WO2021110870A1

WO2021110870A1 - Method for producing a fermentation product

Info

Publication number: WO2021110870A1
Application number: PCT/EP2020/084532
Authority: WO
Inventors: Diethard Mattanovich; Damiano TOTARO; Matthias Steiger; Peter Ertl; Mario ROTHBAUER
Original assignee: Acib Gmbh; Universität Für Bodenkultur Wien
Priority date: 2019-12-05
Filing date: 2020-12-03
Publication date: 2021-06-10

Abstract

A method for producing a fermentation product in a cell culture, comprising: a. determining optimized process parameters for culturing a high producer cell line to produce said fermentation product, wherein the determining step of a. is made upon cell culture employing a multiplexed microfluidic perfusion cell culture device in perfusion mode, and wherein the high producer cell line is optionally selected from a repertoire of cell lines capable of producing said fermentation product; and b. culturing said high producer cell line by an industrial scale method under the selected process parameters to produce said fermentation product.

Description

METHOD FOR PRODUCING A FERMENTATION PRODUCT

FIELD OF THE INVENTION

The present invention refers to a method for producing a fermentation product, wherein a multiplexed microfluidic perfusion cell culture device is applied for screening and selecting producing cell lines or process parameters. The present invention further refers to a multiplexed microfluidic perfusion cell culture device and the manufacturing thereof.

BACKGROUND OF THE INVENTION

Industrial production based on bioprocesses is a well-established method today for a variety of products ranging from chemicals and advanced biofuels in bacterial and yeast cell factories to biopharmaceuticals utilizing mammalian cells and other cell lines. The key challenges in bioprocess development are associated with (a) determining the most suitable cell line for the production and (b) identifying the optimum process parameters, such as pH, oxygen concentration, temperature, cell density and cell culture medium composition and concentration, as well as productivity variations under certain conditions.

Despite these challenges, traditional shake flasks and bench-scale bioreactors (ranging from 10 mL to 10 L volume) are most commonly used during the early stages of bioprocess development, though they are expensive, time-consuming and labour- intensive as well as lacking the throughput for efficient production optimizations.

These limitations pose a serious challenge for the development of a new bioprocess, particularly in the early stages, where large numbers of input factors and response variables need to be analytically assessed to determine the quality of the product and reliability of the selected procedure.

A strategy to reduce time and costs of lab-scale fermentations is based on process automation and utilization of miniaturized technologies. Microfluidic devices have demonstrated reduced reagent/media consumption, rapid mass transfer caused by improved surface-area-to-volume ratio and smaller diffusion distances resulting in an improved mixing, which is an important factor in managing the needs of living cell cultures.

Some attempts have been made concerning the fabrication of microfluidic version of batch bioreactors and microfluidics droplets for cell culture, taking advantage of the physical and mechanical properties of some polymers, such as the high gas permeability of polydimethylsiloxane (PDMS) for the aeration of the culture, or the relative ease of structuring polymers like poly(methyl methacrylate) (PMMA) and PDMS to create fluidic structures. Additionally, they enable the fabrication of disposable devices either as a monolith or in a modular assembly and the high optical transparency of most of these polymers facilitates integration of optical detection methods.

Miniaturized batch reactors of different volumes have been tested, especially on E. coli cultures. A constant effort was made towards downscaling the reactors. Szita et al. disclose microbioreactors with a working volume of 150 pl_, allowing to perform parallel batch culture fermentations (Szita et al., Lab Chip 2005, 5, 819-826). Zanzotto et al. disclose the operation of a batch microbioreactor with a volume of 5-50 pL containing sensors for the measurement of optical density, dissolved oxygen and pH; and compared the results obtained with the batch microbioreactor to a larger scale 500- mL bench-scale bioreactor (Zanzotto et al. Biotechnol. Bioeng. 2004, 87(2), 243-254). Totaro et al. describe a microfluidic platform for downscaling fermentation made of polydimethylsiloxane (D. Totaro et al. „Microfluidic Platform for Downscaling Fermentation and for Screening Engineered Strains”, Poster presentation at European Summit of Industrial Biotechnology, Graz, Austria, 2017).

A perfusion-capable microdevice for the investigation of secondary metabolite production using microalgae cultures is reported by Paik et al. (Bioresour. Technol. 2017, 233, 433-437). Mozdierz et al. disclose a perfusostat device with a working volume of 1 mL featuring sensors for dissolved oxygen and pH measurement (Mozdzierz et al., Lab Chip 2015, 15, 2918-2922). Downscaling of perfusion microbioreactors to a volume of 1.6 pL is disclosed by Vit et al. (J. Chem. Technol. Biotechnol. 2019, 94, 712-720). US7507579B2 and US20060199260 refer to microscale bioreactors in which two vessels are separated by a membrane, and further describe an apparatus for parallel operation of a plurality of such individual microreactors.

WO201 4059035 A1 discloses a method of optimizing a perfusion cell culture system by decreasing the starting perfusion rate, resulting in an increased residence time of cells in the bioreactor and the cell retention device, and increasing the starting bioreactor volume or decreasing the starting cell retention device volume, or both.

W02007008609 A2 discloses a cell culture array for handling cells in microfluidic systems. Small fluidic structures mimic liver vasculature in living tissue, and enable high density tissue-like cultures in defined structures. There is an inlet and an outlet channel and a fluidic connection through small perfusion channels that are smaller than the cells which therefore stay inside the cell culture array.

US7507579 B2 discloses an apparatus and method for simultaneous microfermentors and arrays for culturing cells.

EP3401014 A1 discloses a microfluidic chip and a method for enriching cells in a microfluidic chip.

WO201 700786 A1 discloses vascularized perfusion devices and an adaptable in vitro microcirculation model.

There is a need to provide a small-scale method of selecting process parameters to be applied to cell cultures which can be upscaled to the industrial scale.

SUMMARY OF THE INVENTION

It is the objective of the present invention to provide an improved method for producing a fermentation product in industrial scale by applying process parameters and/or cell lines selected by using a microfluidic cell culture device. It is another objective to provide an improved microfluidic cell culture device that is able to run fermentation processes in parallel for simultaneously screening process parameters and/or cell lines, wherein the selected process parameters and/or cell lines can be used in a large-scale process of producing a fermentation product.

The objective is solved by the claimed subject matter, and as further described herein.

The present invention provides a method for producing a fermentation product in a cell culture, comprising: a. determining optimized process parameters, for culturing a high producer cell line to produce said fermentation product, wherein the determining step of a. is made upon cell culture employing a multiplexed microfluidic perfusion cell culture device in perfusion mode, and wherein the high producer cell line is optionally selected from a repertoire of cell lines capable of producing said fermentation product; and b. culturing said high producer cell line by an industrial scale method or fermenter under the optimized process parameters to produce said fermentation product.

Specifically, the determining step is by selecting process parameters, which were determined as being optimal for individual producer cell lines in the microfluidic perfusion system. Such process parameters surprisingly were found to be suitable when culturing such producer cell lines at large scale.

In one embodiment described herein, said process parameters are selected from the group comprising productivity, temperature, oxygen concentration or pressure, in particular dissolved oxygen concentration, pH value, cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass concentration, metabolites concentration, and carbon dioxide concentration.

Specifically, said process parameters are optimized or selected by culturing said high producer cell line in a multiplexed microfluidic perfusion cell culture device in perfusion mode, wherein said process parameters are selected and/or optimized, according to the amount of the fermentation product produced.

According to one aspect described herein, said high producer cell line is selected by culturing said repertoire of cell lines in a multiplexed microfluidic perfusion cell culture device, wherein said repertoire comprises a variety of cell lines, which differ in their capability of producing said fermentation product.

Specifically, said high producer cell line is selected from said repertoire according to the amount of the fermentation product produced.

A process parameter selected according to the amount of the fermentation product produced may include e.g. productivity to produce a fermentation product, titer or concentration of the fermentation product, or the respective yield. In particular, the amount of the fermentation product is obtained in the cell culture medium or filtrate.

In one embodiment as described herein, said multiplexed microfluidic perfusion cell culture device comprises at least: a. a cell culture chamber unit (1) comprising at least two spatially distinct cell culture chambers (4), wherein each of said cell culture chambers comprises a cell inoculation channel (5), a flow inlet (6) and a flow outlet (7); and b. a microchannel network unit (2) comprising for each of said cell culture chambers (4) a flow input channel (8) connected to the flow inlet (6) and a flow output channel (9) connected to the flow outlet (7); wherein the flow output channel (9) and/or the flow outlet (7) is equipped with a cell retention filter unit (3), preferably wherein the cell retention filter unit comprises a porous filter with a pore size of less than 1 pm.

According to a specific aspect, there is provided a multiplexed microfluidic perfusion cell culture device comprising at least: a. a cell culture chamber unit (1) comprising at least two spatially distinct cell culture chambers (4), wherein each of said cell culture chambers (4) comprises a cell inoculation channel (5), a flow inlet (6) and a flow outlet (7) wherein (5), (6) and (7) are separate elements; and b. a microchannel network unit (2) comprising for each of said cell culture chambers (4) a flow input channel (8) connected to the flow inlet (6) and a flow output channel (9) connected to the flow outlet (7); wherein the flow output channel (9) and/or the flow outlet (7) is equipped with a cell retention filter unit (3), wherein the cell retention filter unit comprises a porous filter with a pore size of less than 1 pm.

Specifically, the device comprises one or more integrated sensors for assessing one or more process parameters. Preferably, each of the cell culture chambers comprises one or more integrated sensors. In one embodiment described herein, one or more integrated sensors are positioned within the microchannel network unit. In one embodiment, the device comprises one or more external detection units connected to each cell culture chamber for assessing one or more process parameters.

Specifically, said one or more integrated sensors are selected from the group consisting of a temperature sensor, an oxygen sensor, a pH sensor, a carbon dioxide sensor, a biomass sensor, a protein sensor, an organic molecule sensor, and a metabolite sensor.

Specifically, said one or more external detection units are connected to each cell culture chamber to determine any one or more of the process parameters selected from the group consisting of cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass density or concentration, and metabolite concentration. In a specific aspect, said multiplexed microfluidic perfusion cell culture device comprises a material selected from the group comprising polydimethylsiloxane (PDMS), glass, polyesters, polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymers (COC), polystyrene (PS), poly-ethylene glycol diacrylate (PEG-DA), polyurethane (PU), polypropylene (PP), and polytetrafluoroethylene (PTFE).

In a specific aspect, the volume of said spatially distinct cell culture chambers is less than 1 ml_, preferably less than 500 pl_, 400 mI_, 300 mI_, 100 mI_, more preferably less than 50 mI_, even more preferably less than 20 mI_, most preferably 15 pL or less.

In one embodiment described herein, said repertoire of cell lines is selected from the group consisting of yeast, bacterial or mammalian cells.

Specifically, the yeast is of a species selected from the group consisting of any of the Saccharomyces genus, Pichia genus, Scheffersomyces genus, Komagataella genus, Yarrowia genus, Hansenula genus, Ogataea genus, or Klyveromyces genus, preferably Saccharomyces cerevisiae, Pichia pastoris, Komagataella pastoris, or Komagataella phaffii.

Specifically, the bacterial cells are selected from the group consisting of Escherichia, Bacillus, Lactobacillus, Clostridium, Pseudomonas, preferably Escherichia coli.

Specifically, the mammalian cells are selected from the group consisting of CHO, BHK, HeLa, HEK293, MDCK, NIH3T3, NS0, PER.C6, SP2/0 and VERO cells.

One aspect as described herein refers to the fermentation product, which is selected from a cell metabolite or a protein.

Specifically, the cell metabolite is selected from the group consisting of organic acids, alcohols and amino acids, preferably lactic acid, itaconic acid, citric acid, succinic acid, glutamic acid, 3-hydroxypropionic acid, adipic acid, ethanol, propane-1 , 3-diol, 1- butanol, 2-butanol, butane-1 ,4-diol, L-glutamic acid, L-lysine, L-threonine.

Specifically, the protein is a secreted protein.

In one aspect, the protein is selected from the group of heterologous proteins, preferably selected from an antigen-binding protein, a therapeutic protein, an enzyme, a peptide, a protein antibiotic, a toxin fusion protein, a carbohydrate - protein conjugate, a structural protein, a regulatory protein, a vaccine antigen, a growth factor, a hormone, a cytokine, a process enzyme. In one aspect described herein, said industrial scale method is a non-perfusion method, preferably a batch, fed-batch or non-perfusion continuous method. In another aspect described herein, said industrial scale method is a perfusion method.

Specifically, the industrial scale method employs a reactor of a volume of at least 1 L, specifically at least 10 L, more specifically at least any one of 50 L, 100 L, 500 L, 1 m³, 10 m³, 100 m³, 300 m³, or 500 m³.

Industrial scale batch or fed-batch fermenters typically have a volume ranging from 1 to 50000 L.

Industrial scale continuous fermenters typically have a volume ranging from 1 to 50000 L.

Industrial scale perfusion fermenters typically have a volume ranging from 1 to 1000 L.

Further disclosed herein is a method for screening one or more process parameters for culturing a cell line and producing a fermentation product from said cell line comprising the steps of: a. providing a cell line capable of producing said fermentation product; b. culturing the cell line in separate microfluidic perfusion cell culture chambers under differing process parameters to produce said fermentation product, wherein said process parameters are selected from the group comprising productivity, temperature, oxygen concentration, pH value, cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass concentration, metabolite concentration, and carbon dioxide concentration; c. determining one or more process parameters optimized according to the fermentation product yield; and d. culturing the cell line in an industrial scale method or fermenter, wherein one or more of the selected process parameters are applied.

A further aspect described herein refers to a method for screening a production cell line and producing a fermentation product from said cell line comprising the steps of: a. providing a repertoire of cell lines comprising a variety of cell lines which differ in their capability of producing said fermentation product; b. culturing each of said cell lines in a separate microfluidic perfusion cell culture chamber under conditions to produce said fermentation product; c. selecting a high producer cell line according to the fermentation product yield; d. optionally selecting one or more process parameters; and e. culturing the selected cell line in an industrial scale method or fermenter under conditions to produce said fermentation product.

Another embodiment described herein relates to the use of process parameters and/or a high producer cell line selected by a method employing a multiplexed microfluidic perfusion cell culture device for producing a fermentation product in an industrial scale process, which may include a non-perfusion or perfusion cultivation system.

FIGURES

Figure 1a: 3D structure design of the multiplexed microfluidic device according to one embodiment described herein, showing a cell culture chamber unit 1, a microchannel network unit 2 and a bottom layer 10.

Figure 1b: 3D structure of one spatially separate cell culture chamber 4 of the device depicted in Figure 1a, showing a cell retention filter unit 3, a flow inlet 6 and flow outlet 7, as well as three separate channels for cell inoculation 5, flow input 8 and flow output 9.

Figure 2a: 3D structure design of the multiplexed microfluidic device according to another embodiment described herein, showing a cell culture chamber unit 1 comprising three layers 16, 17, 18, a microchannel network unit 2 comprising two layers 19, 20, and a cell retention filter unit 3 positioned between the cell culture chamber unit 1 and the microchannel network unit 2, wherein the cell retention filter unit 3 comprises a membrane 11 sandwiched between two adhesive sheets 12 and 13, which are positioned between two glass plates 14 and 15.

Figure 2b: 3D structure of one individual cell culture chamber 4 of the device depicted in Figure 2a, showing a membrane 11, two adhesive sheets 12 and 13 a flow inlet 6 and flow outlet 7, as well as three separate channels for cell inoculation 5, flow input 8 and flow output 9.

Figure 3: Cross section of a cell culture chamber 4 depicted in Figure 1b and Figure 2b, showing a flow inlet 6, a flow outlet 7, a flow input channel 8, a flow output channel 9, a sensor spot 21 which is integrated within the cell culture chamber, and can be used e.g. for oxygen sensing, and a light emitter 22 and a light detector 23 of an external biomass detection unit. Figure 3 further shows cells 25 inside the cell culture chamber 4, substrate 24 of the cell culture medium, and a fermentation product 26.

DETAILED DESCRIPTION

Specific terms as used throughout the specification have the following meaning.

The term “cell culture” or “culturing” or “cultivation” as used herein refers to the maintenance of cells in an artificial environment, under conditions favoring growth, differentiation, continued viability and productivity, in an active or quiescent state of the cells, specifically in a controlled bioreactor according to methods known in the industry.

The term “cell line” as used herein refers to an established clone of a particular cell type that has acquired the ability to proliferate over a prolonged period of time. A cell line is typically used for expressing an endogenous or recombinant gene, or products of a metabolic pathway to produce polypeptides or cell metabolites mediated by such polypeptides. Herein, the term “cell line” is used interchangeably with a strain of cells, also referred to as “cell strain”.

The term “repertoire of cell lines” as used herein is understood as a library or collection of a variety of clones, which may be provided in separate containments, or in a pool. The repertoire may be of a particular cell type. Specific repertoires comprise or consist of a variety of clones which are variants of a parent strain, wherein variation can be achieved by genetic engineering resulting in different phenotypes selectable by their function. The repertoire may comprise or consist of a number of different clones e.g. to cover a diversity of at least any one of 10, 100, or 1000 cell lines, each characterized by a different genotype and/or phenotype. Specific clones contained in a repertoire used for selecting a high producer cell line may differ in their capability to produce a fermentation product.

Such repertoire can be suitably screened and individual clones can be selected according to desired structural or functional properties. The variety of cell lines can be cultured in a pool, or in individual containments. For example, a variety of cell lines may be cultured in the multiplexed perfusion device, wherein a pool of cell lines (e.g. the repertoire in a pool, or in one or more subpools) is cultured in one single cell culture chamber, herein understood as pool screening. Upon such pool screening, a suitable pool (or subpool) can be selected for further screening of a subpool or an individual cell line.

A repertoire of cell lines can be constructed by well-known techniques, involving, for example, CRISPR, golden gate cloning, PCR, electroporation, DNA synthesis, gel electrophoresis, plasmids and bacteriophages construction and amplification, restriction enzymes digestions, DNA sequencing, blotting techniques, chemical gene synthesis, transformation, transfection.

The term “high producer cell line” as used herein shall refer to a cell line characterized by fast growth and/or high amount of a fermentation product. In a microfluidic perfusion device, a typical high producer cell line produces at least any one of 1 g/L, 5 g/L or 10 g/L, when the product is a cell metabolite, or at least any one of 1 mg/L, 5 mg/L or 10 mg/L, when the product is a protein, such as a heterologous protein. In industrial scale, a typical high producer cell line produces at least any one of 10 g/L, 15 g/L or 20 g/L, when the product is a cell metabolite, or at least any one of 100 mg/L, 250 mg/L or 500 mg/L when the product is a protein, such as a heterologous protein.

The term “fermentation product” as used herein refers to the product produced by culturing a cell line in a method as disclosed herein.

The term “heterologous protein” as used herein refers to a protein which is either foreign to a given host cell, i.e. “exogenous”, such as not found in nature in said host cell; or that is naturally found in a given host cell, e.g., is “endogenous”, however, produced by the host cell employing a heterologous expression construct encoding said protein, e.g., employing an exogenous nucleic acid fused or in conjunction with an endogenous nucleic acid, thereby rendering the construct heterologous. A heterologous protein is typically produced by means of a recombinant expression construct, to express a recombinant protein of interest.

The term “recombinant protein” as used herein, includes a protein that is prepared, expressed, created or isolated by recombinant means, such as a protein isolated from a cell line transformed or transfected with a recombinant expression construct to express said protein. In accordance with the present invention conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, (1982). The production method described herein specifically allows for the fermentation on a pilot or industrial scale in a bioreactor. A “bioreactor” can include a fermenter or fermentation unit, or any other suitable reaction vessel.

Specifically, the industrial scale method as described herein is a non-perfusion method or a perfusion method.

The term “non-perfusion method” specifically refers to a batch, fed-batch, or a non-perfusion continuous method.

A “batch method”, or “batch mode” is to be understood as a cell culture process by which a small amount of a cell culture solution is added to a medium and cells are grown without adding an additional medium or discharging a culture solution during culture.

“Fed-batch method” or “fed-batch mode” refers to a culture technique starting with cell growth in the batch phase, followed by a “fed” phase during which the cell culture is in continuous mode wherein the cell culture medium is continuously added (“fed”) to the bioreactor.

“Continuous method”, or “continuous mode” is a cell culture process by which a medium is continuously added and discharged during culture.

The term “non-perfusion continuous” method specifically refers to those culture techniques which allow the continuous discharge of cells together with the cell culture medium or fermentation product. In such case, the cells are not retained in the bioreactor.

In contrast, a “perfusion method” or “perfusion mode” as described herein is a form of the continuous method, wherein the cells are retained in the bioreactor, or cell culture chamber.

The term “process parameter” as used herein refers to a parameter under which cultivation of a cell line is performed. Process-parameters can be measured to any kind of cell cultures. A cell culture can be controlled by applying certain process parameters. Non-limiting examples of process parameters include cell productivity, temperature, oxygen concentration or pressure, in particular dissolved oxygen concentration, pH value, cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass density or concentration, metabolites concentration, and carbon dioxide concentration.

The term “screening step” as used herein refers to testing a variety of process parameters and/or cell lines, and selecting specific process parameters and cell lines, respectively, according to pre-determined selection criteria. The term “selection step” shall refer to choosing one or more process parameters and/or high producer cell lines from the variety of process parameters and cell lines, respectively, for producing a fermentation product.

The term “cell culture medium” as referred to herein is a medium for culturing cells containing substrate and nutrients that maintain cell viability, support proliferation, growth and/or the production of a fermentation product e.g. by biotransformation of a carbon source. The cell culture medium may contain any of the following in an appropriate combination: substrate (carbon/energy source (e.g., glycerol, succinate, lactate, and sugars such as, e.g., glucose, lactose, sucrose, and fructose)), nitrogen source, precursors, and nutrients such as vitamins and minerals, salts, buffer(s), amino acids, antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc.

Cell culture media ordinarily used for particular cell types are known to those skilled in the art.

The multiplexed microfluidic perfusion cell culture device as described herein is also referred to as a “chip”, e.g. with respect to “on-chip” culture, or “microfluidic platform”.

The term “multiplexed” as used herein refers to the arrangement of bioreactors that allow performing or control at least two different fermentation processes in parallel and optionally simultaneously.

The term “cell culture chamber unit” as used herein refers to a unit of a material containing at least two spatially separate cell culture chambers.

The term “cell culture chamber” as used herein refers to a bioreactor e.g., in the form of a well or a vessel, in which cells grow and produce a fermentation product.

The term “channel” as used herein refers to a hole of constant or systematically varied cross-sectional area through a material for transporting a fluid. Generally, a channel has a defined cross-sectional geometry, which may be rectangular, ovoid, circular, or one of these geometries with an imposed finer feature, such as indentations, etc. A "microchannel", has at least one dimension of less than 1000 microns. Typically, the characteristic dimensions of a cross-section of a microchannel (e.g., height and width of a channel with a rectangular cross-section, diameter of a microchannel with a circular cross-section, etc.) will both be less than 1000 microns. It will be understood that the cross-section is to be taken perpendicular to the length of the microchannel and that the length of the microchannel is often greater than 1000 microns. It will further be appreciated that any of the channels in the devices described herein may be, and typically is, a microfluidic channel, for example a “flow input channel” is a channel for transporting a fluid into a cell culture chamber, and a “flow output channel” is a channel for transporting a fluid out of a cell culture chamber.

The term “flow inlet” as used herein refers to the device element on or at the cell culture chamber that connects each cell culture chamber with a flow input channel.

The term “flow outlet” as used herein refers to the device element on or at the cell culture chamber that connects each cell culture chamber with the corresponding flow output channel.

As specific flow inlet or flow outlet may be a hole in one of the walls of the cell culture chamber, optionally including a construction outside the cell culture chamber that is equipped with a connector element, suitable to connect with the flow input channel and flow output channel, respectively.

As used herein, an “integrated sensor” refers to a sensor which is integrated within or part of the device described herein. Preferably an integrated sensor is positioned within the cell culture chamber. An integrated sensor may also be positioned within other parts of the device, such as within the microchannel network unit. Examples include, but are not limited to a temperature sensor, an oxygen sensor, a pH sensor, a carbon dioxide sensor, a protein sensor, an organic molecule sensor, a metabolite sensor. An external detection unit is understood as a detection unit located outside, but connected to, a cell culture chamber to determine any one or more of the process parameters selected from the group consisting of cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass concentration, and metabolite concentration. Examples include, but are not limited to a biomass detection unit based on light scattering. Specifically, integrated sensors and external detection units allow for a convenient and in situ determination and analysis of the effect of different process parameters on cell growth and fermentation product yield, which facilitates process parameter optimization in a screening process. Specifically, any of the sensors may be referred to as an “in-line sensor”, wherein the term “in-line” as used herein refers to the possibility of continuously measuring a process parameter without drawing a sample. In particular, any one of an integrated sensor and/or external sensor and detection unit, respectively, may be used for in-line analysis of one or more process parameters. According to the present invention, a method is provided for producing a fermentation product in a cell culture. The method specifically comprises selecting at least one process parameter for culturing a high producer cell line to produce the fermentation product, wherein the selection step comprises employing a multiplexed microfluidic perfusion cell culture device in perfusion mode. The method may also comprise selection of a high producer cell line from a repertoire or variety of cell lines capable of producing said fermentation product. The method further specifically comprises culturing said high producer cell line by an industrial scale method under the selected process parameters to produce said fermentation product.

Selection of process parameters specifically refers to the selection of e.g. temperature, oxygen concentration or pressure, in particular dissolved oxygen concentration, pH value, cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass concentration, metabolite concentration, and carbon dioxide concentration. These process parameters typically influence cell growth and fermentation product yield and may be optimized in order to increase the fermentation product yield for a given cell line. Thus, according to one embodiment as described herein, a multiplexed microfluidic perfusion cell culture device is used to screen and optimize process parameters. The device allows for a high throughput screening and fast collection of data.

Process parameters optimized by employing a microfluidic perfusion cell culture device in perfusion mode may be applied in an industrial scale-non perfusion or perfusion method for culturing a cell line and producing a fermentation product.

Specifically, the method described herein can be used to select a high producer cell line from a repertoire of cell lines. Bioprocess development often involves screening a large pool or variety of producing cell lines of a particular cell type, which differ in their capability to produce a fermentation product.

Specifically, the method described herein allows screening and selecting a high producer cell line from a pool or variety of cell lines. The selected cell line(s) can be applied in an industrial scale-non perfusion method for culturing a cell line and producing a fermentation product.

Specifically, the screening and selection steps involve applying a multiplexed microfluidic perfusion cell culture device, such as further described herein.

According to a specific embodiment described herein, the multiplexed microfluidic perfusion cell culture device is able to run fermentation processes in parallel for screening various cell lines and/or process parameters. The device described herein is specifically useful for high-throughput screening and allows fast and efficient collection of data, especially when compared to screening methods employing large-scale shake flask methods.

According to a specific embodiment, the multiplexed microfluidic perfusion cell culture device comprises at least: a. a cell culture chamber unit comprising at least two spatially distinct cell culture chambers, wherein each of said cell culture chambers comprises a cell inoculation channel, a flow inlet and a flow outlet; and b. a microchannel network unit comprising for each of said cell culture chambers a flow input channel connected to the flow inlet and a flow output channel connected to the flow outlet; wherein the flow output channel and/or the flow outlet is equipped with a cell retention filter unit.

Specifically, the present invention provides a multiplexed microfluidic perfusion cell culture device comprising at least: a. a cell culture chamber unit comprising at least two spatially distinct cell culture chambers; and b. a microchannel network unit comprising for each of said cell culture chambers a flow input channel connected to the flow inlet and a flow output channel connected to the flow outlet; wherein the flow output channel and/or the flow outlet is equipped with a cell retention filter unit, characterized in that each of said cell culture chambers comprises a cell inoculation channel, a flow inlet and a flow outlet.

Specifically, the device described herein comprises for each cell culture chamber three separate channels, namely for cell inoculation, flow input and flow output.

The technical effect of the separate flow input channel and flow output channel, and the separate flow inlet and flow outlet, respectively, is that the device can be operated in perfusion mode.

The technical effect of the separate cell inoculation channel is that cell inoculation can be conveniently performed individually for each of the cell culture chamber, which is advantageous if a variety of cell lines are cultured in parallel. In addition, the inoculation channel separate from the flow input channel allows clogging the channel after inoculation thereby preventing contamination of the cell culture chamber with cells after inoculation.

The technical effect of the cell retention filter unit is that cells are maintained in the cell culture chamber while the device is operated in perfusion mode. According to one embodiment described herein, the cell culture chamber unit and the microchannel network unit are bonded together or an integrated part of the cell culture device. The technical effect is that such device can be provided as a single unit, which is ready to use and easy to handle without the need to assembly the individual units or chambers before use.

Specifically, said cell retention filter unit comprises a porous filter with a pore size of less than 5 pm, preferably less than 4 pm, 3 pm, 2 pm, most preferably less than 1 pm. The technical effect of the pore size is to retain cells inside the cell culture chamber and to allow passage of molecules (non-cellular molecules, other than cells) contained in the cell culture medium and filtrate though the flow outlet.

In one aspect described herein, said cell retention filter unit comprises any one of a layer, a grid, a membrane, or a unit comprising micro-structured pillars or micro- structured pores, preferably comprising a material selected from the group comprising polyester, polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymers (COC), polystyrene (PS), poly-ethylene glycol diacrylate (PEG-DA), polyurethane (PU), polypropylene (PP), and polytetrafluoroethylene (PTFE). The technical effect of any such material is the fabrication of a filter unit with a suitable pore size for cell retention. The advantage of such materials is a cost-effective manufacturing process for large-scale production, as well as chemical and mechanical properties that makes the fabrication process more robust and versatile.

In one embodiment described herein, said cell retention filter unit is provided as a cell retention layer connecting said cell culture chamber unit with said microchannel network unit. The technical effect of said cell retention layer which is preferably a continuous layer, is the standardization of the device equipment for each of the cell culture chambers with a filter at the flow outlet.

Specifically, said cell retention layer is an integrated part of the microchannel network unit, or of the cell culture chamber unit. The technical effect is a convenient fabrication method of the device described herein and a ready to use and easy to handle device. Specifically, said cell retention layer comprises a membrane, in particular a membrane layer. The membrane is specifically provided as a layer separate from the microchannel network unit and separate from the cell culture chamber unit. According to a specific embodiment, said separate membrane is connected to any one of or both of the microchannel network unit and the cell culture chamber unit within the cell culture device, to become an integrated part of the cell culture device. Specifically, the membrane is a porous filter as further described herein.

According to a specific embodiment described herein, said cell retention layer comprises a membrane that is sandwiched between two adhesive resin sheets and positioned between two glass plates, wherein one of the glass plates is in contact with the cell culture chamber layer, and the other glass plate is in contact with the microchannel network layer, thereby connecting said cell culture chamber unit with said microchannel network unit; wherein said adhesive resin sheets and said glass plates are perforated with inlet holes and outlet holes corresponding to the size of the flow inlet and flow outlet, respectively.

Specifically, the adhesive sheets are comprising or comprised of any one or more of silicone, rubber, acrylic polymer or any other adhesive material. The technical effect of the adhesive sheets is to tightly adhere to any of the adjacent layers, thereby connecting the adjacent layers.

Specifically, the cell culture chamber unit and/or the microchannel network unit comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), glass, polyesters, polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymers (COC), polystyrene (PS), poly-ethylene glycol diacrylate (PEG- DA), polyurethane (PU), polypropylene (PP), and polytetrafluoroethylene (PTFE).

The technical effect of said materials is the provision of biocompatible materials providing favourable chemical and mechanical properties for design and application, which materials allow cost-effective large volume manufacturing and micro-structuring by several techniques. The materials are optionally oxygen permeable.

According to a specific embodiment described herein, said at least two cell culture chambers are formed by assembling two or more layers with an integrated cell culture chamber architecture. The technical effect of assembling two or more layers is a simple fabrication method of the device throughout the multiplexed arrangement of the cell culture chambers, wherein each of the chambers has the same dimensions. Exemplary dimensions of each of the chambers are: length ranging between 0.1 mm and 20 mm; width ranging between 0.1 mm and 5 mm; height ranging between 0.05 mm and 2 mm. According to a specific example, the chamber dimensions are the following: length ranging between 5 mm and 7 mm; width ranging between 1 mm and 3 mm; height ranging between 0.5 mm and 1 mm. Specifically, the length is 7 mm, the width is 2 mm and the height is 1 mm.

Specifically, said microchannel network unit is provided as a layer, which is an integrated part of the device.

Specifically, said microchannel network unit is formed by assembling two or more layers, also referred to as sublayers, such as e.g. at least 2, 3, 4, 5, or 6 sublayers.

According to a specific embodiment, the device described herein comprises one or more integrated sensors for assessing one or more process parameters. Preferably, each of the cell culture chambers (4) comprises one or more integrated sensors. In one embodiment described herein, one or more integrated sensors are positioned within the microchannel network unit.

Specifically, the process parameters are selected from the group consisting of productivity, temperature, oxygen concentration or pressure, in particular dissolved oxygen concentration, pH value, cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass concentration, metabolite concentration, and carbon dioxide concentration.

Specifically, the concentration of biomass is determined e.g. as cell density (cell/volume), or as raw sensor signal indicating the amount of biomass in the medium (such as light intensity, transmittance, absorbance, current, voltage, or impedance).

Specifically, the device described herein comprises one or more detection units connected to each cell culture chamber to determine any one or more of the process parameters selected from the group consisting of cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass concentration, and metabolite concentration. Specifically, said one or more detection units are provided as external detection unit, i.e. a unit which provides for a non-invasive detection of a process parameter e.g., positioned outside the cell culture chamber and/or not in direct contact with the cell culture (indirect measurement). Specifically, said one or more detection units can be integrated within the device described herein, or provided as a separate detection unit outside the device described herein and connected to the device described herein.

The technical effect of an integrated sensor and/or an external detection unit aligned with each of the cell culture chambers is the in-line measurement and of a process parameter during the cell cultivation process and individual control of the cell culture. Specifically, the same sensor used in the device described herein is disposed at or in each of the cell culture chambers and configured to measure the same process parameter for each of the cell cultures within the multiplexed device.

Specifically, the volume of each of said cell culture chambers is less than 1 ml_, preferably less than 500 pl_, 400 mI_, 300 mI_, 100 mI_, more preferably less than 50 mI_, even more preferably less than 20 mI_, most preferably 15 pL or less. The technical effect of the volume of the cell culture chambers is to provide a microfluidic device.

Specifically, the geometry of said cell culture chambers allows a shear stress of no more than any one of 5 x 10⁵ Pa, 4 x 10 ⁵ Pa, 3 x 10⁵ Pa, 2 x 10⁵ Pa, or 1 x 10⁵ Pa as determined by computational Fluid Dynamic (CFD) simulations. The technical effect of such geometry is the low shear stress which allows running the cell culture at small scale in perfusion mode.

The device described herein is a multiplexed microfluidic perfusion cell culture device that is able to run parallel fermentation processes for screening various cell lines and/or process parameters. The device is specifically useful for high-throughput screening. A specific embodiment comprises at least one integrated sensor within the device and cell culture chamber, respectively, which allows in situ measurement of a process parameter.

Specifically, the device described herein is capable of operating in non-perfusion mode and perfusion mode, wherein perfusion mode is preferred. The term “non perfusion mode” as used herein refers to batch mode, fed-batch mode and non perfusion continuous mode.

Specifically, the device described herein comprises a cell culture chamber unit 1 and a microchannel network unit 2, e.g. as displayed in Figures 1a and 2a. The cell culture chamber unit 1 specifically contains at least two spatially distinct cell culture chambers, preferably at least any one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48, 60, 72, 84, or 96 cell culture chambers. Yet, the device may comprise more than one cell culture chamber unit e.g., a series of cell culture chamber units in parallel. In such case, the device may comprise multiples of the number of cell culture chambers contained in one unit. The 3D structure of an exemplary cell culture chamber 4 is depicted in Figure 1b and 2b. Each cell culture chamber 4 may represent a single vessel comprising a volume of less than 1 ml_, preferably less than any one of 500 pl_, 400 mI_, 300 mI_, 200mI_, 100 mI_, 90 mI_, 80 mI_, 70 mI_, 60 mI_, 50 mI_, 40 mI_, 30 mI_, 20 mI_, most preferably 15 mI_ or less.

The geometry of each cell culture chamber may comprise a rectangular cross section.

Specifically, the flow inlet channel 8 and the flow outlet channel 9 are positioned spatially distinct from one another e.g., in close proximity to the opposite ends of one long side of a rectangular cover of the cell culture chamber. Specifically, both channels, flow inlet channel 8 and flow outlet channel 9, are positioned on one side of the cell culture chamber, i.e. both either on the upper side or on the lower side of the cell culture chamber.

The spatially distinct cell culture chambers of the device described herein are specifically fabricated into one single unit, which is herein referred to as the cell culture chamber unit 1. The spatially distinct cell culture chambers can be individually addressable, in particular by suitable control elements, and the cell culture chambers are separated from one another to allow culturing the cells in each of the cell culture chambers differently, e.g. under different conditions. Each cell culture chamber 4 features a cell inoculation channel 5, e.g. to allow inoculating each cell culture chamber with a different inoculum (e.g. different cell lines), a flow inlet 6 and a separate flow outlet 7, e.g. as displayed in Figures 1b and 2b. The flow inlet 6 can be used for supplying the cells 25 in the cell culture chamber with cell culture medium comprising a substrate 24 (e.g. carbon sources for cell growth and/or for biotransformation into fermentation products) and/or nutrients. The separate flow outlet 7 can be used for collecting material, e.g., samples or products. Since the flow outlet is running through a filter unit, the collected material is also referred to as “filtrate”. In specific embodiments, the filtrate may comprise a fermentation product 26.

A cross section of an exemplary cell culture chamber 4 described herein is depicted in Figure 3.

The cells 25 may e.g., be selected from any of yeast, bacterial or mammalian cells. The substrate 24 in the cell culture medium may, for example, comprise a carbon source such as glucose.

The fermentation product may e.g., comprise a cell metabolite or a protein.

The cell inoculation channel 5 described herein is suitably connected to means for cell inoculation to allow importing the cells into the cell culture chamber, and optionally equipped with means to be clogged after inoculation. Cell inoculation can be performed individually for each of the cell culture chambers. Thus, each cell culture chamber 4 can be loaded with a different cell line, making the device suitable for screening a repertoire of different cell lines in parallel, or even simultaneously.

The flow inlet 6 of each culture chamber 4 allows supplying each cell culture chamber 4 with a different cell culture medium, allowing to determine growth and/or productivity of the cells under different process parameters and/or cell culture conditions. For example, for each cell culture chamber 4, the cell culture medium may vary in the composition and concentration of the substrate(s) 24 and/or nutrients, oxygen concentration or pH value. The flow rate of the cell culture medium can be chosen individually for each of the cell culture chambers.

The inlet 6 and outlet 7 are specifically connected to a flow input channel 8 and a flow output channel 9, respectively, which are situated in the microchannel network layer 2, e.g. as displayed in Figures 1a, 1b, 2a and 2b. The flow input channel 8 is herein specifically understood as the channel through which the cell culture medium comprising the substrate(s) 26 and/or nutrients is transported to the inlet of the cell culture chambers. The flow output channel 9 is herein specifically understood as the channel through which the filtrate comprising the fermentation product 26 is transported out of the outlet of each cell culture chamber. The flow output channel allows for the perfusion of the cell culture while retaining the cells within the cell culture chamber, and to collect (e.g. continuously collect) a fermentation product 26.

In an exemplary device described herein, the flow output channel 9 and/or the flow outlet 7 of each cell culture chamber is equipped with a cell retention filter unit 3, e.g. as displayed in Figures 1b, 2a and 2b. In addition, the flow input channel 8 and/or the flow inlet 6 of each cell culture chamber may also be equipped with a cell retention filter unit 3.

The cell retention filter unit 3 described herein is suitable to retain cells inside the cell culture chambers, while allowing the filtrate to leave the cell culture chamber unit. If the flow input channel 8 and/or the flow inlet 6 of each cell culture chamber are equipped with said cell retention filter unit 3, the cell retention filter unit 3 allows the cell culture medium comprising a substrate and nutrients to enter the cell culture chambers.

Specifically, the cell retention filter unit may comprise a filter with a pore size less than 5 pm, preferably less than 4 pm, 3 pm, 2 pm, most preferably less than 1 pm. The pore size is typically smaller than the cell size, and larger than the size of molecules (non-cellular molecules, other than cells) contained in the cell culture medium and filtrate.

Specifically, a suitable cell retention filter unit 3 comprises any one of a layer (e.g. a continuous layer), a grid (e.g. a continuous grid), a membrane, or a unit comprising micro-structured pillars or micro-structured pores.

In a preferred embodiment described herein, the cell retention filter unit 3 is provided as a cell retention layer connecting said cell culture chamber unit 1 with said microchannel network unit 2.

Specifically, the cell retention layer is an integrated part of the microchannel network unit, e.g. as shown in Figure 1b. According to a specific example, the cell retention filter unit 3 may be placed immediately at the edge of the cell culture chamber, within the flow outlet 7 and/or the flow output channel 9, or between the flow outlet 7 and the flow output channel 9.

According to a specific example, the cell retention filter unit 3 may be placed immediately at the edge of the cell culture chamber, within the flow inlet 6 and/or the flow input channel 8, or between the flow inlet 6 and the flow input channel 8.

A specific flow inlet or flow outlet may be a hole in one of the walls of the cell culture chamber 4, optionally including a construction outside the cell culture chamber that is equipped with a connector element, suitable to connect with the flow input channel and flow output channel, respectively. According to a specific aspect, the flow inlet 6 and/or flow outlet 7 is comprised of or composed of a round diffuser within the ceiling or bottom of the cell culture chamber.

Specifically, the cell retention filter unit 3 comprises any one or more of a material selected from the group comprising polyester, polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymers (COC), polystyrene (PS), poly-ethylene glycol diacrylate (PEG-DA), polyurethane (PU), polypropylene (PP), and polytetrafluoroethylene (PTFE).

Specifically, the cell culture chamber unit 1 and/or the microchannel network unit 2 comprise any one or more of a material selected from the group consisting of polydimethylsiloxane (PDMS), glass, polyesters, polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymers (COC), polystyrene (PS), poly-ethylene glycol diacrylate (PEG-DA), polyurethane (PU), polypropylene (PP), and polytetrafluoroethylene (PTFE).

In an exemplary device described herein, the cell culture chamber unit 1 and the microchannel network unit 2 may be bonded together or an integrated part of the cell culture device e.g., to build one single material unit. The cell culture chamber unit 1 and the microchannel network unit 2 may be specifically fabricated separately and bonded together after fabrication. Each of the cell culture chamber unit 1 and the microchannel network unit 2 may be fabricated out of one or more individual layers (including e.g., sublayers), which can be assembled and/or bonded together to build one unit.

In preferred embodiments described herein, the cell culture chamber unit 1 may comprise one or more layers (including sublayers), specifically at least 2, 3, 4, 5, or 6 layers, that may be in contact with the cell culture. In specific embodiments, the cell culture chamber unit 1 may comprise one or more bottom layers representing the bottom of the spatially distinct cell culture chambers, one or more continuous layers with suitable holes forming the walls of the cell culture chambers, optionally wherein one of the walls comprises a hole for the inoculation inlet. In addition, one or more layers may form the cover of the cell culture chambers and may include holes suitable for the flow inlet 6 and flow outlet 7, and optionally the inoculation inlet. Preferably, any one or more, or all, of the sublayers from the cell culture chamber unit 1 may be composed of PDMS or COC, optionally wherein one or two or more additional layers are composed of glass, or any other inert solid carrier material.

In preferred embodiments described herein, the microchannel network unit 2 may comprise one or more layers (including sublayers), specifically at least 2, 3, 4, 5, or 6 layers. Any one or more of layers may contain holes for the flow input channel and flow output channel. Any one or more of the layers of the cell retention layer 2 may comprise or consist of a glass layer or glass plate, e.g. a bottom and/or top glass layer, such as to provide an inert solid carrier material. Any one or layers may be composed of PDMS or COC. Preferably, any one or more, of the layers from the microchannel network unit 2 may be composed of PDMS or COC, optionally wherein one or two or more additional layers are composed of glass, or any other inert solid carrier material.

Specifically, the microchannel network unit 2 comprises the cell retention filter unit 3, with a sublayered structure or not. Specifically, the cell retention layer comprised in the cell retention filter unit 3 may be composed of one or more sublayers, which may be flexible or rigid, such as a sublayer comprising or consisting of a membrane 11 , e.g. as shown in Figure 2a and 2b. In a preferred embodiment described herein, the cell retention layer comprises a membrane 11 and at least one additional sublayer, preferably at least two additional sublayers. The additional sublayers may comprise an adhesive layer, or an adhesive sheet, which is understood as a continuous layer, and which may be placed between the cell culture chamber unit 1 and the membrane 11 and/or between the membrane 11 and the microchannel network unit 2. The layered structure allows a tight integration of the membrane 11 between the cell culture chamber unit 1 and the flow output channel 9 and/or the flow input channel 8 of microchannel network unit 2.

In an especially preferred embodiment described herein, the membrane 11 may be sandwiched between two adhesive resin sheets 12 and 13 and positioned between two glass plates 14 and 15, wherein one of the glass plates 14 is in contact with the cell culture chamber layer 1 , and the other glass plate 15 is in contact with the microchannel network layer 2, thereby connecting said cell culture chamber unit 1 with said microchannel network unit 2; wherein said adhesive resin sheets 12 and 13 and said glass plates 14 and 15 are perforated with inlet holes and outlet holes corresponding to the size to the flow inlet 6 and flow outlet 7, respectively. In this embodiment, which is visualized in Figure 1a, the adhesive resin sheets 12 and 13 ensure tight integration of the membrane 11.

Specific exemplary devices described herein are provided as ready-to-use devices. Specifically, the device described herein may be fabricated by any of a number of microfabrication techniques, or combinations thereof, including but not limited to microinjection molding, hot embossing, 3D printing, electroplating, microelectrode discharge machining, micropattern cutting, lithography including photo-lithography, soft lithography, film deposition, or etching.

According to a specific embodiment, the device may be disposable, or reusable, depending on the particular application.

Specifically, the device described herein allows the measurement of one or more process parameters, wherein said parameters are selected from the group consisting of productivity, temperature, oxygen concentration or pressure, in particular dissolved oxygen concentration, pH value, cell culture medium composition, concentration of cell culture components, flow rate, biomass concentration, cell density, metabolite concentration, and carbon dioxide concentration. A process parameter can e.g., be determined with an integrated sensor. An integrated sensor is herein understood to be integrated within or part of the device and positioned such to determine a cell culture parameter, and optionally monitor or control the cell culture. Preferably an integrated sensor is positioned within the cell culture chamber and in direct contact with the cell culture e.g., to allow determination of the process parameter in situ, i.e. within the cell culture. An integrated sensor may also be positioned within other parts of the device, such as within the microchannel network unit. These sensors may e.g., be selected form the group consisting of temperature sensors, oxygen sensors, pH sensors, carbon dioxide sensors, protein sensors, organic molecule sensors, and metabolite sensors. Further process parameters may be determined by one or more of non-integrated sensors, herein also referred to as “external sensors”, e.g. as integrated part of an external detection unit. Specifically, an external sensor may be part of an external detection unit connected to each cell culture chamber 4 Such external detection units can be suitable to determine any one or more of the process parameters selected from the group consisting of cell culture medium composition, concentration of a cell culture component, flow rate, biomass concentration (cell density), and metabolite(s) concentration.

Specifically, integrated sensors and external detection units allow fora convenient and in situ determination and analysis of the effect of different process parameters on cell growth and fermentation product yield, which facilitates process parameter optimization in a screening process. Specifically, any of the sensors may be referred to as an “in-line sensor”, wherein the term “in-line” as used herein refers to the possibility of continuously measuring a process parameter without drawing a sample. In particular, any one of an integrated sensor and/or external sensor and detection unit, respectively, may be used for in-line analysis of one or more process parameters.

Specific integrated sensors described herein are oxygen sensors, pH sensors and carbon dioxide sensors. Exemplary sensors, e.g. as shown in Figure 3, are based on optical detection. For example, an oxygen sensor spot 21 may be provided by immobilization of an oxygen-sensitive dye inside the cell culture chamber.

A specific external sensor may be provided to determine the cell density or biomass, e.g. by a method of determining light scattering. Measurement of the scattered light may be performed by applying a light emitter 22 e.g. placed on one side of the cell culture chamber, and a light detector 23, such as organic photodiodes, placed on the other side of the cell culture chamber (opposite to the light emitter 22).

Specifically, the device described herein may be connected to one or more specific external detection units, such as a column chromatograph and/or mass spectrometer for further analysis of the filtrate e.g. to determine a fermentation product, or component(s) of the cell culture medium or cellular products.

According to a specific example, the only oxygen supply is through the inflow of cell culture medium. In specific embodiments, an additional oxygen supply is by means of diffusion through a cell chamber wall, e.g. a bottom layer, when using a gas permeable material, such as PDMS.

According to a specific example, such as depicted in Figure 1a and 1b, the device comprises a bottom layer 10 fabricated out of PDMS or plastic. The cell culture chamber unit 1 and the microchannel network unit 2 of the specific example depicted in Figure 1a is each composed of a single layer of microstructured plastic material. In this specific example, the cell retention filter unit 3 is an integrated part of the microchannel network unit 2, as depicted in Figure 1b. According to this specific example, the cell culture chamber unit 1 contains 12 spatially distinct cell culture chambers 4, each cell culture chamber featuring a rectangular cross section. The volume of the cell culture chamber 4 depicted in Figure 1b is 15 pL. Figure 1b shows that for this specific embodiment of the invention, the flow input channel 8 and the flow output channel 9 are arranged parallel to each other and are located on one side of the cell culture chamber, at right angle to the direction of the flow inlet 6 and the flow outlet 7, with filter units at the “knee” positioned between the flow inlet 6 and the flow input channel 8, and between the flow outlet 7 and the flow output channel 9, respectively. Specifically, the cell inoculation channel 5 is positioned on the other side of the cell culture chamber, opposite to the flow input channel 8 and the flow output channel 9 and at right angle to the direction of the flow input channel 8 and the flow output channel 9. In this specific example (Figure 1a and Figure 1b), device assembly includes bonding the bottom layer 10, the cell culture chamber unit 1 and the microchannel network unit 2 to each other, by applying e.g. adhesive bonding, thermal fusion bonding or solvent bonding.

Figures 2a and 2b show an example for another embodiment described herein. According to this specific embodiment, the cell culture chamber unit 1 consists of 3 individual layers made of PDMS, whereas the microchannel network unit 2 consists of two individual layers made of PDMS. In this specific example, the cell retention unit 3 is provided as a continuous cell retention layer connecting said cell culture chamber unit 1 with said microchannel network unit 2. The cell retention layer according to this specific example comprises a separate membrane 11, which is sandwiched between two adhesive resin sheets 12 and 13 and positioned between two glass plates 14 and 15, wherein one of the glass plates (14) is in contact with the cell culture chamber layer 1, and the other glass plate (15) is in contact with the microchannel network layer 2, thereby connecting said cell culture chamber unit 1 with said microchannel network unit 2; wherein said adhesive resin sheets 12 and 13 and said glass plates 14 and 15 are perforated with inlet holes and outlet holes corresponding to the size to the flow inlet 6 and flow outlet 7, respectively. For this specific example, the cell culture chamber unit 1 contains 12 spatially distinct cell culture chambers 4, each cell culture chamber featuring a rectangular cross section. The volume of the cell culture chamber depicted in Figure 2b is 15 mI_. Figure 2b shows that for this specific embodiment of the invention, the flow input channel 8 and the flow output channel 9 are arranged parallel to each other and are located on one side of the cell culture chamber, whereas the cell inoculation channel is located on the opposite side. In this specific example (Figure 2a and 2b), device assembly includes bonding the individual layers of the cell culture chamber unit 1 and the microchannel network unit 2, for example, by air plasma treating followed by annealing. The cell retention filter unit 3 is sandwiched between the cell culture chamber unit 1 and the microchannel network unit 2 in this specific example (Figure 2a and 2b).

Figure 3 depicts a cross section of a cell culture chamber 4 according to the specific examples of Figure 1a and Figure 1b, and Figure 2a and Figure 2b. The cell culture chamber according to these specific examples has a rectangular cross section and a volume of 15 mI_. The cell culture chamber 4 as depicted in Figure 3 includes a liquid comprising cells 25, cell culture medium with substrate 24 and fermentation product(s) 26. The cell culture chamber 4 according to the specific example depicted in Figure 3 further includes an integrated sensor spot 21, which may be an oxygen sensor spot. The specific example depicted in Figure 3 further includes an external detection unit for biomass detection, consisting of a light emitter 22 outside the cover of the chamber with means to direct light into the chamber, and a light detector 23, which may comprise organic photodiodes, placed beneath the cell culture chamber.

According to a specific aspect, a microfluidic device such as described herein, contains at least two spatially distinct cell culture chambers, and is able to run a series of fermentation processes in parallel e.g., at least two, preferably at least any one of 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 18, 24, 48, 60, 72, 86 or 96 fermentation processes in a cell culture chamber unit comprising at least the same number of cell culture chambers.

Thus, such a device is useful for screening one or more process parameters for culturing a cell line and/or for screening a pool or variety of cell lines producing a fermentation product. Specifically, the device allows in situ monitoring of one or more process parameters simultaneously, allowing a fast and efficient screening process.

Specifically, the method for screening process parameters and producing a fermentation product as described herein, can be performed in the multiplexed mode, in particular running multiple fermentations.

According to a preferred embodiment, multiple fermentations are performed in parallel, wherein the same kind of cell line is cultivated in perfusion mode in separate microfluidic perfusion cell culture chambers e.g. applying the same or different conditions, i.e. different process parameters. Performing multiple fermentations in parallel makes it possible to identify one or more improved or optimum parameters for culturing a cell line and producing a fermentation product. For example, a single process parameter may be varied over a range of values, while holding the other parameters constant. According to another aspect, multiple parameters may be simultaneously varied across a range of values.

Parameters that may be varied include, but are not limited to: cell culture medium composition (carbon/energy source (e.g., glycerol, succinate, lactate, and sugars such as, e.g., glucose, lactose, sucrose, and fructose), nitrogen source, precursors, and nutrients such as vitamins and minerals, salts, etc.), temperature, pH value, oxygen concentration or pressure, in particular dissolved oxygen concentration, carbon dioxide concentration, flow rate, biomass concentration, metabolite concentration, redox potential, agitation rate, aeration rate, ionic strength, osmotic pressure, water activity, hydrostatic pressure, etc. Preferably, the cell culture medium may be varied in composition and/or concentration for identifying the optimum cell culture medium. Any of these parameters may be varied in different ways in each cell culture chamber, so that a time-optimal manner of varying the parameters can be identified, e.g., a manner of varying the parameters so as to optimize the process, e.g., to maximize production of a desired fermentation product.

Specifically, a method for screening a cell line further described herein may comprise selecting the cell line from a repertoire of cell lines comprising a variety of cell lines e.g. in a pool or in separate containments. According to a preferred embodiment, multiple fermentations are performed in parallel, wherein different cell lines of the same species or parent strain are cultivated in separate microfluidic perfusion cell culture chambers under the same or different conditions. Performing multiple fermentations in parallel makes it possible to identify an improved or optimum cell line for producing a fermentation product. A production cell line may be selected based on a variety of criteria. For example, a production cell line may be, but is not limited to: a cell line that produces the largest amount of a desired product in a given time, herein also referred to as the “best producer”, a cell line that is stable over a long cultivation time allowing several generations of cells to produce the fermentation product, a cell line that is able to produce a desired product using a particular starting material (e.g., an inexpensive starting material), a cell line which is able to grow in medium lacking particular components; a cell line that is able to tolerate buildup of toxic or inhibitory metabolites in the culture; a cell line that is able to tolerate a wider range of growth conditions such as pH, oxygen concentration, etc.; a cell line that is able to achieve a higher cell density, etc. Preferably, the selected cell line is a high producer cell line. A cell line selected as described herein is typically tested for its capacity to produce a fermentation product in the filtrate e.g., by any of the following tests: ELISA, activity assay, HPLC, or other suitable tests, such as SDS-PAGE and Western Blotting techniques, or mass spectrometry.

A specific selection parameter is cell culture or clone stability over a prolonged time of cultivation. A stable cell culture as described herein is specifically understood to refer to a cell culture maintaining the genetic properties, specifically keeping the production level high, e.g. at least at a pg level, even after about 20 generations of cultivation, preferably at least 30 generations, more preferably at least 40 generations, most preferred of at least 50 generations. Specifically, stability can be determined when culturing in the microfluidic perfusion device, which allows screening and selecting a clone that is stable in large scale fermentations.

In bioprocess development, screening and selection of process parameters or cell lines is typically performed in shake flask experiments, involving time consuming and tedious processes and off-line analysis. Screening process parameters and/or cell lines using a microfluidic device such as described herein provide a simple and effective way of collecting a large amount of data in a short time frame. In particular, performing multiple fermentations in perfusion mode under different conditions for the same cell line, or under identical conditions for varying cell lines is particularly efficient, as described in more detail in the Examples section.

A method for producing a fermentation product in a cell culture as described herein is suitable for any type of cells including prokaryotic and/or eukaryotic cells. In particular, cell lines may be selected from yeast, bacterial, mammalian, fungi and/or plant cells. Preferably, the cells are any one of yeast, bacterial or mammalian cells. In certain preferred embodiments described herein the cells are of a type currently used in commercial bioprocesses.

In one aspect described herein, a cell line is selected from a repertoire of cell lines of the same species, or derived from the same parent strain. Such repertoire may contain cell lines which are naturally occurring variants, which may also be referred to as mutants, and/or may contain engineered cell lines. For example, cell line variants or mutants may be prepared by introducing appropriate nucleotide changes into the organism's DNA. The changes may include, for example, deletions, insertions, or substitutions of, nucleotides within a nucleic acid sequence of interest. The changes may also include introduction of a DNA sequence that is not naturally found in the cell line.

In a specific embodiment described herein, the cell culture is yeast and the fermentation product is an organic acid or a protein. In a preferred embodiment the yeast is of the Saccharomyces genus, preferably S. cerevisiae, and the fermentation product is an organic acid, preferably lactic acid. In another preferred embodiment, the yeast is of the Komagataella genus, preferably P. pastoris, and the fermentation product is a protein, preferably human serum albumin (HSA).

In a specific embodiment described herein, the cell culture is a bacterial cell culture, and the fermentation product is an organic acid. Preferably, the bacteria are Escherichia, more preferably E. coli, and the fermentation product is an organic acid, preferably itaconic acid.

In another specific embodiment described herein, the cell culture is a mammalian cell. Preferably the mammalian cell is CHO, and the fermentation product is a protein, preferably a recombinant protein, more preferably an antibody, most preferably IgG.

According to a specific example, a yeast strain is cultured by an industrial-scale process in batch mode to produce a fermentation product which is a cell metabolite, such as an organic acid, alcohol or amino acid.

According to another specific example, a yeast strain or mammalian cell line is cultured by an industrial-scale process in fed-batch mode to produce a fermentation product which is a polypeptide or protein, such as antibodies or antibody fragments, HSA, enzymes, hormones, or vaccines.

According to another specific example, a bacterial or yeast strain or mammalian cell line is cultured by an industrial-scale process in continuous mode to produce a fermentation product which is a polypeptide or protein, or a cell metabolite.

Unless stated otherwise herein, the industrial-scale method can include any desired volume or production capacity including but not limited to bench-scale, pilot- scale, and full production scale capacities.

Moreover, and unless stated otherwise herein, the industrial-scale method can include any suitable reactor(s) including but not limited to stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and/or spouted bed bioreactors. As used herein, “reactor” can include a fermenter or fermentation unit, or any other reaction vessel and the term “reactor” is used interchangeably with “fermenter.” For example, in some aspects, an example bioreactor unit can perform one or more, or all, of the following: feeding of nutrients and/or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH level, agitation (e.g., stirring), and/or cleaning/sterilizing. Example reactor units, such as a fermentation unit, may contain multiple reactors within the unit, for example the unit can have 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100, or more bioreactors in each unit and/or a facility may contain multiple units having a single or multiple reactors within the facility. In various embodiments, the bioreactor can be suitable for batch, semi fed-batch, fed- batch, and/or a continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor can have a volume between about 1 L and about 50,000 L. Non-limiting examples include a volume of 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and/or 50,000 liters. Additionally, suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be formed of any suitable material including metal alloys such as stainless steel and Inconel, plastics, and/or glass.

In embodiments and unless stated otherwise herein, the industrial-scale method described herein can also include any suitable unit operation and/or equipment not otherwise mentioned, such as operations and/or equipment for separation, purification, and isolation of such products. Any suitable facility and environment can be used, such as traditional stick-built facilities, modular, mobile and temporary facilities, or any other suitable construction, facility, and/or layout. For example, in some embodiments modular clean-rooms can be used. Additionally, and unless otherwise stated, the devices, systems, and methods described herein can be housed and/or performed in a single location or facility or alternatively be housed and/or performed at separate or multiple locations and/or facilities.

Suitable techniques may encompass culturing in a bioreactor combining batch, fed-batch and continuous cultivation methods, and/or any combination thereof.

According to specific examples, a repertoire of engineered S. cerevisiae cell lines was screened using the microfluidic device described herein, and a high producer cell line for producing lactic acid as a fermentation product was selected, which high producer cell line can be applied in a large-scale batch process for producing lactic acid.

According to specific examples, screening in said microfluidic device was performed in batch mode and perfusion mode. Surprisingly, the screening in perfusion mode provided suitable screening results which could be applied to the large-scale batch fermentation process. The results were about the same as obtained from screening in larger scale batch mode shake flask experiments. The results could be obtained significantly faster than in shake flask experiments, demonstrating that screening performed in the microfluidic perfusion device is significantly more efficient than shake flask screening, as further shown in the Examples section.

According to further specific examples described herein, a repertoire of engineered P. pastoris cell lines was screened using the microfluidic perfusion cell culture device described herein operated in perfusion mode. Surprisingly, the screening in perfusion mode provided suitable screening results which could be applied to the large-scale fed-batch fermentation process. The results were about the same as compared to screening in larger scale 1 -litre fed-batch bioreactors. These specific examples demonstrate that a high producer cell line for producing HSA as a fermentation product was efficiently selected with the microfluidic device described herein, which high producer cell line could be applied in a large-scale fed-batch method for producing a protein such as HSA.

According to further specific examples described herein, a repertoire of engineered E. coli cell lines was screened using the microfluidic perfusion cell culture device described herein operated in perfusion mode, and a high producer cell line for producing itaconic acid as a fermentation product was efficiently selected with the microfluidic device described herein, which high producer cell line can be applied in a large-scale method for producing organic acids, such as itaconic acid.

According to further specific examples described herein, a repertoire of CHO cell lines was screened using the microfluidic perfusion cell culture device described herein operated in perfusion mode, and a high producer cell line for producing IgG as a fermentation product was efficiently selected with the microfluidic device described herein, which high producer cell line can be applied in a large-scale method for producing antibodies, such as IgG, or antibody fragments.

As described herein, lactic acid overproducing engineered S. cerevisiae strains were cultivated in the microfluidic device under perfusion and non-perfusion modes and exhibited after one day fermentation the same lactic acid titer and specific productivity if compared with shake flasks. Such results were also consistent with batch cultures.

As further described herein, HSA overproducing engineered P. pastoris strains were cultivated in the microfluidic device under perfusion mode and they exhibited after two-day experiments similar divergences in productivity that were consistent with fed- batch cultures performed in 1 L bioreactor, proving that it was possible to screen the best performer, in terms of specific productivity, by means of the microfluidic device.

According to specific examples, the fabrication of a 12-chamber array microfluidic device is disclosed. The cell culture chamber is made of PDMS, chosen because of its ease of manipulation and therefore useful for rapid prototyping of laboratory-made devices. Moreover, the high gas permeability of PDMS avoids oxygen limitation inside the cell culture chambers. The usage of silicon-based adhesive material allows an easy integration of a porous filter enabling perfusion-mode miniaturized cultures. The specific properties of such device, its size, the reduced amount of cell numbers required and the higher throughput due to the possibility to run many parallel experiments make said device more convenient than benchmark tools like shake flasks. Nevertheless, other materials can be considered for an easier integration in the workflow of large-volume manufacturing processes; a simplification of the design can be thus considered, allowed by switching from PDMS to a thermoplastic material.

Specifically, the low volume of the device, the small amount of cell mass required for each culture and the possibility to integrate in-line sensor for real-time monitoring of cell culture parameters proved the efficacy and versatility of the device, that could provide meaningful data for the development and upscaling of the bioprocess much faster than benchmark tools.

EXAMPLES

The Examples which follow are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to limit the scope of the invention in any way. The Examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art.

Example 1 : Fabrication of a silicon-based multiplexed microfluidic perfusion cell culture device with integrated optical sensors for process parameter monitoring

In order to have a high-throughput platform able to provide information on many parallel microbial cultures of industrially relevant engineered strains, a silicon-based multiplexed microfluidic perfusion cell culture device (herein referred to as MmR) comprising an array of cell culture chambers was fabricated by employing micromachining techniques.

MmR was designed as a silicon- and glass-based monolith device with an area of 76 mm x 26 mm featuring 12 spatially distinct cell culture chambers 4. In order to create the 3D microfluidic architecture, 10 different layers were designed by employing the graphic software AutoCAD (AutoCAD 2017, AutoDesk, USA). The 2D representation of each layer is reported in Figure 2a.

According to this example, the cell culture chamber unit 1 consists of 3 layers. The first layer 16 of the cell culture chamber unit 1 is composed of a 250-pm thick silicon sheet (Superclear silicone sheets, MVQ Silicones GmbH, Germany), representing the bottom of the cell culture chambers. The second (17) and third (18) layer of the cell culture chamber unit 1 are each composed of a 500 pm-thick silicon sheet (Superclear silicone sheets, MVQ Silicones GmbH, Germany), forming the walls of the cell culture chambers (chamber dimensions: 7 mm length, 2 mm width). According to this example, the cell retention filter unit 3 consists of 5 layers. The first layer of the cell retention filter unit 3 is a 1-mm thick drilled glass plate 14 containing the inlet and outlet holes of the chambers (1 mm diameter). The next three layers of the cell retention filter unit 3 consist of a 0.4 pm pore size porous filter membrane 11 (Polyester Membrane Filters, Sterlitech Corporation, WA, USA) sandwiched between silicon adhesive resin sheets 12 and 13 (80-pm thick silicon adhesive resin, ARcare8259, Adhesive Research, Ireland). The fifth layer of the cell retention filter unit 3 is a 1-mm thick drilled glass plate 15 containing holes connecting inlet and outlet of the chambers to, respectively, pump and sample collection tubes.

According to this example, the microchannel network unit 2 consists of two layers. The first layer 19 of the microchannel network unit 2 is a 250-pm thick silicon sheet (Superclear silicone sheets, MVQ Silicones GmbH, Germany) containing a microchannel array for medium supply and sample collection. The second layer 20 of the microchannel network unit 2 is a 3 mm punched polydimethylsiloxane (PDMS) layer containing 0.5 mm diameter punched holes that enabled interfacing micro-channels with the outer tubing.

The micro-patterns designed on AutoCAD file were structured into layers 17, 18, 14, 13 and 19 by using a GS-24 vinyl cutter (Roland DGA Corporation, Germany). The 10 2-D layers were then assembled so that three intermediate units were formed: a cell culture chamber unit 1 featuring the 12 spatially distinct cell culture chambers 4, a cell retention filter unit 3 and a microchannel network unit 2 for medium supply and permeate collection, which is connected to pump and sampling tubes through PEEK tubing (1/32" outer diameter, 250-pm inner diameter). The silicon layers that build up the cell culture chamber unit 1 were air plasma treated (Harrick Plasma, High Power, 2 min) and the cell retention filter unit 3 was sandwiched between the cell culture chamber unit 1 and the microchannel network unit 2. Similarly, the first (19) and second (20) layer of the microchannel network unit 2 were air plasma treated and put into contact. All plasma- treated layers were annealed at 70°C for 10 min in order to increase bonding strength. Inlet and outlet holes with 0.9 mm diameter were drilled through the glass plates 14 and 15 of the cell retention filter unit 3 in order to connect the cell culture chamber unit 1 with the microchannel network unit 2. The cell retention filter unit 3 was assembled by sandwiching the 0.4 pm pore-size porous filtration membrane 11 between the silicon adhesive resin sheets 12 and 13. These latter ones were put into contact and bonded by applying a gentle pressure between the two drilled glass plates 14 and 15. The oxygen sensors were prepared by embedding the metal complex platinum (II) meso-tetra (4-fluorophenyl) tetrabenzoporphyrin (PtTPTBPF) inside amine- functionalized polystyrene beads. The synthesis of the oxygen-sensitive particles, their characterization and calibration were performed as disclosed by Ehgartner et al. (Anal. Chem. 2016, 88, 9796-9804; Sensors Actuators B Chem. 2016, 228, 748-757). Oxygen monitoring was carried out at a sampling frequency of 1 Hz using a FireSting02 optical oxygen meter (Pyroscience, Germany) connected to optical fibers (length 1 m, outer diameter 2.2 mm, fiber diameter 1 mm)³. The oxygen sensor spots were integrated inside the device by immobilizing the particles on the top of each chamber 21 , based on a procedure reported in literature (Sticker et al, ACS Appl. Mater. Interfaces 2019, 11, 9730-9739). The oxygen sensor spots were positioned at the bottom of the glass plate 14, whose surface represents the top of the cell culture chambers (Figure 2a). Finally, device assembly was completed by bonding the cell culture chamber unit 1 and the microchannel network unit 2 to, respectively, the lower and the upper glass plates 14 and 15 of the microchannel network unit 3 by using oxygen plasma and a final annealing step at 70°C for 10 min.

Each individual 15-pL cell culture chamber 4 of the microfluidic device (resulting from the assembly of the three intermediate units, whose 3D final design can be observed in Figure 2a and the cross-section in Figure 2b was composed by one main body equipped with a cell inoculation channel 5, a flow inlet 6 for medium supply and a flow outlet 7 for filtrate collection.

Afterwards, the sensors were calibrated using a CO2/O2 oxygen controller (C02/02-Controller 2000, Pecon GmbH, Germany) equipped with integrated zirconium oxide oxygen sensors. Specifically, a two-point calibration was carried out, by constructing a calibration curve after registering the signal at 0% dissolved oxygen content (% DO) and 100% DO.

To gain a deeper understanding of cell culture conditions within the micro-device, an in silico characterization strategy was employed to determine apparent shear forces in the micro-bioreactor array. Finite element software COMSOL Multiphysics 5.2 was used to mathematically study the hydrodynamic properties and oxygen concentration in the microfluidic devices. Two physics modules, Laminar Flow (spf) and Transport of Diluted Species (tds) in COMSOL Multiphysics 5.2 were adopted and three dependent variables - velocity u, pressure p and molar concentration of oxygen c - were considered. A time-dependent simulation during a period of 24 hours at 10 min intervals was built, and the Navier-Stokes and the continuity equations were coupled with transport theory including diffusion and convection to solve the problem. The following boundary conditions were implemented: (A) specific inlet flow rate and no pressure at the outlet; (B) non-slip condition at the chamber/channel wall; (C) incompressible fluidic; (D) specific oxygen concentration on the surfaces exposed to the atmosphere (external surface of the device, inlet). The diffusion coefficients (at 30°C) and initial oxygen concentration are listed in Table 1 (Evenou et al., Tissue Eng. Part C Methods 2010, 16, 311-318). Three specific flow rates were considered for the calculations: 3.75 pl_ h ¹ (that, based on cell culture chamber design, corresponded to a perfusion rate P = 0.25 h ¹), 7.5 mI_ tv¹ (P = 0.5 tv¹), and 15 mI_ tv¹ (P = 1 tv¹).

Table 1. Physical properties applied in the simulations. Oxygen mass diffusivity in the aqueous medium and inside the PDMS matrix (DO2). Dissolved oxygen concentration in the medium in the medium at the inlet of the chamber (Initial O2).

Computational fluid dynamic (CFD) simulation revealed that, in the presence of increasing perfusion rates, only very low shear stress was exerted to yeast cells in the cell culture chamber with shear values ranging from 9.2 10⁶ Pa, to 1.8 10⁵ Pa and to 3.7 10⁵ Pa, respectively (Table 2).

Table 2. CFD simulations of flow velocity profile and shear stress during on- chip yeast perfusion culture modes. Calculations were based on three different flow rates ranging from 3.75 pL-h ¹ to 15 pL-h ¹.

Example 2: Evaluation of alternative materials and fabrication approaches for the development of the multiplexed microfluidic platform. Fabrication of COC- based multiplexed microfluidic platform (COC-MpP)

Two aspects were taken into account during the development of the microfluidic platform: the materials employed for the fabrication and the fabrication methods, which may impact not only the scientific and technical applications, but also the upscaling cost. PDMS and silicones have been widely employed in microfluidics (especially in academic projects) since the beginning of this field thanks to many useful properties - e.g. biocompatibility, ease of manipulation and gas permeability.

Thermoplastics such as poly(methyl methacrylate) (PMMA), polycarbonate (PC), cyclic olefin copolymer (COC), and poly(ethylene terephthalate) (PET), are attracting attentions for chemical and biological microfluidic applications. Thermoplastics possess inherent robustness to mechanical deformation and resistance to chemicals. Moreover, thermoplastics allow for high-volume fabrication of microchips using microinjection molding and hot embossing. Multi-layer thermoplastic chips can be assembled through a single-step thermal fusion bonding in which all layers have similar glass transition temperatures.

The fabrication of the multiplexed microfluidic platform described in Example 1 is therefore replicated by employing thermoplastic materials, while keeping the same design. Specifically, all glass and silicon layers are replaced by COC-made layers, except of the first layer 16 of the cell culture chamber unit that is made of polystyrene, chosen because of its higher O2 permeability than COC, a property that can be useful for submerged cultures. Thermoplastic-made layers can be sealed together by applying heat and pressure. An important consideration for this second device is the incorporation of a quick-connect, leak-free and high-pressure manifold for the inlets and outlets on the lid, which makes the fluidic interfaces much easier than for PDMS-based devices. Example 3: Design simplification for industrial-scale fabrication of plastic- made devices

A simpler design of the device can lead on to a reduced number of fabrication and assembly steps, which could make such device suitable for a cost-effective large- volume manufacturing.

The design of the device is simplified to be composed of only 3 layers as reported in Figure 1a and Figure 1b. The cell retention filter unit 3 is integrated into the microchannel network channel unit 2, therefore in a single layer, and the rest of the device is composed by cell culture chamber unit 1 and bottom layer 10 (which can be a either a plastic thin layer or a PDMS-based thin layer). The 2-D structure and layout of 2 and 1 are designed by AutoCAD or another common graphical software. Subsequently, the 3-D microstructure can be reproduced by employing two different fabrication techniques: hot embossing or micro-injection moulding. Both of them require a template containing the inverse features that will be transferred to the final device and that can be reused for many manufacturing cycles. The materials listed in Example 2, section b) are suitable for both techniques. Templates for 1 and 2 are therefore fabricated.

Common templates for hot embossing are made of silicon, metals, epoxy resins, photoresist or glass. The material is micro-structured by photo-lithographic methods. The surface is covered by a photo-sensitive material (photoresist), which is then exposed to a pattern light that cause a chemical change. Such light-induced modification allows to remove some part of the photoresist. The surface then undergoes an etching step, wherein a liquid or plasma chemical agent removes the uppermost layer of the substrate in the areas that are not protected by photoresist, thus obtaining the desired micro-structures.

The template is then put in contact with the plastic material, and both of them are heated up to a temperature that depends on the glass transition temperature of the plastic material. Once the desired temperature is reached, a moderate pressure is applied over time, so that the micro-structured features of the template can be imprinted on the surface of the thermoplastics. The procedure is repeated for both 1 and 2.

The template employed for injection moulding fabrication are usually based on metals (i.e. aluminium, steel, nickel) and they require specific fabrication techniques such as LIGA based (lithography, electroplating, molding) technologies, silicon wet bulk micromachining and deep reactive ion etching, whose choice depends on several aspect like on geometry, surface quality, aspect ratio. The micro-structured mould is placed inside a cavity wherein the melted plastic is then injected, so that it could fill all the micro cavities and the microstructures could be formed on the surface once it is completely cooled down and solid. The procedure is repeated for both 1 and 2.

The final step of the manufacturing process is device assembly through bonding 1, 2 (fabricated either by injection moulding or hot embossing) and 10. Several bonding strategies can be used for this purpose: the most common one are adhesive bonding, thermal fusion bonding and solvent bonding. Adhesive bonding consists in applying a thin layer of an adhesive liquid on the surfaces that have to put in contact, and the bond is then formed after curing by UV light irradiation. In thermal bonding, the surfaces of the two layers are put into contact and then heated to a temperature near or above the glass transition of the material, while applying a pressure, thus creating an intimate contact. In solvent bonding, an appropriate solvent is applied on the surfaces of the layers, softening the material; the application of a pressure ensures the formation of a strong interaction among the polymer chain of the two layers. 1 and 2 are bonded by employing one of the mentioned methods and, subsequently, they are bonded to 10 through the same approach if 10 is made out of plastic. If 10 is made out of PDMS, chemical modification of the surfaces is needed. The surfaces of 2 and 10 are modified with silanol groups, and then they can be bonded by thermal bonding as the application of optimal temperature and pressure allow the formation of covalent bonds among the silanol chains exposed on the surfaces.

Example 4: Characterization of the platform in terms of biocompatibility and microenvironment provided for microbial growth

MmR was tested by running cultures of a S. cerevisiae wild type strain, a model organism which is easy to handle under different experimental conditions, therefore helpful to evaluate the biocompatibility of the novel cultivation system and cell behaviour under the different physical-chemical environment. Moreover, such preliminary step could provide evidence that the platform is capable of monitoring fast changes in relevant process parameters during process optimization and strain selection.

The S. cerevisiae wild type strain CBS7962 (Centraalbureau of Schimmelcultures, Netherlands) was inoculated from cryo-vials on Petri dishes containing YPD agar medium and incubated at 30°C for 48 hours. Subsequently, cells were transferred in shake flasks containing fresh medium (YNB-3G) with 30 g L^_1 glucose (D(+)-glucose monohydrate, Carl Roth GmbH + Co. KG, Germany), 4.54 g L^_1 urea (Carl Roth GmbH + Co. KG, Germany), and 3.4 g- L^_1 yeast nitrogen base without amino acids and ammonium sulphate (YNB, Becton, Dickinson and Company, France) and incubated at 28°C, 180 rpm. After 24 h, cultures were centrifuged (2000 g, 5 min), supernatant was discarded and the pellet re-suspended in the same medium (YNB-3G).

S. cerevisiae wt biomass concentration during on-chip batch cultures was monitored using the external biomass detection unit consisting of a light emitter 22 and Organic Photodiodes as a light detector (23, see Figure 3). Prior to the experiments, a calibration was carried out in order to correlate optical density Oϋboo (a common parameter in microbiology experiments) to the signal recorded by the instrument. The system setup of light scattering measurements for biomass concentration monitoring has been described elsewhere (Charwat et al. 2013, Anal. Chem. 85, 11471-11478). Optical light scattering measurements were conducted using a computer-controlled shutter that timed laser exposure (one opening per minute). The scattered light was analysed by Organic Photodiodes (OPDs) 23, which were kept at a reverse bias of -5 V. OPD currents were voltage converted and amplified by an operational amplifier (LM6132AIM/NOPB from National Semiconductor Operation), digitally converted by a microcontroller (ATmega32, Atmel), and readout by a Labview program. The optical setup was calibrated by using S. cerevisie 15-pL aliquots - obtained from the overnight pre-cultures previously described - that were diluted in YNB-3G at different biomass concentrations ranging from Oϋboo = 0.01 to Oϋboo = 8. The voltage was recorded three times for each optical density point, and then mean value and standard deviation were calculated. Such data was used to construct a calibration curve correlating OD to recorded voltage (Table 3).

Table 3. Calibration data of the optical setup for biomass detection. The optical density of the samples (OD column) was correlated to the voltage recorded by the light scattering station. For each Oϋboo point, mean value and standard deviation (sd) were calculated.

Prior to inoculation, the microfluidic devices were treated with 70% ethanol for 30 min, and subsequently with Dl water in order to remove solvent residues. All devices were allowed to dry at 35°C overnight ensuring absence of ethanol residues. After the 24-h pre-culture step, cells were re-suspended in fresh YNB-3G medium at a biomass concentration of Oϋboo = 0.5. 15-pL aliquots were then injected inside the chambers of the MmR by means of a GC-gastight glass syringe and the device was placed inside the light scattering measuring station. The scattered light signal was then constantly recorded for 24 hours, and the experiment was carried out in duplicate. The voltage recorded was approximately stable around from 3.8 V to 4.2 ± 0.44

V for the first 5 hours and then increased for the following 13 hours up to 9.4 ± 0.21 V. A parallel negative control experiment was performed (also in duplicate), by measuring the scattered light from a chamber with only medium without yeast inoculum: a constant voltage around 3.1 V was recorded throughout 24 hours, suggesting that the analytical setup was effective at detecting biomass growth on chip (Table 4). Based on the previously reported calibration, it was possible to distinguish a 5-hour lag phase with a biomass concentration of 0.63 ± 0.23 Oϋboo, and a subsequent 13-hour exponential phase with a growth up to 3.6 ± 0.1 Oϋboo (Table 5).

Table 4. Characterization of the multiplexed microfluidic platform with in-line optical sensor for evaluation of biomass growth. Voltage (V) (related to scattered light) registered by the light scattering measuring station. Comparison between signals recorded during S. cerevisiae wild type strain cultures and negative control experiments (medium only). For each time point, mean value and standard deviation (sd) were calculated.

Table 5. Characterization of the multiplexed microfluidic platform with in-line optical sensor for evaluation of biomass growth. Growth curve recorded during the on-chip static culture of the S. cerevisiae wild type strains expressed in terms of Oϋboo, based on the calibration reported. For each time point, mean value and standard deviation (sd) were calculated.

Concurrently, oxygen biosensing was performed using integrated oxygen microsensors (PtTPTBPF-dye impregnated polystyrene particles) located in the center of the cell culture chamber to monitor oxygen availability throughout on-chip batch yeast cultivations. After the 24-h pre-culture step, cells were re-suspended in fresh YNB-3G medium at a biomass concentration of Oϋboo = 0.5. 15-pL aliquots were then injected inside the chambers of the multiplexed microfluidic platform by means of a GC-gastight glass syringe. Dissolved oxygen content was then constantly monitored for 18 hours in each cell culture chamber. The experiments were performed in triplicates. The acquired signal was correlated to dissolved oxygen percentage (% DO) based on the calibration previously reported.

A constant decrease in DO was recorded, from 86.3 ± 0.87 % to 9.7 ± 0.49 % during the cultivation. A parallel negative control experiment was performed, by measuring the DO content in a chamber with only medium without yeast inoculum: a constant value around 98% was recorded throughout 24 hours (a deviation from 100% could be due to small variation in the morphology of the sensors spot from one chamber to another) (Table 6). The results suggested the effectiveness of this optical analytical setup at detecting dissolved oxygen concentration and oxygen consumption during on- chip cultures.

Table 6. Characterization of the multiplexed microfluidic platform with in-line optical sensor for evaluation of oxygen consumption. On-chip dissolved oxygen content recorded through fluorescence lifetime measurements. Comparison between signals recorded during S. cerevisiae wild type strain cultures and negative control experiments (medium only). For each time point, mean value and standard deviation (sd) were calculated.

These results demonstrated that the micro-culture environment provided by the device was optimal for yeast cell growth and that the integration of optical and opto- chemical oxygen sensors allowed to continuously investigate down-scaled 15 pi yeast cultures in a non-invasive, real-time manner. In-line sensors (integrated sensors) may continuously acquire data regarding e.g. biomass and oxygen which allows an efficient screening phase setup, in terms of saved time and experiment quality. Example 5: Evaluation of lactic acid production in two S. cerevisiae engineered strains cultivated under batch mode in shake flasks

Two lactic acid overproducing Saccharomyces cerevisiae engineered strains were studied in the MmR, evaluating its effectiveness at monitoring the bioprocess and identifying the best producer. For this aim, a comparative study was necessary in order to discuss the results from the microfluidic setup by using a reference system. Thus, the two strains were cultivated under batch mode in shake flasks, as they are the most common cultivation tool employed in the early stages of bioprocess development, and they will represent the benchmark for the reported lactic acid production screening experiments.

Saccharomyces cerevisiae strains described in WO2017182403A1 were used. These strains are based on strain CBS7962, and carry following modifications: Apdd Apdc5 Apdc6, and transformed with a plasmid carrying the gene encoding L-lactate dehydrogenase of Lactobacillus plantarum (protein sequence ID: YP_004888540.1) under the control of the yeast TP11 -promoter. Two strains of different productivities were isolated and used here (LACp: strain with lower productivity and LACe: strain with higher productivity).

To improve lactic acid production and the tolerance to low pH and high glucose concentrations, the strain was subjected to adaptive laboratory evolution over three months (around 250 generations). The cells were diluted every 24 h to Oϋboo = 0.5 in fresh medium containing 150 g- L ¹ glucose (D(+)-glucose monohydrate, Carl Roth GmbH + Co. KG, Germany), 5 g L^-1 ethanol (ethanol 96%, Merck Millipore, Germany), 4.54 g L^-1 urea (Carl Roth GmbH + Co. KG, Germany), and 3.4 g- L^-1 yeast nitrogen base without amino acids and ammonium sulfate (YNB, Becton, Dickinson and Company, France) and grown in shake flasks at 30°C, 180 rpm. At the end of each one- day cultivation period, the cell suspension reached a lactic acid concentration of around 5 g L^-1 and a pH of 2.7-2.9. In the reported work, the evolved strain, named LACe, was compared to the non-evolved parental strain LACp.

The two S. cerevisiae engineered strains, LACp and LACe, were inoculated from cryo-vials on Petri dishes containing agar medium with 3.4 g L-1 YNB, 4.54 g L-1 urea and 5 g L-1 ethanol and maintained at 30°C for 72 h. Subsequently, cells were transferred into 10 mL YNB+E medium containing 3.4 g L-1 YNB, 4.54 g L-1 urea, 5 g L-1 ethanol and incubated at 30°C, 180 rpm and a relative humidity (RH) of 70%. A 1 :10 volume ratio (volume of liquid to volume of shake flask) was kept constant for every pre-culture. After 24 h, cells were centrifuged (2000 g, 5 min), supernatant was discarded and pellet re-suspended in fresh YNB+E at a biomass concentration of Oϋboo = 0.7. The same procedure for biomass propagation was repeated twice again, for another 48 hours. On the fourth day, cells were centrifuged (2000 g, 5 min), supernatant was discarded and pellet resuspended at a biomass concentration of Oϋboo = 40 in fresh YNB+10G medium containing 3.4 g L-1 YNB, 4.54 g L-1 urea and 100 g L-1 glucose, that represented the medium for triggering lactic acid bio-production.

All shake flask experiments for lactic acid production were performed in triplicates, with 30 ml_ culture volume inside 100 ml_ shake flaks that were incubated at 30°C, 180 rpm and RH = 70%. Samples were drawn after 6, 12 and 24 hours from inoculation for biomass concentration, pH measurements and organic compounds detection by HPLC. Optical density for biomass concentration was determined by appropriate dilution of culture broth to an absorbance of 0.1 -0.7 at 600 nm measured by a photometer (Biochrom WPA C08000 Cell Density Meter).

The concentrations of residual glucose and lactate were determined by HPLC analysis using a method for the detection of carboxylic acids and sugars previously established (Pfliigl et al., 2012, Bioresour. Technol. 119, 133-140). In short, the concentrations of residual glucose and lactate were determined by HPLC analysis (Shimadzu, Korneuburg, Austria) with an Aminex HPX-87H column (300 mm x 7.8 mm; Biorad) equipped with a Micro-Guard Cation H Cartridge (30 mm x 4.6 mm, Biorad). The column was operated at 60 °C and a flow rate of 0.6 mL min ¹ with 4 mM H2SO4 as mobile phase. A refraction index detector (RID-10A, Shimadzu, Korneuburg, Austria) and a UV-VIS photodiode array detector (SPD-M20A, Shimadzu, Korneuburg, Austria) were used for quantification. From now on, the reported HPLC method will be named as “HPLC-Method_1”. The quantitative analysis by HPLC was used to calculate the biomass specific lactate production rate (q , reported as mg lactic acid produced per g biomass per hour) as follow using Equation 1.

Equation 1:

where c_lCLC is the lactic acid concentration (g L ¹) measured in the sample, At is the time elapsed from the inoculation and c_x is the biomass concentration (g L ¹). Due to metabolic properties, it was expected that the two strains (LACe and LACp) could not grow on glucose media. Off-line optical density measurements were performed to monitor cell growth: Oϋboo was constant over 24 hours culture for both LACe (evolved strain) and LACp (parental strain), proving that biomass concentration was stable. Thus, the process can be described as a growth-decoupled bioconversion of glucose into lactic acid. HPLC analysis showed that LACp strain showed a concentration of 11.7 ± 0.6 g L ¹ (Table 7) and LACe strain yielded 21.3 ± 0.5 g L ¹ lactic acid (Table 8). By looking at the process kinetics (Table 9), a similar trend was observed for both strains: the highest value was reached after 6 hours culture (qiac.max), and then slightly decrease in the following 18 hours. Specifically, LACe exhibited a qiac.max = 92.2 ± 5.0 mg g^-1 h ¹, which then dropped to 84.9 ± 5.5 mg g^-1 h ¹ at the end of the experiment. LACp exhibited a qiac.max = 63.3 ± 4.1 mg g^-1 h ¹, which then dropped to 47.9 ± 3.3 mg g^-1 h ¹. The observed differences in production behavior and resulting the decrease in the qiac was most likely caused by rapid pH decrease within the first few hours from 3.6 to 2.7 as a result of the accumulation of lactic acid in culture medium (Table 7 and Table 8). Moreover, a low pH is known to promote intracellular acidification, LDH inhibition and cell death resulting in a further decrease in production yield.¹⁰ The observations are also in line with previous reports on decreased cell metabolism in the presence of lactic acid and pH below 3¹¹-¹³. As it was reported in the description of the experiments, the main drawback of conventional bioprocess optimization is the time required (e.g. days to weeks) to first expand and then select the strain with highest productivity, a problem that can be overcome by the miniaturization of the process.

Table 7. Off-line monitoring of process parameters and metabolites profile during S. cerevisiae LACp shake flask batch cultures. Optical density (Oϋboo), pH, glucose and lactic acid concentration inside the medium at each time point are reported. For each time point, mean value and standard deviation (sd) were calculated.

Table 8. Off-line monitoring of process parameters and metabolites profile during S. cerevisiae LACe shake flask batch cultures. Optical density (Oϋboo), pH, glucose and lactic acid concentration inside the medium at each time point are reported. For each time point, mean value and standard deviation (sd) were calculated.

Table 9. Calculated biomass specific lactic acid production rates (reported as milligram lactic acid produced per g biomass per hour) based on lactic acid produced by LACp and LACe during shake flask batch cultures. For each time frame, mean value and standard deviation (sd) were calculated.

Example 6: Evaluation of lactic acid production in two S. cerevisiae engineered strains cultivated under batch mode in the MmR

The two S. cerevisiae engineered strains, LACp and LACe, were cultivated under the same experimental conditions reported in the Example 5 to evaluate whether cells were able to produce lactic acid in the novel microenvironment and how fast it was possible to distinguish the best producer.

For start of cultivation, cells were inoculated from cryo-vials on Petri dishes containing agar medium with 3.4 g L ¹ YNB, 4.54 g L ¹ urea and 5 g L ¹ ethanol and maintained at 30°C for 72 h. Subsequently, cells were transferred into 10 mL YNB+E and incubated at 30°C, 180 rpm and a relative humidity of 70%. A 1 :10 volume ratio (volume of liquid to volume of shake flask) was kept constant for every pre-culture. After 24 h, cells were centrifuged (2000 g, 5 min), supernatant was discarded and pellet re suspended in fresh YNB+10G at a biomass concentration of Oϋboo = 40.

Prior to inoculation, the microfluidic devices and tubing were treated with 70% ethanol for 30 min and Dl water in order to remove solvent residues. All devices were allowed to dry at 35°C overnight ensuring absence of ethanol residues. 15-pL culture aliquots from the previously described culture (YNB+G medium, initial Oϋboo = 40) were injected inside the chambers of the microfluidic device with the aid of a GC-gastight glass syringe. The device was incubated under constant conditions of 30°C and 70% relative humidity to reduce evaporation through PDMS¹⁴. Due to the low volume imprint of the microfluidic device, for quantification of glucose and lactic acid, four individual 15-pL cultures were pooled for 6, 12 and 24 hours in quadruplicates for each time point.

The concentrations of residual glucose and lactate were determined by HPLC- Method_1 , and the quantitative analysis was used to calculate the biomass specific lactate production rate (q ), as follow using Equation 1.

HPLC analysis showed that LACp yielded a final titer of 12.8 ± 0.68 g L ¹ (Table 10) and LACe yielded a final titer of 25.2 ± 1.2 g L ¹ (Table 10). The kinetics of the bioprocess was found to be similar as the one in shake flask: the highest value was reached after 6 hours culture (qiac.max), and then slightly decreased in the following 18 hours. Specifically, LACe exhibited a qiac.max = 119.3 ± 13.0 mg g^-1 h ¹, which then dropped to 98.0 ± 7.3 mg g^-1 h ¹ at the end of the experiment. LACp exhibited a qiac.max = 74.0 ± 8.2 mg g^_1 h ¹, which then dropped to 41.9 ± 4.4 mg g^_1 h ¹ (Table 11). The observed cell behavior in terms of metabolic activity can be related to the same causes discussed in Example 5.

Table 10. Off-line monitoring of metabolites profile during S. cerevisiae LACp and LACe on-chip batch cultures. Glucose and lactic acid concentration inside the medium at each time point are reported. For each time point, mean value and standard deviation (sd) were calculated.

Table 11. Calculated biomass specific lactic acid production rates (reported as milligram lactic acid produced per g biomass per hour) based on lactic acid produced by LACp and LACe during on-chip batch cultures. For each time frame, mean value and standard deviation (sd) were calculated.

An additional advantage of using complementary on-chip sensing strategies such as light scatter detection of biomass and embedded oxygen sensors is the ability to gather more meaningful information about metabolic status of producing strains, concurring to provide an overview of the whole bioprocess (all process parameters have to be taken into account when scaling-up the bioprocess). As previously mentioned and as proved by off-line optical density measurements, LACe and LACp don’t grow under the reported experimental conditions. Thus, a continuous non-invasive light scattering measurement was run over 24 hour to monitor biomass concentration. After the 24-h pre-culture step, cells were re-suspended in fresh YNB+10G medium at a biomass concentration of Oϋboo = 4. 15-pL aliquots were then injected inside the chambers of the multiplexed microfluidic platform by means of a GC-gastight glass syringe and the device was placed inside the light scattering measuring station. The scattered light signal was then constantly recorded for 24 hours. A constant voltage around 7.2 ± 0.02 V was observed for LACe and 6.9 ± 0.09 V for LACp (Table 12). Therefore, it can be stated that the biomass was stable during on-chip batch cultures and the growth-decoupled biochemical lactic acid bio-production occurred also under these experimental conditions.

Table 11. Real-time non-invasive biomass monitoring during S. cerevisiae on-chip batch cultures. Voltage (V) (related to scattered light) registered by the light scattering measuring station. Comparison between signals recorded during l_ACp and LACe on- chip cultures.

Concurrently, non-invasive oxygen monitoring was performed during the process. After the 24-h pre-culture step, cells were re-suspended in fresh YNB-10G medium at a biomass concentration of Oϋboo = 40. 15-pL aliquots were then injected inside the chambers of the multiplexed microfluidic platform by means of a GC-gastight glass syringe and the fluorescent lifetime measurements were constantly run over 24 hours. Each experiment was carried out in duplicates. The registered DO was constant around 20% within the first 6 hours both for LACe and LACp. Subsequently, DO in LACp cultures rapidly and constantly increased in the next hours up to 90.0 ± 0.63 %. Also LACp cultures showed an increase in DO content after 6 hours, although the curve had a less steep slope and the final value reached was 66.6 ± 0.85 % (Table 13. Therefore, DO monitoring showed a different oxygen consumption for the strain tested, confirming, together with the off-line lactic acid quantification, the different metabolic behaviours of the two strains under the present experimental conditions.

Table 12. Real-time non-invasive dissolved oxygen monitoring during S. cerevisiae on-chip batch cultures. Comparison between signals recorded during LACp and LACe on-chip cultures. For each time point, mean value and standard deviation (sd) were calculated. Overall, such comparative study allowed evaluating the effect of physical- chemical condition in micro-culture environment: with high surface-to-volume ratio and reduced distances compared to mass transfer coefficients, diffusion is the predominant phenomenon in terms glucose and oxygen supply. The results of lactic acid production suggested not only that yeast biochemical activities is at same level of the one registered in shake flasks in absolute terms, but also that the strains’ performances diverged similarly when compared on the small scale. Specifically, a statistically significant difference between the two strains was found after 6 hours: LACe productivity was 1.5- times higher than LACp (p-value = 0.003) after shake flasks, and 1.6-times higher in batch cultures on chip (p-value = 2.7 10⁶).

Example 7: Evaluation of lactic acid production in two S. cerevisiae engineered strains cultivated under perfusion mode in the MmR

Therefore, the exemplary microfluidic platform described herein was integrated with a filtration system for performing continuous flow culture in perfusion mode. Such strategy allows studying cells in a more complex setup while keeping, at the same time, a simple design if compared with large-scale systems. The flow-through setup was therefore tested by running LACp and LACe perfusion cultures, and comparing the results with on-chip batch and shake flask cultures previously described.

For on-chip perfusion cultures, the inlet ports of the device were connected through PEEK tubing (1/32" outer diameter, 250 pm inner diameter) to plastic syringes filled with sterile YNB+G; the outlet ports of the device were connected through other PEEK tubing to safe-lock tubes where the effluent coming from the chamber was collected. The syringes were placed inside an infusion pump (PHD ULTRA infuse/withdraw programmable pump, Hugo Sachs Elektronik-Harvard Apparatus GmbH, Germany) and 6 pL medium was pumped inside the channels at 4 pL min ¹ flow rate so that the whole volume of the inlet channels (up to the chamber filter) could be filled with liquid removing all the air. Afterwards, 15 mI_ culture aliquots (YNB-10G, ODeoo= 40 prepared after 24-h pre-culture step) were injected inside the chambers with the aid of a GC-gastight glass syringe through the inoculation channels, which were then sealed, and the pump was turned on again to start the perfusion. The device was then incubated at 30°C and RH = 70% to avoid evaporation and bubble formation. Samples were collected from the outlet tubes after 3, 6, 12 and 24 h for organic compounds quantification, always replacing the collection tube with a new sterile one. Three different flow rates of 15, 7.5 and 3.75 pl_ h ¹, corresponding to hourly perfusion rates of P = 1 , 0.5 and 0.25 h ¹, were tested.

The concentrations of residual glucose and lactate were determined by HPLC- Method_1 , and the quantitative analysis was used to calculate the lactic acid mass produced by each chamber (pg) accumulated in the effluent at each time point and the specific lactate production rate (qi_ac, reported as mg lactic acid per g biomass per hour, mg g^-1 h ¹) using Equation 2 as follows.

Equation 2.

where m_lac is the mass (pg) of lactic acid accumulated in the effluent per chamber and m_x is the biomass in the corresponding microfluidic cell culture chamber.

LACe and LACp behavior was investigated under different perfusion rates to evaluate how the constant replenishing of medium could influence strain productivity. Specifically, the two strains were cultivated in the microfluidic platform under three different YNB+10G medium constant flow rates: 3.75, 7.5 and 15 pL h ¹, corresponding to 0.25, 0.5 and 1 h ¹ perfusion rates and lactic acid collected outside the device was quantified. Throughout 24 h, LACp showed a total lactic acid production ranging from 466 ± 71 pg for P = 0.25 h ¹, to 718 ± 31 pg for P=0.5 h ¹ and 887 ± 41 pg for P =1 h ¹ (Table 14). LACe exhibited a total lactic acid production ranging from 554 ± 40 pg for P = 0.25 h ¹, to 841 ± 31 pg for P=0.5 lr¹ and 1106 ± 72 pg for P =1 lr¹ (Table 15). Based on this data, it can be stated that perfusion setup triggered a higher lactic acid production for both strains if compared to batch cultures (351 ± 21 g L^_1 for LACe and 183 ± 9.0 g L ¹ for LACp in 24 hours experiments, as showed in Table 14 and Table 15. Moreover, lactic acid production increased together with the perfusion rate, as the maximum was recorded under P =1 h ¹ for both strains. In more detail, LACe showed a 2-fold increase, LACp a 1.9-fold increase when switching from 0.25 to 1 h ¹. Moreover, the lactic acid specific production rate constantly increased during every experiment for both strains and for every perfusion rate, with its highest value after 24 h. Specifically, in LACp cultures, qiac.max ranged from 209.8 ± 13.2 mg g^_1 lr¹ for P = 0.25 h ¹, to 278.2 ± 26.1 mg g-¹ lr¹ for P = 0.5 lr¹ and 435.0 ± 23.0 mg g^~1 lr¹ for P = 1 lr¹ (Table 16). In LACe cultures, qiac.max ranged from 273.3 ± 23.6 mg g^-1 h ¹ for P = 0.25 h ¹, to 362.9 ± 14.2 mg g^_1 h ¹ for P = 0.5 h ¹ and to 448.9 ± 40.1 mg g^_1 h ¹ for P = 1 h ¹ (Table 17). This trend was different from the one registered in shake flasks and on-chip batch cultures: a maximum 4-fold productivity increase was registered for LACe when switching from batch cultures to perfusion cultures; a maximum 6-fold productivity increase was registered for LACp when switching from batch cultures to perfusion cultures. This result proves that the perfused setup was effective at keeping the culture micro-environment optimal for the biochemical process thanks to the constant replenishing of fresh medium. The beneficial effect of perfused setup can be better observed by comparing the biomass specific lactic acid production rates of all cultivation modes tested and calculated over the whole lactic acid produced in 24 hours. In shake flask culture, LACp yielded a daily qiac = 61.2 ± 3.5 mg g^-1 h ¹ and LACe a daily qiac = 89.3 ± 2.6 mg g^-1 h ¹. Such values constantly increased in microfluidic perfused cultures together with the perfusion rate, ranging from qu\c = 140.0 ± 7.9 mg g^-1 h ¹ for LACp and qiac = 244.2 ± 19.2 mg g^-1 h ¹ for LACe under P = 0.25 h^-1 and qiac = 363.2 ± 11.6 mg g^-1 h ¹ for LACp and qiac = 436.6 ± 25.2 mg g^-1 h ¹ for LACe under P = 1 h ¹ (Table 18). Thus, the results highlighted that not only miniaturized perfusion cultures could provide quick results about strains performance, but also that they could represent a versatile tool to investigate more in detail cells metabolic activity and requirements, useful data for the upscaling phase of bioprocess development.

Table 13. Monitoring of lactic acid mass accumulated in the permeate coming out of the chip at different time points and different perfusion rates during LACp cultures. Lactic acid mass produced in a single microfluidic chamber during a batch culture and comparison with lactic acid produced by a single chamber under the perfusion rates of 0.25 h ¹, 0.5 h ¹ and 1 h ¹. For each time point, mean value and standard deviation (sd) were calculated.

Table 14. Monitoring of lactic acid mass accumulated in the permeate coming out of the chip at different time points and different perfusion rates during LACe cultures. Lactic acid mass produced in a single microfluidic chamber during a batch culture and comparison with lactic acid produced by a chamber under the perfusion rates of 0.25 h ¹, 0.5 h ¹ and 1 h ¹. For each time point, mean value and standard deviation (sd) were calculated.

Table 15. Calculated biomass specific lactic acid production rates (reported as milligram lactic acid produced per g biomass per hour) based on lactic acid produced by LACp during on-chip perfusion cultures under the perfusion rates of 0.25 h ¹, 0.5 h ¹ and 1 h ¹. For each time frame, mean value and standard deviation (sd) were calculated.

Table 16. Calculated biomass specific lactic acid production rates (reported as milligram lactic acid produced per g biomass per hour) based on lactic acid produced by LACe during on-chip perfusion cultures under the perfusion rates of 0.25 h ¹, 0.5 h ¹ and 1 h^-1. For each time frame, mean value and standard deviation (sd) were calculated.

Table 18. Calculated biomass specific lactic acid production rates (reported as milligram lactic acid produced per g biomass per hour) based on lactic acid produced by LACe and LACp Comparison among daily biomass specific lactic acid production rates (lactic acid produced throughout the 24-h experiment) of all the cultivation systems reported. For each cultivation mode, mean value and standard deviation (sd) were calculated. A further investigation was carried out in order to understand the differences between perfused and batch micro-cultures environment, by analyzing and quantifying the parameters that have a major impact on the culture: glucose concentration inside the chamber and in the collected spent medium, and lactic acid concentration inside the chamber. Glucose concentration in the output flow was maintained constantly close the maximum value in every experiment, as the minimum was 95.4 ± 1.1 g L^-1 for LACe under P = 0.25 h ¹ (Table 19 and Table 20). Concerning the glucose concentration inside the chamber, at the end of the experiment its value was also close to the input one, as the minimum registered was 90.1 ± 3.3 g L^-1 for LACe under P = 1 h ¹ (Table 20, therefore always higher than batch cultures, with a glucose concentration that dropped to 60 ± 2.3 g L ¹ (Table 10). Consequently, the system was able to provide constantly a high substrate concentration while triggering at the same time a high production level. In addition, the lactic acid content inside the cell culture chambers was quantified at the end of each experiment, when the process kinetic was at its maximum. As it can be seen in Table 21, in LACp cultures, lactic acid concentration inside the chamber was 4.78 ± 0.55 g L ¹ for P = 0.25 h ¹, 3.77 ± 0.09 g L ¹ for P = 0.5 lr¹ and 2.68 ± 0.10 g L ¹ for 1 h ¹. In LACe cultures, lactic acid concentration inside the chamber was 6.16 ± 0.89 g L ¹ for P = 0.25 tv¹, 4.89 ± 0.32 g L·¹ for P = 0.5 tv¹ and 2.94 ± 0.34 g L ¹ for 1 tv¹. Such values are lower than the ones reached in batch cultures (25.2 ± 1.2 g L·¹ for LACe and 12.7 ± 0.7 g L ¹ for LACp), proving that the perfused setup is able to constantly remove lactic acid from the micro-culture environment, which is advantageous as it represents a toxic compound for the cells and can drastically slow down biochemical processes. This suggests that the perfused micro-environment, thanks to constant replenishing of culture medium, can improve strain productivity in both strains. Still, when the two strains are compared, a statistically significant difference was found during perfusion in the productivity between LACe and LACp strains already after 3 hours from the inoculum both under P = 0.5 h^-1 and P = 1 h ¹ (p-value = 0.028 for both).

Table 17. Monitoring of glucose accumulated in the permeate at each time point during LACp on-chip perfusion cultures under the three perfusion rates of 0.25 hr¹, 0.5 hr¹ and 1 hr¹. For each time point, mean value and standard deviation (sd) were calculated.

Table 20. Monitoring of glucose accumulated in the permeate at each time point during LACe on-chip perfusion cultures under the three perfusion rates of 0.25 hr¹, 0.5 hr¹ and 1 hr¹. For each time point, mean value and standard deviation (sd) were calculated.

Table 18. Quantification of glucose and lactic acid concentrations inside the micro-cell culture chamber of the platform after 24-h on-chip perfusion cultures under the three perfusion rates of 0.25 hr¹, 0.5 hr¹ and 1 hr¹. Comparison between results from LACp and LACe. For each time point, mean value and standard deviation (sd) were calculated. Furthermore, using non-invasive sensing approaches, it was possible to continuously monitor dissolved oxygen content in the cultures at a constant biomass concentration. DO detection indicates a different behaviour of this setup if compared with batch mode: in LACp cultures, DO was constant around an average value of 11% for approximately 11 hours under the three perfusion rates, then increased up to 56 ± 0.8 % for P = 0.25 tv¹, to 22 ± 0.7 % for P = 0.5 tv¹ to 20 ± 0.5 % for P = 1 tv¹ (Table 22). In LACe cultures, DO was constant around an average value of 8% for approximately 12 hours under the three perfusion rates, then increased up to 37 ± 2.5 % for P = 0.25 h ¹, slightly decreased to 5.0 ± 3.3 % for P = 0.5 h ¹ and increased up to 12.2 ± 9.1 % for P = 1 h^-1 (Table 23). Such data is in line with the different metabolic activity of the two strains in the perfused systems highlighted by lactic acid quantification, and represent another process variable that is crucial in the study of a new bioprocess.

Table 19. Real-time non-invasive dissolved oxygen monitoring during LACp on- chip perfusion cultures under the three perfusion rates of 0.25 h ¹, 0.5 h ¹ and 1 h ¹. For each time point, mean value and standard deviation (sd) were calculated.

Table 20. Real-time non-invasive dissolved oxygen monitoring during LACe on- chip perfusion cultures under the three perfusion rates of 0.25 h ¹, 0.5 h ¹ and 1 h ¹. For each time point, mean value and standard deviation (sd) were calculated.

Example 8: Evaluation of HSA production in two P. pastoris engineered strains cultivated under perfusion mode in the MmR

The previous experiments proved that the MmR could provide an optimal cultivation environment for yeast cell growth and metabolic activity. Moreover, the productivity of engineered yeast strains was found to be similar (or even higher) to shake flask cultures thus proving that it is effective at screening the best performer and at collecting meaningful data during the development of a new bioprocess. Another proof- of-principal experiment was run, in order to evaluate whether the MmR could also represent an optimal environment for recombinant protein producing engineered yeast strains, and how fast such results could be available compared to common lab-scale cultivation systems.

The expression strain used in this study was P. pastoris CBS7435 wild-type strain (wt) (Centraal Bureau voor Schimmelcultures, The Netherlands). A puzzle vector carrying the human serum albumin gene (HSA) under control of the PGAP promoter was constructed as described by Prielhofer et al. (Prielhofer, R., Maurer, M., Klein, J. et al. Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris. Microb Cell Fact 12, 5 (2013) doi:10.1186/1475-2859-12-5). The vector was linearized prior to the transformation of P. pastoris wt. Selection was based on Zeocin resistance for the HSA. 3pg of linearized and purified (by Wizard SV Gel and PCR Cleanup System, Promega Corp) plasmid vector was used for the electrotransformation. Subsequently, cells were plated on YPD-agar plates (10 g L-1 yeast extract, 20 g L ¹ soy peptone, 20 g L ¹ glucose, 20 g L ¹ agar-agar) with 50, 100 or 500 mg ml_ ¹ Zeocin. Two randomly selected clones were used for the experiments reported below, and they were named as HSA#4 and HSA#15.

For start of cultivation, cells were inoculated from cryo-vials on YPD-agar plates (10 g L-1 yeast extract, 20 g L ¹ soy peptone, 20 g L ¹ glucose, 20 g L ¹ agar-agar, 100 pg mL ¹ Zeocin) and then maintained at 25°C for 72 h. For biomass formation, liquid YPD medium containing 10 g L-1 yeast extract, 20 g L ¹ soy peptone, 20 g L ¹ glucose, 100 pg mL ¹ Zeocin was used. Cells were transferred into 15 mL YPD medium and incubated at 25°C, 180 rpm for 24 hours. Subsequently, cells were centrifuged (2000 g, 5 min), supernatant was discarded and pellet re-suspended at a biomass concentration of Oϋboo = 40 in minimal ASM medium (3.2 g L ¹ (NH^HPC , 0.4 g L ¹ (NH )₂S04, 0.25 g L ¹ MgS0₄-7H₂0, 1.32 g L ¹ KCI, 0.027 g L ¹ CaCI₂-2H₂0, 11 g L ¹ citric acid monohydrate, 0.74 mL L ¹ PTM0 trace metals, 0.4 mg L ¹ biotin, 10 mL L ¹ NH4OH (25%); pH set to 6.5 with KOH).

For on-chip perfusion cultures, the inlet ports of the device were connected through PEEK tubing (1/32" outer diameter, 250 pm inner diameter) to plastic syringes filled with sterile ASM; the outlet ports of the device were connected through other PEEK tubing to safe-lock tubes where the effluent coming from the chamber was collected. The syringes were placed inside an infusion pump (PHD ULTRA infuse/withdraw programmable pump, Hugo Sachs Elektronik -Harvard Apparatus GmbH, Germany) and 6 pL medium was pumped inside the channels at 4 pL min ¹ flow rate so that the whole volume of the inlet channels (up to the chamber filter) could be filled with liquid removing all the air. Afterwards, 15 pL culture aliquots were injected inside the chambers with the aid of a GC-gastight glass syringe through the inoculation channels, which were then sealed, and the pump was turned on again to start the perfusion. Given the genetic properties of the two strains, a glucose-limiting environment was needed for triggering HSA production. From previous bioreactor fed-batch experiments, it was found that the glucose uptake rate of the strains (expressed as g glucose per g biomass per hour) is 0.18 g g ¹ h ¹. Based on this data, two different ASM medium were prepared so that, even at different flow rates, the same glucose-feeding rate of 0.18 g g^-1 h^-1 could be ensured in every experimental setup. One medium contained 6.6 g L^-1 glucose (ASM- G.1), and another one 16 g L ¹ glucose (ASM-G.2). Two different perfusion rates were applied: P = 0.25 h-1 for ASM-G.1 and P = 0.1 h-1 for ASM-G.2. The device was then incubated at 25°C and RH = 50% to avoid evaporation and bubble formation. Samples were collected from the outlet tubes after 12, 24, 36 and 48 h for HSA quantification, always replacing the collection tube with a new sterile one. At the end of the 48-h cultures, cell suspensions were drawn from the cell culture chambers to measure optical density. Each experiment was carried out in quadruplicates.

HSA from culture supernatant was quantified by the Human Albumin Elisa Quantitation Kit (catalog no. E80-129, Bethyl Laboratories Inc. Montgomery, TX, USA) following the suppliers instruction manual. The HSA standard was used with a starting concentration of 400 pg L^-1. Samples were diluted accordingly in dilution buffer (50 mM Tris-HCI, 140 mM NaCI, 1% (w/v) BSA, 0.05% (v/v) Tween20, pH 8.0). ELISA plates were coated with anti-HSA antibody diluted 1:1000 in coating buffer (34.3 mM NaHC03, 15.7 mM Na2C03, pH 9.6) at room temperature (RT) on a rotatory shaker for 1.5 h. After washing with the appropriate washing buffer (50 mM Tris-HCI, 140 mM NaCI, 0.05% (v/v) Tween20, pH 8.0), the plates were incubated with blocking solution (50 mM Tris- HCI, 140 mM NaCI, 1% (w/v) BSA, pH 8.0) at RT on a rotary shaker for 20 min. After washing, HSA standards and samples were applied to the plates and incubated at RT for 1 h on a rotatory shaker. Plates were washed and incubated with HRP-conjugate secondary antibody (1 :30,000 dilution in dilution buffer) for 1 hour at RT on a rotatory shaker. Plates were then washed and TMB substrate was added. The detection reaction was stopped after 2 min by the addition of 2 M H2SO4 and absorbance was measured at 450 nm. HSA#4 and HSA#15 behaviour was investigated under two different perfusion rates (but constant glucose-feeding rate) to evaluate how the constant replenishing of medium could influence strain productivity and if such experimental setup could provide meaningful data about the performance of recombinant protein production consistent with the lab-scale experiments. Throughout 48 hours, HSA#4 showed a total HSA production ranging from 145.7 ± 10.7 ng HSA for P = 0.25 h ¹ (Table 24), to 360.6 ±

141.8 ng HSA for P = 0.1 h ¹ (Table 25). HSA#15 showed a total HSA production ranging from 85.5 ± 13.7 ng HSA for P = 0.25 lr¹ (Table 24), to 144.7 ± 10.5 ng HSA for P = 0.1 h^-1 (Table 25). Calculated biomass specific HSA production rate in HSA#4 cultures ranged from 16.8 ± 1.2 pg g^_1 h ¹ under P = 0.25 h ¹, to 21.1 ± 7.1 pg g^_1 h ¹ under P = 0.1 h ¹. Calculated qHSA in HSA#15 cultures ranged from 6.2 ± 1.0 pg g^_1 h ¹ under P = 0.25 h^-1, to 7.0 ± 0.5 pg g ¹ h ¹ under P = 0.1 h ¹ (Table 26). HSA#4 proved to be a better HSA producer than HSA#15, both in absolute terms (mass of protein produced) and of process kinetics. Moreover, it seemed that a lower perfusion rate (in this case, 0.1 rather than 0.25 h ¹) could trigger a higher protein production, although this aspect would require a further investigation of metabolic and genetic properties of the strains, which is beyond the intentions of the reported work. A time-resolved monitoring of protein production showed that a difference in protein production could be observed much earlier than 48 hours: in experiments under P = 0.25 h ¹, it was possible to observe a divergence in HSA production after 12 h, with 22.6 ± 6.3 ng produced by HSA#4 and 2.0 ± 0.5 ng by HSA#15. Moreover, under both experimental conditions, it was possible to observe a divergence in HSA production after 24 h, with 54.5 ± 12.3 ng (HSA#4) and

31.8 ± 8.1 ng (HSA#15 protein produced in the whole time frame 0 - 24 h under P = 0.25 h ¹, and 56.0 ± 14.5 ng (HSA#4) and 29.1 ± 10.3 ng (HSA#15 under P = 0.1 h ¹. It can be concluded that the MmR was able to provide the necessary environment for cultivation of P. pastoris engineered strains and for maintaining the experimental conditions needed for protein production. The system was able to trigger different level of productivity by tuning the perfusion rate. At the end of 48-h cultures, it was possible to distinguish the productivity levels of the two strains, which were comparable to the results obtained from lab-scale cultures, proving the effectiveness of the MmR at screening yeast engineered strains for recombinant production.

Table 21. Monitoring of HSA accumulated in the permeate coming out of the chip at different time points during P. pastoris perfusion cultures. Comparison between the two P. pastoris engineered strains, HSA#4 and HSA#15, cultivated under the perfusion rates of 0.25 h ¹. For each time point, mean value and standard deviation (sd) were calculated.

Table 22. P = Monitoring of HSA accumulated in the permeate coming out of the chip at different time points during P. pastoris perfusion cultures. Comparison between the two P. pastoris engineered strains, HSA#4 and HSA#15, cultivated under the perfusion rates of 0.1 h ¹. For each time point, mean value and standard deviation (sd) were calculated.

Table 23. Calculated biomass specific HSA production rates (reported as microgram HSA produced per g biomass per hour) based on the total protein collected in 48 hours during HSA#4 and HSA#15 perfusion cultures. Comparison between two perfusion rates, P = 0.1 and P = 0.25 h ¹. For each perfusion rate, mean value and standard deviation (sd) were calculated. Example 9: Evaluation of HSA production in two P. pastoris engineered strains cultured under fed-batch mode in 1 -litre bioreactor cultures

For start of cultivation, cells were inoculated from cryo-vials on YPD-agar plates (10 g L-1 yeast extract, 20 g L ¹ soy peptone, 20 g L ¹ glucose, 20 g L ¹ agar-agar, 100 pg ml_ ¹ Zeocin) and then maintained at 25°C for 72 h. For biomass formation, liquid YPD medium containing 10 g L^-1 yeast extract, 20 g L^-1 soy peptone, 20 g L^-1 glucose, 100 pg ml_ ¹ Zeocin was used. Cells were transferred into 15 ml_ YPD medium and incubated at 25°C, 180 rpm for 24 hours. The biomass accumulated in this phase was then used as inoculum for bioreactor cultures.

The fed batch fermentations of HSA#4 and HSA#15 were done in a DASGIP Parallel Bioreactor System (Eppendorf AG, Germany) with a final working volume of 0.7 L. Strains with the GAP promoter were cultivated at the growth rate of p = 0.05 h ¹. Approximately 18 h batch phase (300 ml_ batch volume) was followed by a fed batch phase with a glucose medium addition (550 g glucose, 10.4 ml_ PTM0 trace salts stock solution and 10.4 ml_ biotin stock solution per liter). Feeding medium was supplied through an exponential flow based on the equation y = 0.994 e° ^05x. Experiments were terminated when the feeding addition led to the maximum reactor volume and it happened after approximately 74 hours. All the fermentations were performed at 25°C, the dissolved oxygen was maintained at DO = 30% by controlling the stirring speed, and the pH 5.0 was controlled by adding NH4OH (25%). The media used for cultivation was BSM and consisted of 11.48 g L¹ H3PO4, 0.5 g L¹ CaCl2-2H20, 7.5 g L¹ MgS04-7H₂0, 9 g L·¹ K2SO4, 2 g L·¹ KOH, 40 g L·¹ glycerol, 0.25 g L·¹ NaCI, 4.35 ml_ L^-1 PTM0, 0.87 mg L^-1 biotin, 0.1 ml_ L^-1 Glanapon 2000, pH set to 5.5 with 25% NH4OH. PTM0 consisted of 6.0 g L·¹ CuS04-5H₂0, 0.08 g L·¹ Nal, 3.36 g L·¹ MnS0₄ ^*H₂0, 0.2 g L·¹ Na₂Mo04-2H₂0, 0.02 g L·¹ H3BO3, 0.82 g L·¹ C0CI2, 20.0 g L ¹ ZnCI₂, 65.0 g L ¹ FeS04-7H₂0, 5 ml_ L ¹ H2SO4 (95% - 98%).

HSA was quantified using a Caliper Labchip-DS microfluidic instrument (Perkin Elmer) with BSA as standard protein. Protein quantification in samples from bioreactor cultures showed that, after 74-h fermentation, HSA#4 achieved a final titre of 227.9 ± 18.6 mg L^-1 whereas, under the same experimental conditions, HSA#15 achieved a final titre of 168.7 ± 14.2 mg L ¹, 1.4-times lower than the previous strains (Table 27). The calculation of specific protein production rate showed also a higher average productivity for HSA#4, with 6.7 ± 0.6 mg g^-1 h ¹, than for HSA#15, which exhibited 4.9 ± 0.4 mg g^-1 h ¹, thus confirming that the strain HSA#4 is the best producer under such experimental conditions and could be considered for an upscaling of the process. Such data were therefore consistent with that achieved by the miniaturized cultures performed in the MmR, confirming its potential as screening tool for industrially relevant engineered strains.

Table 27. Monitoring of HSA accumulated in the fermentation medium at different time points during P. pastoris fed-batch bioreactor cultivation. Comparison between the two P. pastoris engineered strains, HSA#4 and HSA#15. For each time point, mean value and standard deviation (sd) were calculated.

Example 10: Evaluation of itaconic acid production in E. coli engineered strains cultured under perfusion mode in the multiplexed microfluidic platform Itaconic acid is a dicarboxylic acid with a high potential as a biochemical building block. A proof-of-principal application test is run to confirm the possibility of application of the MmR with E. coli cultures, and bacterial cultures in general, as they cover a broad market segment in biotechnological industry.

An E. coli strain is genetically engineered with a plasmid vector containing the CAD (cis-aconitate decarboxylase) gene from Aspergillus terreus, whose overexpression would allow achieving the production of itaconic acid into the bacterial cell. Starting from Escherichia coli MG1655 (DSMZ, German Collection of Microorganisms and Cell Cultures), different clones, able to overexpress the gene encoding cis-aconitate decarboxylase (CAD; GenBank ID: BAG49047.1) gene from Aspergillus terreus are selected and tested.

E. coli engineered strains are cultivated under perfusion mode in the MmR by employing the experimental setup described in Example 7. E. coli pre-cultures are injected inside the chambers via the inoculation channel that is clogged afterwards. A glucose medium is then pumped and the effluent is collected outside the chip. Sample can be then analysed by HPLC (with the same method described in Example 7) for itaconic acid quantification

The E. coli strains are also cultivated in shake flasks. The cultures are run starting from a pre-culture inoculum into a glucose medium. At the end of experiments, samples are analysed by HPLC in order to quantify itaconic acid production. Such data is compared with the one obtained by miniaturized cultures into MmR to evaluate its effectiveness to screen the best conditions for bacteria-based bioprocess development.

Example 11: Evaluation of IgG production in engineered CHO cells cultured under perfusion mode in the multiplexed microfluidic platform

Chinese hamster ovary (CHO) cells represent the most frequently applied host cell system for industrial manufacturing of recombinant protein therapeutics, especially monoclonal antibodies, whose market is worth tens billion dollars every year and has constantly increased over the last decade. The integration of the presented MmR in the workflow of biopharmaceutical industry would represent a huge asset in the development of novel therapeutics.

A CHO K1 cell line (from the European Collection of Authenticated Cell Cultures (ECACC)) is genetically engineered with a plasmid vector containing the gene for the overexpression of an Immunoglobulin G (IgG). Different clones are selected and tested.

CHO engineered strains are cultivated under perfusion mode in the MmR by employing the experimental setup described in Example 7. Aliquots form pre-cultures are injected inside the chambers via the inoculation channel that is clogged afterwards. A glucose medium is then pumped and the effluent is collected outside the chip. Sample can be then analysed by ELISA kit for IgG quantification.

The CHO strains are also cultivated in lab-scale bioreactors. The cultures are run starting from a pre-culture inoculum into a glucose medium. At the end of fermentation, samples are analysed by ELISA in order to quantify IgG production. Such data is compared with the one obtained by miniaturized cultures into MmR to evaluate its effectiveness to screen the best strain for antibody production in mammalian cell cultures.

Claims

1. A method for producing a fermentation product in a cell culture, comprising: a. determining optimized process parameters for culturing a high producer cell line to produce said fermentation product, wherein the determining step of a. is made upon cell culture employing a multiplexed microfluidic perfusion cell culture device in perfusion mode, and wherein the high producer cell line is optionally selected from a repertoire of cell lines capable of producing said fermentation product; and b. culturing said high producer cell line by an industrial scale method under the selected process parameters to produce said fermentation product.

2. The method of claim 1 , wherein said process parameters are selected from the group comprising productivity, temperature, oxygen concentration, pH value, cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass concentration, metabolites concentration, and carbon dioxide concentration.

3. The method of claim 1 or 2, wherein said optimized process parameters are determined by culturing said high producer cell line in a multiplexed microfluidic perfusion cell culture device in perfusion mode, wherein optimized process parameters are selected according to the amount of the fermentation product produced.

4. The method of any one of claims 1 to 3, wherein said high producer cell line is selected by culturing said repertoire of cell lines in a multiplexed microfluidic perfusion cell culture device, wherein said repertoire comprises a variety of cell lines, which differ in their capability of producing said fermentation product.

5. The method of any one of claims 1 to 4, wherein said multiplexed microfluidic perfusion cell culture device comprises at least: a. a cell culture chamber unit (1) comprising at least two spatially distinct cell culture chambers (4), wherein each of said cell culture chambers (4) comprises a cell inoculation channel (5), a flow inlet (6) and a flow outlet (7), wherein (5), (6) and (7) are separate elements; and b. a microchannel network unit (2) comprising for each of said cell culture chambers (4) a flow input channel (8) connected to the flow inlet (6) and a flow output channel (9) connected to the flow outlet (7); wherein the flow output channel (9) and/or the flow outlet (7) is equipped with a cell retention filter unit (3), wherein the cell retention filter unit comprises a porous filter with a pore size of less than 1 pm.

6. The method of claim 5, wherein said device comprises one or more integrated sensors for assessing one or more process parameters, preferably wherein each of the cell culture chambers (4) comprises one or more integrated sensors.

7. The method of any one of claims 5 or 6, wherein the volume of said spatially distinct cell culture chambers is less than 100 pL, preferably less than 50 pL, more preferably less than 20 pL.

8. The method of any one of claims 1 to 7, wherein said repertoire of cell lines is selected from the group consisting of yeast, bacterial or mammalian cells.

9. The method of any one of claims 1 to 8, wherein the fermentation product is selected from a cell metabolite or a protein.

10. The method of claim 9, wherein the cell metabolite is selected from the group consisting of organic acids, alcohols and amino acids, preferably lactic acid, itaconic acid, citric acid, succinic acid, glutamic acid, 3-hydroxypropionic acid, adipic acid, ethanol, propane-1 , 3-diol, 1 -butanol, 2-butanol, butane-1 ,4-diol, L-glutamic acid, L- lysine, or L-threonine.

11. The method of claim 10, wherein the protein is selected from the group of heterologous proteins, preferably selected from an antigen-binding protein, therapeutic protein, an enzyme, a peptide, a protein antibiotic, a toxin fusion protein, a carbohydrate - protein conjugate, a structural protein, a regulatory protein, a vaccine antigen, a growth factor, a hormone, a cytokine, a process enzyme.

12. The method of any one of claims 1 to 11, wherein said industrial method is a non-perfusion method, preferably a batch, fed-batch or non-perfusion continuous method.

13. A method for screening one or more process parameters for culturing a cell line and producing a fermentation product from said cell line comprising the steps of: a. providing a cell line capable of producing said fermentation product; b. culturing the cell line in separate microfluidic perfusion cell culture chambers under differing process parameters to produce said fermentation product, wherein said process parameters are selected from the group comprising productivity, temperature, oxygen concentration, pH value, cell culture medium composition, concentration of a cell culture medium component, flow rate, biomass concentration, metabolite concentration, and carbon dioxide concentration; c. determining one or more process parameters optimized according to the fermentation product yield; and d. culturing the cell line in an industrial scale method, wherein one or more of the selected process parameters are applied.

14. A method for screening a production cell line and producing a fermentation product from said cell line comprising the steps of: a. providing a repertoire of cell lines comprising a variety of cell lines which differ in their capability of producing said fermentation product; b. culturing each of said cell lines in a separate microfluidic perfusion cell culture chamber under conditions to produce said fermentation product; c. selecting a high producer cell line according to the fermentation product yield; d. optionally selecting one or more process parameters; and e. culturing the selected cell line in an industrial scale method under conditions to produce said fermentation product.

15. Use of process parameters and/or use of a high producer cell line selected by a method employing a multiplexed microfluidic perfusion cell culture device, for producing a fermentation product in an industrial scale process.