RESUMO
INTRODUCTION: In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not always correspond with the highest quality of the produced protein. While some post-translational modifications, such as disulphide bonds, are required to achieve the biologically active conformation, others may have a negative impact on the product's activity, effectiveness, and/or safety. Therefore, they are classified as product associated impurities, and they represent a crucial quality parameter for regulatory authorities. RESULTS: In this study, fermentation conditions of two widely employed industrial E. coli strains, BL21 and W3110 are compared for recombinant protein production of a single-chain variable fragment (scFv) in an industrial setting. We found that the BL21 strain produces more soluble scFv than the W3110 strain, even though W3110 produces more recombinant protein in total. A quality assessment on the scFv recovered from the supernatant was then performed. Unexpectedly, even when our scFv is correctly disulphide bonded and cleaved from its signal peptide in both strains, the protein shows charge heterogeneity with up to seven distinguishable variants on cation exchange chromatography. Biophysical characterization confirmed the presence of altered conformations of the two main charged variants. CONCLUSIONS: The findings indicated that BL21 is more productive for this specific scFv than W3110. When assessing product quality, a distinctive profile of the protein was found which was independent of the E. coli strain. This suggests that alterations are present in the recovered product although the exact nature of them could not be determined. This similarity between the two strains' generated products also serves as a sign of their interchangeability. This study encourages the development of innovative, fast, and inexpensive techniques for the detection of heterogeneity while also provoking a debate about whether intact mass spectrometry-based analysis of the protein of interest is sufficient to detect heterogeneity in a product.
Assuntos
Proteínas de Escherichia coli , Anticorpos de Cadeia Única , Escherichia coli/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Proteínas Recombinantes , Proteínas de Escherichia coli/metabolismo , Dissulfetos/metabolismoRESUMO
Inclusion bodies (IBs) often emerge upon overexpression of recombinant proteins in E. coli. From IBs, refolding is necessary to generate the native protein that can be further purified to obtain pure and active biologicals. This work focusses on refolding as a significant process step during biopharmaceutical manufacturing with an industrial perspective. A theoretical and historical background on protein refolding gives the reader a starting point for further insights into industrial process development. Quality requirements on IBs as starting material for refolding are discussed and further economic and ecological aspects are considered with regards to buffer systems and refolding conditions. A process development roadmap shows the development of a refolding process starting from first exploratory screening rounds to scale-up and implementation in manufacturing plant. Different aspects, with a direct influence on yield, such as the selection of chemicals including pH, ionic strength, additives, etc., and other often neglected aspects, important during scale-up, such as mixing, and gas-fluid interaction, are highlighted with the use of a quality by design (QbD) approach. The benefits of simulation sciences (process simulation and computer fluid dynamics) and process analytical technology (PAT) for seamless process development are emphasized. The work concludes with an outlook on future applications of refolding and highlights open research inquiries.
Assuntos
Produtos Biológicos , Corpos de Inclusão , Produtos Biológicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Corpos de Inclusão/metabolismo , Redobramento de Proteína , Proteínas Recombinantes/biossínteseRESUMO
Fusion protein technologies improve the expression and purification of recombinant proteins, but the removal of the tags involved requires specific proteases. The circularly permuted caspase-2 (cpCasp2) with its specific cleavage site, efficiently generates the untagged protein. While cleavage with cpCasp2 is possible before all 20 proteinogenic amino acids, cleavage before valine, leucine, isoleucine, aspartate and glutamate suffers from slow, and before proline extremely slow, turnover. To make the platform fusion protein process even more general such that any protein with an authentic N-terminus can be produced with high efficiency, the bacterial selection system PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection) was used to evolve cpCasp2 into a variant with a catalytic turnover two orders of magnitude higher and the ability to cleave before any amino acid. The high specificity and the stability of the original circularly permuted protease was fully retained in this mutant, while the high manufacturability was mostly retained, albeit with decreased soluble titer. Four point-mutations are responsible for this change in activity, two of which are located in or near the binding pocket of the active site. This variant was named CASPON enzyme and is a major component of the CASPase-based fusiON (CASPON) platform technology. Applicability for the production of recombinant proteins was demonstrated by enzymatic removal of the CASPON tag from five model proteins. The CASPON tag enables high soluble expressions, affinity purification and good accessibility for cleavage. The five industry-relevant proteins of interest were FGF2, TNF, GH, GCSF and PTH.
Assuntos
Aminoácidos , Caspase 2 , Cromatografia de Afinidade , Proteínas Recombinantes de Fusão/metabolismo , Proteínas RecombinantesRESUMO
Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at -20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.
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Caspase 2/biossíntese , Cisteína Endopeptidases/biossíntese , Escherichia coli/química , Proteínas Recombinantes de Fusão/biossíntese , Caspase 2/isolamento & purificação , Caspase 2/metabolismo , Clonagem Molecular , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/metabolismo , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Process analytical technology combines understanding and control of the process with real-time monitoring of critical quality and performance attributes. The goal is to ensure the quality of the final product. Currently, chromatographic processes in biopharmaceutical production are predominantly monitored with UV/Vis absorbance and a direct correlation with purity and quantity is limited. In this study, a chromatographic workstation was equipped with additional online sensors, such as multi-angle light scattering, refractive index, attenuated total reflection Fourier-transform infrared, and fluorescence spectroscopy. Models to predict quantity, host cell proteins (HCP), and double-stranded DNA (dsDNA) content simultaneously were developed and exemplified by a cation exchange capture step for fibroblast growth factor 2 expressed in Escherichia coliOnline data and corresponding offline data for product quantity and co-eluting impurities, such as dsDNA and HCP, were analyzed using boosted structured additive regression. Different sensor combinations were used to achieve the best prediction performance for each quality attribute. Quantity can be adequately predicted by applying a small predictor set of the typical chromatographic workstation sensor signals with a test error of 0.85 mg/ml (range in training data: 0.1-28 mg/ml). For HCP and dsDNA additional fluorescence and/or attenuated total reflection Fourier-transform infrared spectral information was important to achieve prediction errors of 200 (2-6579 ppm) and 340 ppm (8-3773 ppm), respectively.
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Cromatografia por Troca Iônica/métodos , Fator 2 de Crescimento de Fibroblastos/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/genética , Fator 2 de Crescimento de Fibroblastos/genética , Modelos Químicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Regulação para CimaRESUMO
Escherichia 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.
Assuntos
Escherichia coli/citologia , Corpos de Inclusão/química , Fracionamento Celular/economia , Fracionamento Celular/métodos , Escherichia coli/química , Escherichia coli/genética , Corpos de Inclusão/genética , Miniaturização/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Fatores de TempoRESUMO
A simultaneous crystallization and aqueous two-phase extraction of a single chain antibody was developed, demonstrating process integration. The process conditions were designed to form an aqueous two-phase system, and to favor crystallization, using sodium sulfate and PEG-2000. At sufficiently high concentrations of PEG, a second phase was generated in which the protein crystallization occurred simultaneously. The single chain antibody crystals were partitioned to the top, polyethylene glycol-rich phase. The crystal nucleation took place in the sodium sulfate-rich phase and at the phase boundary, whereas crystal growth was progressing mainly in the polyethylene glycol-rich phase. The crystals in the polyethylene glycol-rich phase grew to a size of >50 µm. Additionally, polyethylene glycol acted as an anti-solvent, thus, it influenced the crystallization yield. A phase diagram with an undersaturation zone, crystallization area, and amorphous precipitation zone was established. Only small differences in polyethylene glycol concentration caused significant shifts of the crystallization yield. An increase of the polyethylene glycol content from 2% (w/v) to 4% (w/v) increased the yield from approximately 63-87%, respectively. Our results show that crystallization in aqueous two-phase systems is an opportunity to foster process integration.