Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/145—Clostridium

Definitions

• the present invention relates to a liquid culture medium comprising yeast extract, glycine, arginine, glutamine, serine, threonine, magnesium ions, calcium ions, selenium (e.g., in the form of sodium selenite) and water. It further relates to a liquid feed composition for use as feed in a fed-batch process comprising at least yeast extract, threonine, serine and water. It relates to a kit of parts comprising the liquid culture medium and the liquid feed composition, the use of the liquid culture medium and the liquid feed composition for cultivating Hathewaya histolytica (previously Clostridium histolyticum) and obtaining one or more proteases and methods concerning the cultivation.
• Hathewaya histolytica previously Clostridium histolyticum
• Collagen is the major structural constituent of mammalian organisms and makes up a large portion of the total protein content of skin and other parts of the animal body. In humans, it is particularly important in the wound healing process and in the process of natural aging. Various skin traumas such as bumps, surgery, infection and accident are often characterized by the erratic accumulation of fibrous tissue that is rich in collagen and having increased proteoglycan content. In addition to the replacement of the normal tissue which has been damaged or destroyed, excessive and disfiguring deposits of new tissue sometimes form during the healing process.
• Collagen-mediated diseases Numerous diseases and conditions are associated with excess collagen deposition and the erratic accumulation of fibrous tissue rich in collagen. Such diseases and conditions are collectively referred to herein as "collagen-mediated diseases".
• Collagenase is an enzyme that has the specific ability to digest collagen. Collagenase has been used to treat a variety of collagen-mediated diseases such as Peyronie's disease and Dupuytren's disease, and to remove necrotic tissue from wounds.
• collagenase and other proteases are used in vitro for tissue dissociation and isolation of a variety of cells, such as e.g. pancreatic islet cells, hepatocytes and tumor cells. These cells find multiple applications in research and clinics.
• proteases such as e.g. neutral protease and/or clostripain
• pancreatic islet cells such as pancreatic islet cells, hepatocytes and tumor cells.
• hepatocytes and tumor cells find multiple applications in research and clinics.
• One common source of crude collagenase or mixtures of proteases is from a bacterial fermentation process, especially from the fermentation of Hathewaya histolytica (previously Clostridium histolyticum).
• Hathewaya is a genus of gram-positive bacteria. Hathewaya (Clostridium) bacteria are anaerobic and are common in the soil, water and in the intestinal tracts of humans and other animals.
• the species Hathewaya histolytica (Clostridium histolyticum) is capable of producing collagen degrading enzymes and other enzymes with proteolytic activity, such as e.g. collagenases (EC 3.4.24.3), neutral protease and clostripain (EC 3.4.22.8).
• Important products of the cultivation process are the bacterial collagenases which possess proteolytic activity against collagen.
• a growth medium for Hathewaya histolytica (Clostridium histolyticum) is disclosed that comprises water, fish gelatin and a peptone from a non-mammalian source, whereby fish peptone is excluded.
• the only examples for a peptone from a non-mammalian source are plant peptones.
• This growth medium suffers from the fact that fish gelatin and a peptone from a non-mammalian source are complex ingredients that comprise a large variety of compounds, most of which are not known. Moreover, they are prepared from natural sources and are therefore subject to natural fluctuations in their contents.
• EP 2 865 748 Al discloses animal product-free culture media for bacteria of the genus Clostridium (now Hathewaya), especially Clostridium histolyticum (now Hathewaya histolytica) comprising water, non-animal origin peptone, or its derivatives, yeast extract and the amino acids cysteine and arginine.
• the nonanimal origin peptones are derived from plants (plant peptones).
• Vegetable peptones are used according to WO 2007/089851 Al for the fermentation of Clostridium histolyticum (now Hathewaya histolytica) to produce collagenase I and collagenase II.
• Plant peptones have the disadvantages that they are complex and undefined mixtures and that the production of the raw materials is often subject to large batch variability, especially if weather conditions or planting regions change.
• the composition of plant peptones may significantly vary depending on the production process and the supplier. Thus, it is difficult or just not possible to obtain a constant product quality or to find alternative suppliers. For these reasons, plant oeotones are not desired as ingredient for most industrial processes for the cultivation of bacteria.
• Hathewaya histolytica (Clostridium histolyticum) and the production of proteases.
• Hathewaya histolytica (Clostridium histolyticum) secretes collagenase I, collagenase II, clostripain and neutral protease during cultivation.
• the activity of all these enzymes in the supernatant is strongly influenced by the compositions of the plant peptones used for cultivation and may vary by a factor 5 or more, depending on the source or raw material of a plant peptone, the manufacturer, the production process, and on the batch. As outlined above, such product variability and dependency on a single supplier are suboptimal for an industrial production process, especially in the pharmaceutical industry.
• the aim of this invention was to provide a liquid medium and/or a liquid feed composition which (i) supports growth of the bacteria and (ii) stimulates secretion of enzymes with proteolytic activity into the culture medium and (iii) reduces product and process variability. It is particularly preferred that the medium stimulates a high volume activity (enzyme activity per culture volume) in the culture supernatant.
• An additional purpose of the present invention is to provide a new type of culture medium that allows the cultivation of bacteria without addition of animal-derived components and/or plant derived components.
• the medium should furthermore have as few components as possible, in order to avoid any contamination with undesirable substances and to avoid overly complex influences on the fermentation process.
• such culture medium allows for the effective cultivation of the species Hathewaya histolytica (Clostridium histolyticum) and for the production of one or more proteases, e.g. collagenase I, collagenase II, neutral protease and/or clostripain. It is a further object of the present invention to provide a supernatant of a Hathewaya histolytica (Clostridium histolyticum) liquid culture, which may be used forthe isolation of the one or more proteases comprised therein. It is also an object of the present invention to provide a process for the production of proteases wherein the degradation of the proteases is low.
• the culture medium may provide the following benefits: a) higher batch reliability than culture media comprising animal peptones or plant peptones; b) all components can be obtained in suitable and constant quality from multiple suppliers; c) the activity of collagenase in the supernatant of Hathewaya histolytica (Clostridium histolyticum) is 400 U / L or more (PZ activity).
• the present invention concerns a liquid culture medium comprising, essentially consisting of, or consisting of
• magnesium ions e.g. 100 to 3000 mg/L of magnesium sulfate heptahydrate (MgSC x 7 H2O),
• composition (a) necessarily includes the listed ingredients and (b) is open to unlisted ingredients that do not materially affect the basic and novel properties of the composition.
• the liquid culture medium according to the present invention is suitable as culture medium for bacteria of the genus Hathewaya (Clostridium) and especially as culture medium for Hathewaya histolytica (Clostridium histolyticum; e.g. Clostridium histolyticum Gil and/or ATCC® 21000TM). It provides nutrients required for sufficient growth of the bacteria and - optionally in combination with a feed composition according to the invention - for the effective production of one or more proteases, such as e.g. collagenase I, collagenase II, neutral protease and/or clostripain.
• proteases such as e.g. collagenase I, collagenase II, neutral protease and/or clostripain.
• the activity of collagenase in the supernatant is 400 PZ units/L or more, in another embodiment 600 PZ units/L or more, in another embodiment 1200 PZ units/L or more.
• 1 PZ Unit according to Wuensch catalyzes the hydrolysis of 1 pmol 4-phenylazobenzyl- oxycarbonyl- L-prolyl-L-leucyl- glycyl-L-prolyl-D-arginine (PZ) per minute at 25 °C, pH 7.1 (Wuensch, E. & Heidrich, H.G. (1963) Hoppe-Seyler's Z. Physiol. Chem. 333, 149-51).
• the concentration of yeast extract in the liquid culture medium is in the range of 2.5 to 80, 5.0 to 60 or 10 to 30 g /L. In another embodiment, the concentration of yeast extract in the liquid culture medium is in the range of 17 to 25 g /L. In some embodiments, the liquid culture medium comprises 20 g/L yeast extract.
• yeast extracts can be used.
• yeast extracts and methods for the preparation are well known in the art. Contrary to animal peptones or plant peptones, yeast extracts are produced by better controlled industrial processes which include fermentation of the yeast in an industrial bioreactor. For this reason, batch reliability is higher than for plant- or animal- derived components. Different batches of yeast extract produced by the same process provide a similar performance in terms of bacterial growth and production of proteases. Furthermore, yeast extracts produced by different processes and/or different producers can substitute each other and provide similar performance in terms of bacterial growth and production of proteases. In summary, yeast extracts provide increased process robustness over animal peptones and plant peptones.
• any commercially available yeast extract may be used.
• Non-limiting examples are "BD Yeast Extract Technical” (Becton Dickinson and Company, Miami, FL, USA, Art. -No.: 288610), “BD Yeast Extract” (Becton Dickinson, Art. -No.: 212730), facedBD Bacto Yeast Extract” (Becton Dickinson, Art. -No.: 212730).
• "BD Yeast Extract Technical” (Becton Dickinson, Art.- No.: 288610) provides a high yield of collagenases and other proteases and fermentations with this yeast extract are well reproducible.
• Further suitable yeast extracts are e.g. Difco Yeast Extract, low-dusting (LD) Art. -No.: 210933", yeast extracts produced by Ohly, the Kerry HY-Yeast series, and the Yeast Extract Art. -No.: A1552 from AppliChem.
• the yield and concentration of collagenase is measured by the PZ-activity of the supernatant.
• the activity of neutral protease in the supernatant can be measured e.g. according to Lin, Y.-C. et al. (1969) J. Biol. Chem. 244, 789- 93; or Kunitz, M. 1947. Crystalline soybean trypsin inhibitor. II. General properties. J. Gen. Physiol. 30:291-310; or as described in Example 1.
• the activity of clostripain in the supernatant can be measured e.g. as described in Kzedy, F. J. et al. Biochemistry, 1965, 4, 2303-2308; or as described in Example 2.
• the inventive liquid culture medium comprises glycine or its pharmaceutically acceptable salts in a concentration of 1.0 to 30 g/L.
• concentration of glycine in the inventive liquid culture medium is in the range of 5 to 8 g/L.
• concentration of glycine in the liquid culture medium is in the range of 5 to 7 g/L.
• the liquid culture medium comprises 6.5 g/L glycine.
• the inventive liquid culture medium comprises arginine or its pharmaceutically acceptable salts in a concentration of 1.0 to 35 g/L.
• concentration of arginine in the inventive liquid culture medium is in the range of 5 to 8 g/L.
• concentration of arginine in the inventive liquid culture medium is in the range of 6 to 7 g/L.
• the liquid culture medium comprises 6.7 g/L arginine.
• the inventive liquid culture medium comprises glutamine or its pharmaceutically acceptable salts in a concentration of 0.20 to 0.80 g/L.
• Glutamine has a positive influence on bacterial growth and on the PZ-activity in the supernatant.
• concentration of glutamine in the inventive liquid culture medium is in the range of 0.25 to0.40 g/L.
• concentration of glutamine in the inventive liquid culture medium is in the range of 0.30 to 0.35 g/L.
• the liquid culture medium comprises 0.32 g/L glutamine.
• the inventive liquid culture medium comprises serine or its pharmaceutically acceptable salts in a concentration of 0.20 to 1.0 g/L.
• Serine has a positive influence on bacterial growth, but a negative influence on productivity.
• Productivity is the ratio of PZ-activity to bacterial growth.
• Bacterial growth can be determined by known methods, e.g. by measuring the turbidity. In some embodiments, bacterial growth is determined by measuring the optical density at 600 nm. The use of lower concentrations than 0.20 g/L of serine is in principle possible, but may result in lower bacterial growth. Serine concentrations higher than 1.0 g/L may reduce productivity.
• the concentration of serine in the inventive liquid culture medium is in the range of 0.37 to 0.50 g/L. In another embodiment, the concentration of serine in the inventive liquid culture medium is in the range of 0.37 to 0.47 g/L. In some embodiments, the liquid culture medium comprises 0.42 g/L serine.
• the inventive liquid culture medium comprises threonine or its pharmaceutically acceptable salts in a concentration of 0.3 to 3.0 g/L.
• the lower concentrations of threonine within this range provide higher PZ-activity in the supernatant than the higher concentrations within this range.
• the use of lower concentrations than 0.3 g/L of threonine will result in lower bacterial growth. Higher concentrations of threonine than 3.0 g/L may reduce the productivity.
• the concentration of threonine is not higher than 3.0 g/L.
• the concentration of threonine in the inventive liquid culture medium is in the range of 0.5 to 1.50 g/L.
• the concentration of threonine in the inventive liquid culture medium is in the range of 0.8 to 1.2 g/L.
• the liquid culture medium comprises 1 g/L threonine.
• the liquid culture medium according to the invention comprises 0.4 to 12 mmol/L magnesium ions (Mg 2+ ), e.g. 100 to 3000 mg/L of magnesium sulfate heptahydrate (MgSC x 7 H2O).
• the liquid culture medium according to the invention comprises 1.2 to 6.9 mmol/L magnesium ions (Mg 2+ ).
• the concentration of magnesium sulfate heptahydrate in the liquid culture medium is in the range of 300 to 1700 mg/L.
• the liquid culture medium according to the invention comprises 2.8 to 5.3 mmol/L magnesium ions (Mg 2+ ).
• the magnesium sulfate heptahydrate concentration is in the range of 700 to 1300 mg/L.
• the liquid culture medium comprises 995 mg/L magnesium sulfate heptahydrate.
• the liquid culture ccording to the invention comprises 0.34 to 3.4 mmol/L caf to 500 mg/L of calcium chloride dihydrate (CaCI 2 x 2 H 2 O).
• the liquid culture medium according to the invention comprises 0.68 to 2.0 mmol/L calcium ions (Ca 2+ ).
• the concentration of calcium chloride dihydrate in the liquid culture medium is in the range of 100 to 300 mg/L.
• the liquid culture medium according to the invention comprises 1.0 to 2.0 mmol/L calcium ions (Ca 2+ ).
• the calcium chloride dihydrate concentration is in the range of 150 to 300 mg/L.
• the liquid culture medium comprises 260 mg/L calcium chloride dihydrate.
• the anions for the magnesium and calcium ions may be selected from the group consisting of chloride, sulfate, phosphate and acetate may be used.
• magnesium sulfate may be used for magnesium ions and/or calcium chloride may be used for calcium ions.
• the liquid culture medium according to the invention comprises 0.00095 to 0.0152 mmol/L selenium, e.g.
• the liquid culture medium according to the invention comprises 0.0027 to 0.0061 mmol/L selenium.
• the concentration of sodium selenite pentahydrate in the liquid culture medium is in the range of 0.70 to 1.60 mg/L.
• the liquid culture medium according to the invention comprises 0.0038 to 0.0053 mmol/L selenium.
• the sodium selenite pentahydrate concentration is in the range of 1.00 to 1.40 mg/L.
• the liquid culture medium comprises 1.17 mg/L sodium selenite pentahydrate.
• sodium selenite Na 2 SeO3 or its pentahydrate is used to provide selenium to the inventive liquid culture medium, i.e., the medium comprises sodium selenite.
• Other selenium comprising compounds or compositions may be used. It is well within the knowledge of the expert in the art to choose suitable forms of selenium. It is well known that the selenium introduced into the media may be in a suitable form that has sufficient bioavailability. This makes the selenium comprised in the media available to the bacteria. Whether a compound or a composition provides sufficient selenium to the media can simply be determined by comparison of the yield of the desired proteases in a fermentation. This yield can be compared with the yield obtained through use of sodium selenite. If necessary, the amounts of compounds or composition other than sodium selenite or its pentahydrate used to provide selenium could be adjusted so that the same or similar results are obtained.
• the inventive liquid culture medium as described herein may comprise water, for example, purified water or water for injection.
• Purified water is water that has been mechanically filtered or processed to remove impurities and make it suitable for use, especially pharmaceutical use. Water for injection is even more purified and is further defined e.g. in the European or United States Pharmacopeia.
• the liquid culture medium comprises water in the amount of at least 50, 60, or 70 percent by weight based on the whole weight of the liquid culture medium.
• the liquid culture medium comprises water in the amount of at least 78 % by weight, or in the amount of at least 90% by weight, or in the amount of at least 95 % by weight.
• water is the remainder of the liquid culture medium.
• the liquid culture medium comprises purified water or water for injection according to the European or United States Pharmacopeia, e.g. in the above soecified amounts.
• the liquid culture medium is used for the fermentation of Hathewaya histolytica (Clostridium histolyticum) and the production of at least one protease selected from the group consisting of collagenase I, collagenase II, neutral protease and clostripain.
• the liquid culture medium comprises, essentially consists of, or consists of 10 to 30 g /L of yeast extract, 5 to 8 g/L of glycine, 5 to 8 g/L of arginine, 0.25 to 0.40 g/L of glutamine, 0.37 to 0.50 g/L of serine, 0.50 to 1.50 g/L of threonine, 1.2 to 6.9 mmol/L magnesium ions (Mg 2+ ), 0.68 to 2.0 mmol/L calcium ions (Ca 2+ ), 0.0027 to 0.0061 mmol/L selenium, and water.
• the liquid culture medium comprises, essentially consists of, or consists of 10 to 30 g /L of yeast extract, 5 to 8 g/L of glycine, 5 to 8 g/L of arginine, 0.25 to 0.40 g/L of glutamine, 0.37 to 0.50 g/L of serine, 0.50 to 1.50 g/L of threonine, 300 to 1700 mg/L of magnesium sulfate heptahydrate (MgSC x 7 H2O), 100 to 300 mg/L of calcium chloride dihydrate (CaCIz x 2 H2O), 0.70 to 1.60 mg/L of sodium selenite pentahydrate (Na2SeC>3 x 5 H2O), and water.
• yeast extract essentially consists of, or consists of 10 to 30 g /L of yeast extract, 5 to 8 g/L of glycine, 5 to 8 g/L of arginine, 0.25 to 0.40 g/L of glutamine, 0.37
• the liquid culture medium comprises, essentially consists of, or consists of 17 to 25 g /L of yeast extract, 5 to 7 g/L of glycine, 6 to 7 g/L of arginine, 0.30 to 0.35 g/L of glutamine, 0.37 to 0.47 g/L of serine, 0.8 to 1.2 g/L of threonine, 2.8 to 5.3 mmol/L magnesium ions (Mg 2+ ), 1.0 to 2.0 mmol/L calcium ions (Ca 2+ ), 0.0038 to 0.0053 mmol/L selenium, and water.
• the liquid culture medium comprises, essentially consists of, or consists of 17 to 25 g /L of yeast extract, 5 to 7 g/L of glycine, 6 to 7 g/L of arginine, 0.30 to 0.35 g/L of glutamine, 0.37 to 0.47 g/L of serine, 0.8 to 1.2 g/L of threonine, 700 to 1300 mg/L of magnesium sulfate heptahydrate, 150 to 300 mg/L of calcium chloride dihydrate, 1.00 to 1.40 mg/L of sodium selenite pentahydrate, and water.
• the liquid culture medium comprises, essentially consists of, or consists of 20 g/L yeast extract, 6.5 g/L glycine, 6.7 g/L arginine, 0.32 g/L glutamine, 0.42 g/L serine, 1 g/L threonine, 995 mg/L magnesium sulfate heptahydrate, 260 mg/L calcium chloride dihydrate, and 1.17 mg/L sodium selenite pentahydrate, and water (e.g. purified water).
• the inventive liquid culture medium provides a stable and reproducible culture medium with a high batch stability, which provides for a high process robustness for the cultivation of Hathewaya histolytica (Clostridium histolyticum) and the production of proteases, such as e.g. collagenase I, collagenase II, neutral protease and clostripain.
• the liquid culture medium is free from any components derived from animals or plants. Such animal or plant derived ingredients may comprise adventitious agents and/or their ingredients may vary between manufacturers and even between different batches of the same manufacturer.
• Using a liquid culture medium without animal or plant derived ingredients for the cultivation of Hathewaya histolytica (Clostridium histolyticum) and the production of proteases provides more safety for the end user of the proteases and improves reliability of the inventive liquid culture medium in fermentations.
• derived from animals or “animal derived” or “animal-derived” as used herein shall mean any substance of animal origin.
• the terms “derived from plants” or “plant derived” or “plant-derived” as used herein shall mean any substance of plant origin and any substance of non-plant origin processed using one or more plant-derived substances (e.g. plant-derived enzymes).
• the liquid culture medium or the liquid feed composition does not comprise glucose. In some embodiments, the liquid culture medium or the liquid feed composition does not comprise a sugar selected from the group consisting of glucose, galactose, lactose, mannose, raffinose, fucose, sucrose and arabinose. In some embodiments, the liquid culture medium or the liquid feed composition does not comprise any sugar.
• the liquid culture medium comprises an anti-foaming agent.
• Antifoaming agents suppress the development of foam in the culture medium and during fermentation. Any commercially available anti-foaming agent may be used.
• One suitable antifoaming agent is XIAMETERTM ACP-1500 (EU) Antifoam Compound from Dow.
• the anti- foaming agent may be added in an amount suitable to suppress foaming to an acceptable level. Fermentations in small scale may be conducted without the addition of anti-foaming agent. In large-scale fermentations anti-foaming agent may be added.
• the liquid culture medium has a pH value in the range of 6.5 to 8.2. In some embodiments, the pH value of the liquid culture medium is in the range of 7.2 to 8.1 or 7.5 to 8.0.
• the pH value may be regulated by any known method, e.g. by addition of a buffer with a suitable buffer capacity or by addition of acids or bases. In some embodiments, for example in pre-cultures and in fermentations with supernatants with a volume of about 0.5 liter or less, buffer may be added. For example, a 3-(N-morpholino)propanesulfonic acid buffer (MOPS buffer) may be used.
• MOPS buffer 3-(N-morpholino)propanesulfonic acid buffer
• the pH value may be continuously measured and acids or bases (or both in the time course of a cultivation process) are added to keep the pH value in the desired range.
• acids or bases or both in the time course of a cultivation process
• phosphoric acid and/or sodium hydroxide may be used.
• any of the two methods may be used.
• the liquid culture medium is sterilized.
• the composition is free from any self-replicating organism. Sterilization can be achieved by standard methods known to the skilled person, e.g. by heat treatment, such as, for example, autoclaving.
• the sterilized liquid culture medium comprises an inoculum of Hathewaya histolytica (Clostridium histolyticum).
• the liquid culture medium is free from any self-replicating organism but Hathewaya histolytica ' " m histolyticum).
• the present invention concerns a liquid feed composition, for example for use as feed in a fed-batch process for the cultivation of Hathewaya histolytica (Clostridium histolyticum), comprising, essentially consisting of, or consisting of at least 20 g/L of yeast extract, at least 3 g/L of threonine, at least 1.5 g/L of serine, and water.
• Hathewaya histolytica Clotridium histolyticum
• fed-batch culture shall mean an operational technique in biotechnological processes where one or more nutrients are fed (supplied) to a container, e.g. a bioreactor, during cultivation and in which the product(s) remain in the container until the end of the run.
• a container e.g. a bioreactor
• the product(s) remain in the container until the end of the run.
• the liquid feed composition and the liquid culture medium are different compositions.
• the liquid feed composition comprises at least 30, 40, or 50 g/L yeast extract.
• the liquid feed composition comprises at least 4, 5, 6, 7, 8, 9, or 10 g/L threonine.
• the liquid feed composition comprises at least 2.0, 2.5, 3.0, 4.5, 5.0 g/L serine.
• the liquid feed composition comprises, essentially consists of, or consists of 50 to 200 g/L yeast extract, 3 bis 60 g/L threonine and 1.5 to 40 g/L serine, and water.
• the liquid feed composition comprises, essentially consists of, or consists of 70 to 130 g/L yeast extract, 3 bis 10 g/L threonine and 1.5 to 10 g/L serine, and water.
• the liquid feed composition comprises, essentially consists of, or consists of 100 g/L yeast extract, 5 g/L threonine and 2.5 g/L serine, and water.
• the inventive liquid feed composition as described herein comprises water, for example purified water or water for injection, in the amount of at least 50, 60, or 65 percent by weight based on the whole weight of the liquid feed composition.
• the liquid feed composition comprises water, for example purified water or water for injection, in the amount of at least 70 percent by weight, or at least 80 percent by weight, or at least 95 % by weight. In some embodiments, water is the remainder of the liquid feed composition.
• the liquid feed composition has a pH value in the range of 6.5 to 8.2.
• the liquid feed composition is sterilized.
• the composition is free from any self-replicating organism. Sterilization can be achieved by standard methods known to the skilled person, e.g., by heat treatment, such as, for example, autoclaving.
• the present invention concerns a kit of parts comprising a liquid culture medium of the present invention and a liquid feed composition of the present invention.
• the - u: — 4.: on j s use f u
• a fourth aspect of the present invention is a supernatant of a Hathewaya histolytica (Clostridium histolyticum, e.g. Clostridium histolyticum Gil and/or ATCC® 21000TM) liquid culture, comprising a liquid culture medium of the present invention and/or a liquid feed composition of the present invention and one or more proteases.
• the one or more proteases may be selected from the group consisting of collagenase I, collagenase II, neutral protease and clostripain.
• the supernatant of a Hathewaya histolytica (Clostridium histolyticum) liquid culture prepared by use of a liquid culture medium and/or a liquid feed composition of the present invention has a collagenase activity of at least 400 PZ Units/L (Wuensch units), or at least 500 PZ Units/L, or at least 600 PZ Units/L, or at least 700 PZ Units/L, or at least 800 PZ Units/L, or at least 900 PZ Units/L, or at least 1000 PZ Units/L, or at least 11000 PZ Units/L, or at least 1200 PZ Units/L.
• PZ Units/L Wiensch units
• An embodiment of this aspect of the invention is a culture supernatant of Hathewaya histolytica (Clostridium histolyticum) comprising one or more proteases obtainable by the method of (a) providing a sterilized liquid culture medium according to the invention with an inoculum of Hathewaya histolytica (Clostridium histolyticum), (b) cultivating the bacteria, whereby the bacteria secrete the one or more proteases into the liquid phase; (c) separating solids, e.g. cellular and other particulate matter, from the liquid phase; and thereby obtaining the culture supernatant from Hathewaya histolytica (Clostridium histolyticum) comprising one or more proteases.
• the supernatant is obtained from a liquid fed- batch culture. Accordingly, in some embodiments, the supernatant is obtainable by the method of (a) providing a sterilized liquid culture medium according to the invention with an inoculum of Hathewaya histolytica (Clostridium histolyticum), (b) cultivating the bacteria in a fed-batch operation, (c) adding the liquid feed composition of the invention, (d) separating solids, e.g. cellular and other particulate matter, from the liquid phase; and thereby obtaining the culture supernatant from Clostridium histolyticum comprising one or more proteases.
• the one or more proteases may be selected from the group comprising collagenase I, collagenase II, neutral protease, and clostripain.
• the present invention concerns the use of a liquid culture medium of the present invention for cultivating Hathewaya histolytica (Clostridium histolyticum; e.g. Clostridium histolyticum Gil and/or ATCC® 21000TM) and obtaining at least one protease from the culture supernatant, e.g. a protease with collagenase activity, such as collagenase I and/or collagenase II, and/or neutral protease and/or clostripain.
• the present invention concerns the use of a liquid feed composition of the present invention for cultivating Hathewaya histolytica (Clostridium histolyticum; e.g.
• Clostridium histolyticum Gil and/or ATCC® 21000TM and obtaining at least one protease from the culture supernatant, e.g. a protease with collagenase activity, such as collagenase I and/or collagenase II, and/or neutral protease and/or clostripain.
• a protease with collagenase activity such as collagenase I and/or collagenase II, and/or neutral protease and/or clostripain.
• the present invention concerns a method compris 1 — 7 cultivating Hathewaya histolytica (Clostridium histolyticum; e.g. Clostridium histolyticum Gil and/or ATCC® 21000TM) in a liquid culture medium of the present invention and obtaining at least one protease from the culture supernatant.
• Hathewaya histolytica Clostridium histolyticum; e.g. Clostridium histolyticum Gil and/or ATCC® 21000TM
• collagenase I, collagenase II, neutral protease and/or clostripain are obtained with the aforementioned method.
• the pH value of the culture is controlled such that it is in the range of 6.5 to 8.2. In some embodiments, the pH value of the culture is controlled such that it is in the range of 7.2 to 8.1 or 7.5 to 8.
• the cultivation is preferably performed in a fed-batch operation by use of a sterilized liquid feed composition as described herein as feed.
• the pH value of the culture is controlled such that it is in the range of 7.2 to 7.8.
• the method according to the present invention comprises the steps of
• the culture or fermentation process may be monitored continuously or at one or more time points during cultivation, e.g. by determining turbidity and/or by determining protease activity in a sample of the culture supernatant (which may be taken at one or more time points during cultivation).
• the addition of the liquid feed composition is started when bacterial growth rate has reached the exponential phase.
• the addition of the liquid feed composition is started when bacterial growth rate has reached about 40%, 50%, 60%, or 70% of the turbidity maximum.
• the bacteria may be cultivated for 15 to 19 hours. Since the enzyme ratio changes over time, the cultivation time may be adapted according to the one or more target enzymes. Suitable methods to monitor the cultivation and bacterial growth phases are e.g.
• step (d) may comprise one or more purification steps such as e.g. filtration and/or column chromatography.
• WO 2020/164721 Al describes some example procedures for obtaining one or more proteases from a culture supernatant of Hathewaya histolytica (Clostridium histolyticum).
• 1 PZ unit activity according to Wuensch is the activity that catalyzes the hydrolysis of 1 pmol 4- phenyl- azobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine per minute at 25 °C, pH 7.1. All values of Wuensch units herein are given in PZ Units/liter (U/L), i.e. Wuensch units per liter of culture medium, if not otherwise stated.
• the turbidity was measured off-line in the Mettler Toledo UV-VIS spectrophotometer UV5Nano at 600 nm (OD 600). It is measured against an empty sample (medium without inoculum). If necessary, the solution must be diluted so that the measured value is in the measuring range of up to 0.6 OD 600.
• ATCC® 21000TM Clostridium histolyticum Gil and/or ATCC® 21000TM were used. ATCC® 21000TM is available at the ATCC (American Type Culture Collection). As yeast extract "BD Yeast Extract” (Becton Dickinson, Art. -No.: 288610) was used. In the below described experiments all cultures were inoculated with 2 volume percent of a pre-culture.
• the pre- culture was prepared by Inoculation of the liquid culture medium with the contents of 1 vial (the vials were defined to contain a minimum of 10 6 CFU; according to analysis the vials contained 35 x 10 6 CFU/Vial.) of lyophilized Hathewaya histolytica (Clostridium histolyticum Gil or ATCC® 21000TM) mixed with 1 mL of the liquid culture medium and anaerobic incubation at 37 °C in shake flasks. As bioreactors Multifors bioreactors (from Infors GmbH, Einsbach, Germany) were used. Further materials, reagents and devices are described in the Examples below.
• Example 1 Activity assay of neutral protease using a continuous photometric assay with the substrateN-(3-[furyl] acryloyl)-glycine leucine amide (FAGLA).
• One FAGLA unit (1U) is defined as the hydrolysis of lpmol of N-[3-(2- furyl)acryloyl]-glycine-L- leucine amide per minute.
• temperature controlled UV-VIS spectrophotometer e.g. Cary 50, 60 or 100, Agilent
• circular shaker e.g. Scientific Industries, Vortex Genie2
• UV cuvettes semi-micro e.g. Brand, Art. No. 759150
• semi-micro quartz cuvettes e.g. Hellma
• PD-10 Desalting Columns e.g. GE Healthcare, Art. No. 17-0851-01).
• Reagents purified water, Ph. Eur., 2-Morpholinoethanesulfonic acid monohydrate, abbrev. MES (e.g. Merck, Art. No. 7 1.060126), Calcium chloride dihydrate (e.g. Merck, Art. No. 1.02382), Dimethyl sulfoxide for spectroscopy, abbreviation DMSO (e.g. Merck, Art. No. 1.02950.), Triton X-100 (e.g. Sigma-Aldrich, Art. No. 23,472-9, 2-propanol (e.g. Merck, Art. No. 1.01040), 1 mol/l Sodium hydroxide solution (Merck, Art. No.
• Reagent solutions a) MES buffer pH 6.5: 10.67 g (0.05 mol) of 2-morpholinoethanesulfonic acid monohydrate and 0.735 g (0.005 mol) of calcium chloride dihydrate were dissolved in approximately 800 ml of purified water. Then 10 ml of 2- propanol (1% (v/v)) was added to the buffer mixture. The pH was adjusted to 6,5 at room temperature with 1 mol/l sodium hydroxide solution and filled up to 1000 ml with purified water. 0.1 ml of Triton X-100 were added to the buffer while stirring by using a 100 pl piston-stroke pipette to slowly pipette the highly viscous Triton X- 100 solution (0.01% (v/v).
• Reference substance Approximately 12-20 mg of the reference substance Neutral Protease NB (Nordmark Biochemicals, Uetersen, Germany) was weighed and adjusted to a concentration of 10 mg/ml with MES buffer pH 6.5 as described under a). This solution was stored on ice. Further dilutions (within the valid working range, as described below) were prepared from this solution with MES buffer pH 6.5. Approximately 2.5 to 3.6 mg/ml of reference substance was used. The absorbance changes must lie within the valid working range. The analyte solutions were prepared shortly before the determination with MES buffer pH 6.5 at RT and measured promptly. The activity of the reference substance was measured in duplicate for at least one dilution in the valid working range.
• Tris buffer pH 9.5: 0.606 (0.005 mol) tris(hydroxymethyl)aminomethane and 0.37 g (0.0025 mol) calcium chloride dihydrate was dissolved in approximately 400 ml purified water. The pH was adjusted to 9.5 with 1 mol/l hydrochloric acid at room temperature and filled up to 500 ml with purified water. The resulting buffer had a concentration of tris(hydroxymethyl)aminomethane of 0.01 mol/l and of Calcium chloride of 0.005 mol/l).
• At least one duplicate determination was carried out. Depending on the activity, the samples were diluted to be within the valid working range (see below). Samples from the concentrate of a cell-free culture supernatant were re-buffered with a PD- 10 column equilibrated with Tris buffer pH 9.5 before measurement.
• the test was performed at +30°C and a wavelength of 345 nm in a temperature-controlled UV-VIS spectrophotometer.
• the absorbance change at 345 nm was determined for a time interval of 5 min.
• the FAGLA substrate solution was tempered for approx. 30 min in a water bath at +30°C before the actual measurement.
• the semi-micro cuvettes incl. 0.8 ml substrate solution can be tempered directly in the UV-VIS photometer for at least 10 min before measurement.
• the photometer was calibrated against MES buffer pH 6.5 without substrate solution.
• 0.8 ml of tempered substrate solution was placed in the cuvettes.
• 0.2 ml sample solution was added, immediately premixed by drawing up with the pipette (approx, three piston strokes) and the entire reaction mixture was mixed with a disposable stirring spatula. Then the measurement was started immediately.
• the final volume in the test was 1 ml.
• a prerequisite for an evaluable activity measurement is an absorbance change per minute (A A 345nm / min) in the valid working range from - 0.01 to - 0.035 and the course of the negative slope must be linear. If the values are too high (A A 345nm / min), stronger dilutions must be used and if the values are too low, lower dilutions are to be used.
• the current reference substance was evaluated. First, a target value and acceptance criteria were defined. The activities of the reference substance and the samples from the mean values of the duplicate determination were calculated. To calculate the negative absorbance change (AA/ min) of the measurement, values from 0 to 5 min were evaluated. Acceptance criteria for evaluation: A A 345nm / min of all measured values must lie in the linear working range. The maximum deviation may be ⁇ 8% of the mean value. If there were major deviations, the measurements were repeated.
• a A Absorption change per minute at 3456nAi VF
• V 1 ml (total volume)
• v 0.2 ml (sample volume)
• Example 2 Determination of clostripain activity in culture supernatant from Clostridium histolyticum
• the enzyme clostripain catalyzes the hydrolysis of benzoyl-L-arginine ethyl ester (BAEE) in the presence of calcium and the reducing agent dithiothreitol (DTT), as measured photometrically at 255 nm (Kezdy FJ, Lorand L, Miller KD. Titration of active centers in thrombin solutions. Standardization of the enzyme. Biochemistry 1965;4:2302-8).
• the increase in absorbance per unit time is a direct measure of enzyme concentration when the reaction is of pseudo-zero order, that is, when it is constant.
• One unit is the amount of enzvmatic activity that catalyzes the hydrolysis of 1 pmol BAEE per minute at 25°C and pH 7.8 in the presence of 2.5 mM DTT.
• Spectrophotometer Cary 50 Varian
• Vortexer Vortexer
• Rotator Quartz cuvettes
• d 10 mm.
• Reagents Purified water, Ph. Eur. and USP, Dithiothreitol (DTT) (Serva, Art. No. 20710), Na- Benzoyl-L-arginine-ethyl ester HCI (BAEE) (Serva, Art. No. 14600), Sodium dihydrogen phosphate monohydrate (Merck, Art. No. 6346), 1 N Sodium hydroxide solution (Merck, Art. No. 09137), Calcium acetate hydrate (Kraft, Art. No. 15208)
• Reagent solutions 7.5 mM DTT solution: 57.8 mg DTT were dissolved to 50 ml purified water. The solution was prepared fresh daily. 1.5 mM BAEE solution: 51.8 mg BAEE were dissolved to 100 ml purified water. The solution was prepared fresh daily. 75 mM sodium phosphate buffer, pH 7.8: 10.35 g of sodium dihydrogen phosphate was dissolved in approximately 800 ml of purified water and the pH was adjusted accurately to pH 7,8 with sodium hydroxide solution at room temperature. The solution was then filled up to 1000 ml with purified water.
• Enzyme solvents Solution A (without activation): 1.0 mM Ca(OAc)z: 40 mg calcium acetate hydrate were dissolved to 250 ml purified water.
• Collagenase NB1 (Nordmark Biochemicals, Uetersen, Germany) as reference substance for identity was also analyzed.
• Sample Preparation The protein concentration in the cell-free culture supernatant was determined by measuring absorbance at 280 nm by spectrophotometry. Three samples of the cell-free culture supernatant where taken and each sample was diluted with Solution B to a concentration of 20 mg of the sample per ml of Solution B.
• Sample X and Sample Y are incubated between 40 minutes and 4 hours at +4° C and then 100 pl of Sample X are further diluted with 900 pl of Solution C and 200 pl of Sample Y are further diluted with 200pl of Solution A to obtain Samples X and Y as ready for measurement ("Sample Xm" and "Sample Ym").
• the activated Sample Xm and the non-activated Sample Ym were measured in parallel. In addition a zero measurement was performed. The following amounts of reagents (pretempered to 25°C) were pipetted into quartz cuvettes:
• Example 3 Preparation of a liquid culture medium
• Example 4 Preparation of a liquid culture medium for pre-cultures
• the flask was subsequently filled with purified water under stirring up to an overall volume of the liquid culture medium of one liter and the pH was adjusted to 7.9 by addition of a 10 M sodium hydroxide solution in water. Finally the liquid culture medium was sterilized in an autoclave.
• Example 5 Preparation of a liquid feed composition
• a 100 mL Erlenmeyer flask was filled with 100 mL medium according to Example 4. About 1 mL of the medium was removed from the flask and the contents of one vial of lyophilized Clostridium histolyticum Gil (about 35 x 10 6 CFU/Vial) or ATCC® 21000TM was mixed therewith. The medium was inoculated with this mixture. The culture thus prepared was grown without shaking for 24 hours ( ⁇ 1 hour). Growth took place either in an anaerobic pot with exclusion of oxygen (Anaerocult, manufacturer Merck) in an incubator or in an anaerobic workbench with anaerobic gas atmosphere (nitrogen 85%, carbon dioxide 10% and hydrogen 5%). The ambient temperature was kept at 37 ° C.
• a 100 mL Erlenmeyer flask was filled with 100 mL medium according to Example 4.
• the medium was inoculated with 5 mL of the first pre-culture (which corresponds to 5% of the second pre-culture medium volume).
• the culture was grown without shaking for 12 hours ( ⁇ 0.5 hours). Growth took place under anaerobic conditions as disclosed in Example 6 and at an ambient temperature of 37 °C.
• the second pre-culture was prepared in a 1000 mL flask filled with 1000 mL medium according to Example 4.
• the medium was inoculated with 50 mL of the first preculture (which corresponds to 5% of the second pre-culture medium volume).
• the culture was grown without shaking for 12 hours ( ⁇ 0.5 hours). Growth took place under anaerobic conditions as disclosed in Example 6 and at an ambient temperature of 37 °C.
• Example 8 Main culture in a bioreactor
• the start volume of the medium (0,6 L) was inoculated with 12 mL of the second pre-culture (which corresponds to 2% of the medium volume).
• the start volume of the medium 27 L was inoculated with 540 mL of the second pre-culture (which corresponds to 2% of the medium volume).
• the liquid culture medium was introduced into the bioreactor and sterilized (the Multifors in an autoclave; the Techfors in the reactor itself). Subsequently the bioreactor was flushed with nitrogen and stirred for at least 3 h before inoculation. 2 volume percent of inoculum of were used.
• the corresponding amount of the second pre-culture of Example 7 was removed from the second pre-culture using a sterile syringe and the main culture was inoculated via the inoculation port of the bioreactor.
• the pre-culture was pumped into the bioreactor via a hose (the container with the pre-culture was placed on a scale in order to determine the correct amount of inoculum).
• the culture time was between 15 and 19 hours, mainly 17 hours and addition of the liquid feed composition according to Example 5 was started after 9 hours of fermentation.
• Clostridium histolyticum ATCC® 21000TM Clostridium histolyticum ATCC® 21000TM:

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