482 research outputs found
Mathematical modeling of diafiltration
The main objective of this study is to provide a general mathematical model in a compact form for batch diafiltration techniques. The presented mathematical framework gives a rich representation of diafiltration processes due to the employment of concentration-dependent solute rejections. It unifies the existing models for constant volume dilution mode, variable volume dilution mode, and concentration mode operations. The use of such a mathematical framework allows the optimization of the overall diafiltration process. The provided methodology is particularly applicable for decision makers to choose an appropriate diafiltration technique for the given separation design problem
Organic solvent nanofiltration membrane cascades for solvent exchange and purification
Imperial Users onl
Digital twins for scFv production in Escherichia coli
Quality-by-Design (QbD) is demanded by regulatory authorities in biopharmaceutical production. Within the QbD frame advanced process control (APC), facilitated through process analytical technology (PAT) and digital twins (DT), plays an increasingly important role as it can help to assure to stay within the predefined proven acceptable range (PAR).This ensures high product quality, minimizes failure and is an important step towards a real-time-release testing (RTRT) that could help to accelerate time-to-market of drug substances, which is becoming even more important in light of dynamical pandemic situations. The approach is exemplified on scFv manufacturing in Escherichia coli. Simulation results from digital twins are compared to experimental data and found to be accurate and precise. Harvest is achieved by tangential flow filtration followed by product release through high pressure homogenization and subsequent clarification by tangential flow filtration. Digital twins of the membrane processes show that shear rate and transmembrane pressure are significant process parameters, which is in line with experimental data. Optimized settings were applied to 0.3 bar and a shear rate of 11,000 s−1. Productivity of chromatography steps were 5.3 g/L/d (Protein L) and 2167 g/L/d (CEX) and the final product concentration was 8 g/L. Based on digital twin results, an optimized process schedule was developed that decreased purification time to one working day, which is a factor-two reduction compared to the conventional process schedule. This work presents the basis for future studies on advanced process control and automation for biologics production in microbials in regulated industries
Hybrid modeling of a biorefinery separation process to monitor short-term and long-term membrane fouling
Membrane filtration is commonly used in biorefineries to separate cells from fermentation broths containing the desired products. However, membrane fouling can cause short-term process disruption and long-term membrane degradation. The evolution of membrane resistance over time can be monitored to track fouling, but this calls for adequate sensors in the plant. This requirement might not be fulfilled even in modern biorefineries, especially when multiple, tightly interconnected membrane modules are used. Therefore, characterization of fouling in industrial facilities remains a challenge. In this study, we propose a hybrid modeling strategy to characterize both reversible and irreversible fouling in multi-module biorefinery membrane separation systems. We couple a linear data-driven model, to provide high-frequency estimates of trans-membrane pressures from the available measurements, with a simple nonlinear knowledge-driven model, to compute the resistances of the individual membrane modules. We test the proposed strategy using real data from the world's first industrial biorefinery manufacturing 1,4-bio-butanediol via fermentation of renewable raw materials. We show how monitoring of individual resistances, even when done by simple visual inspection, offers valuable insight on the reversible and irreversible fouling state of the membranes. We also discuss the advantage of the proposed approach, over monitoring trans-membrane pressures and permeate fluxes, from the standpoints of data variability, effect of process changes, interaction between module in multi-module systems, and fouling dynamics
Improving downstream processing for viral vectors and viral vaccines
Viral vectors are playing an increasingly important role in the vaccine and gene
therapy elds. The broad spectrum of potential applications, together with
expanding medical markets, drives the e orts to improve the production processes
for viral vaccines and viral vectors. Developing countries, in particular, are
becoming the main vaccine market. It is thus critical to decrease the cost per
dose, which is only achievable by improving the production process. In particular
advances in the upstream processing have substantially increased bioreactor yields,
shifting the bioprocess bottlenecks towards the downstream processing. The
work presented in this thesis aimed to develop new processes for adenoviruses
puri cation. The use of state-of-the-art technology combined with innovative
continuous processes contributed to build robust and cost-e ective strategies for
puri cation of complex biopharmaceuticals.(...
Flexible Manufacturing Facility for Biopharmaceuticals
Monoclonal antibodies (mAbs) have the potential to treat a wide range of diseases. They possess the ability to bind target molecules in a highly specific and effective manner. Recently, great technological advances have been made to enhance the therapeutic effects of these drugs, making treatment cheaper, easier, and more effective while allowing companies to profit significantly. As a Contract Manufacturing Organization (CMO) for these products, we offer the newest technology and many flexible options for producing these proteins. Our facility is designed to produce protein products in Chinese Hamster Ovary (CHO) cells, followed by modification and purification steps. We present the option to cleave smaller antigen binding fragments (Fab) from the mAb product, removing the crystallizable fragment (Fc) which can interfere with the binding specificity of the drug. We also offer the option for polyethylene glycosylation (PEGylation), which has been shown to improve the effectiveness of these drugs. The attachment of a polyethylene glycol (PEG) molecule to the protein enhances its circulation time in the human body so that less frequent doses are needed.
To demonstrate the capabilities of this flexible facility, we have modeled the production of an innovative PEGylated anti-TNF-α mAb. Celltech and Pfizer currently have similar products in Phase III clinical trials; and UCB Incorporated’s Cimzia® recently received FDA approval for the first humanized PEGylated anti-TNF-α Fab’ therapeutic protein. Many non- PEGylated TNF-α inhibitor mAbs are currently on the market to treat pathologies including rheumatoid arthritis and Crohn’s disease. PEGylated products have a clear advantage over these drugs.
Our facility can produce up to 55 batches of protein product a year for a maximum yield of 993 kg. For economic analysis of this product, sales from the first year of Cimzia® were considered since this product is almost identical to the one being modeled. Producing at 75% of the total design capacity, this facility has a NPV of $1,319,592,100, an IRR of 33.51% and an ROI of 53.0%. This level of production would leave a significant amount of time remaining for other products to be manufactured as well. The additional products that the facility will produce will be mAbs of all forms (i.e. cleaved, uncleaved, PEGylated, non-PEGylated) that are protected under IP for small biotech firms that do not have the capital to build such facilities. Currently, small biotech firms are producing 81 mAbs and are looking to license production for their Phase III molecules. Clearly, this will become a very profitable CMO as we would be able to capture much of this demand. A major threat looming over the mAb market, however, is the production of small molecular inhibitors, which are currently in Phase I and Phase II clinical trials. Such molecules may be able to capture the full market since they would not only have significant delivery advantages over TNF-α inhibitors, which require injection, but also might have an enhanced side effect profile compared with biologics
Production of a SARS-CoV-2 Spike Protein Vaccine Using the Baculovirus Expression Vector System
Various COVID-19 vaccines are currently in development, as the COVID-19 pandemic has created an unmet need for protection against the SARS-CoV-2 virus. While there are many different types of vaccines, we focused on developing one that would be safe, affordable, and quickly available for emergency use. A vaccine synthesized using recombinant proteins utilizes a reliable and well-studied technological platform, avoids the safety risks inherent to viral vectors, and provides a cost-effective, scalable method of production of antigen used to induce an immune response. Other vaccines on the market notably include Pfizer’s and Moderna’s mRNA based vaccines. Although these are widely used, there is still a large demand for an inexpensive yet safe and effective vaccine. Herein, we propose the production of 500 million doses of a recombinant spike protein-based COVID-19 vaccine in a quick time frame and cost-effective manner, using the baculovirus expression vector system (BEVS). Our upstream process involves a three-stage cellular scale-up from shake flasks to WAVE bioreactors to perfusion to production bioreactors, as well as an additional two-stage viral amplification from flasks to WAVE bioreactors. Our downstream process involves a six-stage protein recovery with depth filtration, his-tag chromatography, viral inactivation, ion-exchange chromatography, viral filtration, and diafiltration. We will be partnering with a contract manufacturing organization (CMO) for this project, as we do not have the time to quickly build a plant to get these vaccines out for emergency use. This arrangement makes this process highly profitable. Selling each dose for 2 billion and an extremely high IRR due to the lack of permanent and fixed costs other than our rental fee. The IRR for the CMO is estimated to be at least 16% with the NPV of the plant at $855,000 and an ROI of 18%
Scalable mRNA machine for regulatory approval of variable scale between 1000 clinical doses to 10 million manufacturing scale doses
The production of messenger ribonucleic acid (mRNA) and other biologics is performed primarily in batch mode. This results in larger equipment, leaning/sterilization volumes, and dead times compared to any continuous approach. Consequently, production throughput is lower and capital costs are relatively high. Switching to continuous production thus reduces the production footprint and also lowers the cost of goods (COG). During process development, from the provision of clinical trial samples to the production plant, different plant sizes are usually required, operating at different operating parameters. To speed up this step, it would be optimal if only one plant with the same equipment and piping could be used for all sizes. In this study, an efficient solution to this old challenge in biologics manufacturing is demonstrated, namely the qualification and validation of a plant setup for clinical trial doses of about 1000 doses and a production scale-up of about 10 million doses. Using the current example of the Comirnaty BNT162b2 mRNA vaccine, the cost-intensive in vitro transcription was first optimized in batch so that a yield of 12 g/L mRNA was achieved, and then successfully transferred to continuous production in the segmented plug flow reactor with subsequent purification using ultra- and diafiltration, which enables the recycling of costly reactants. To realize automated process control as well as real-time product release, the use of appropriate process analytical technology is essential. This will also be used to efficiently capture the product slug so that no product loss occurs and contamination from the fill-up phase is <1%. Further work will focus on real-time release testing during a continuous operating campaign under autonomous operational control. Such efforts will enable direct industrialization in collaboration with appropriate industry partners, their regulatory affairs, and quality assurance. A production scale-operation could be directly supported and managed by data-driven decisions
Process design and optimization towards digital twins for HIV-gag VLP production in HEK293 cells, including purification
Despite great efforts to develop a vaccine against human immunodeficiency virus (HIV), which causes AIDS if untreated, no approved HIV vaccine is available to date. A promising class of vaccines are virus-like particles (VLPs), which were shown to be very effective for the prevention of other diseases. In this study, production of HI-VLPs using different 293F cell lines, followed by a three-step purification of HI-VLPs, was conducted. The quality-by-design-based process development was supported by process analytical technology (PAT). The HI-VLP concentration increased 12.5-fold while >80% purity was achieved. This article reports on the first general process development and optimization up to purification. Further research will focus on process development for polishing and formulation up to lyophilization. In addition, process analytical technology and process modeling for process automation and optimization by digital twins in the context of quality-by-design framework will be developed
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