3D printing technology: Supply chain independent single-use plastic ware and bioreactors for cell culture

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Building the future with on demand 3D printing

The current shorts in single use (SU) supply chains show how dependent both industry and academia are from only a few vendors worldwide. This is severely hindering fundamental research and process development for the pandemic response. With 3D printing technology we can manufacture SU equipment on demand and on site. In this study we investigated different commercially available low-cost materials and their compatibility for cell culture. We identified poly lactic acid (PLA) as perfect candidate for 3D printed parts for cell culture applications. The worldwide supply chain issues for SU shaking flasks and reactors gave us the incentive to develop 3D printed counterparts to maintain our HEK293 cell culture. The shake flasks were designed in Autodesk inventor 3D CAD. The materials tested represent the market of different 3D printing technologies and materials, ranging from UV-polymerizing resin printers to thermoplastic printers. We included different manufacturers, plant derived and water washable resins as well as medical Class IIa resins. Whereas resin printed shaking flasks needed washing, curing and sterilization using isopropyl alcohol, the thermoplastic flasks were directly autoclaved. The different materials were tested with HEK293 cells under standard conditions. Cell growth and viability were monitored daily. Please click Additional Files below to see the full abstract

High cell density culture for VLP production in latent virus-free insect cell line

Continuous virus-like particle (VLP) production in insect cells with the baculo virus expression system faces numerous problems: The presence of defective interfering particles (DIPs) hinders continuous infection, cell growth is slower than virus replication, cell retention membranes and hollow fibres retain VLPs. High cell densities (HCD) can nevertheless enable VLP process intensification in small reactor vessels. We were able to achieve a HCD with the Tnms42 insect cell line in both shaking flasks with pseudo perfusion and in the bioreactor utilizing an alternating tangential flow (ATF) filtration. Compared to some commercially available insect cell lines, this cell line has no persistent adventitious viral infection and is equally well suited to produce HA-GAG VLPs as HighFiveTM cells. With daily medium exchange the exponential growth phase could be elongated and the total cell concentration (TCC) increased by a factor of two in the shaking flask (SF) and four in the bioreactor (BR) to ~40x106 cells/mL in comparison to reference batch processes (~9 Figure 1). Such HCD can be used to optimize and investigate cell concentration at infection (CCI), multiplicity of infection (MOI) and cell state at infection to reach higher volumetric VLP titers. Please click Download on the upper right corner to see the full abstract

Integration of continuous ethanol precipitation and flocculation into manufacturing of antibodies

Precipitation and flocculation are optimal unit operations for continuous capture of proteins from culture supernatant. Precipitation and flocculation can be operated in a real continuous manner. The methods can be combined and after this capture step the precipitate can be stored as a concentrated solution or even as a precipitate. We have developed several precipitation/ flocculation protocols for capture of antibodies from culture supernatant. These include the combinations of CaCl2 flocculation with either cold ethanol precipitation or PEG precipitation and octanoic acid precipitation with PEG precipitation. The precipitation conditions have been screened in microtiter plates or in case of cold ethanol precipitation in small scale reactors. For cold ethanol precipitation a combination with CaCl2 flocculation is best suited. In the first step the high molecular mass impurities can be removed by flocculation with CaCl2 while in the second step the antibody is precipitated by addition of ethanol and low molecular mass impurities are removed. The whole procedure can be repeated and then final polishing can be performed by an anion exchange step in flow through mode. Octanoic acid precipitation is also a very efficient step but an additional phase can be formed which is difficult to remove. In Figure 1 a bench top reactor for cold ethanol precipitation is depicted. The rector consists of two sections (Figure 1). First a concurrent cooling is installed to ensure a constant cooling rate while ethanol is added to the culture supernatant or pre-treated supernatant. In the second section a countercurrent cooling is installed to keep the reactors at the requested temperature. The industry standard for antibody capture is protein A affinity chromatography. Thus the properties of the antibody after cold ethanol precipitation have been compared to protein A purified material. No significant difference could be observed (Figure 2) in either composition or structure. Further purification has been tested by anion-exchange monoliths in order to remove further host cell proteins. The different protocols will be compared to other standard platforms for antibody purification. The possibilities to integrate in-process control and maintenance of steady state will be discussed. The economics of such a process will be discussed for the different scenarios of clinical phase manufacturing and how strategies can be developed when antibodies become commodities or when oral delivery becomes reality. Next steps will be shown how to scale up such a process

Continuous protein precipitation – A robust antibody purification method without the need for steady state conditions during continuous integrated production

Antibody precipitation as capture step is an alternative technology to chromatography, because it is a fully continuous method. Bioreactor effluent from a perfusion reactor can be directly processed without surge tanks and the method can be combined with flocculation. We hypothesized that a continuous precipitation and flocculation reactor can be operated under non-steady state conditions. This will allow the design of reactors with extremely short residence time. In batch precipitation steady stats is achieved when the floc size does not change over time. We developed a continuous capture step for antibodies by protein precipitation with PEG 6000. Precipitation and re-solubilization conditions were established by a high throughput approach in batch mode. Continuous precipitation was realized by a tubular reactor design containing static mixers. The particle size distribution of the product precipitate was on-line monitored by Focused Beam Reflectance Measurement (FBRM) in a flow cell. Various hold times (2 to 20 minutes) of the cell culture harvest and different product concentrations were used to simulate changes during continuous fermentation. Precipitate was collected and separation was performed by centrifugation. High molecular weight impurities were reduced by a factor of 5 to 10, yields of 90% and purity increase by a factor of 2.5 were achieved according to size exclusion chromatography. Using FBRM we were able to demonstrate that different residence times during precipitation do not significantly change the particle size distribution. Stable performance concerning product quality (yield, purity and high molecular weight impurities) for different residence times were demonstrated for up to 90 minutes runtime. The influence of product concentration on the precipitate was quantified by FBRM. We demonstrated that protein precipitation is a robust and feasible capture step for mAb purification that does not require steady state conditions and that such continuous reactors are not compulsory operated at steady state conditions. Optimization potential was identified and can be realized during upscale, and higher yields and better performance can be reasonably expected for larger scales

Residence time distribution of continuous protein a chromatography

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Elution profile from periodic counter current capture step as an on-line monitoring and control tool for perfusion bioreactors

Current trends in bioprocessing move towards the implementation of more on-line sensors such as Raman spectroscopy for titer monitoring in perfusion bioreactors. However, process performance data from one downstream unit operation can also be used to monitor and control the unit operation directly upstream. Despite several authors demonstrated a successful integration of continuous up-and downstream processes little attempts have been made to leverage the information derived from downstream processing as a real time feedback loop for upstream processing. We have developed a simple and robust approach in which protein A periodic counter current chromatography (PCCC) can function as an on-line monitoring tool for protein titer in continuous upstream fermentations. For a proof of concept, we exploit the fact that performance and binding capacities of state of the art protein A chromatography material do not significantly decrease throughout hundreds of cycles. Therefore, it is possible to predict the concentration of antibodies in the feed material from the elution pool and the volume loaded onto the column. We use the breakthrough curve during the interconnected phase of the PCCC, which is key for this approach. In the interconnected phase, the first column was loaded to 80% breakthrough, and the breakthrough curve modelled for a number of different concentrations in the feed material. Using the breakthrough curve, the time of the breakthrough can be modelled against the increase of product present in the feed stream, allowing the prediction of the concentration of antibody in the perfusion fermentation. This information feedback loop through the integration of PCCC and fermentation into effectively one single unit operation makes the titer determination in the fermentation obsolete, using the PCCC effectively as online monitoring tool itself. Future work after this proof of concept will include the prediction of protein A binding capacities through the lifetime of the resin and determination of accuracy and quantification limits the of interconnected units. Please click Additional Files below to see the full abstract

On demand, in-situ media and buffer preparation in continuous integrated biomanufacturing to reduce water consumption and CO2 footprint

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Scaleable microscale perfusion systems for yeast and mammalian cells to accelerate process development of bioproducts

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Continuous protein precipitation – A robust antibody purification method without the need for steady state conditions during continuous integrated production.

Antibody precipitation as capture step is an alternative technology to chromatography, because it is a fully continuous method. Bioreactor effluent from a perfusion reactor can be directly processed without surge tanks and the method can be combined with flocculation. We hypothesized that a continuous precipitation and flocculation reactor can be operated under non-steady state conditions. This will allow the design of reactors with extremely short residence time. In batch precipitation steady stats is achieved when the floc size does not change over time. We developed a continuous capture step for antibodies by protein precipitation with PEG 6000. Precipitation and re-solubilization conditions were established by a high throughput approach in batch mode. Continuous precipitation was realized by a tubular reactor design containing static mixers. The particle size distribution of the product precipitate was on-line monitored by Focused Beam Reflectance Measurement (FBRM) in a flow cell. Various hold times (2 to 20 minutes) of the cell culture harvest and different product concentrations were used to simulate changes during continuous fermentation. Precipitate was collected and separation was performed by centrifugation. High molecular weight impurities were reduced by a factor of 5 to 10, yields of 90% and purity increase by a factor of 2.5 were achieved according to size exclusion chromatography. Using FBRM we were able to demonstrate that different residence times during precipitation do not significantly change the particle size distribution. Stable performance concerning product quality (yield, purity and high molecular weight impurities) for different residence times were demonstrated for up to 90 minutes runtime. The influence of product concentration on the precipitate was quantified by FBRM. We demonstrated that protein precipitation is a robust and feasible capture step for mAb purification that does not require steady state conditions and that such continuous reactors are not compulsory operated at steady state conditions. Optimization potential was identified and can be realized during upscale, and higher yields and better performance can be reasonably expected for larger scales