Projects

The main objective of this project is to investigated filtration behaviour of flexible particles and components (e.g. gel particles, droplets, cells), and to design membrane separation processes in such a way that they take this as a starting point of design. The deformability of these particles under actual filtration conditions is far from understood, and likely is influenced by bulk and surface characteristics. For this the following techniques will be used: 

1) Microfluidic tools with which membrane structures can be emulated will be used to investigate the effect of pore geometry, and orientation, on retention.

2) Modelling of the permeation behaviour of flexible particles based on general bulk and interfacial properties.

3) Design or select membranes based on the identified behaviours, together with the industrial partners.

4) Show a proof of principle using an application system relevant for one of the industrial partners. 

Preparative scale cross-flow filtration devices typically contain several layers of soft materials. Different membrane and spacer materials are stacked to achieve optimal retention and capacity for the respective target molecules. These stacks are arranged in cassettes to achieve high surface areas at low footprints. The devices usually have complex internal geometries with channels and flow redirections to direct the liquid flow and hold the filter material in place. Non-homogeneous flow along the filter surface, local compression of the filter material and hydrodynamic bypasses are unavoidable by design. Flow resistance and mechanical compression versus defects due to overstress need to be analysed and balanced. Special attention will be given to scalability of the filtration process and to the transferability of the findings between laboratory and production scales. 

Enrolment in Doctoral degree: RWTH Aachen University

Persons involved: Matteo Truzzi (PhD student), Patrice Bacchin (Supervisor), Clémence Coetsier (Supervisor), Christel Causserand (Supervisor), Olivier Lorain (Representative of the industrial partner, Polymem)

Duration: 15/09/2024 – 14/09/2027

Keywords: Membranes, biomolecules, proteins, perfusion, nanostructure

Introduction: Recombinant proteins, such as monoclonal antibodies, are among the best-selling drugs on the market. Although they are typically produced using fed-batch processes, recently there has been a growing interest in their continuous manufacturing using perfusion bioreactors, that offer higher productivity and flexibility.

A key component of these systems is the cell retention device, which typically consists of membrane modules. However, these membranes are highly susceptible to fouling: cells, cell debris and other components of the culture broth can either accumulate on the membrane surface, forming a cake, or get captured in the membrane pores, blocking them. This significantly reduces product transmission and permeate flux.

Technological Challenges: The problem of membrane fouling has been analyzed for a long time through numerous studies, leading to theories and models presented by several authors over the course of the years. Nevertheless, it remains an open issue, especially in an evolving field such as biomanufacturing. The particular conditions adopted in perfusion bioreactors, characterized by low and constant permeate fluxes, can lead to specific fouling mechanisms. Gaining a deeper understanding of these mechanisms, and of the transfer of biomolecules through the membrane under such conditions, represents one of the main challenges of the present project, and is the key to the development of membrane materials and structures exhibiting better performances.

Research Goal: This project aims to fill this research gap by developing new membranes with controlled pore size, pore shape and surface chemistry, optimized for the utilization in perfusion bioreactors. These membranes will be the result of a detailed study and modeling of the fouling mechanisms, carried out with the help of micro- and millifluidic devices.

The objective of this PhD project is to design separation units with novel electrospun membranes for in-situ virus removal from simulated bio-fluids. Specifically, this project aims to:

1) Screen through biocompatible materials that can form mechanically strong thin-film structure, fabricate a series of nanofibrous membranes with selected polymers as the blank control.

2) Fabricate a series of composite membrane by blending the selected polymer with pre-functionalized copolymers to a selected virus. The formation of membrane morphology will be optimized. Characterize the membrane morphology, chemistry and conduct biocompatibility testing.

3) Assemble the as-fabricated membranes into the housing to form a separation unit, whereby the hydrodynamics will be optimized. 

4) Set-up and evaluate the separation performance of the membrane unit using synthetic feedstocks, examine the viral removal efficiency.

5) Establish a mathematical model to understand the transport mechanisms of the membranes by examining the synergistic separation effects; examine the structure-chemistry-fluid relationship. Develop a mechanistic model by integrating factors associated with the mass transport and flow dynamics, verify the model via experimental data.

Persons involved: Vidianos Giannitsis, Xing Yang

Duration: 01/2025-01/2029

Keywords: Protein recovery, membrane crystallization, electric field-assisted crystallization, conductive membranes, non-solvent induced phase separation (NIPS), classical nucleation theory (CNT)

Technological Challenges: Proteins are high value biomolecules with a wide variety of applications. One of the main factors hindering them is the high cost associated with their separation, even with commercial techniques such as Protein A Chromatography. Crystallization is one of the most cost effective separation processes, and it has been widely researched as an economic alternative. However, protein crystallization is seen as a challenging process. Due to the size of the protein, the energy barrier for stable crystal nuclei to be formed is very high. This results in high supersaturation being necessary to produce crystals. But, proteins are very sensitive molecules, significantly limiting the ways in which we can achieve said supersaturation. Furthermore, the mechanism of protein crystallization is not fully understood, especially in the case of heterogeneous nucleation (crystals are formed on a surface, rather than in the solution), leading to it being a mostly empirical process, which is not always reproducible.

Introduction: Membrane Crystallization is a highly researched process for improving crystallization performance. The membrane has a dual function in this process. It acts as a surface to enable heterogeneous nucleation, which significantly lowers the energy barrier for crystal nuclei formation. Furthermore, it acts as a selective barrier for solvent removal, which increases the supersaturation of the solution, and does so in a manner that is safe for the protein (osmotic flow). As a combination, these 2 factors, both of which are easily controllable, make membrane crystallization much easier than traditional solution crystallization, as well as more reproducible.

Another process which is widely cited in literature to improve crystallization performance is electric field-assisted crystallization. When charged molecules - like proteins far from their isoelectric point - enter an electric field, their motion is controlled by it. This creates a high local supersaturation near the electrode which attracts the protein. Furthermore, due to the different permittivity of the protein in the liquid and solid phase, the electric field can thermodynamically favor the phase transition and further decrease the energy barrier for nucleation.

Despite work in both of these sectors, there is a lack of studies showing the integration of these 2 processes, which however is expected to yield promising results.

Research Goal: The goal of this project is to design a protein crystallization process, making use of both a membrane and an electric field to intensify the process. Initially, the processes will be studied separately to understand their effects and then their synergy will be studied in detail to try and maximize the yield of protein crystallization. Furthermore, the project will have a modelling part, where we will try to mechanistically understand the process better and be able to make informed, rather than empirical decisions to optimize the process further.

The project aims to apply innovative and sustainable methodologies to recover high added-value compounds from dairy wastes. The use of whey, the most relevant cheese by-product, allows to minimize the environmental impact of dairy industry by recovering greatly exploitable value-added proteins. In more details, the idea is to use whey proteins to develop an edible antimicrobial coating to increase food shelf life and prevent spoilage. In order to reach the proposed objective, the research activity will be based on the development of an integrated membrane-system that will consists of sequential membrane separation processes coupled with hydrolysis bioreactors, to recover and hydrolyse whey proteins. Whey protein enzymatic hydrolysis will be carried out in a membrane bioreactor (EMBR), using proteolytic enzymes. The bioactive peptides obtained from whey proteins hydrolysates will be then purified and fractionated by membrane chromatography. The use of functionalized membrane adsorbers for peptides fractionation is an attractive alternative to chromatographic resins, since they are less expensive, scalable and can operate at fast flow rates. Since most antimicrobial peptides from whey proteins are positively charged at neutral pH, they can be fractionated using cation exchange membranes by tuning the elution gradient. Whey-derived antimicrobial peptides will be characterized and tested in different recipes to obtain an edible coating for a semi- seasoned cheese. The formulation will be tested during cold shelf storage with the aim to reduce biofilm formation and the persistence of spoilage bacteria in food environment.

Membrane adsorbers have smaller footprints and can be operated at higher flow rates as compared to packed bed chromatography which typically provides higher capacities. New membrane materials with micro-porous gel elements and competitive capacities are recently developed at Sartorius. They can be functionalized to facilitate affinity, ion-exchange and mixed-mode chromatography. The combined design of porous structures, functional groups and operating conditions opens a wide portfolio of applications for membrane adsorbers. For example, field flow fractionation can potentially be replaced by chromatography and ultrafiltration. Rapid cycling of single and tandem units or simulated moving bed processes allow moving towards continuous processing. Numerous impact factors pose a challenge for device characterization and process development. Process models similar to the general rate model for packed bed chromatography will be used for tackling this challenge and exploring the applicability of porous membranes in particular for antibodies and small particles. Digital tools will also be made available for smart operation and control of automated systems.

Enrolment in Doctoral degree: RWTH Aachen University

Persons involved: Pablo Almendras Flores, Rob Lammertink, Joris de Grooth, Anurag Arun

Duration: 2024-2028

Keywords: Membrane technology, sustainability assessment, process feasibility, life cycle analysis, techno-economic analysis, scenario analysis

Introduction: In concordance with several EU/global initiatives (SDG, EU Green Deal) it is necessary to move towards more sustainable practices to reach sustainable development, and within the membrane technology field it has also become a major concern in recent years. This project focuses on assessing the sustainability of membrane-based processes, tackling the analysis from the 3 main pillars of sustainability: environmental, economic and social.  By integrating process economy and Life Cycle Analysis (LCA) and using scenario analysis based on real data, literature and experimental inputs from the others DCs, a comprehensive assessment framework will be developed that can be used to help membrane-based emerging technologies to reach industrial viability while being sustainable at the same time.

Technological Challenges: Achieving a balance between membrane performance, cost-effectiveness and sustainability may represent a challenge due to high reliance on fossil based raw materials and this issue can compromise the processes scalability and reliability in industrial applications.

Research Goal: To develop a methodology for evaluating the sustainability of membrane-based processes through scenario analysis, integrating economic, environmental and technical aspects to enhance the development of novel separation technologies. 

Enrolment in Doctoral degree: University of Twente

The project will focus on the development of novel units for membrane chromatography. Packed column chromatography is one of the main separation processes in the purification of biomolecules, and membrane chromatography is an emerging technology that shows great promise. However, there are still challenges with the implementation of membrane chromatography in industrial bioprocesses, mainly due to the lack of suitable equipment that can provide reliable performance at different scales. As with conventional packed columns, uniform mobile phase distribution and reduced residence time distribution within the stationary phase are essential to ensure efficiency and resolution in membrane processes. From this perspective, the geometry and architecture of the module play a critical role. Membrane modules can be classified according to the flow path within the bed: axial flow or radial flow. Regardless of the flow path, membrane chromatography modules are characterized by a bed with a high surface area to length ratio, uneven residence time distribution and a high dead volume fraction. These parameters and their effects on the process performance vary significantly with the module size and cannot be accurately predicted based on experimental results obtained on small modules. As the module size increases, the uniformity of fluid distribution deteriorates, the residence time distribution increases, and the ratio of dead volume to membrane volume alters. 

Persons involved: Chih-Ching Huang, Patrice Bacchin, Yannick Hallez

Duration: 3 years

Keywords: Membrane formation, Microfluidics, Diffusiophoresis, Field flow fractionation (FFF)

Introduction: This project will focus on fabricating membranes within microfluidic systems using liquid induced phase separation (LIPS) for bioseparation applications.

Microfluidics, which enables the manipulation of fluids at the microscale, offers several advantages for bioseparation applications, including precise control, reduced reagent consumption, and the potential for integration and automation. These features make it highly suitable for applications in biosensing, protein analysis, diagnostics, and drug delivery. Microfluidics has already demonstrated its potential in enhancing separation processes through miniaturization and local control of mass transport. However, in all of these applications, the use of membranes in microfluidics system is relatively limited, especially for separation. Therefore, it is interesting to developing microfluidics with membranes to explore new separation applications.

Currently, we have successfully fabricated permeable membranes within microfluidic channels using photopolymerization techniques. This approach has allowed us to integrate separation elements directly on microfluidic chips. But photopolymerized membranes are limited in terms of material diversity, tunability of pore size, and chemical resistance. These limitations restrict our ability to tailor membrane properties for specific separation tasks and to study the membrane formation process in detail.

To address these limitations, this project proposes a novel strategy. To fabricate membranes inside microfluidic channels via LIPS. By collaborating with our industrial partner Polymem, who has already commercialized LIPS membrane technologies, this approach enables the creation of a broader range of membrane chemistries with tunable pore sizes and enhanced resistance to solvents. More importantly, performing phase inversion at the microscale will allow for the investigation of local dynamics during membrane formation, which remains poorly understood in conventional systems.

In the second phase of the project, these in situ fabricated membranes will be used to perform bioseparation directly within the microfluidic system. In addition to conventional filtration, we will integrate novel separation mechanisms such as diffusiophoresis and field-flow fractionation (FFF). These techniques exploit solute gradients and particle diffusivity to separate objects based on charge or size without the need for external pressure, offering promising advantages for the selective handling of complex biological mixtures.

Overall, this project aims to establish a microfluidic platform that not only enables controlled membrane fabrication via phase inversion but also demonstrates its application in microfluidic chip bioseparation. Combining membrane science with microscale fluid handling, the project seeks to explore both fundamental aspects of membrane formation and the development of new separation strategies suitable for complex biological systems.

Technological Challenges: Controlled membrane formation in microchannels. Due to the geometry at the microscale, it is a huge challenge to control the dope solution (casting solution) and water.

Research Goal: To successfully fabricate membranes inside microfluidic chips, and subsequently use these chips for bio-object separation in collaboration with other DCs.

To provide a better understanding of the conditions that have an effect in membrane harvesting of different microorganisms from culture broth, specifically:

1) Trained to grow various cells (e.g. Escherichia coli, Saccharomyces cerevisiae, Blakeslea trispora, mammalian cells) in different scales and bioreactors (e.g. shaking flasks, small bioreactors, perfusion systems).

2) Screen a number of four available commercial MF and/or four UF membranes with different configurations (e.g., flat sheets, tubular, hollow fibre, etc.) and material (e.g., PES, PVDF, etc) performance initially with a bacterium (E. coli) and a cheap and relatively simple culture broth, such as Luria–Bertani medium.

3) Evaluate and optimize the cell separation conditions (temperature, transmembrane pressure, flow rate, optimum surface area to broth load, etc.) Detailed evaluation of the filtration performance of the two best membranes material/configuration under the optimum operating conditions with different E. coli culture media and culture ages.

4) Evaluate the performance of the two best performing membranes under the optimum conditions derived from the previous stages with S. cerevisiae (yeast) and Blakeslea trispora (fungi) cells and relatively simple and appropriate broth in each case.

5) Compare the different microorganism behaviour and try to get a universal model for the filtration behaviour. Characterize the membrane surface by various spectrophotometric techniques, such as FITR, Raman, etc.

The current PhD main objective is to develop a novel and low-energy simultaneous production of intracellular, carotenoids, and extracellular, hydrolytic enzymes, by using the fungi Blakeslea trispora in a membrane bioreactor using agri- food waste as carbon resource, specifically:

1) Screen a number of available juice industry waste pulp material (e.g., beetroot, apple, citrus etc.) in shake flasks to evaluate fungus yields in the two target products (carotenoids and hydrolytic enzymes). These products yields will be mainly evaluated using the existing previous knowledge and appropriate protocols that are in place.

2) Evaluate in custom made small volume (500 mL) batch bioreactors the short-term performance of submerged MF/UF tubular/hollow fibre membranes. Optimize in these small bioreactors the growth conditions (e.g., carbon source, product and cells removal and cells recycling ratio etc) and the in situ physical cleaning of membrane by backwashing for prolonging the operating periods and achieving the higher possible productivities.

3) Set-up and evaluate long –term performance in a larger capacity (3 L) custom-made membrane bioreactor and appropriate automation and control. The membrane bioreactor will be equipped with a conductivity probe, turbidity probe probe, and an online gas analyser able to record in real time all the products formation providing crucial information for the system kinetics and performance.

4) Use of a second side UF membrane system that will allow the hydrolytic extracellular enzymes to reach higher concentrations.

5) Techno-economical evaluation of the proposed combined extracellular and intracellular biotechnological products formation in comparison with other existing processes.