Protein purification

Abstract

The present invention relates to a method for purifying proteins produced by homologous or heterologous expression in a ciliate host from non-chromatographically purified harvested cell culture fluid (HCCF), comprising the steps of incubating the harvested cell culture fluid with a kosmotropic agent, efflorescence of the protein by the formation of crystals, and optionally harvesting the efflorescent protein (Figure 1).

Description

[Technical field]

[0001] This application relates to the purification of proteins. [Background technology]

[0002] Proteins produced in biological systems require purification before they can be administered to patients. A variety of approaches exist for this purpose, each with different advantages and disadvantages. In many cases, increasing purity comes at the expense of higher costs and lower throughput, and vice versa.

[0003] Furthermore, different production methods and administration routes have different requirements regarding purity: proteins for external use need to be less purified than proteins for intravenous administration.

[0004] Generally, regardless of which host (bacteria, yeast, mammalian cells) is chosen for homologous or heterologous expression of a protein, the cell culture supernatant contains unwanted components such as endotoxins, antibiotics, residues, metabolic products, etc. in addition to the components of the cell culture medium before harvest and the produced protein.

[0005] One of the standard methods for protein purification is chromatography. However, mass transfer limitations necessitate the design of large chromatography columns, which in turn require large amounts of resin. Resins have a limited life span and are therefore consumables. Furthermore, resins are expensive, especially affinity chromatography resins and immobilized metal chromatography resins.

[0006] Generally, protein purification using chromatographic purification steps such as chromatography is costly due to the need for large amounts of sophisticated equipment, consumables, and expensive buffers. Furthermore, chromatographic purification steps often have limited throughput and require parallel processing to obtain sufficient yields, resulting in increased costs.

[0007] Nevertheless, for proteins intended for human consumption or used therapeutically, chromatography remains the method of choice to meet the high demands on product purity.

[0008] However, there are therapeutic applications that require large amounts of protein, especially for some enzyme replacement therapies such as pancreatic enzyme replacement therapy (PERT), but also for other oral enzyme replacement therapies, where chromatographic purification would drive up costs beyond acceptable limits.

[0009] On the other hand, there are protein expression systems, such as ciliate expression systems, that have relatively low production costs per gram of enzyme produced. Chromatographic purification of the produced enzymes will impact the economic benefits that these systems can offer.

[0010] Therefore, one object of the present invention is to provide a purification method which overcomes the above mentioned drawbacks.

[0011] These and other objects are achieved according to the invention by the methods and means set forth in the independent claims. The dependent claims relate to specific embodiments. Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention provides modified lipase enzymes. [Means for solving the problem]

[0013] The general advantages of the invention and its features are explained in detail below. [Brief description of the drawings]

[0014] [Figure 1]Figure 1 shows an overview of N-glycosylation patterns in various taxa. In general, the term "N-glycosylation" refers to the glycosylation of the amino acid residue asparagine (N), where an oligosaccharide chain is attached by oligosaccharyltransferase to an asparagine residue present in the tripeptide sequence Asn-X-Ser or Asn-X-Thr. It is clear that ciliates are characterized by a very simple and small N-glycosylation pattern compared to other taxa such as mammals, yeasts, and transgenic plants, whereas prokaryotes do not have any N-glycosylation at all. [Diagram 2] FIG. 2 is a micrograph (40x) of lipase crystals formed after incubation of non-chromatographically purified harvested cell culture fluid (HCCF) with 1000 mM ammonium sulfate for 130 minutes. [Diagram 3] FIG. 3 is a photomicrograph (40x) of lipase crystals formed after incubation of non-chromatographically purified harvested cell culture fluid (HCCF) with 850 mM ammonium sulfate for 130 minutes. [Figure 4] FIG. 4 is a photomicrograph (40x) of lipase crystals formed after incubation of non-chromatographically purified harvested cell culture fluid (HCCF) with 700 mM ammonium sulfate for 130 minutes. [Diagram 5] FIG. 5 is a photomicrograph (40x) of harvested cell culture fluid (HCCF) that was not chromatographically purified after incubation with 3000 mM sodium chloride for 130 minutes. [Figure 6] FIG. 6 is a micrograph (10×) of harvested cell culture fluid (HCCF) that was not chromatographically purified after incubation with 300 mM ammonium sulfate for 3 hours. [Figure 7] FIG. 7 is a micrograph (10x) of harvested cell culture fluid (HCCF) that was not chromatographically purified after incubation with 300 mM ammonium sulfate for 20 hours. [Figure 8]FIG. 8 shows the results of a rhodamine-triglyceride-agarose assay to measure the lipolytic activity of the supernatant and crystals after exposing cell culture medium containing lipase to various crystallization conditions. [Figure 9] FIG. 9 shows the results of a BCA assay to measure the total protein concentration in the supernatant and crystals after exposing harvested cell culture fluid to different crystallization conditions. [Figure 10] Figure 10 shows the protein crystallization scheme. The steps taken during the crystallization process are diagrammed. Lipase enzyme was produced in a ciliate expression system as discussed elsewhere herein. Protein crystallization was initiated with 0.3 M ammonium sulfate. An increase in ammonium sulfate to 0.7 M was performed after 20 hours of incubation at room temperature. The protein crystals were then washed twice and the supernatant was discarded. The remaining wet lipase crystals were stored at -20°C. [Figure 11] Crystals of α-amylase from T. thermophila obtained after 93 hours using 6% (w / v) PEG4000 as a crystallization agent. Magnification: Fig. 11: 100x, Fig. 12: 400x. Bars indicate approximately 100 μm. [Figure 12] Crystals of α-amylase from T. thermophila obtained after 93 hours using 6% (w / v) PEG4000 as a crystallization agent. Magnification: Fig. 11: 100x, Fig. 12: 400x. Bars indicate approximately 100 μm. [Figure 13] FIG. 13 shows the results of a rhodamine-triglyceride assay measuring the lipolytic activity of the supernatant and crystallized product after exposing a cell culture solution containing lipase to different crystallization conditions using NaH2PO4 as a crystallizing agent. [Figure 14] Figure 14 shows the crystals of lipase 0120 derived from T. thermophila obtained after 115 hours using 1.25M NaH2PO4 as a crystallizing agent. The bar indicates approximately 100 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Before describing the invention in detail, it is to be understood that the invention is not limited to the particular component parts of the devices described, or to the process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is to be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural references unless the context clearly dictates otherwise. Furthermore, when a parameter range is given that is bounded by numerical values, it is to be understood that the range is deemed to be inclusive of these limits.

[0016] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate embodiments unrelated to each other. Features discussed in one embodiment are meant to be disclosed in relation to other embodiments shown herein. In one case, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, a person skilled in the art will understand that this does not necessarily mean that the feature is not meant to be disclosed in the other embodiment. A person skilled in the art will understand that it is the intent of the present application to disclose the features in other embodiments as well, but this has not been done merely for the sake of clarity and to keep the present specification to a manageable volume.

[0017] Furthermore, the contents of the prior art documents referred to in this specification are incorporated by reference, in particular those prior art documents that disclose standard or conventional methods, in which case the incorporation by reference has the primary purpose of providing a fully enabling disclosure and avoiding lengthy repetition.

[0018] According to a first aspect of the present invention, there is provided a method for purifying a protein, the protein being produced by homologous or heterologous expression in a ciliate host and obtained from harvested cell culture fluid (HCCF) that has not been chromatographically purified.

[0019] The method includes the steps of: a) incubating the harvested cell culture with a cosmotropic agent; b) efflorescing the protein by the formation of crystals, and c) Optionally, harvesting the effloresced protein.

[0020] This method does not include cross-linking the protein before or after efflorescence.

[0021] The inventors have shown that such a method, which does not involve a chromatographic purification step or a protein cross-linking step, gives superior results in terms of purity and yield compared to, for example, chromatographic purification, and furthermore has substantial cost advantages.

[0022] The term "ciliate host" as used herein refers to the scientific phylum Ciliophora, which are unicellular eukaryotic organisms ("protozoa" or "protists") characterized, among other things, by a relatively large size (some species can reach 2 mm in length), a cell surface covered with cilia, and two types of nuclei: a small diploid micronucleus and a large diploid macronucleus (used for protein expression). The latter is generated from the micronucleus by genome amplification and heavy editing. Ciliates, particularly Tetrahymena thermophila, have been used as expression hosts for heterologous or homologous protein expression production, as described, for example, in Weide et al. 2006 and in EP1350838 (US6962800), the contents of which are incorporated herein by reference.

[0023] As used herein, the term "pre-harvest cell culture fluid (CCF)" refers to a liquid medium containing ciliate host cells, and in particular to a medium containing an enzyme produced by homologous or heterologous expression.

[0024] As used herein, the term "Harvested Cell Culture Fluid (HCCF)" refers to liquid medium obtained from Harvested Cell Culture Fluid (CCF) by centrifugation, filtration, and / or similar separation methods.

[0025] As used herein, the term "non-chromatographically purified" is intended to mean that the harvested cell culture fluid has not been subjected to chromatography-based purification, and does not exclude simple size-exclusion purification steps such as filtration, or density-based purification steps such as centrifugation.

[0026] As used herein, the term "efflorescing" refers to the process by which proteins precipitate through the formation of crystals. The terms "efflorescing" and "precipitation through the formation of crystals" are sometimes used interchangeably. Originally, the term "efflorescence" referred to the crystalline precipitates that form when water is present on the surfaces of brick, concrete, stone, plaster, and other building materials. However, as used herein, the meaning refers to the process described above.

[0027] As used herein, an "efflorescent protein" is a protein obtained by a process such as precipitation and crystal formation.

[0028] That is, an efflorescent protein is a protein that exists in the form of crystals obtained according to the above-mentioned method. In this sense, the terms "efflorescent protein" and "crystallized protein" are used interchangeably herein.

[0029] As used herein, the term "kosmotropic agent" refers to an agent that contributes to the stability and structure of water-water interactions in a solution. Kosmotropes favorably interact with water molecules and stabilize intramolecular interactions of macromolecules such as proteins, whereas chaotropes, on the other hand, disrupt the structure of water, increase the solubility of non-polar solvent particles, and destabilize aggregates of solutes.

[0030] Ionic kosmotropes tend to be small or have a high charge density. Ionic kosmotropes include CO3 2- , SO4 2- , HPO4 2- , Mg 2+ , Li + , Zn 2+ and Al 3+ See the Hofmeister series or calculate the hydrogen bond free energy (ΔG HB ) can be used to establish a scale for quantifying the degree of hydrogen bonding of ions in water. For example, kosmotrope CO3 2- , and O.H. - ΔG HB is 0.1-0.4 J / mol, whereas the chaotrope SCN - ΔG HB is -1.1 to -0.9.

[0031] Other suitable kosmotropes include non-ionic kosmotropes, which have no net charge but are highly soluble and highly hydrated. Such kosmotropes include carbohydrates such as trehalose and glucose, polyhydric alcohols, proline, and tert-butanol.

[0032] The inventors surprisingly showed that the enzyme produced in a ciliate host by homologous or heterologous expression does not require prior chromatographic purification but can be simply and easily purified by efflorescence and crystal formation after treatment with kosmotrope.

[0033] This is quite surprising since in the prior art literature, before crystallizing proteins, especially lipases, the proteins always undergo purification steps such as chromatographic purification (see, for example, Hekmat (2015) who discusses large-scale crystallization of proteins for purification and formulation).

[0034] However, although the authors understand that crystallization can replace one or more chromatographic steps in the protein purification process, all of the proteins studied and ultimately crystallized were purified, i.e., they had undergone chromatographic purification steps before being used.

[0035] Moreover, the inventors have surprisingly shown that the enzymes so produced do not need to be cross-linked, as suggested for example in some prior art documents.

[0036] For example, US6359118B2 discloses the preparation and use of glycoprotein crystals cross-linked with carbohydrate chains. Cross-linking is preferably achieved by using a diamine cross-linking agent such as hexamethylenediamine, diaminooctane, or ethylenediamine. The glycoprotein is preferably a lipase, more preferably Candida rugosa lipase. Such cross-linked glycoprotein crystals are -Maintaining the structural and functional integrity of the glycoprotein backbone - Better stability in harsh environmental conditions It is said that, On the other hand, cross-linking methods are said to offer significantly higher achievable yields even in concentrated solutions where the glycoprotein concentration is close to the theoretical packing limit achievable for a molecule of a given size.

[0037] Without being bound by theory, one of the reasons why the method according to the invention gives good results without prior cross-linking of the enzymes may be the fact that by using suitable promoters, proteins including homologous and heterologous enzymes, especially lipases, can be expressed in large amounts in ciliates. In this way, high yields can be obtained and a cross-linking step (to increase the yield after efflorescence) can be made unnecessary.

[0038] Furthermore, without being bound by theory, one of the reasons why the method according to the invention gives excellent results without prior chromatographic purification of the enzyme may be due to the fact that ciliates are capable of secreting large amounts of proteins into the cell-free supernatant, allowing crystallization without prior chromatography and / or cross-linking.

[0039] Furthermore, without being bound by theory, one of the reasons why the method according to the invention gives excellent results without prior chromatographic purification of the enzyme may be due to the fact that ciliates produce proteins characterized by a very simple and uniform glycosylation pattern, see FIG. 1 for an illustration.

[0040] Thus, in one embodiment, the protein is a glycosylated protein.

[0041] In general, many proteins, including enzymes, require glycosylation to maintain activity and stability. Tetrahymena lipase 120 (discussed elsewhere herein) for example has two potential n-glycosylation sites (N61, motif NV; N67, motif NST).

[0042] In this regard, for example, Chang et al. (2007) argue that structural genomics poses special problems because glycosylation is often required for correct folding, whereas chemical and conformational heterogeneity generally inhibits crystallization.

[0043] Interestingly, Chang et al. proposed using glycosylation processing inhibitors during protein expression to avoid glycosylation and promote crystallization. Such processing can have significant effects on the chemical-physical and physiological properties of the processed enzyme, resulting in undesirable loss of activity and / or stability.

[0044] Of course, enzymes can also be produced in prokaryotic expression systems. These systems do not glycosylate the proteins at all, and proteins produced in this way are likely to pose fewer problems when crystallizing. However, the complete lack of glycosylation can have a significant effect on the chemical, physical and physiological properties of the enzymes produced in this way, resulting in undesirable losses in activity and / or stability.

[0045] In this light, and without being bound by theory, the fact that ciliates produce proteins characterized by a very simple and uniform glycosylation pattern (see Figure 1) may certainly facilitate crystallization and make unnecessary previous chromatographic purification steps or the use of glycosylation processing inhibitors. According to one embodiment of the present invention, the cosmotropic agent is Ammonium sulfate, Sodium dihydrogen phosphate, and / or Polyethylene glycol It has at least one of the following.

[0046] According to one embodiment of the present invention, the polyethylene glycol used has a size range between >50 and <10000 g / mol.

[0047] In further embodiments, the polyethylene glycol used is 50 g / mol or more; 100 g / mol or more; 200 g / mol or more; 300 g / mol or more; 400 g / mol or more; 500 g / mol or more; 600 g / mol or more; 700 g / mol or more; 800 g / mol or more; 900 g / mol or more; 1000 g / mol or more; 1200 g / mol or more; 1400 g / mol or more; 1 600g / mol or more;1800g / mol or more;2000g / mol or more;2200g / mol or more;2400g / mol or more;2600g / mol or more;2800g / mol or more;3000g / mol or more;3200g / mol or more;3400g / mol or more;3600g / mol or more;3800g / mol or more;4000g / mol or more;4200g / mol or more;4400g / mol or more Upper;4600g / mol or more;4800g / mol or more;5000g / mol or more;5200g / mol or more;5400g / mol or more;5600g / mol or more;5800g / mol or more;60 00g / mol or more;6200g / mol or more;6400g / mol or more;6600g / mol or more;6800g / mol or more;7000g / mol or more;7200g / mol or more;7400g / m ol or more;7600g / mol or more;7800g / mol or more;8000g / mol or more;8200g / mol or more;8400g / mol or more;8600g / mol or more;8800g / mol or more;9000g / mol or more;9200g / mol or more;9400g / mol or more;9600g / mol or more;9800g / mol or more and / or 10000g / mol or more.

[0048] In further embodiments, the polyethylene glycol used is 10000 g / mol or less; 9800 g / mol or less; 9600 g / mol or less; 9400 g / mol or less; 9200 g / mol or less; 9000 g / mol or less; 8800 g / mol or less; 8600 g / mol or less; 8400 g / mol or less; 8200 g / mol or less; 8000 g / mol or less; 7800 g / mol or less; 7 600g / mol or less; 7400g / mol or less; 7200g / mol or less; 7000g / mol or less; 6800g / mol or less; 6600g / mol or less; 6400g / mol or less; 6200g / mol or less;6000g / mol or less;5800g / mol or less;5600g / mol or less;5400g / mol or less;5200g / mol or less;5000g / mol or less;4800g / mol or less Bottom;4600g / mol or less;4400g / mol or less;4200g / mol or less;4000g / mol or less;3800g / mol or less;3600g / mol or less;3400g / mol or less;32 00g / mol or less;3000g / mol or less;2800g / mol or less;2600g / mol or less;2400g / mol or less;2200g / mol or less;2000g / mol or less;1800g / m ol or less; 1600g / mol or less; 1400g / mol or less; 1200g / mol or less; 1000g / mol or less; 900g / mol or less; 800g / mol or less; 700g / mol or less; 600g / mol or less; 500g / mol or less; 400g / mol or less; 300g / mol or less; 200g / mol or less; 100g / mol or less and / or 50g / mol or less.

[0049] According to one embodiment of the invention, the harvested cell culture fluid contains between ≧0.05 and ≦200 g / l protein.

[0050] In some embodiments, the collected cell culture solution contains proteins at 0.05 g / l or more; 0.1 g / l or more; 0.15 g / l or more; 0.2 g / l or more; 0.3 g / l or more; 0.4 g / l or more; 0.5 g / l or more; 0.6 g / l or more; 0.7 g / l or more; 0.8 g / l or more; 0.9 g / l or more; 1 g / l or more; 2 g / l or more; 3 g / l or more; 4 g / l or more; 5 g / l or more; 6 g / l or more; 7 g / l or more; 8 g / l or more; 9 g / l or more; 10 g / l or more; 15 g / l or more; 20 g / l or more; 25 g / l or more; 30 g / l or more; 35 g / l or more; 40 g / l or more; 45 g / l or more; 50 g / l or more; 55 g / l or more; 60 g / l or more; 65 g / l or more; 70 g / l or more; 75 g / l or more; 80 g / l or more; 85 g / l or more; 90 g / l or more; 95 g / l or more; 100 g / l or more; 110 g / l or more; 120 g / l or more; 130 g / l or more; 140 g / l or more; 150 g / l or more; 160 g / l or more; 170 g / l or more; 180 g / l or more; 190 g / l or more; or 200 g / l or more.

[0051] In some embodiments, the collected cell culture solution contains proteins at 200 g / l or less; 190 g / l or less; 180 g / l or less; 170 g / l or less; 160 g / l or less; 150 g / l or less; 140 g / l or less; 130 g / l or less; 120 g / l or less; 110 g / l or less; 100 g / l or less; 95 g / l; 90 g / l or less; 85 g / l or less; 80 g / l or less; 75 g / l or less; 70 g / l or less; 65 g / l or less; 60 g / l or less; 55 g / l or less; 50 g / l or less; 45 g / l or less; 40 g / l or less; 35 g / l or less; 30 g / l or less; 25 g / l or less; 20 g / l or less; 15 g / l or less; 10 g / l or less; 9 g / l or less; 8 g / l or less; 7 g / l or less; 6 g / l or less; 5 g / l or less; 4 g / l or less; 3 g / l; 2 g / l or less; 1 g / l or less; 0.9 g / l or less; 0.8 g / l or less; 0.7 g / l or less; 0.6 g / l or less; 0.5 g / l or less; 0.4 g / l or less; 0.3 g / l or less; 0.2 g / l or less; 0.15 g / l or less; 0.1 g / l or less; or 0.05 g / l or less.

[0052] According to some embodiments of the present invention, to effloresce a protein, The pH of the medium is set between 5 and 8, and / or The temperature is set between 10℃ and 40℃.

[0053] According to one embodiment of the present invention, for incubation of the harvested cell culture fluid, the kosmotropic salt concentration is set in the range of 50 to 2500 mM.

[0054] In some embodiments, the cosmotropic salt concentration is set to be 50 mM or more; 100 mM or more; 150 mM or more; 200 mM or more; 250 mM or more; 300 mM or more; 350 mM or more; 400 mM or more; 450 mM or more; 500 mM or more; 550 mM or more; 600 mM or more; 650 mM or more; and 700 mM or more.

[0055] In some embodiments, the cosmotropic salt concentration is set to be 2500 mM or less; 2300 mM or less; 2100 mM or less; 2000 mM or less; 1900 mM or less; 1800 mM or less; 1700 mM or less; 1600 mM or less; 1500 mM or less; 1400 mM or less; 1300 mM or less; 1200 mM or less; 1100 mM or less; and 1000 mM or less.

[0056] In some embodiments, the cosmotropic salt concentration is set between 50 mM or more and 2500 mM or less; between 100 mM or more and 2300 mM or less; between 150 mM or more and 2100 mM or less; between 200 mM or more and 2000 mM or less; between 250 mM or more and 1900 mM or less; between 300 mM or more and 1800 mM or less; between 350 mM or more and 1700 mM or less; between 400 mM or more and 1600 mM or less; between 450 mM or more and 1500 mM or less; between 500 mM or more and 1400 mM or less; between 550 mM or more and 1300 mM or less; between 600 mM or more and 1200 mM or less; between 650 mM or more and 1100 mM or less; between 700 mM or more and 1000 mM or less.

[0057] Preferably, these concentration ranges apply to ammonium sulfate and / or sodium dihydrogen phosphate.

[0058] In one embodiment, for the crystallization of amylase, a kosmotropic salt concentration of 100 mM or more is used. In one embodiment, for the crystallization of lipase, a kosmotropic salt concentration of 300 mM or more is used. Preferably, these concentration ranges apply to ammonium sulfate and / or sodium dihydrogen phosphate.

[0059] In some embodiments, the kosmotropic salt concentration is set in the range of 700 mM to 1 M. Preferably, these concentration ranges apply to ammonium sulfate and / or sodium dihydrogen phosphate.

[0060] According to one embodiment of the present invention, for incubation of the harvested cell culture medium, the polyalcohol concentration is set in the range of ≧2 to ≦25 w / v %.

[0061] In some embodiments, the polyalcohol concentration is 2 w / v% or more; 2.5% or more; 3% or more; 3.5% or more; 4% or more; 4.5% or more; 5% or more; 5.5% or more; 6% or more; 6.5% or more; 7% or more; 7.5% or more; 8% or more; 8.5% or more; 9% or more; 9.5% or more; 10% or more; 10.5% or more; 11% or more; 11.5% or more; 12% or more; 12.5% ​​or more; 13% or more; 13.5% or more; 14% or more; 14.5% or more; 15% or more; 15.5% or more; 16% or more; 16.5% or more; 17% or more; 17.5% or more; 18% or more; 18.5% or more; 19% or more; 19.5% or more and / or 20 w / v% or more.

[0062] In some embodiments, the polyalcohol concentration is set to be 25 w / v% or less; 24.5% or less; 24% or less; 23.5% or less; 23% or less; 22.5% or less; 22% or less; 21.5% or less; 21% or less; 20.5% or less; 20% or less; 19.5% or less; 19% or less; 18.5% or less; 18% or less; 17.5% or less; 17% or less; 16.5% or less; 16% or less; 15.5% or less; 15% or less; 14.5% or less; 14% or less; 13.5% or less; 13% or less; 12.5% ​​or less; 12% or less; 11.5% or less; 11% or less; 10.5% or less and / or 10 w / v% or less.

[0063] Preferably, these concentration ranges apply to polyethylene glycol.

[0064] In some embodiments, the polyhydric alcohol is PEG 4000 (4000 g / mol), preferably at a concentration of 6 w / v %.

[0065] In some embodiments, the polyhydric alcohol is PEG 6000 (6000 g / mol), preferably at a concentration of 6 w / v %.

[0066] The above concentration ranges are by no means binding, since the inventors have noticed that the higher the protein concentration in the medium, the lower the concentration of cosmotropic agent required.

[0067] According to one embodiment of the present invention, the incubation of the harvested cell culture medium with the cosmotropic agent is carried out for a period of 10 minutes to 36 hours.

[0068] According to one embodiment of the invention, the pre-harvested cell culture fluid is subjected to one or more filtration and / or centrifugation steps prior to step a) to obtain the harvested cell culture fluid.

[0069] Such filtration includes at least one of the following: Microfiltration to remove cells from CCF, e.g. hollow fiber cross-flow filtration systems (0.4-0.8 μm size exclusion) · Ultrafiltration, optionally followed by diafiltration, to remove smaller peptides and impurities.

[0070] Such centrifugation includes the use of a centrifuge or centrifugal separator, such as those provided by Westfalia Separator, In one embodiment, at least one filter is applied having a molecular weight cut-off (MWCO) compatible with the respective protein to be purified.

[0071] In one embodiment, at least one filter is applied having a molecular weight cut off (MWCO) of 50 kd or less.

[0072] In a further embodiment, the at least one filter applied is 49 kDa or less; 48 kDa or less; 47 kDa or less; 46 kDa or less; 45 kDa or less; 44 kDa or less; 43 kDa or less; 42 kDa or less; 41 kDa or less; 40 kDa or less; 39 kDa or less; 38 kDa or less; 37 kDa or less; 36 kDa or less; 35 kDa or less; 34 kDa or less; 33 kDa or less; 32 kDa or less; 31 kDa or less; 30 kDa or less; 29 kDa or less; 28 kDa or less; 27 kDa or less; 26 kDa or less; 25 kDa or less; 24 kDa or less; 23 kDa or less; 22 kDa or less; 21 kDa or less; 20 kDa or less; 19 kDa or less; 18 kDa or less; 17 kDa or less; 16 kDa or less; 15 kDa or less; 14 kDa or less; 13 kDa or less; 12 kDa or less; 11 kDa or less; 10 kDa or less; 9 kDa or less, preferably 8 kDa or less.

[0073] In this context, it is important to understand that the molecular weight cutoff is adapted to the molecular weight of the protein without the signal peptide. Furthermore, it has been shown in the past that enzymes can form enzymatically active fragments that retain their enzymatic activity, for example the protease CCP5 (which forms an active fragment of 12 kDa).

[0074] According to one embodiment of the invention, the ciliate host is a Tetrahymena sp.

[0075] In one embodiment, the ciliate host is Tetrahymena thermophila, which in this context has been used as an expression host for heterologous or homologous protein expression production, for example as described in Weide et al. 2006 and EP1350838 (US6962800), the contents of which are incorporated herein by reference.

[0076] In general, methods for transformation of ciliates, including Tetrahymena, that can be used in the context of the present invention consist inter alia of microinjection, electroporation and particle bombardment and are described, for example, in Tondravi & Yao (1986), Gaertig & Gorovsky (1992) and Cassidy-Hanley et al (1997).

[0077] Methods for transformation and heterologous protein expression have been described for a few protists (WO 00 / 58483 and WO 00 / 46381). The generation of division-stable transformants of the ciliate Tetrahymena thermophila can be achieved following transfection of either the somatic macronucleus or the generative micronucleus by microinjection, electroporation, or biolistic bombardment.

[0078] Selection of transformants can be performed using different selection markers such as neomycin resistance (Weide et al. 2006, BMC) and stable thymidine auxotrophic Tetrahymena cells can be obtained by integrating heterologous genes by homologous DNA recombination (Weide et al. 2006, BMC). Also, blasticidin S (Weide et al. 2007, BMC) or paclitaxel (WO 00 / 46381) resistance has been considered.

[0079] Suitable promoters for protein expression in ciliates are disclosed, for example, in commonly assigned US2008261290A1, the contents of which are incorporated herein by reference, which discloses heat-inducible promoters and metallothionein promoters that can also be used for the purposes of the present invention.

[0080] According to one embodiment of the invention, homologous or heterologous expression is Metallothionein gene (MTT1) inducible promoter, and / or Heat-inducible promoter is under control.

[0081] Said MTT1 inducible promoter is disclosed, for example, in WO2003006480A1 (US20030027192A1), the contents of which are incorporated herein for enablement purposes.

[0082] Said heat-inducible promoters are disclosed, for example, in EP1902138B1 (US7723506B2), the contents of which are incorporated herein for enablement purposes.

[0083] According to one embodiment of the invention, the protein is an enzyme.

[0084] According to a further embodiment, the enzyme is Lipase Proteases ·amylase ·Glutenase Glutamine-specific cysteine ​​protease Prolyl endopeptidase Saccharase, and / or Lactase At least one selected from the group consisting of:

[0085] Lipase, amylase or protease can be used, for example, in pancreatic enzyme replacement therapy (PERT) for the treatment of exocrine pancreatic insufficiency, which has the following causes: JPEG2024513114000002.jpg100170

[0086] In such treatments, patients often require large amounts of the enzyme, which are administered orally. The standard treatment is porcine pancreatic homogenate (i.e. a very complex product with variable lipase content) granulated and delivered as capsules, with patients having to take an average of 2.5 g (8 capsules of 300 mg each) or more per day (reports suggest that some patients must take up to 50 capsules). This amounts to approximately 1 kg of product per year, highlighting the fact that purification by chromatography would unduly increase the cost of the product. According to one embodiment of the invention, the enzyme is: a) Tetrahymena lipase as set forth in any one of SEQ ID NOs: 1 to 3 or 8 to 10; b) Tetrahymena amylase as set forth in SEQ ID NO: 4 or 11; c) a Tetrahymena protease as set forth in any one of SEQ ID NOs: 5-7, 12-14 or 15-17, or d) (i) a variant of any one of the above enzymes having at least 90% amino acid sequence identity with any one of the above enzymes, wherein the variant retains enzymatic function; and / or (ii) A mutant of any one of the above enzymes in which one to three amino acid residues have been removed or added at the N-terminus and / or C-terminus.

[0087] SEQ ID NO:1 is the sequence of Tetrahymena lipase 120 published at UniProt Identifier: Q237S4 (TTHERM_00320120). It has already been discussed, inter alia, in WO2016116600A1 (US10590402B2) and is currently in preclinical evaluation for pancreatic enzyme replacement therapy (PERT).

[0088] Further enzymes are detailed below: SEQ ID NOs: 1-7 each refer to enzymes with a signal peptide; SEQ ID NOs: 8-14 refer to enzymes without a signal peptide; SEQ ID NOs: 15-17 refer to protease enzymes without a propeptide. JPEG2024513114000003.jpg122170

[0089] As used herein, the "percentage of sequence identity" is determined by comparing two optimally aligned biological sequences (amino acid sequences or polynucleotide sequences) over a comparison window, where the corresponding sequence portions in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence that does not contain additions or deletions due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0090] The term "identical" or percent "identity" refers to two or more sequences or subsequences that are the same sequence, with respect to two or more nucleic acid or polypeptide sequences. Two sequences are "substantially identical" if they have a specified percentage of the same amino acid residues or nucleotides (i.e., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity over a specified region, or, if not specified, the entire reference sequence) when compared and aligned for maximum correspondence over a comparison window or specified region, using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection. The present disclosure provides polypeptides that are substantially identical to the polypeptides exemplified herein. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, in some cases at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, in some cases at least about 150, 200 or 250 amino acids in length, or over the entire length of the reference sequence. For shorter amino acid sequences, for example sequences of 20 or fewer amino acids, substantial identity exists if one or two amino acid residues are conservatively substituted in accordance with conservative substitutions as defined herein.

[0091] According to one embodiment, the method further comprises a step of solubilizing the efflorescent protein. The protein thus purified is transferred back into solution. This may be useful for applications where soluble protein is required, such as parenteral administration. For other applications, such as enteral administration, the efflorescent form of the protein may be used.

[0092] According to another aspect of the present invention, there is provided a pharmaceutical intermediate obtained by the method according to the above description. By general definition, a pharmaceutical intermediate is a raw drug or prodrug obtained by synthesis or biofermentation, which is used as a raw material for producing pharmaceutical preparations that can be administered to patients. For this purpose, the pharmaceutical intermediate must undergo further molecular transformation or processing.

[0093] In the context of the present invention, such intermediates are therefore defined as products obtained directly or indirectly by the process according to the above description, but which are not yet ready to be administered to a patient as such. In other words, they are used to prepare pharmaceutical preparations, which in some respects can also be called prodrugs. Such intermediates can be in solubilized form as described above, or in efflorescent (=crystallized) form.

[0094] Thus, according to one embodiment, the pharmaceutical intermediate comprises a solubilized form of the efflorescent protein or the efflorescent protein itself.

[0095] According to another aspect of the present invention there is provided a purified protein or pharmaceutical intermediate obtained by the method according to the above description.

[0096] According to another aspect of the present invention, there is provided a purified and crystallized protein or pharmaceutical intermediate comprising a ciliate-type glycosylation pattern. As discussed herein, ciliates produce proteins that are characterized by a very simple and uniform glycosylation pattern, which is unique compared to other taxa.

[0097] In particular, the ciliate glycosylation pattern is at least one of the following: No fucose No xylose -Mannose-poor No galactose No N-acetylneuraminic acid, and / or No N-glyclylneuraminic acid For an illustration, see Figure 1. Such proteins can be obtained, for example, by the methods described above.

[0098] A variant of any one of the above enzymes, intermediates or proteins, according to one embodiment of the invention, is or comprises at least one of the following: a) Tetrahymena lipase as set forth in any one of SEQ ID NOs: 1 to 3 or 8 to 10; b) Tetrahymena amylase as set forth in SEQ ID NO: 4 or 11; c) a Tetrahymena protease as set forth in any one of SEQ ID NOs: 5-7, 12-14 or 15-17, or d) (i) a variant of any one of the above enzymes having at least 90% amino acid sequence identity with any one of the above enzymes, wherein said variant retains enzymatic function, and / or (ii) a variant of any one of the above enzymes having one to three amino acid residues removed or added at the N-terminus and / or C-terminus.

[0099] According to another embodiment of the invention, the intermediate or protein is or comprises an enzyme selected from the group shown in the table below: JPEG2024513114000004.jpg46170

[0100] These enzymes are used in oral enzyme replacement therapy and, similar to pancreatic enzyme replacement therapy, are required in large quantities, making cost-effective expression and purification techniques, such as the crystallization process described here, advantageous.

[0101] According to one embodiment of the invention, the intermediate or protein is provided in crystalline form.

[0102] According to one embodiment of the invention, the intermediate or protein has a purity grade of 80% or greater.

[0103] According to one embodiment of the present invention, the protein crystals of the intermediate or protein have a length between 2 and 1000 μm.

[0104] According to one embodiment of the invention, the protein crystals of the intermediate or protein have a diameter between 0.05 μm and 10 μm.

[0105] According to another aspect of the present invention there is provided a pharmaceutical formulation comprising an intermediate or a protein according to the above description, optionally further comprising one or more pharma- ceutically acceptable excipients.

[0106] According to some embodiments of the invention, the protein is an enzyme.

[0107] According to a further embodiment of the invention, the enzyme is at least one selected from the group consisting of: Lipase Proteases ·amylase ·Glutenase Glutamine-specific cysteine ​​protease Prolyl endopeptidase Saccharase, and / or Lactase.

[0108] In a further embodiment, the enzyme a) Tetrahymena lipase as set forth in any one of SEQ ID NOs: 1 to 3 or 8 to 10; b) Tetrahymena amylase as set forth in SEQ ID NO: 4 or 11; c) a Tetrahymena protease as set forth in any one of SEQ ID NOs: 5-7, 12-14 or 15-17, or d) (i) a variant of any one of the above enzymes having at least 90% amino acid sequence identity with any one of the above enzymes, wherein the variant retains enzymatic function, and / or (ii) a variant of any one of the above enzymes having one to three amino acid residues removed or added at the N-terminus and / or C-terminus.

[0109] The modular assembly of lipase, protease and amylase activities allows the formulation to be adapted to patients with different pathologies and therefore different dosing requirements. For example, a high amylase content is undesirable for children with mucovishidosis. Proteases are contraindicated in patients with acute or active chronic pancreatitis. And fourthly, in contrast to fungal lipases, the formulation is activated by physiological concentrations of bile acids.

[0110] According to another aspect of the invention, an enzyme or pharmaceutical preparation as described above is provided for use (for the manufacture of a medicament) in the treatment of a human or animal subject diagnosed with, suffering from, or at risk of developing a disorder caused by enzyme deficiency or dysfunction.

[0111] This language is to be regarded as embracing both the Swiss-type claim language accepted in some countries (in which case the brackets are to be regarded as not including them) and the EPC 2000 language (in which case the brackets and any content within them are to be regarded as not including them).

[0112] According to another aspect of the present invention, there is provided a method for treating or preventing a disorder caused by an enzyme deficiency or dysfunction, the method comprising administering to a human or animal subject a therapeutically sufficient dose of an enzyme or pharmaceutical preparation according to the above description.

[0113] According to a further embodiment, the disorder caused by an enzyme deficiency or dysfunction is Lipid indigestion and / or digestive disorders Disorders caused by enzyme deficiency or dysfunction. Exocrine pancreatic insufficiency that may be due to: Chronic pancreatitis Cystic fibrosis Main pancreatic duct obstruction Pancreatic resection ·Gastrectomy ·Short bowel syndrome Hereditary hemochromatosis Celiac disease Zollinger-Ellison syndrome At least one selected from the group consisting of:

[0114] In one embodiment, in said use, the digestive disorder is exocrine pancreatic insufficiency (EPI), which may be caused, inter alia, by cystic fibrosis, pancreatic duct obstruction, or pancreatectomy.

[0115] According to another embodiment, the inflammatory condition is chronic inflammation of the pancreas (pancreatitis) or inflammatory bowel disease.

[0116] Other gastrointestinal disorders include steatorrhea, celiac disease, and indigestion.

[0117] EPI refers to the inability to properly digest food due to a lack of digestive enzymes produced by the pancreas. EPI is seen in people with cystic fibrosis and Shwachman-Diamond syndrome and is caused by the gradual loss of pancreatic cells that make digestive enzymes; the loss of digestive enzymes results in poor digestion and prevents nutrients from being absorbed through the normal digestive process. Chronic pancreatitis is the most common cause of EPI in humans.

[0118] Steatorrhea is the presence of excess fat in the stool. The stool may also be floating, appear oily, and have a particularly foul odor due to the excess lipids. Oily anal leakage and some degree of fecal incontinence may occur. Increased fat excretion may occur, as measured by measuring the level of fat in the stool. There is no standard definition of how much fecal fat constitutes steatorrhea.

[0119] Dyspepsia is a condition in which patients experience abdominal bloating, gas, and a feeling of fullness after a fatty meal. Because such symptoms are commonly associated with irritable bowel syndrome (IBS), some researchers have speculated that pancreatic enzymes might help treat IBS symptoms. However, no studies have been done.

[0120] In one embodiment, in said use, the lipid digestion disorder is lipoprotein lipase deficiency, a condition caused by a mutation in the gene encoding lipoprotein lipase.

[0121] According to yet another embodiment of the present invention there is provided the use of a combination of two or more lipase enzymes according to the above description, or a pharmaceutical preparation containing the latter, for the treatment of cystic fibrosis.

[0122] Cystic fibrosis is an inherited disease that causes the body to produce abnormally thick, sticky mucus. Patients often suffer from nutritional deficiencies because the mucus prevents pancreatic enzymes from reaching the intestine. Taking lipase can help these patients improve the nutrition they get from food.

[0123] According to a further embodiment, the disorder caused by an enzyme deficiency or dysfunction is Celiac Sprue Sucrose intolerance or hereditary sucrose isomaltase deficiency (GSID), and / or ·Lactose intolerance At least one selected from the group consisting of:

[0124] As noted elsewhere herein, these diseases can be advantageously treated by oral enzyme replacement therapy using the respective enzymes produced by the methods disclosed herein.

[0125] According to another aspect of the present invention there is provided a dosage unit comprising a protein or pharmaceutical preparation according to the above description.

[0126] In one embodiment, such dosage units are provided in a form suitable for oral administration.

[0127] In some embodiments, the dosage unit is a capsule, tablet, or sachet. EXAMPLES

[0128] While the present invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered as illustrative or exemplary and not restrictive, and the present invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0129] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'. 1. Crystallization of T. thermophila lipase using ammonium sulfate Materials and Methods Culture medium Standard Complex Medium Paromomycin concentration for fermentation: 2 μg / ml paromomycin buffer Ammonium sulfate stock solution 20mM Phosphate-Citrate 4M Ammonium Sulfate pH 6.0 Washing buffer 20mM Phosphate-Citrate 0.7M Ammonium Sulfate pH 7.0 Citrate / Phosphate Buffer 20mM Phosphate-Citrate pH 7.0 machine: JPEG2024513114000005.jpg115170 Software JPEG2024513114000006.jpg24170

[0130] Transformation For the generation of the cytosubstrate (Tt_pAX_hn_MTT1_Ttherm_00320120_K12), the episomal vector pAX_hn_MTT1_Ttherm_00320120 (provided by Cilian AG) was inserted into the conjugative parental cells T. thermophila B 1868.7 and T. thermophila SB 1969 by the method of biological transformation, which was performed as previously described in Cassidy-Hanley et al. (1997).

[0131] Cell Culture / Fermentation Batch fermentation of T. thermophila was carried out at a 600-liter scale using a Techfors T900 fermenter (Infors, Bottmingen-Basel, Switzerland). 3 Cells / ml were inoculated and cultured in SPP medium. Temperature was maintained at 30°C, and pO2 was controlled at 20% of air saturation by agitator speed (50-105 rpm) and air flow rate (0.1-0.3 vvm). pH was adjusted to 7.0 during fermentation. 10 μg / ml cadmium chloride was added to the logarithmic phase culture to induce lipase expression.

[0132] Collection and Filtration / USP 24 hours after induction, a hollow fiber module (Spectrum PES, lumen 0.5 mm, area 2.9 m) was 2From the 600 l culture, 550 l of supernatant was obtained by filtration using a 0.5 μm cutoff filter (Spectrum Laboratories Inc., Rancho Dominguez, CA, USA). The filtrate was concentrated to 6 l using a Sartocon Slice Hydrosart tangential flow cassette (30 kDa cutoff; Sartorius, Göttingen, Germany). After concentration, the concentrate was washed with buffer (citrate-phosphate, pH 7.0) and immediately frozen at -80 °C.

[0133] Crystallization experiments Lipase protein crystallization was performed by adding appropriate crystallization reagents to the protein concentrate. The protein concentration of the concentrated and diafiltered fermentation broth was 46.69 mg / ml. The concentrate was mixed with ammonium sulfate stock solution to a final ammonium sulfate concentration of 0.3 M (Figure 1). The suspension was incubated at room temperature for 20 h. The ammonium sulfate concentration of the suspension was then increased to 1 M. The prepared suspension was further incubated at room temperature for 2 h. The crystallization broth was centrifuged at 1,900 x g for 10 min (Sorvall Lynx 6000 Centrifuge; Thermo Scientific TM The supernatant was discarded and the remaining crystalline pellet was laminated and loosened in wash buffer. The suspension was centrifuged again at 1,900 x g for 10 min. In all, the crystalline pellet was washed twice and the supernatant was discarded. A final centrifugation was performed at 2,500 x g for 20 min. The wet crystalline pellet was stored at -20 °C for further investigation.

[0134] Micrographs were performed using a Motic microscope AE2000 and the corresponding evaluation software Motic Images Plus 3.0. 20 μl of the crystal suspension was transferred onto a microscope slide and covered with a glass slide. The prepared suspension was visually inspected with several magnifications (10x, 20x, 40x). Length and width were measured using the evaluation software.

[0135] Rhodamine assay Lipolytic activity was measured based on the rhodamine-triglyceride-agarose assay published by Jette et al. In contrast to the original protocol, the triglyceride-agarose mixture is replaced by olive oil as substrate. Active lipase breaks down triglycerides into glycerol and free fatty acids. These fatty acids bind to rhodamine B and can be detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The samples must be diluted to allow acceptable quenching. The thus diluted samples are mixed with the rhodamine mixture in olive oil in a microtiter plate. Afterwards, the samples were incubated for 20 min at 30 °C in a microtiter plate reader (Synergy H1; BioTek Instruments, Inc). Fluorescence was measured every 50 s for 20 min. Finally, the measured activity was calculated according to the activity of the reference standard.

[0136] Lambert-Beer assay Assuming high purity of the lipase protein, the protein concentration was determined based on Beer-Lambert law. The absorbance was measured at 280 nm, and the protein concentration was calculated based on equation (i). The extinction coefficient was determined using the in silico prediction tool Protparam. JPEG2024513114000007.jpg6170 expression 1:ε λ = molar absorptivity; c=molar concentration; d=layer thickness

[0137] 2. Crystallization of α-amylase from T. thermophila using PEG 4000 a-Amylase from T. thermophila (TTHERM_00411610) was crystallized from an amylase-rich protein concentrate (95 g / L) buffered with 20 mM phosphate-citrate (pH 7) obtained by fermentation of recombinant T. thermophila with PEG 4000 and subsequent DSP. Crystallization was initiated by slowly adding 25% (v / v) PEG 4000 stock solution (40% (w / v) PEG 4000 in 20 mM phosphate-citrate buffer, pH 7) to the gently stirred amylase concentrate, resulting in a final PEG 4000 concentration of 6% (w / v). After the solution was properly mixed, the mixture was incubated at room temperature (20-22 °C) for 93 h without stirring. Samples were removed every day and examined under a microscope for the formation of crystals. No crystallization was observed during the first 3 days, but after 93 h, large needle-shaped crystals up to about 650 μm in length and 5 μm in width were observed (Figures 11 and 12). Crystals were pelleted by centrifugation (10 min, 1500 rcf, RT) and redissolved in H2O. The crystal solution and centrifugation supernatant were assayed for amylase activity using the Phadebas Amylase Test (Phadebas AB, Kristianstad, Sweden) according to the manufacturer's recommendations. Approximately 67% of the total amylase activity was found in the degraded crystal pellet, and approximately 33% remained in the supernatant.

[0138] 3. Crystallization of T. thermophila lipase using sodium dihydrogen phosphate buffer NaH2PO4 stock solution 20 mM K2HPO4 2.5 M NaH2PO4 pH 7.0 Lysis buffer 10 mM K2HPO4 pH 7.0

[0139] The harvested cell culture fluid (HCCF) containing overproduced Tetrahymena lipase 120 was filtered to remove undissolved components and concentrated to 55 g / L (approach 1), 40 g / L (approach 2), 25 g / L (approach 3) and 16 g / L (approach 4). The four samples were adjusted to final concentrations of 0.3 M NaH2PO4 (approach 1), 0.5 M NaH2PO4 (approach 2 and 3) and 0.7 M NaH2PO4 (approach 4). After 25 h, the NaH2PO4 concentration was increased to 0.5 M (approach 1), 0.75 M (approach 2) and 1 M (approach 3 and 4). After 48 h, the concentration in approaches 3 and 4 was increased to 1.25 M. The final samples were taken after 115 h. To determine the ratio of crystallized to uncrystallized lipase, samples were taken after 25, 48, and 115 hours and centrifuged (10,000 rcf, 5 min). The total lipase activity in the supernatant represents uncrystallized lipase, and the total lipase activity in the redissolved pellet represents crystallized lipase. The results are shown in Figures 13 and 14.

[0140] References: JPEG2024513114000008.jpg169170

[0141] array The following sequences form part of the disclosure of this application. A WIPO ST 25 compatible electronic sequence listing is also provided with this application. For the avoidance of doubt, in case of any discrepancy between a sequence in the table below and a sequence in the electronic sequence listing, the sequence in this table shall be considered correct. Each of SEQ ID NOs: 1 to 7 refers to an enzyme with a signal peptide. SEQ ID NOs: 8 to 14 (not shown in this list but shown in the electronic sequence listing) refer to an enzyme without a signal peptide. SEQ ID NOs: 15 to 17 (not shown in this list but shown in the electronic sequence listing) refer to a protease enzyme without a signal peptide. JPEG2024513114000009.jpg233170JPEG2024513114000010.jpg67170

Claims

1. A method for purifying a protein from harvested cell culture fluid (HCCF), wherein the protein is produced by homologous or heterologous expression in a ciliate host, and the harvested cell culture fluid has not been purified by chromatography; The method a) incubating the harvested cell culture with a cosmotropic agent; b) efflorescing the protein by the formation of crystals; and c) Optionally, harvesting the efflorescent protein. Including, The method does not include a step of cross-linking the protein before or after efflorescence.

2. The method of claim 1 , wherein the protein is a glycosylated protein.

3. Cosmotropic agent ammonium sulfate, sodium dihydrogen phosphate, and / or Polyethylene glycol The method of claim 1 , wherein the at least one of

4. 2. The method of claim 1, wherein the harvested cell culture fluid contains from 3 to 200 g / l of protein.

5. To effloresce proteins, The pH in the medium is set to 5 or higher and 8 or lower, and / or - The temperature is set to 10°C or higher and 40°C or lower. The method of claim 1.

6. The method according to claim 1, wherein the kosmotropic salt concentration is set in the range of 50 to 2500 mM for incubation of the harvested cell culture fluid.

7. The method according to claim 1, wherein the polyhydric alcohol concentration is set in the range of 2 to 25 w / v % for incubation of the harvested cell culture fluid.

8. 2. The method of claim 1, wherein the incubation of the harvested cell culture medium with the cosmotropic agent is carried out for a period of at least 10 minutes and not more than 36 hours.

9. 2. The method of claim 1, wherein the pre-harvested cell culture fluid is subjected to one or more filtration and / or centrifugation steps prior to step a) to obtain the harvested cell culture fluid.

10. 2. The method of claim 1, wherein the ciliate host is Tetrahymena sp.

11. The homologous or heterologous expression - the metallothionein gene (MTT1) inducible promoter, and / or - Heat-inducible promoter The method of claim 1 , wherein the control is

12. The method of claim 1 , wherein the protein is an enzyme.

13. Protein, Lipase Protease ·amylase ・Glutenase Glutamine-specific cysteine ​​protease Prolyl endopeptidase Saccharase, and / or Lactase The method of claim 12, wherein the at least one selected from the group consisting of:

14. The enzyme, a) a Tetrahymena lipase according to any one of SEQ ID NOs: 1 to 3 or 8 to 10; b) Tetrahymena amylase according to SEQ ID NO: 4 or 11; c) a Tetrahymena protease according to any one of SEQ ID NOs: 5 to 7, 12 to 14 or 15 to 17, or d) (i) a variant of any one of the above enzymes having at least 90% amino acid sequence identity with any one of the above enzymes, wherein said variant retains enzymatic function, and / or (ii) a variant of any one of the above enzymes having one to three amino acid residues removed or added at the N-terminus and / or C-terminus. The method of claim 12, wherein 15. The method of claim 1, further comprising a step of solubilizing the efflorescent protein.

16. A pharmaceutical intermediate obtained by the method according to claim 1.

17. 17. The pharmaceutical intermediate of claim 16, comprising a solubilized form of the efflorescent protein or the efflorescent protein itself.

18. A purified protein obtained by the method of claim 1.

19. A purified and crystallized protein or pharmaceutical intermediate containing a ciliate-type glycosylation pattern.

20. 20. An intermediate according to any one of claims 16 or 17 or a protein according to any one of claims 18 or 19, which is or comprises at least one of the following: a) a Tetrahymena lipase as set forth in any one of SEQ ID NOs: 1-3 or 8-10; b) Tetrahymena amylase as set forth in SEQ ID NO: 4 or 11; c) a Tetrahymena protease as set forth in any of SEQ ID NOs: 5-7, 12-14 or 15-17; or d) (i) a variant of any one of the above enzymes having at least 90% amino acid sequence identity with any one of the above enzymes, wherein said variant retains enzymatic function, and / or (ii) a variant of any one of the above enzymes having one to three amino acid residues removed or added at the N-terminus and / or C-terminus.

21. 20. The intermediate of claim 16 or 17 or the protein of claim 18 or 19, provided in crystalline form.

22. 20. The intermediate of any one of claims 16 or 17 or the protein of any one of claims 18 or 19, having a purity of 80% or more.

23. An intermediate described in any one of claims 16 or 17 or a protein described in any one of claims 18 or 19, wherein the protein crystal has a length of 2 or more and 1000 μM or less.

24. A pharmaceutical formulation comprising an intermediate according to claim 16 or 17 or a protein according to claim 18 or 19, and optionally further comprising one or more pharma- ceutically acceptable excipients.

25. 25. The pharmaceutical formulation of claim 24, wherein the protein is an enzyme.

26. The enzyme, Lipase Protease ·amylase ・Glutenase Glutamine-specific cysteine ​​protease Prolyl endopeptidase Saccharase, and / or Lactase 26. The pharmaceutical formulation of claim 25, which is at least one selected from the group consisting of:

27. 27. The pharmaceutical formulation of claim 26, wherein the enzyme is or comprises: a) a Tetrahymena lipase according to any one of SEQ ID NOs: 1 to 3 or 8 to 10; b) Tetrahymena amylase according to SEQ ID NO: 4 or 11; c) a Tetrahymena protease according to any one of SEQ ID NOs: 5 to 7, 12 to 14 or 15 to 17, or d) (i) a variant of any one of the above enzymes having at least 90% amino acid sequence identity with any one of the above enzymes, wherein said variant retains enzymatic function, and / or (ii) a variant of any one of the above enzymes having one to three amino acid residues removed or added at the N-terminus and / or C-terminus.

28. 20. Use of a protein according to claim 18 or 19 in the manufacture of a medicament for the treatment of a human or animal subject diagnosed with, suffering from or at risk of developing a disorder caused by enzyme deficiency or dysfunction, or for the prevention of such a condition.

29. 27. The pharmaceutical formulation of claim 26 for the treatment or prevention of a disorder caused by an enzyme deficiency or dysfunction.

30. Disorders caused by enzyme deficiency or dysfunction Lipid indigestion and / or digestive disorders - Disorders caused by enzyme deficiency or dysfunction - Exocrine pancreatic insufficiency, the causes of which may be: ・Chronic pancreatitis Cystic fibrosis - Main pancreatic duct obstruction Pancreatic resection ・Gastrectomy ・Short bowel syndrome Hereditary hemochromatosis Celiac disease Zollinger-Ellison syndrome ・Celiac Sprue - sucrose intolerance or hereditary sucrose isomaltase deficiency (GSID), and / or ・Lactose intolerance 30. The pharmaceutical formulation of claim 29, which is at least one selected from the group consisting of:

31. 27. A dosage unit comprising the pharmaceutical formulation of claim 26.

Images