Manufacturing method with feedback control

Abstract

The present disclosure generally relates to a method of making a biological product (e.g., an enzyme) using fermentation and subsequent purification of the biological product. By utilizing online biomass data for feedback control in the fermenter, efficient and stable cross flow microfiltration operations can be performed with active cells.

Description

Technical Field

[0001] The present disclosure generally relates to methods of making bioproducts using fermentation and subsequent purification of the bioproducts. Background Art

[0002] Microbial expression to produce products through fermentation is a common method for producing various biomaterials, such as enzymes, as described, for example, in patent publication WO2021170799A1.

[0003] After fermentation is complete, the desired product needs to be separated from the fermentation broth. This is typically accomplished by first separating the aqueous phase from the cell material in a filtration step, followed by extraction or adsorption of the product from the filtrate. However, such filtration steps are often associated with significant losses of the desired product. This is primarily because the filter cake cannot be adequately washed off, resulting in a significant amount of product remaining in the filter cloth. In practice, it has been observed that the efficiency and capacity of the filtration process are strongly dependent on the quality of the fermentation broth.

[0004] In order to overcome the above-mentioned problems in processing fermentation broth, patent publication US Pat. No. 6,515,11B1 describes other filtration methods, such as membrane filtration.

[0005] However, such cross-flow filtration methods have disadvantages, primarily due to the rapid, uncontrolled increase in biomass concentration. At a critical cell concentration, the microfiltration membrane can become clogged and hinder the recovery process.

[0006] Therefore, there is a need for improved manufacturing methods. Summary of the Invention

[0007] The present disclosure provides an improved manufacturing method for extracting products from fermentation broth using a cross-flow filtration device in combination with an online biomass probe and a feedback loop to continuously dilute the fermentation broth.

[0008] This has advantages in several respects, such as the ability to operate at the highest possible biomass concentration, providing high product concentrations in the fermentate / retentate. High product concentrations can also be achieved in the retentate, thereby providing high product titers in the permeate. This results in faster and more efficient cross-flow operation and good recovery rates. Furthermore, in a fed-batch fermentation setup, the product, such as an enzyme, is secreted into the fermentation broth. The product, such as an enzyme, is obtained by cross-flow microfiltration while the fed-batch fermentation process remains operational to avoid cleavage and contamination by unwanted enzymes, such as proteases.

[0009] According to a first aspect of the present invention, a method for producing a product by fermenting a host cell is disclosed, comprising the following steps:

[0010] a) adding the host cell culture to a fermentor;

[0011] b) transferring water or buffer from a buffer tank to a fermentor, wherein the buffer tank is operably connected to the fermentor via a pump or a valve;

[0012] c) conveying the fermentate from the fermentor to a cross-flow microfiltration unit;

[0013] d) transferring the biomass from the cross-flow microfiltration unit back to the fermentor; and

[0014] e) delivering the permeate from the cross-flow microfiltration unit to a product container;

[0015] A biomass probe is present in the fermenter to measure the cell concentration. The process control system receives data on the cell concentration and controls a pump or valve to deliver water or buffer to the fermenter so that a specific predetermined cell concentration is maintained.

[0016] In one embodiment, the method further comprises operatively connecting a feed medium tank to the fermentor.

[0017] According to a second aspect of the present invention, a product obtainable by the method according to the first aspect is disclosed.

[0018] In one embodiment the product obtainable by the process according to the first aspect is an enzyme.

[0019] In a further embodiment, the product obtainable by the method according to the first aspect is a naturally occurring enzyme or an enzyme produced by a natural microorganism or expression host. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a schematic diagram of an embodiment.

[0021] Figure 2 is a diagram showing online and offline biomass according to one embodiment.

[0022] Figure 3 is a diagram showing online and offline biomass according to one embodiment.

[0023] Figure 4 is a graph showing dilution ratios according to one embodiment.

[0024] Figure 5 is a graph showing product concentrations according to one embodiment.

[0025] Figure 6 is a diagram showing online and offline biomass according to one embodiment.

[0026] Figure 7 is a diagram showing fermentor volume and water addition according to one embodiment.

[0027] Figure 8 is a graph showing product concentrations according to one embodiment.

[0028] Figure 9 is a schematic diagram of an embodiment. DETAILED DESCRIPTION

[0029] The present disclosure relates to efficient product recovery from high cell density fermentations using cross-flow microfiltration. In one embodiment, the product is an enzyme.

[0030] Developed integrated upstream and downstream processes. Figure 1 and 9 A schematic diagram of a process setup (100) is shown. Water or buffer is held in a buffer tank (101), and a feed medium solution is held in a feed medium tank (102). Both the buffer / water and the feed medium solution, such as a sugar solution, are transported to a fermentor (103), where cells proliferate and the product is expressed. From the fermentor, the feed is directed to a cross-flow microfiltration unit (104), where the permeate is separated from the biomass. The biomass is transported back to the fermentor (103), and the permeate is transported to a product container (105). Thus, the cross-flow microfiltration unit is used to separate the enzyme from the fermentation broth containing the cells.

[0031] To avoid cell lysis in the fermentor (103) and the release of proteases and other by-products, the culture is continued while the product is harvested from the fermentation broth. Product stability in the fermentor (103) is achieved by directly connecting the fermentor to the cross-flow filtration unit and by controlling the pH, temperature and oxygen supply within the desired ranges.

[0032] In addition, in order to be able to control the biomass at the highest possible concentration, an online biomass probe (106) is used in the fermentor (103) to measure the biomass concentration. The biomass probe (106) sends data to the process control system (107) which interprets the data. A feedback loop is created when the process control system (107) adjusts the pump or valve (108) that supplies water or buffer from the buffer tank (101) to the fermentor (103). This enables continuous dilution of the cell-containing fermentation broth based on the data from the biomass probe (106).

[0033] Thanks to this feedback control, the biomass concentration can be maintained at an optimal level. Otherwise, the biomass concentration would increase rapidly and uncontrollably. At a critical cell concentration, the microfiltration membrane would clog and hinder the recovery process.

[0034] The further advantages of online biomass feedback control are many. For example, this scheme can operate under the highest possible biomass concentration, thereby provides high product concentration in fermentation product / retentate. It is also possible to realize high product concentration in retentate, provide high product titer in permeate. This results in faster and more effective cross-flow operation and good recovery. In addition, in the fed-batch fermentation setting, the product, for example, enzyme is secreted into the fermentation liquid. Obtain the product, for example enzyme, by cross-flow microfiltration, while the fed-batch fermentation process keeps running to avoid cracking and by unwanted enzyme, for example protease pollution.

[0035] In one embodiment, the cell concentration measured by the biomass probe (106) can also be compared to the biomass content (packed cell volume (PCV), vol %) estimated in an offline sample.

[0036] In one embodiment, the product is an enzyme. Applications for continuous harvesting of enzyme products using a process setup (100) according to an embodiment are particularly advantageous because enzyme products are typically not present during the inactivation period of a GMO production host strain because they are unstable at the required pH and / or temperature. A further advantage of a process setup (100) according to an embodiment is that the release of proteases caused by cell lysis is minimized and the release of GMO DNA caused by cell lysis is minimized, thereby reducing the need for separate complex DNA capture methods. A further advantage of a process setup (100) according to an embodiment is that the efficiency of operating with a continuous cross-flow filter at optimal process parameters is increased because membrane fouling caused by biomass getting stuck and clogging the cross-flow filter is minimized and controlled. The higher product titers obtained at reduced permeate volumes can avoid the need for ultrafiltration concentration and enzyme capture chromatography.

[0037] Cloning and expression

[0038] Any product suitable for microbial expression can be produced using the process setup (100) according to one embodiment. A specific system for expressing enzymes is disclosed in patent publication WO2021170799A1.

[0039] Suitable expression hosts are readily known to those skilled in the art. Examples of expression hosts are pichia pastoris or yeast species, yeast saccharomyces cerevisiae, fission yeast species, Candida species, Candida cylindrica, Kluyveromyces species, Hansenula polymorpha, Fusarium species, Fusarium oxysporum, Aspergillus species, Aspergillus oryzae or Aspergillus niger, Trichoderma species, Escherichia coli or Bacillus species, Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, alkaliphilic Bacillus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus luminescent, Bacillus megaterium, Bacillus thuringiensis, Streptomyces species, Streptomyces lividans or Streptomyces murinus, Corynebacterium species, Yarrowia, preferably Aspergillus oryzae, Aspergillus niger or Bacillus subtilis.

[0040] Recombinant cloning and expression systems suitable for expression of the desired product can be further introduced into host cells, e.g., recombinant host cells, using standard transformation or transfection protocols known to those skilled in the art and explained in the literature (e.g., J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory manual, 2nd edition, vols. 1-3, Cold Spring Harbor Laboratory Press).

[0041] Example

[0042] Example 1

[0043] In the first experiment, biomass concentration was measured online (in pF / cm) and offline (PCV, in vol%). Fermentation volume loss due to permeate outflow was manually adjusted by adding water in an attempt to maintain a constant fermentor volume. Without automated control, clogging of the microfiltration membrane was prevented, but a stable biomass concentration could not be achieved as crossflow parameters (transmembrane pressure, CF overflow, permeate flow) fluctuated and biomass concentration increased.

[0044] Figure 2 is a graph showing online and offline biomass (in terms of PCV, packed cell volume) measurements in a fermenter during a manually controlled cross-flow microfiltration process. The efficient penetration of the enzyme through the membrane depends on the biomass concentration and biofouling of the membrane.

[0045] Example 2

[0046] The second experiment showed that too high a biomass concentration inhibited the penetration of the product into the permeate.

[0047] Figure 3is a graph showing online and offline biomass measurements in a fermenter during a PID controlled cross-flow microfiltration process; the controller set point was 28 pF / cm. Figure 4 is a graph showing the fermenter volume and water addition during the culture time.

[0048] The feedback loop allows flexible adjustment of the biomass concentration resulting from permeate extraction and continued growth. The increase in the fermenter is due to the addition of more volume via feed and water dilution than is withdrawn via permeate.

[0049] Figure 4 Figure 2 is a graph showing the dynamically changing dilution rate during enzyme DSP. The controller starts too late, so the dilution is too slow and the set point of 28 pF / cm is reached too late.

[0050] Therefore, very high biomass overshoot (>30 pF / cm, 60 vol% PCV) leads to biofouling of the membrane and reduces enzyme penetration. Figure 5 Seen in.

[0051] Figure 5 is a graph showing the product concentration in the retentate (fermentate) and the permeate. It can be seen that there is a low breakthrough through the cross-flow membrane.

[0052] Example 3

[0053] The third experiment describes the ideal DSP. A biomass concentration of 26 pF / cm was selected as the set point. The PID controller parameters were optimized to maintain this set point within ±0.37 pF / cm. The corresponding PCV values ​​were within the range of 51.7 ± 1.0 vol%.

[0054] Figure 6 is a graph showing online and offline biomass measurements in a fermenter during a PDI-controlled cross-flow microfiltration process; the controller set point was 26 pF / cm.

[0055] Figure 7 is a graph showing the fermenter volume and water addition during the culture time.

[0056] Figure 8 is a graph showing the product concentration in the retentate (fermentate) and the permeate. Optimal breakthrough through the cross-flow membrane is evident.

[0057] The dynamic dilution rate ensures stable CFF operation and high enzyme penetration (also at high product concentrations) over several hours.

[0058] Biomass viability is ensured by continuous fed-batch fermentation during enzyme harvesting, minimizing cell lysis, protease release, and product cross-contamination. Online control, based on the Hamilton InCyte signal in the fermenter, dilutes the biomass to an optimal value by adding water, ensuring stable in-situ downstream processing. Membrane fouling is thus controlled by establishing a defined cell cake (too high a cell density blocks the filter membrane) that results in low transmembrane retention of the target enzyme.

[0059] By utilizing online biomass data for feedback control in the fermenter, efficient and stable cross-flow microfiltration operation can be performed with active cells. This is advantageous because the product is obtained in high concentration and without significant by-products.

[0060] The process setup according to one embodiment can be used for a variety of enzymes without the need for additional purification and concentration steps.

[0061] The present invention has been described with reference to various embodiments, aspects, examples, etc. These elements are not intended to be read separately from each other. Therefore, the present disclosure provides a combination of two or more embodiments, aspects, examples, etc.

[0062] All embodiments described herein are intended to be within the scope of the disclosed invention. These and other embodiments of the invention will be readily apparent to those skilled in the art from the accompanying detailed description of the preferred embodiments, with reference to the entire specification, the invention not being limited to any preferred embodiment disclosed.

Claims

1. A method for producing a product by fermenting a host cell, comprising the following steps: a) adding the host cell culture to a fermentor; b) transferring water or buffer from a buffer tank to the fermentor, wherein the buffer tank is operatively connected to the fermentor via a pump or a valve; c) conveying the fermentate from the fermentor to a cross-flow microfiltration unit; d) transferring the biomass from the cross-flow microfiltration unit back to the fermentor; and e) delivering the permeate from the cross-flow microfiltration unit to a product container; Wherein a biomass probe is present in the fermenter, the biomass probe measures the cell concentration, and the process control system receives data on the cell concentration and controls a pump or valve to deliver water or buffer to the fermenter so that a specific predetermined cell concentration is maintained.

2. The method of claim 1, further comprising a feed medium tank operably connected to the fermentor.

3. A product obtainable by the method according to claim 1 or 2.

4. A product obtainable by the method of claim 1 or 2, wherein the product is an enzyme.

5. The product obtainable by the process according to claim 4, wherein the product is a naturally occurring enzyme and / or an enzyme produced by a natural microorganism.

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