Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein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 currently produced as IBs. To obtain correctly folded, soluble product, IBs have to processed, namely, harvested, solubilized, refolded. Several years ago, it was discovered that, depending on cultivation conditions properties, contain partially folded...

In bioprocess engineering the Qualtiy by Design (QbD) initiative encourages use of models to define design spaces. However, clear guidelines on how for QbD are validated still missing. this review we provide a comprehensive overview validation methods, mathematical approaches, and metrics currently applied in modeling. The methods cover analytics data used modeling, model training selection, measures predictiveness, uncertainties. We point out general issues calibration different types put...

Optical density (OD) is a critical process parameter during fermentation, this being directly related to cell density, which provides valuable information regarding the state of process. However, measure OD, sampling fermentation broth required. This particularly challenging for high-throughput-microbioreactor (HT-MBR) systems, require robotic liquid-handling (LiHa) systems control tasks, such as pH regulation or carbon feed additions. Bioreactor volume limited and automated at-line occupies...

Monolithic columns are a special type of chromatography column, which can be used for the purification different biomolecules. They have become popular due to their high mass transfer properties and short times. Several articles already discussed monolith manufacturing, as well characteristics. In contrast, this review focuses on applied aspect monoliths discusses most relevant biomolecules that successfully purified by them. We describe success stories viruses, nucleic acids proteins...

The methylotrophic yeast Pichia pastoris is a well-studied host organism for recombinant protein production, which usually regulated either by constitutive promoter (e.g. of glyceraldehyde-3-phosphate dehydrogenase; PGAP) or an inducible alcohol oxidase 1; PAOX1). Both systems have several advantages and disadvantages; with one the main disadvantages being their lack tunability. Various novel systems, are de-repressed, allowing higher degrees freedom, been reported. Recently, bi-directional...

The methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders use proteins from in most biopharmaceutical applications. Glyco-engineered strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology protein controlled environment bioreactor scarce. A...

Cell disruption is a key unit operation to make valuable, intracellular target products accessible for further downstream operations. Independent of the applied cell method, each process must be evaluated with respect efficiency and potential product loss. Current state-of-the-art methods, like measuring total amount released protein plating-out assays, are usually time-delayed involve manual intervention making them error-prone. An automated method monitor at-line not available date.In...

Recombinant protein production in E. coli often leads to the formation of inclusion bodies (IBs). Although downstream processing IBs has reputation being a great hurdle, advantages can be substantial. Highly pure recombinant with possibility correctly folded structures and an easy separation from cell matter are decisive factors that make IB processes so interesting. Product yield, purity biological activity refolded responses evaluate process. The objective this case study was develop...

Escherichia coli is one of the most commonly used host organisms for production recombinant biopharmaceuticals. E. usually characterized by fast growth on cheap media and high productivity, but drawback its intracellular product formation. Product recovery from bioprocesses requires tedious downstream processing (DSP). A typical DSP an starts with a cell disruption step to access product. Different methods exist, scalable process achieved pressure homogenization (HPH). The protocols HPH are...

The bacterium Escherichia coli is a well-studied recombinant host organism with plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced E. coli. However, the high metabolic burden during protein production can lead to cell death, consequent lysis, undesired product loss. Thus, fast precise analyzers monitor bioprocesses retrieve key process information, optimal time point harvest, needed....

During recombinant protein production with E. coli, the integrity of inner and outer membrane changes, which leads to product leakage (loss integrity) or lysis integrity). Motivated by current Quality Design guidelines, there is a need for monitoring tools determine leakiness in real-time. In this work, we assessed novel approach coli cell attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. Various preprocessing strategies were tested combination regression...

Monitoring process variables in bioprocesses with complex expression systems, such as filamentous fungi, requires a vast number of offline methods or sophisticated inline sensors. In this respect, cell viability is crucial variable determining the overall performance. Thus, fast and precise tools for identification key deviations transitions are needed. However, reliable monitoring still scarce to date require equipment. study, we used commonly available size exclusion chromatography (SEC)...

Downstream process development for recombinant glycoproteins from yeast is cumbersome due to hyperglycosylation of target proteins. In a previous study, we purified three Pichia pastoris using simple two-step flowthrough mode approach monolithic columns. this investigated novel automated data science identifying purification conditions such We performed sets design experiments in analytical scale determine the separation efficiency columns different horseradish peroxidase (HRP) isoenzymes....

Horseradish peroxidase (HRP), a versatile heme-containing glycoprotein frequently used in medical diagnostics, is primarily isolated from plant. This process involves filtration, salt precipitation and several chromatography steps, which expensive, renders low yields gives isoenzyme mixtures. Although single isoenzymes can be recombinantly produced Pichia pastoris, they get hyper-glycosylated the yeast rendering downstream cumbersome thus not competitive. In this study, we analyzed...

UV chromatographic data in combination with multivariate analysis (MVDA) has been extensively used for bioprocess monitoring. However, they are usually attributed to shifts along the retention time and require preprocessing. Misaligned result inconsistent MVDA models. Numerous preprocessing techniques available, each varying number of meta-parameters optimize, complexity computational time. Therefore, we aimed at developing a generic workflow screen techniques. We chose four datasets...

Intermittent bolus feeding for E. coli cultivations in minibioreactor systems (MBRs) profoundly affects the cell metabolism. Bolus leads to temporal substrate surplus and transient oxygen limitation, which triggers formation of inhibitory byproducts. Due high demand right after injection substrate, dissolved tension (DOT) signal exhibits a negative pulse. This contribution describes analyzes this DOT response cultivations. In addition gaining information on culture conditions, unique...

Bi-directional static load testing for piles has been around long time. However, the loading mechanism is different from conventional pile method, many practicing engineers have concern over its of top settlement. Its proponents believe it safer, easier, and capable achieving higher loads than traditional top-loading methods. The methods including kentledge constraints performing loads, which may lead to fatal accidents. India seen a lot development in field infrastructure recent past strive...