals

An Octave 10 System (Semba Biosciences, Madison, WI, USA) was used for SMB experiments controlled by SembaPro software version 3.7.2 pre-release 2. An ÄKTA Avant 25 (GE Healthcare, Uppsala, Sweden) controlled by UNICORN software version 6.1 was modified for use with continuous IB filtration and process monitoring. All chemicals were purchased from Sigma (Steinheim, Germany) and Merck (Darmstadt, Germany), if not otherwise stated.

N^pro fusion technology

Two N^pro fusion peptides were used as model proteins. One was

Continuous dissolution reactor for IBs

The design of the overall process and in particular of the continuous dissolution reactor is based on rapid and efficient dissolution of inclusion bodies by NaOH. Dissolution conditions were determined in small scale batch experiments. IBs were completely dissolved by a 1:5 dilution in NaOH and subsequent reduction with MTG in approximately 1 min at final NaOH and MTG concentrations of 75 and 15 mM, respectively. These reaction conditions were the basis for the design of the continuous

Discussion

The experiments included herein proved the concept that a combination of continuous IB dissolution with NaOH and continuous refolding by SMB was a successful process for the production of prepurified protein product. The primary objective was to recycle buffer by a closed-loop SMB configuration and by concentrating the raffinate with TFF to substantially reduce the buffer consumption. Buffer consumption is a major cost driver in downstream processing of protein expressed as inclusion bodies.

Conclusions

On a small scale, we have demonstrated that it is possible to combine continuous inclusion body dissolution, continuous refolding, and buffer recycling by closed-loop SMB. This procedure was demonstrated with real IB suspensions produced in E. coli. Such a design allows the recycling of refolding buffer in the range of >98%. In addition, the refolding reaction is enhanced which further increased productivity of this method. This integrated process is a further contribution to the continuous

Acknowledgements

We thank the group of G. Striedner for fermentation of the N^pro proteins and A. Trefilov for isolation of IBs. This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT – Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG.

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Citation Excerpt :
Low target product concentrations in the refolding step result in large processing volumes at manufacturing scale, necessitating the use of large tanks and requiring proper buffer handling. Therefore, either model-based approaches in refolding [74] or continuous refolding could become a future perspective, reducing the scale and buffer consumption of the process step [75]. Usually, soluble proteins are expressed into the periplasm (E. coli) or extracellularly (P. pastoris), hence no cell disruption is necessary [35].
Current trends in the biopharmaceutical market such as the diversification of therapies as well as the increasing time-to-market pressure will trigger the rethinking of bioprocess development and production approaches. Thereby, the importance of development time and manufacturing costs will increase, especially for microbial production.
In the present review, we investigate three technological approaches which, to our opinion, will play a key role in the future of biopharmaceutical production. The first cornerstone of process development is the generation and effective utilization of platform knowledge. Building processes on well understood microbial and technological platforms allows to accelerate early-stage bioprocess development and to better condense this knowledge into multi-purpose technologies and applicable mathematical models. Second, the application of verified scale down systems and in silico models for process design and characterization will reduce the required number of large scale batches before dossier submission. Third, the broader availability of mathematical process models and the improvement of process analytical technologies will increase the applicability and acceptance of advanced control and process automation in the manufacturing scale. This will reduce process failure rates and subsequently cost of goods. Along these three aspects we give an overview of recently developed key tools and their potential integration into bioprocess development strategies.

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Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling

Highlights

Abstract

Introduction

Section snippets

Equipment and chemicals

Npro fusion technology

Continuous dissolution reactor for IBs

Discussion

Conclusions

Acknowledgements

J. Biotechnol.

Trends Biotechnol.

Curr. Opin. Biotechnol.

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

Protein Expr. Purif.

J. Chromatogr. B: Anal. Technol. Biomed. Life Sci.

Protein Expr. Purif.

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

Chem. Eng. Sci.

Process Biochem.

Process Biochem.

J. Chromatogr. A

N^pro fusion technology